chowdhury, n. s. (2014) effect of probiotics on the regulation of the

Effect of Probiotics on the Regulation of the Inducible NitricOxide Synthase (iNOS) Pathway in Cultured Cells

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Authors Chowdhury, N. S. (Nasima Sultana)

Citation Chowdhury, N. S. (2014) Effect of Probiotics on theRegulation of the Inducible Nitric Oxide Synthase (iNOS)Pathway in Cultured Cells. Unpublished PhD Thesis.Teesside University

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Effect of Probiotics on the Regulation of the Inducible Nitric Oxide Synthase (iNOS)

Pathway in Cultured Cells

Nasima Sultana Chowdhury

A Thesis Submitted to the Teesside University in Partial Fulfilment of the Requirements of the Degree of

Doctor of Philosophy

June 2014

iii

Abstract

Inducible nitric oxide synthase (iNOS)-induced nitric oxide (NO) and its reactive

metabolite peroxynitrite (ONOO־) has been implicated as an important mediator in

the pathogenesis of inflammatory bowel disease (IBD). Immune cells

macrophages produce NO and ONOO־ to provide the host defence and play

important roles in the regulation of inflammatory response. Several in vitro and In

vivo studies have reported the role of probiotics and probiotic-released metabolites

in the treatment and prevention of IBD. The study was therefore started with the

hypothesis that the metabolites released by probiotics present in the formulation

VSL#3 may have the effect on the pro-inflammatory mediator NO and its

regulatory enzyme iNOS in the immune cells.

Murine macrophage J774 cells were used in the study as these cells are readily

induced by bacterial lipopolysaccharide (LPS) and produce pro-inflammatory

mediators nitric oxide (NO) and prostaglandin E2 (PGE2) by the expression of

enzymes iNOS and cyclooxygenase-2 (COX-2) respectively through the Nuclear

Factor-κappa B (NF-B) signalling pathway. The cells were exposed to VSL#3-

conditioned medium (VSL#3-CM) in the absence and presence of LPS and the

inhibitors of a number of enzymes and signalling molecules. To exclude the

possibility of endotoxin contamination in VSL#3-CM, experiments were also

carried out in the absence and presence of an endotoxin neutralising compound

polymyxin B (PmB).

VSL#3-CM increased the basal NO production through the induction of iNOS on a

concentration dependent manner. On the other hand, it suppressed the LPS-

induced NO production but caused slight inhibition on the LPS-induced iNOS

expression. PmB suppressed the LPS-induced NO production but not that caused

by VSL#3-CM suggests the effects of the later were not LPS mediated. Similarly,

VSL#3-CM at a higher concentration induced the basal COX-2. However, it

caused slight inhibition on the LPS-induced COX-2 expression.

iv

Dexamethasone has partially inhibited the VSL#3-CM-induced NO and iNOS

expression. Bisindolylmaleimide (BIM) and SB203580, inhibitor of protein kinase C

(PKC) and p38 MAPK, a member of the mitogen activated protein kinases

(MAPKs) family respectively, have markedly inhibited VSL#3-CM-induced iNOS

expression and NO production. Slight inhibition was made by LY2904002 and

CAY10470, inhibitors of Phosphatidylinocitol 3-kinase (PI3K) and NF-B

respectively. Akt inhibitor XIII and MG132 the inhibitors of the protein kinase B

(Akt) and proteasome showed no inhibition on VSL#3-CM-induced iNOS and NO

production.

A balanced expression of iNOS and NO production is necessary for the regulation

of normal cellular functions. In this study, it has been shown that VSL#3 has the

ability to regulate the function of iNOS and thus the over production of NO in

presence of LPS. This action may explain, in part, the proposed anti-inflammatory

effects of VSL#3 and thus its potential benefits in IBD. Interestingly, VSL#3 may

also induce iNOS expression and NO production through the activation of PKC,

p38 MAPK and PI3K pathways but only when applied alone under control

conditions. Whether this is specific to macrophages or a common effect of VSL#3

is currently not clear. It would not be considered normal however to recommend

VSL#3 for routine use in healthy individuals. The ability of VSL#3 to downregulate

the expression and function of iNOS induced by endotoxin however, provides a

good rational for its use in inflammatory disease states including IBD.

v

I dedicate this thesis to my loving parents

Md. Siraj Uddin Chowdhury

&

Mrs. Sara Taifura Chowdhury (Leena).

Their inspiration, continuous support, encouragement and enthusiasm for

education, equally for boys and girls, gave me the determination and strength to enter post-graduate study leading to PhD Degree

vii

Acknowledgement

In the Name of Allah, the Most Gracious, the Most Merciful.

I would like to thank my Director of Studies Prof. Janey Henderson for her

suggestions, help and co-operation during my research, and with the submission

of the Thesis.

I am most grateful and indebted to my Second Supervisor Prof. Anwar R Baydoun

for his kindness, and help all through the study, and for having me in his laboratory

at the University of Hertfordshire. It would have never been possible for me to

come to this stage of my research without his guidance, support, enthusiasm and

patience.

I thank Dr. Mosharraf Sarker for introducing me to the Teesside University and

helping me to develop the Cell Culture Laboratory at the Teesside University. I am

also thankful to Dr. Shahrzad Connolly for giving me the mental support and

strength at some most difficult moments in the early years of my research work.

I very much appreciate Dr. Shori Thakur for her friendly support and advice in the

laboratory at the University of Hertfordshire.

My special thanks go to Dr. Jagadish Chakraborty for his inspiring words from the

beginning of my study.

To my family – my parents, brothers Rafiq Chowdhury and Hedayet Chowdhury,

sisters Nilufa Chowdhury and Shamima Chowdhury, brother-in-laws Badrul Alam

and Towhidur Rahman Siddique, and my lovely sister-in-law Farida Yeasmeen

Neela – your endless love and support brought me this far; your continuous

inspiration strengthened my resolution to undertake postgraduate research in the

UK and keep going to this day.

I have no words to thank my husband Dr. Sk Masood Ahmed for his patience and

understanding, especially during the last few trying years. Without his support I

would not have been able to continue my research work and bring it to completion.

To my loving son Wahid Masood – I hope, one day you will appreciate why we

both had to miss so much in your childhood.

viii

I would like to thank my Uncle Dr. Lutfor Rahman and Aunt Mrs. Ruh Afza

Rahman for their help during my study in the UK, my teacher Prof. Shahabuddin

Kabir Choudhuri and my friends for their words of encouragement from far away

Bangladesh. I appreciate Mr Shariful Islam, his wife Mrs Momotaj Islam Shoma

and Mr Sheikh Abdul Hamid for their kindness from the very first day of our arrival

in Middlesbrough. I would like to thank Mr Mamun Rashid for his friendly support

during my stay in Middlesbrough.

The friendship and collaboration of my fellow students in Prof Baydoun‟s

laboratory, Peter, Arturo, Ashish, Rahul, Edmund, Tamer, Marzieh, Ashish, Hema,

Iffat, Proveen, Anupama, Pablo, Nimer, Mahdi made my time in that laboratory

enjoyable and fruitful – thank you all.

Finally, I am grateful to both the Teesside University and the University of

Hertfordshire for support, making their facilities available for my research, and to

their staff for all help concerning my work.

ix

Table of Contents

xi

Contents Page No

Abbreviations xxxi

Chapter -1 ...................................................................................................... 1

1.0. Introduction .............................................................................................. 5

1.1. Probiotics ................................................................................................. 5

1.2. Concept of Probiotics ............................................................................... 5

1.3. Functions of probiotics ............................................................................. 6

1.4. Inflammatory bowel disease (IBD) ........................................................... 6

1.5. Gastro-intestinal tract ............................................................................... 9

1.5.1. Intestinal microbial ecology ................................................................... 9

1.5.2. Immunity and intestinal epitheliu ........................................................... 9

1.6. How does probiotic work in intestinal immunity ........................................ 12

1.7. Probiotic-mediated intra-cellular signalling ............................................... 14

1.8. Use of Probiotics in clinical practice ......................................................... 17

1.9. VSL#3 ...................................................................................................... 18

1.10. Role of Probiotics in inflammatory bowel disease (IBD) ........................ 22

1.11. Inflammation .......................................................................................... 23

1.12. Role of Macrophages in Inflammation ................................................... 24

1.13. Nitric oxide (NO) and its chemical properties ......................................... 28

1.13.1. Biological properties of nitric oxide (NO) ............................................. 28

1.13.2. Biosynthesis of nitric oxide (NO) ......................................................... 30

1.13.3. Nitric oxide synthases (NOS) .............................................................. 31

1.14. Role of nitric oxide (NO) in immune response ........................................ 33

1.15. Role of Cyclooxygenase-2 and prostaglandin E2 in immune

response ................................................................................................ 34

1.16. Role of iNOS and COX-2 in inflammatory bowel disease (IBD) ............ 35

xii

1.17. Role of Probiotics in cancer ................................................................... 36

1.18. Aims of the project ................................................................................. 38

Chapter – 2 ..................................................................................................... 39

2.0. Materials and methods ............................................................................. 41

2.1. Culture of probiotic bacteria ..................................................................... 41

2.2. VSL#3 ...................................................................................................... 42

2.2.1. Construction of bacterial growth curve ........................................... 42

2.3. Preparation of Sample ............................................................................. 43

2.3.1. Freeze-dried probiotic culture supernatant ..................................... 43

2.3.2. Probiotic-conditioned cell culture medium ...................................... 43

2.3.3. Sonicated probiotic in cell culture medium ..................................... 43

2.4. Cell culture ............................................................................................... 44

2.4.1 Culture and sub-culture of J774 …………………………………….. 44

2.4.2. Culture and Sub-culture of epithelial cells ...................................... 44

2.4.2.1. Trypsinisation of the cell monolayer .................................. 45

2.5. Plating of cells for experimentation .......................................................... 45

2.6. Determination of cell number ................................................................... 46

2.7. Optimisation of the activation of J774 cells .............................................. 46

2.8. Measurement of total nitrite using Griess assay ...................................... 47

2.8.1. Preparation of nitrite standard curve using sodium nitrite

(NaNO2) ........................................................................................ 49

2.8.2. Procedure ..................................................................................... 50

2.9. Interference of bacterial culture medium (MRS broth) on the

Griess assay ............................................................................................ 51

xiii

2.10. Measurement of total protein in cell lysates using bicinchoninic

acid (BCA) protein assay ....................................................................... 52

2.10.1. Preparation of protein standard curve using bovine Serum

albumin (BSA) ............................................................................ 53

2.10.2. Procedure .................................................................................. 54

2.11. Detection of protein expression by Western blot assay ........................ 55

2.11.1. Preparation of gel ...................................................................... 55

2.11.2. Separation of protein ................................................................. 56

2.11.3. Transfer of protein from gel to membrane ................................. 56

2.11.4. Immunoblotting and enhanced chemiluminescence detection

of proteins ................................................................................... 56

2.11.5. Role of β-actin in Western blot assay ........................................ 57

2.11.6. Quantification of western blots by scanning densitometry ........ 57

2.12. Treatment of J774 cells with probiotics and measurement of nitric

oxide (NO) production followed by the detection of inducible nitric

oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression ..... 58

2.13. Detection of Nitrite in human intestinal epithelial cell culture

medium ................................................................................................ 59

2.13.1. Conversion of nitrate to nitrite ................................................... 59

2.14. Determination of cell viability and cytotoxicity by MTT assay ................ 60

2.15. Treatment of cells with the inhibitors of various signalling

pathways .............................................................................................. 61

2.16. Statistical analysis ................................................................................. 61

xiv

Chapter – 3 ................................................................................................... 63

3.0. Results - Development of in vitro inflammatory model using human

intestinal epithelial cell lines ................................................................... 65

3.1. Introduction ............................................................................................ 67

3.2. Methods ................................................................................................. 68

3.2.1. Activation of cells with individual and combinations of different

cytokines ....................................................................................... 68

3.2.2. Detection of total NO production in cadmium treated culture

medium ......................................................................................... 68

3.2.2.1. Standardization of the cadmium catalysed conversion of

nitrate to nitrite ................................................................ 68

3.3. Results ................................................................................................... 70

3.3.1. Activation of HT-29 cells with lipopolysaccharide (LPS),

interferon- (IFN-) interleukin-1 (IL-1) and tumour necrosis

factor- (TNF-) alone and in combinations .................................. 70

3.3.2. Activation of Caco-2 cells with lipopolysaccharide (LPS) alone

and in combination with tumour necrosis factor- (TNF- 75

3.3.3. Activation of SW-620 cells with lipopolysaccharide (LPS) alone

and in combinations with tumour necrosis factor- (TNF- 77

3.3.4. Activation of Hela cells with different concentration of

lipopolysaccharide (LPS) alone and in combinations with tumour

necrosis factor- (TNF- 79

3.3.5. Conversion of nitrate to nitrite in cadmium treated sodium

nitrate solution ............................................................................... 81

3.3.6. Effects of LPS and TNF- on nitrite production in SW-620

cells ............................................................................................... 84

xv

3.3.7. Effects of LPS on nitrite production in Hela cells ........................... 85

3.3.8. Effects of LPS and TNF- on nitrite production in Hela

cells ............................................................................................... 86

3.4. Discussion ............................................................................................... 87

Chapter – 4 .................................................................................................... 93

4.0. Results - The effect of probiotic culture supernatant and probiotic-

conditioned medium on nitric oxide production and iNOS/COX-2

expression in J774 macrophages ............................................................ 95

4.1. Introduction .............................................................................................. 97

4.2. Methods ................................................................................................... 98

4.2.1. Construction of the bacterial growth curve ..................................... 98

4.2.2. Preparation of Sample ................................................................... 98

4.2.2.1. Freeze-dried LGG culture supernatant ............................ 98

4.2.2.2. Probiotic conditioned cell culture medium ........................ 98

4.2.3. MTT assay ..................................................................................... 98

4.2.4. Activation of macrophages J774 by LPS ....................................... 99

4.2.5. Effects of MRS broth on the sensitivity of the Griess assay ........... 99

4.3. Results ..................................................................................................... 100

4.3.1. Growth phases of bacteria and change in pH ....................................... 100

4.3.2. Probiotic conditioned cell culture medium ............................................. 103

4.3.3. Effect of Freeze-dried MRS broth and Lactobacillus rhamnosus GG

(LGG) culture supernatant (collected from the log phase) on basal

and lipopolysaccharide (LPS)-induced nitric oxide (NO) production

in J774 cells ........................................................................................... 105

xvi

4.3.4. Effect of Freeze-dried MRS broth and Lactobacillus rhamnosus GG

(LGG) culture supernatant (collected from the stationary phase)

on basal and lipopolysaccharide ........................................................... 107

4.3.5. Effects of MRS broth on the sensitivity of the Griess assay ................. 109

4.3.6. Effect of MRS broth on the viability of J774 Macrophages ................... 111

4.3.7. Effects of LGG-CM on basal and lipopolysaccharide (LPS)-induced

nitric oxide (NO) production in J774 macrophages ............................... 113

4.3.8. Effects of LGG-CM medium on basal and lipopolysaccharide

(LPS)-induced cyclooxynase-2 (COX-2) expression in J774

macrophages ....................................................................................... 115

4.3.9. Effect of VSL#3-CM medium on basal and lipopolysaccharide (LPS)-

induced nitric oxide (NO) production and inducible nitric oxide

synthase (iNOS) expression in J774 macrophages .............................. 117

4.3.10. Effects of VSL#3-conditioned medium on basal and

lipopolysaccharide (LPS)-induced cyclooxynase-2 (COX-2)

expression .......................................................................................... 119

4.4. Discussion ............................................................................................... 121

Chapter – 5 .................................................................................................... 125

5.0. Results - Investigation of the signalling pathways involved in

VSL#3-CM-induced NO production and iNOS expression

in J774 Macrophages ............................................................................ 127

5.1. Introduction ............................................................................................. 129

5.2. Methods .................................................................................................. 132

xvii

5.2.1. Preparation of VSL#3-conditioned cell culture Medium

(VSL#3-CM) ................................................................................... 132

5.2.2. Preparation of sonicated probiotic in cell culture medium ............. 132

5.2.3. Cell viability assay - MTT assay .................................................... 132

5.2.4. Treatment of J774 cells with probiotics and measurement of

nitrite production followed by the detection of inducible nitric

oxide synthase (iNOS) and cyclooxigenase-2 (COX-2)

expression ..................................................................................... 133

5.2.5. Treatment of cells with the antibiotic polymysxin B (PmB) ............ 133

5.2.6. Treatment of cells with the inhibitors of various signalling

pathways ...................................................................................... 133

5.3. Results 135

5.3.1. Effects of VSL#3-CM on nitric oxide (NO) production in

non-stimulated and lipopolysaccharide (LPS)-stimulated J774

macrophages ................................................................................ 135

5.3.2. Effects of VSL#3-CM on inducible nitric oxide synthase (iNOS)

expression in basal and lipopolysaccharide (LPS)-stimulated J774

macrophages ................................................................................. 137

5.3.3 Effects of VSL#3-CM on cyclooxigenase-2 (COX-2) expression in

basal and lipopolysaccharide (LPS)-stimulated J774

macrophages ................................................................................. 139

5.3.4. Effect of Polymyxin B (PmB) on VSL#3-CM-induced nitric oxide

(NO) production in J774 macrophages ......................................... 141

5.3.5. Effect of VSL#3-CM on the viability of J774 macrophages ............ 143

5.3.6. Comparison of the effects of VSL#3-CM and sonicated bacteria on

basal nitric oxide (NO) production and inducible nitric oxide

xviii

synthase (iNOS) expression in J774 macrophages ....................... 144

5.3.7. Effect of dexamethasone on VSL#3-CM-induced nitric oxide

(NO) production and inducible nitric oxide synthase (iNOS)

expression in J774 macrophages ................................................. 147

5.3.8. Effect of dexamethasone on the cell viability ................................. 149

5.3.9. Role of the enzyme protein kinase C (PKC) on VSL#3-CM-induced

nitric oxide (NO) production and inducible nitric oxide synthase

(iNOS) expression in J774 macrophages ...................................... 150

5.3.10. Effect of protein kinase C (PKC) inhibitor bisindolylmaleimide

(BIM) on the cell viability ............................................................. 154

5.3.11. Role of phosphatidylinocitol 3-kinase (PI3K) on VSL#3-CM-induced


(iNOS) expression in J774 macrophages .................................... 155

5.3.12. Effect of phosphatidylinocitol 3-kinase (PI3K) inhibitor LY294002

on the cell viability ........................................................................ 158

5.3.13. Effect of protein kinase B (AKT) inhibitor on VSL#3-CM-induced


(iNOS) expression in J774 cells .................................................. 159

5.3.14. Effect of AKT inhibitor XIII on the cell viability ............................. 162

5.3.15. Role of mitogen-activated protein kinase enzyme p38 MAPK on

VSL#3-CM-induced nitric oxide (NO) production and inducible

nitric oxide synthase (iNOS) expression in J774 macrophages .... 163

5.3.16. Effect of P38 inhibitor SB203580 on the cell viability ................... 166

5.3.17. Role of Proteasome on VSL#3-CM-induced nitric oxide (NO)

production and inducible nitric oxide synthase (iNOS) expression

in J774 macrophages .................................................................. 167

xix

5.3.18. Effect of Proteasome inhibitor MG132 on the cell viability .......... 170

5.3.19. Role of nuclear factor kappa B (NFκB) signalling pathway on

VSL#3-CM-induced nitric oxide (NO) production and inducible

nitric oxide synthase (iNOS) expression in J774 macrophages ... 171

5.3.20. Effect of NFκB inhibitor CAY10470 on the cell viability ................ 174

5.3.21. Effect VSL#3-CM on basal and lipopolysaccharide (LPS) induced


(iNOS) expression in rat aortic smooth muscle cells (RASMC) .... 175

5.4. Discussion ............................................................................................... 177

Chapter – 6 ................................................................................................... 187

6.0. General discussion ................................................................................ 189

6.1. Summary and conclusion ...................................................................... 201

6.2. Future work ........................................................................................... 205

Chapter – 7 .................................................................................................. 207

7.0. References ............................................................................................ 209

xxi

List of Figures

xxiii

Contents Page No

Figure 1: Functions of probiotics - protective, structural and metabolic

effects on the intestinal mucosa ..................................................... 7

Figure 2: Immunosensory detection of intestinal bacteria .............................. 11

Figure 3: Role of NO in the killing of microbes by macrophages ................... 25

Figure 4: The binding of LPS to the Toll-like receptor (TLR) present on

the cell membrane leads to the activation of the intracellular

signalling cascades ......................................................................... 27

Figure 5: Regulatory, protective and deleterious biological effects of

nitric oxide (NO) ............................................................................. 29

Figure 6: Schematic presentation of nitric oxide (NO) generation from

L-arginine ........................................................................................ 31

Figure 7: Monolayer of macrophages J774 cells prior to treatment with

drug ................................................................................................. 47

Figure 8: Chemistry of the Griess Reaction .................................................... 48

Figure 9: A representative standard curve of Sodium Nitrite ......................... 50

Figure 10: Reaction of BCA associated with the detection of protein

concentration in the cell lysates .................................................... 52

Figure 11: A representative standard curve of protein (BSA) ......................... 54

Figure 12: Metabolism of MTT to purple formazan salt by the cleavage

of the tetrazolium ring by mitochondrial reductase in viable

cells ................................................................................................ 61

Figure 13: Induction of iNOS in HT-29 cells and J774 macrophages ............ 72

Figure 14: Cytokine stimulated induction of iNOS in HT-29 cells ................... 74

Figure 15: LPS and TNF-α induced iNOS expression in Caco-2 cells ........... 76

Figure 16: LPS and TNF-α induced iNOS expression SW-620 cells .............. 78

xxiv

Figure 17: LPS and LPS-TNF-α induced iNOS expression in Hela cells ...... 80

Figure 18: Development of cadmium catalysed conversion of nitrate to

nitrite ............................................................................................. 82

Figure 19: Effects of LPS and TNF-α on nitrite production by SW-620

human intestinal epithelial cells .................................................... 84

Figure 20: Effects of LPS on nitrite production in Hela uterine

endometrial cells ........................................................................... 85

Figure 21: Effects of LPS and TNF-α on nitrite production in Hela

uterine endometrial cells ............................................................... 86

Figure 22: Bacterial growth curves and changes in pH with growth .............. 101

Figure 23: LPS-induced NO production and iNOS expression in J774

macrophages ................................................................................. 103

Figure 24: Effect of freeze-dried MRS broth and Lactobacillus rhamnosus

GG (collected from the log phase) on basal and

lipopolysaccharide (LPS)-induced NO production in J774

macrophage ................................................................................. 106

Figure 25: Effect of freeze-dried MRS broth and Lactobacillus rhamnosus

GG (collected from the stationary phase) on basal and

lipopolysaccharide (LPS)-induced NO production in J774

macrophage .................................................................................. 108

Figure 26: Effects of MRS broth on the sensitivity of the Griess assay .................... 110

Figure 27: Effect of MRS broth on viability of macrophage J774 cells ........... 111

Figure 28: Effects of LGG-CM on the basal and lipopolysaccharide

(LPS)-induced nitric oxide (NO) production in J774

macrophage ................................................................................. 113

xxv

Figure 29: Effects of LGG-CM on basal and LPS-induced cyclooxynase-2

(COX-2) expression in J774 macrophage .................................... 115

Figure 30: Effects of VSL#3-CM on basal and LPS-induced nitric oxide

(NO) production and inducible nitric oxide synthase (iNOS)

expression in J774 macrophages ................................................. 118

Figure 31: Effects of VSL#3-CM on basal and lipopolysaccharide

(LPS)-induced cyclooxynase-2 (COX-2) expression in J774

macrophage ................................................................................. 119

Figure 32: Effects of VSL#3-CM on NO production in non-stimulated

and LPS-stimulated J774 macrophages ....................................... 135

Figure 33: Effects of VSL#3-CM on iNOS expression in basal and

LPS-stimulated J774 macrophages .............................................. 137

Figure 34: Effects of VSL#3-CM on COX-2 expression in basal and

LPS-stimulated J774 macrophages .............................................. 139

Figure 35: Effect of Polymyxin B (PmB) on VSL#3-CM-induced NO

production in J774 macrophages .................................................. 141

Figure 36: Effect of VSL#3-CM on the viability of J774 macrophages ........... 143

Figure 37: Effects of VSL#3-CM and sonicated bacteria on basal NO

production and iNOS expression in J774 macrophages ............... 145

Figure 38: Effect of dexamethasone on VSL#3-CM-induced NO

production and iNOS expression in J774 macrophages ............... 148

Figure 39: Effect of dexamethasone on the cell viability ................................. 149

Figure 40: Role of protein kinase C (PKC) on VSL#3-CM-induced NO

production and iNOS expression in J774 macrophages .............. 152

Figure 41: Effect of protein kinase C (PKC) inhibitor bisindolylmaleimide

(BIM) on the cell viability ............................................................... 154

xxvi

Figure 42: Role of phosphatidylinocitol 3-kinase (PI3K) on

VSL#3-CM-induced NO production and iNOS expression in

J774 macrophages ....................................................................... 157

Figure 43: Effect of phosphatidylinocitol 3-kinase (PI3K) inhibitor

LY294002 on the cell viability ....................................................... 158

Figure 44: Effect of protein kinase B (AKT) inhibitor on VSL#3-CM-

induced NO production and iNOS expression in J774 cells ........ 161

Figure 45: Effect of AKT inhibitor XIII on the cell viability ............................... 162

Figure 46: Role of p38 MAPK inhibitor SB203580 on VSL#3-CM-

induced NO production and iNOS expression in J774

macrophages ................................................................................ 165

Figure 47: Effect of P38 inhibitor SB203580 on the cell viability .................... 166

Figure 48: Effect of Proteasome inhibitor MG132 on VSL#3-CM-


macrophages ................................................................................. 169

Figure 49: Effect of Proteasome inhibitor MG132 on the cell viability ............ 170

Figure 50: Effect of NFκB inhibitor CAY10470 on VSL#3-CM-


macrophages ................................................................................ 173

Figure 51: Effect of NFκB inhibitor CAY10470 on the cell viability ................ 174

Figure 52: Effect VSL#3-CM on basal and lipopolysaccharide (LPS)

(100 µg/ml) induced NO production and inducible nitric oxide

synthase (iNOS) expression in rat aortic smooth muscle cells

(RASMC) ...................................................................................... 176

xxvii

List of Tables

xxviii

xxix

Contents Page No

Table 1: Signalling pathways modulated in intestinal epithelial cells by

bacterial strains of VSL#3 ................................................................ 20

Table 2. Signalling pathways modulated in macrophages by bacterial

strains of VSL#3 ............................................................................... 21

Table 3: Tissue distribution of different isoforms of nitric oxide synthase

(NOS) ................................................................................................. 32

Table 4: Tissue distribution of different isoforms of cyclooxygenase

(COX) ................................................................................................. 35

Table 5: Composition of MRS broth ................................................................ 41

Table 6: Composition of the complete culture medium .................................. 44

Table 7: Preparation of serial dilution of NaNO2 ............................................. 49

Table 8: Preparation of serial dilution of BSA ................................................. 53

Table 9: Effects of LPS, IFN- and TNF- on NO production in HT-29 and

J774 cells .......................................................................................... 71

Table 10: NO production in HT-29 cells after treatment with different

combinations of LPS, INF-, IL-1, and TNF- 73

Table 11: Effects of LPS and TNF- on NO production in CaCo-2 cells ....... 75

Table 12: Effects of LPS and LPS-TNF-α on NO production in

SW-620 cells ..................................................................................... 77

Table 12: Effects of LPS and TNF-a on NO production in Hela cells ............. 79

xxxi

Abbreviations

Abs – Absorbance

ATF – activator transcription factor

Akt – Protein kinase B

AP-1 – Activator protein-1

ATP – Adenosine 5'-triphosphate

BCA – Bicinchoninic acid

BH4 – Tetrahydrobiopterin

BSA – Bovine serum albumin

Caco-2 –Human colorectal adenocarcinoma cell line

CaM – Calmodulin

cAMP – Cyclic adenosine monophosphate

CAT2 – Cationic amino acid transporter 2

CD – Crohn‟s disease

CD14 – Cluster of differentiation 14

cDNA – Complementary deoxyribonucleic acid

cGMP – Cyclic guanosine monophosphate

COX – Cyclooxygenase

COX-1 – Cyclooxygenase-1

COX-2 – Cyclooxygenase-2

DC – Dendritic cells

DDW – Double distilled water

DMEM – Dulbecco‟s modified Eagle‟s medium

DNA – Deoxyribonucleic acid

ECL – Enhanced chemiluminescence

EDRF – Endothelium-derived relaxing factor

EDTA – Ethylenediamine tetraacetic acid

eNOS – Endothelial nitric oxide synthase

ERK – Extracellular signal-regulated kinases

E. coli – Escherichia coli

FAD – Flavin adenine dinucleotide

FAO/WHO – Food and Agriculture Organization/World Health Organization

http://en.wikipedia.org/wiki/Cluster_of_differentiation

xxxii

FBS – Foetal bovine serum

FMN – Flavin mononucleotide

GIT – Gastrointestinal tract

HASMC – Human aortic smooth muscle cells

hBD – Human beta-defensins

HDL – High density lipoprotein

HEK 293 – Human embryonic kidney epithelial cell line

HeLa – Human uterine endometrial carcinoma cell line

HEPES – 4-(2-hydroxyethy)-1-peperazine ethanesulphonic acid

HRP – Horseradish peroxidase

HSP – Heat shock proteins

HT-29 – Human colorectal adenocarcinoma cell line

IBD – Inflammatory bowel disease

IBS – Irritable bowel syndrome

IEC – Intestinal epithelial cell

IFN-γ – Interferon-gamma

IgG – Immunoglobulin G

IL-1 – Interleukin-1


IL-1β – Interleukin-1beta


iNOS – Inducible nitric oxide synthase

IκB – I kappa B - Inhibitory protein of NF-кB

IKK – IκB kinase

IRAK – IL-1 receptor associated kinase

J774 – Murine macrophage cell line

JNK – c-Jun N-terminal Kinases

kDA – Kilodaltons

LBP – LPS-binding protein

LDL – Low density lipoprotein

LGG – Lactobacillus rhamnosus GG

LPS – Lipopolysaccharide

mA – Milliampere

MAP – Mitogen activated protein

xxxiii

MAPK – Mitogen activated protein kinases

MCP-1 – Monocyte chemotactic protein-1

M-CSF – Macrophage colony-stimulating factor

MEK – Mitogen-activated protein/extracellular signal-regulated kinase kinase

MIF – Macrophage inhibitory factor

MKP-1 – MAPK phosphatase 1

mRNA – Messenger ribonucleic acid

MTT – 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MYD88 – Myeloid differentiation primary response gene 88 (adaptor protein)

NADPH – Nicotinamide adenine dinucleotide phosphate

NF-κB – Nuclear factor-κappa B

NHLA – NG-hydroxy-L-arginine

nNOS – neuronal nitric oxide synthase

NO – Nitric oxide

NOD – Nucleotide-binding oligomerization domain receptors

NOS – Nitric oxide synthase

O2- - Superoxide anion

ONOO- – Peroxynitrite

p38 MAPK – p38 Mitogen Activated Protein Kinase

PAMP – Pathogen-associated molecular patterns

PBS – Phosphate Buffered Saline

Peroxisome proliferator-activated receptors (PPARs)

PG – Prostaglandin

PGE2 – Prostaglandin E2

PI3K – Phosphatidylinositol-3 kinase

PKB – Protein Kinase B

PKC – Protein Kinase C

PRR – Pattern-recognition receptors

PS – Penicillin/Streptomycin

PVDF – Polyvinyldene difluoride

RASMC – Rat Aortic Smooth Muscle Cells

RNA – Ribonucleic acid

RNS – Reactive Nitrogen Species

ROS – Reactive Oxygen Species

xxxiv

S.E.M. – Standard Error Mean

SD – Standard Deviation

SDS – Sodium Dodecyl Sulphate

SDS-PAGE – Sodium Dodecyl Sulphate – Poly Acrylamide Gel Electrophoresis

sGC – soluble Guanylyl Cyclase

SCFA – Short-chain fatty acid

SMC – Smooth Muscle Cells

SW-620 – Human Caucasian colon adenocarcinoma

TEMED – NNN‟N‟-Tetramethylethylenediamine

TGF-β – transforming growth factor β

TLR – Toll-like receptors

TNF-α – Tumor Necrosis Factor-alpha

TPA – 12-O-tetradecanoylphorbol 13-acetate

TRAF6 – TNF receptor associated factor 6

TREs – TPA responsive elements

TRIS Base – Tris(hydroxymethyl)aminoethane

UC – Ulcerative colitis

UV – Ultra violet

VSL#3 – Probiotic formulation - composed of eight probiotic bacteria

VSL#3-CM – VSL#3-conditioned medium

1

Chapter 1 Introduction

3

Effect of probiotics on the regulation of the inducible nitric oxide synthase (iNOS)

pathway in cultured cells

5

1.0. Introduction

1.1. Probiotics

Probiotics are live, non-pathogenic bacteria which are normally present in the

gastro-intestinal tract (GIT). GIT is colonised by a large number of different

species that includes bacteria, fungi, bacteriophages and viruses, and more than

15,000 different strains of microorganisms (Scaldaferri et al., 2013; Schmidt and

Stallmach, 2005). Among these, probiotics are ingestible microorganisms which

produce health beneficial effects to their hosts (Yoon & sun, 2011). The joint report

of Food and Agriculture Organization / World Health Organization (FAO/WHO,

2001) described probiotics as „Live microorganisms which when administered in

adequate amounts confer a health benefit on the host‟. They improve the health of

the host by altering its intestinal microbial balance. These bacteria are known to

stimulate the body‟s immune system and play an important role in maintaining a

healthy gut (Amrouche et al., 2006; Fioramonti et al., 2003).

1.2. Concept of Probiotics

The beneficial role of probiotics such as Lactobacillus and Bifidobacterium in

enhancing intestinal health has been proposed for many years. The concept of

probiotics has been developed from the work of Nobel Prize-winning Russian

scientist Eli Metchnikoff (Metchnikoff, 1908). He first suggested in 1908 that

consumption of fermented milk products increased the longevity of Bulgarian

peasants (Fioramonti et al., 2003).

The name „probiotic‟ has been derived from the Greek word, meaning “supporting

or favouring life”. Lilly & Stillwell first used the term „probiotic‟ in 1965. They

defined these as „substances secreted by one microorganism, which stimulate the

growth of another‟. Since then different scientists have modified the definition

according to the effect produced by these microbes. More recently, Salminen, et

al., (1998), defined probiotics as foods, which contain live bacteria and are

beneficial to health. In 2002, Marteau, et al. defined probiotics as microbial cell

preparation or microbial cells components, which produce beneficial effect on

health. From then, other definitions, developed through the years, were restricted

6

by specification of mechanisms, site of action, delivery format, method or host

(Rachmilewitz et al., 2004; Sanders, 2008).

1.3. Functions of probiotics

The main functions of probiotics include metabolic activities translating into energy

and nutrients uptake, host protection against invasion by foreign microorganisms

and development and homeostasis of the immune system. Lactic acid producing

probiotics known as lactic acid bacteria (LAB) generate antimicrobial factors such

as bacteriocin that protects tissues against the attachment, overgrowth and action

of pathogenic microorganisms (Marteau & Shanahan., 2003; O'Hara and

Shanahan, 2006) (Figure 1). In addition, probiotics reduce lactose-intolerance

symptoms, increase the resistance of intestines to diseases, inhibit the

proliferation of cancer cells, modulate the concentration of plasma cholesterol,

improve the functions of the digestive system and stimulate body‟s immune

defence system that increases the capacity of the host to fight against infection

(Amrouche et al., 2006; Commane et al., 2005).

Based on the role of probiotic bacteria in the host, probiotic formulations are now

widely available as food supplements with a claim that they may be beneficial in

conditions of various gastrointestinal disorders mainly inflammatory bowel disease

(Bibiloni et al., 2005; Pagnini et al., 2010).

1.4. Inflammatory bowel disease (IBD)

Inflammatory bowel disease or IBD is characterised by chronic, uncontrolled

inflammation of the intestinal mucosa. The two main types of IBD are Crohn‟s

disease (CD) and ulcerative colitis (UC) (Abraham and Cho, 2009; Papadakis and

Targan, 2000). Crohn‟s disease generally involves the ileum and colon. It is

characterised by discontinuous inflammation of the epithelial lining and deep

ulcers but can affect any region of the gastro-intestinal tract. In contrast, ulcerative

colitis involves rectum but may affect part or entire colon. It is characterised by

superficial inflammation typically confined to the mucosa (Abraham and Cho,

2009; Bernstein et al., 2005).

7

Figure 1: Functions of probiotics - protective, structural and metabolic effects on the intestinal mucosa. Adopted from O'Hara and Shanahan, 2006.

The rationale for the use of probiotic supplement in IBD is that, there is a strong

association between the pathogenesis of IBD and a reduction of intestinal

microflora (Sanchez et al., 2010). Normal intestinal microflora such as Lactobacilli

and Bifidobacteria have been reported to be displaced by pathogenic microflora in

patients with IBD (Hansen et al., 2010; Triantafillidis et al., 2011). A reduced

production of short-chain fatty acid (SCFA), particularly butyrate by normal

microflora has also been shown in IBD patients (Fava and Danese, 2011). In

addition, a reduction of mucosa-associated Bifidobacteria and increase of E. coli

and clostridia in IBD reported by Mylonaki et al. (2005) supports that an imbalance

between beneficial and pathogenic bacteria may contribute to the pathogenesis of

IBD.

Probiotics

Metabolic function

Structural function

Protective function

Metabolize dietary carcinogen Synthesize vitamins (i.e., Vit K,

biotin, folate) Absorb ions (Mg2+, Ca2+, Fe2+) Ferment non-digestible dietary

residue

Pathogen displacement Nutrient competition Receptor competition Produce anti-microbial factors (i.e., bacteriocins, lactic acids)

Develop host‟s immune system Improve barrier function

8

A reduced production of the mucosal peptide antibiotics defensins by the secretory

epithelial cells Paneth cells has also been reported as a contributing factor in the

pathogenesis of IBD (Simms et al., 2008; Wehkamp et al., 2005a). The defensins

which include -defensins, human -defensin-1 (HBD-1) and human -defensin-2

(HBD-2) exert broad spectrum antimicrobial activity and offer the first line of

defence against pathogens (Boman 2003; Kluver et al., 2006; O'Neil et al., 1999;

Salzman et al., 2003; Zasloff, 2002; Wehkamp et al., 2005b). Murine models

deficient in the ability to produce mature α-defensins peptides have been shown to

be highly susceptible to challenges with orally administered bacterial pathogens

(Wilson et al., 1999).

Probiotic bacteria has been reported to regulate the production of human -

defensin-2 (HBD-2) in intestinal epithelial cells (Schlee et al., 2007; Schlee et al.,

2008; Wehkamp et al., 2004, Habil et al., 2014). Besides, the results from different

studies also suggest that the intestinal microflora plays an important role in the

aetiology and pathogenesis of IBD (Darfeuille–Michaud, 2002; Darfeuille–Michaud

et al., 2004; Garrett et al., 2010; Qin et al., 2010; Rosenfeld and Bressler, 2010;

Sokol et al., 2008).

The use of antibiotics in the treatment of IBD is based on the fact that the

pathogenic microflora is involved in the development of IBD (Triantafillidis et al.,

2011; Wehkamp et al., 2005b). However, in contrast to the antibiotics, that cause

resistance (Beckler et al., 2008; McFarland, 2008) and antibiotic associated colitis

(Issa et al., 2007; Lamont et al., 1980) together with the pathogenic microbiocidal

activity (Lilly &, Stillwell, 1965), resident microflora increase the number and

improve the intestinal microbial balance. That is why these bacteria are called

probiotic which is based on their function and are proposed as an alternative

treatment in gastro-intestinal disorders (Marchesi & Shanahan, 2007).

9

1.5. Gastro-intestinal tract

1.5.1. Intestinal microbial ecology

The gastrointestinal tract (GIT) is one of the key organs of the human body. The

mucosal surface of GIT is about 200-300 m2 and is colonized by bacteria of 400

different species and subspecies (Hao & Lee, 2004). The colonization of intestinal

tract with appropriate microflora and their by-products contribute to the normal

intestinal homeostasis (Drisko et al., 2003). The intestinal epithelium consists of a

layer of single cells, lining the intestinal lumen. The main functions of intestinal

epithelium is to act as a physical barrier to prevent the passage of harmful luminal

antigen, microorganisms and toxins (Blikslager et al., 2007; Podolsky, 1999), and

act as a filter to allow the essential dietary nutrients, electrolytes and water from

the intestinal lumen into circulation (Broer, 2008; Ferraris and Diamond, 1997;

Kunzelmann and Mall, 2002).

Disruption of cellular integrity in the intestinal epithelium may be caused by

pathogenic bacteria, stress and injury through the production of pro-inflammatory

cytokines (Limdi et al., 2006). This results in increased intestinal permeability

which may initiate and exacerbate intestinal inflammation. Impaired intestinal

permeability has been reported in inflammatory bowel diseases (IBD), such as

Crohn‟s disease (Berkes et al., 2003; Bjarnason et al., 1995) and there is a body

of data implicating increased intestinal permeability as a primary etiologic factor

contributing to the pathogenesis of IBD (Gerova et al., 2011; Groschwitz and

Hogan‟ 2009).

1.5.2. Immunity and intestinal epithelium

Intestinal epithelium provides the first line of defence to prevent the invasion of

host by the pathogens. The mucosal surface or mucous layer which covers the

intestinal epithelium represents primary interface between the external

environment and immune system where the pathogens and antigens interact with

the host (Madsen et al., 2001; Winkler et al., 2007). It contains various protective

and antimicrobial substances secreted by epithelial cells and plays a major role in

protection through interaction with the immune systems. Immune cells

http://www.ncbi.nlm.nih.gov/pubmed/?term=Gerova%20VA%5BAuthor%5D&cauthor=true&cauthor_uid=21633531

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10

macrophages, intra-epithelial dendritic cells (DCs), and epithelial cells are in

continuous contact with the environment and coordinate defences for the

protection of mucosal tissues (Tlaskalova-Hogenova et al., 1995; Winkler et al.,

2007).

Intestinal epithelial cells (IEC) produce various receptors called pattern-recognition

receptors (PRRs). They are called PRR because these receptors can distinguish

between the host tissue and pathogens, and can recognise the pathogens,

thereby known as pathogen-associated molecular patterns (PAMPs). Two receptor

families called Toll-like receptors (TLRs) and nucleotide-binding oligomerization

domain receptors (NODs) present on the cell surface, play important role in the

detection of PAMPs. TLRs are the transmembrane proteins with an extracellular

domain made of leucien-rich repeats (LRRs) and an intra-cytoplasmic domain

containing the highly conserved Toll/IL-1 receptor (TIR) domain which is formed by

TLR together with interleukin-1 receptor (Lopez-Boado et al., 2001). Expression of

TLRs increases with the exposure to bacterial products or pro-inflammatory

cytokines (Muzio et al., 2000).

Stimulation of TLRs by PAMPs initiates the signalling cascade after which the cells

of the intestinal epithelium provide a series of anti-microbial peptides which limit

the access of various potentially pathogenic microorganisms (Kindon et al., 1995).

They generate potent anti-microbial peptides human beta-defensins (hBD). These

positively charged peptides bind to the negatively charged bacterial membrane

and exhibit broad-spectrum anti-microbial activities (Sahly et al., 2006; Weinberg

et al., 1998). Among the different types of hBD, hBD-1 is normally expressed in

intestinal epithelial cells, whereas, hBD-2 and hBD-3 are expressed only in cases

of intestinal inflammation. In vitro study of probiotics has been shown to induce

hBD-2 in Caco-2 intestinal epithelial cells. In addition, Intestinal microflora plays

significant role in maintaining intestinal epithelial integrity through the regulation of

the induction of defensins (Bengmark, 2007; Ogushi et al., 2001; Vanderpool et

al., 2008; Wehkamp et al., 2004).

11

Figure 2 below shows the role of intestinal epithelial cells, which secrete many

immune mediators in response to the antigens such as antibacterial peptides,

immunoglobulin A (IgA) and chemokines. Specialized epithelial cells, termed as M

cells, transport antigens to the antigen-presenting cells, which subsequently

process antigens and present them to the native T cells. The pattern recognition

receptors (PRRs) expressed by the dendritic cells (DCs) and epithelial cells

mediate the detection of bacterial antigens and DCs modulate the immune

response by promoting either the effectors or regulatory T cells.

Figure 2: Immunosensory detection of intestinal bacteria. Adopted from

O'Hara and Shanahan, 2006.

12

1.6. How does probiotic work in intestinal immunity

The function of intestinal epithelial barrier is considered as an important part of

immune defence of the host. Dysfunctional epithelial barrier causes inappropriate

access of antigens to the mucosal immune system which is considered as an

important factor in the pathophysiology of IBD. In a recent report, probiotic

mediated cytoprotection has been mentioned as a therapeutic approach that

regulates the homeostasis of intestinal epithelial cells through maintaining cell

survival and enhancing barrier function (Hausmann, 2010). These non-pathogenic

microorflora have been used for the treatment of Crohn‟s disease (Bousvaros et

al., 2005; Malchow, 1997; Yan & Polk, 2012) and ulcerative colitis (Kruis et al.,

2004; Soo et al., 2008). The proposed mechanisms of action through which these

bacteria produce effects include bacterial interference and cytoprotective effects.

Bacterial interference – it is the condition in which one bacterial strain inhibits the

colonization of another by competing with it. They prevent the colonization of the

pathogenic bacteria in the intestinal tract through the production of growth

inhibitors which include bacteriocins (Klaenhammer, 1988; Klaenhammer, 1993)

and microcins (Patzer et al., 2003). Probiotic bacteria compete for the adhesion

sites on intestinal epithelial lining and replace intestinal pathogens (Lee et al.,

2000; Lee et al., 2003). The anti-apoptotic effect of probiotics has been reported

due to the soluble factors such as microcin produced by specific bacteria (Denou

et al., 2009; Patzer et al., 2003). Probiotic-secreted anti-invasive soluble

component has been reported to induce β-defensin-2 in intestinal epithelial cells

which act as broad spectrum antibiotic against gram-negative and gram-positive

bacteria, may strengthen the mucosal barrier through limiting the bacterial

adherence and invasion (Hausmann, 2010; Schlee et al., 2007; Schlee et al.,

2008).

Cytoprotective effects – Increased cytoprotection is related to the decreased

apoptosis of epithelial cells. The cytoprotection happens through the interaction of

three components in the gastrointestinal tract which include - competing bacteria,

intestinal epithelial cells and mucosal immune cells (Hausmann, 2010; Yan and

13

Polk, 2002). Probiotic bacteria has been reported to induce the expression of heat

shock proteins (HSPs) which produce cytoprotective effect on epithelial cells (Liu

et al., 2003; Musch et al., 1996; Musch et al., 1999; Petrof et al., 2004). Probiotic

formulation VSL#3 induces HSP25 and HSP72 (Petrof et al., 2004; Ropeleski et

al., 2003) of which HSP25 preserves the cytoskeletal and tight junction functions

of epithelial layer (Mounier et al., 2002; Urayama et al., 1998). HSP72 protects the

cellular proteins and epithelial cell lines against oxidant injury (Urayama et al.,

1998).

Mucosal immune cells, macrophages, play diverse roles in host defence against

invasive pathogens in intestinal epithelium by phagocytosis, and killing pathogens

through the production of reactive oxygen species (ROS) and reactive nitrogen

species (RNS) (Weber et al., 2009) (Figure 3). However, mucosal macrophages

only ingest and kill pathogens but do not mediate strong pro-inflammatory

response which is essential for maintaining a healthy intestine (Rogler et al, 1998).

This is how they differ from other tissue macrophages which are able to secret a

variety of cytokines and pro-inflammatory mediator that modulate migration and

activation of other immune cells to the sites of inflammation (Gordon, 2007).

However, the tolerance of macrophages to the normal intestinal microflora has

been reported to be lost in inflammatory bowel disease (IBD) and continuous

activation of intestinal immune responses by normal intestinal microflora is thought

to cause IBD (Podolsky, 2002).

Probiotic bacteria regulate the intracellular signalling pathways for the activation of

mammalian immune cells to produce antimicrobial factors, reactive oxygen

species and pro-inflammatory cytokines (Britton and Versalovic, 2008), and help

preventing intestinal tract from the invasion of pathogenic microorganism. Probiotic-derived metabolite has been reported to activate peroxisome proliferator-

activated receptors (PPARs) which are expressed on the surface of the immune

cells and regulate the expression of various signalling pathways involved in

intestinal inflammation (Bassaganya-Riera et al., 2012a).

14

1.7. Probiotic-mediated intra-cellular signalling

A growing body of data suggest that probiotics can modulate systemic and

mucosal immune function, alter intestinal microbial ecology, improve intestinal

barrier function, improve inflammatory bowel disease, diarrhoea, irritable bowel

syndrome, and exert metabolic effects on the host (Alberda et al., 2007; Caballero-

Franco et al., 2007; Dykstra et al., 2011; Ewaschuk et al., 2008; Mack et al., 2003;

Mondel et al., 2009; Resta-Lenert and Barrett KE, 2003; Schlee et al., 2008;

Sekirov et al., 2010; Shanahan, 2010; Ukena et al., 2007). These effects in the

gastro-intestinal tract have been mediated through the stimulation of the

appropriate signalling pathways by probiotics and probiotic-secreted metabolites.

Several studies have shown that the key biological signalling pathways (Figure 4)

in Intestinal epithelial cells and immune cells can be modulated by individual or

combination of probiotic strains (Table 1 and 2). The pathways involved are

discussed below.

Nuclear factor kappa B (NF-B) is one of the key signalling pathways to activate

the immune response and play a central role in inflammation. It has the ability to

induce pro-inflammatory genes in response to inflammatory stimulus. NF-B exists

in the cytoplasm as an inactive form associated with a protein inhibitor called IB.

Upon cellular stimulation, IB is phosphorylated, thus releasing NF-B to

translocate into the nucleus where it binds to the DNA-binding site (B site) and

induces the expression of different genes including pro-inflammatory genes

(Baldwin, 1996, Tak and Firestein, 2001). Phosphorylated IB is ubiquitinated and

subjected to proteasomal degradation (Wehkamp et al., 2004).

Several probiotics have been reported to block the ubiquitination and proteasomal

pathway of IB degradation and thereby inhibit the activation of NF-B (Kumar et

al., 2007; Lin et al., 2009; Petrof, 2009; Petrof et al., 2004; Zhang et al., 2005). In

contrast, there is report that some probiotic can stimulate the activation of NF-B

and increase cytokine secretion (Ruiz et al., 2005).

15

Activator protein-1 (AP-1) is another transcription factor that plays a crucial role

in the expression of inflammatory genes. AP-1 is a homo and/or heterodimeric

protein complex composed of proteins belonging to Jun (c-Jun, JunB and JunD),

Fos (c-Fos FosB and Fra-1/2) and activator transcription factor (ATF) families

(Hess et al., 2004). The formation and activation of AP-1 is mediated through the

activation of mitogen-activated protein kinases (MAPK) signalling pathways in

response to a variety of stimuli that includes cytokines, stress, growth factors and

bacterial infection (Ding et al., 2008; Reddy et al., 2002; Vesely et al., 2009; Young

et al., 2003). The activated AP-1 translocates into the nucleus where it binds to its

DNA-binding site (TRE site) and regulates the expression of different genes

including pro-inflammatory genes (Nakamura et al., 1991; Smeal et al., 1989; Van

Dam & Castellazzi, 2001). Many compounds that activate NF-B also activate AP-

1 (Karin et al., 1997; Wehkamp et al., 2004). Probiotics have been reported to

suppress LPS-induced inflammatory mediator tumour necrosis factor- (TNF-)

production through the inhibition of AP-1 activation and this may explain part of

their anti-inflammatory and potentially protective role in human monocytes and

monocyte-derived macrophages (Lin et al., 2008).

Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear

hormone receptor proteins that work as transcription factors and regulate the

expression of various genes. PPAR- plays an important role in the regulation of

the intestinal inflammation and is also a target for probiotics (Michalik et al., 2006;

Voltan et al., 2008). PPAR-has the ability to inhibit the activity of NF-B thereby

inhibit the expression of the inflammatory gene. Impaired expression of PPAR-

has been reported in ulcerative colitis. Probiotics have been reported to modulate

the PPAR-dependent pathway in intestinal inflammation (Are et al., 2008;

Dubuquoy et al., 2003; Kelly et al., 2004; Ortu et al., 2012). Report has shown that

probiotics increase the expression of PPAR-in human colonocytes and colon

epithelial cell lines (Lee et al., 2005) and reduce inflammation by up-regulating the

expression of PPAR- Dubuquoy et al., 2003; Mencarelli et al., 2011).

16

Mitogen-activated protein kinases (MAPK) are a group of enzymes responsible

for phosphorylating serine and threonine amino acids in many proteins, and

consist of three separate signalling cascades regulated by the p38 MAPK,

extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinase or

stress-activated protein kinases (JNK/SAPK). MAP kinases are involved in various

signal transduction pathways including inflammatory response (Lee and Young,

1996; Lee et al., 1994). Probiotics have been reported to suppress the MAPK

signalling pathways such as p38 MAPK and extra cellular- related kinase 1/2 (ERK

1/2) to inhibit the secretion of pro-inflammatory cytokines (Jijon et al., 2004; Resta-

Lenert and Barrett, 2006) and this may also be critical in mediating their anti-

inflammatory actions.

Protein kinase C (PKC) is a family of serine-threonine-specific protein kinases

that can phosphorylate a wide variety of proteins and involved in diverse cellular

signalling pathways (Loegering and Lennartz, 2011). Several studies have

implicated the role of PKC in the immune cell activation and inflammatory

responses (Baxter et al., 1992; Castrillo et al., 2001; Diaz-Guerra et al., 1996;

Larsen et al., 2000; Lin and Chang, 1996; Shapira et al., 1997; Valledor et al.,

2000). Activation of PKC is essential for the epithelial integrity. Cario et al.

reported on the activation of PKC isoforms PKC- and PKC- through the

stimulation of TLR2 in intestinal epithelial cell lines Caco-2 and HT-29. Probiotic-

secreted extracellular proteins have been reported to improve the gut barrier

function and IBD through the activation of PKC-dependent pathway (Cario et al.,

2004; Rao et al., 2009; Seth et al., 2008).

Phosphatidylinositol 3-kinase (PI3K) are a family of kinase enzymes involved in

different cellular responses (Toker and Cantley, 1997). The PI3K signalling

pathway plays a central role in the regulation of inflammatory response and

inappropriate regulation of this pathway can lead to various diseases including

cancer (Cahill et al., 2011; Duan et al., 2005). PI3K pathway is another target for

probiotics and their metabolites (Thomas and Versalovic, 2010).

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17

Protein kinase B (PkB) also known as AKT is a serine/threonine specific protein

kinase, which plays important roles in various cellular processes including NF-B-

dependent transcription and inflammatory cytokine production (Rajaram et al.,

2006). Probiotic-released proteins have been reported to produce the anti-

inflammatory effect through the inhibition of PI3K/AKT pathway (Dai et al., 2013;

Yan F et al., 2007).

Thus, from the discussions above, it would appear that specific targeting of key

signalling pathways may be critical for the actions and beneficial effects of

probiotics under conditions associated with inflammation.

1.8. Use of Probiotics in clinical practice

In recent years, the proposed health benefits of probiotics have undergone

thorough scientific evaluation and showed strong evidence for their beneficial

effects in various human diseases (Boyle et al., 2006; Marteau 2002). Meta-

analysis of randomized controlled trials has shown the major clinical effects of

probiotics in gastro-intestinal disorders. The combination of different probiotics

have been reported effective in preventing antibiotic-associated diarrhoea

(D'Souza et al., 2002), infective diarrhoea in both adults and children (Allen et al.,

2004) and inflammatory bowel disease (Gionchetti et al., 2000; Weizman et al.,

2005). On the basis of this, probiotics have been recommended for the prevention

and treatment of a diverse range of intestinal disorders including inflammatory

bowel disease.

Probiotics are now commercially available as a dietary supplement in the form of

tablet, capsule or powders and as fermented dairy products such as yogurt and

milk. The available probiotic products on the markets are based on their evaluation

in controlled human studies. The most commonly used probiotic strains belong to

the genera of Lactobacillus and Bifidobacterum which are used individually or in

combination. There is no fixed number of probiotic bacteria that must be ingested

to achieve a beneficial effect. However, to increase the chance of adequate gut

colonisation, probiotic preparation must contain the live and viable

18

microorganisms, and several billion microflora is recommended to be ingested

(Cremonini et al., 2002; ICH Expert Working Group, 2006; Kopp et al., 2008;

Sutton, 2008; US Food and Drug Administration, 2006; Williams, 2010). Some of

these products include Lactobacillus rhamnosus GG or LGG (Culturelle, Amerifit

Brand, Fairfield, NJ), Saccharomyces Boulardii (Florastor, Biocodex, Inc.,

Beauvais, France), Bifidobacterum infantis 35624 (Align, JB Laboratories, Holland,

MI), and VSL#3 (Sigma-Tau Pharmaceuticals, Inc, Gaithersburg, MD).

1.9. VSL#3

VSL#3 is a commercially available probiotic preparation, which a mixture of eight

different strains of probiotic bacteria contain 450 billion, freeze-dried, live lactic

acid producing Lactobacillus and Bifidobacterium.

The effects of probiotics on intestinal inflammation are strain specific. Different

probiotics produce effects in different health conditions and act through different

mechanisms (Reid et al., 2003; Rioux et al., 2005; Venturi et al., 1999). They

produce their effects on epithelial cells and macrophages through increasing the

mucin production (Mack et al., 2003; Mack et al., 1999) and barrier function

(Czerucka et al., 2000; Otte and Podolsky, 2004), inducing the anti-microbial and

heat shock proteins (Schlee et al., 2008; Wehkamp et al., 2004), interfering with

pathogenic organisms (Castagliuolo et al., 1996; Chen et al., 2006) and

modulating signalling pathways (Bai et al., 2004; Eun et al., 2007; Petrof et al.,

2009; Resta-Lenert and Barrett, 2006).

On the basis of the effects produced by individual probiotic bacteria through the

scientific studies (Table 1 and Table 2), VSL#3 has been formulated with a view

that the unique blend of bacteria in VSL#3 will work by colonising the gastro-

intestinal tract to sustain a favourable balance. In vitro studies have shown lactic

acid bacteria to be effective in removing pathogenic bacteria. Probiotic released

metabolites such as lactic acid lowers the pH that has a role in the inhibition of the

growth of pathogenic bacteria in the gastro-intestinal tract.

http://www.medscape.com/viewarticle/719654_11

19

The probiotic mixture VSL#3 has been reported to increase the cytoprotection of

epithelial cells and used as an effective treatment in ulcerative colitis. An in vitro

study has shown that VSL#3 conditioned medium inhibits the activation of

transcription factor NF-B responsible for the expression of inflammatory proteins,

and thereby reduce inflammation (Hausmann, 2010). In this regard, there are

indications that this may in part be associated with the regulation of the enzymes

such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)

which play crucial roles in regulating cellular responses through the production of

nitric oxide (NO) and prostaglandin E2 (PGE2) respectively during inflammation.

Thus their inhibition may abate inflammation.

20

Table 1. Signalling pathways modulated in intestinal epithelial cells by bacterial strains of VSL#3

Probiotic strains

Model system

Signalling pathway

Probiotic effects References

Lactobacillus

acidophilus ATCC 4356

IECs MAPKs Activation of ERK1/2

and p38MAPK

Resta-Lenert and

Barrett, 2006

Lactobacillus

acidophilus ATCC 4356

IECs NF-B Decreases IκBα

phosphorylation

Resta-Lenert and

Barrett, 2006

Lactobacillus bulgaricus IECs NF-B Decreases p65

translocation

Bai et al., 2004

Lactobacillus casei DN-

114 001

IECs NF-B Prevents IκBα

degradation

Tien et al., 2005

Lactobacillus casei IECs PPAR- Increases PPAR-

mRNA

Eun et al., 2007,

Bifidobacterium longum IECs NF-B Decreases p65

translocation

Bai et al., 2004;

Bai and Quyang,

2006

Streptococcus

thermophilus ATCC

19258

IECs MAPKs Activation of ERK1/2

and p38

Resta-Lenert and

Barrett, 2006

Streptococcus

thermophilus ATCC

19258

IECs NF-B Decreases IBα

phosphorylation

Resta-Lenert and

Barrett, 2006

VSL#3 IECs PPAR- Enhanced expression

of PPAR-

Ewaschuk et al.,

2006

Abbreviations: ERK, extracellular signal-regulated kinases; IEC, intestinal epithelial cell; IBα,

inhibitor of NF-B; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-

activated protein kinase; NF-B, nuclear factor-kappaB; PPAR-, peroxisome proliferator activated

receptor-gamma. Adopted from Thomas and Versalovic, 2010.

21

Table 2. Signalling pathways modulated in macrophages by bacterial strains of VSL#3

Probiotic strains Model system

Signalling pathway

Probiotic effects References

Lactobacillus casei Shirota

Macrophages NF-B Inhibits IBα phosphorylation

Watanabe et al., 2009

Lactobacillus casei Shirota

Macrophages MAPKs Inhibits ERK1/2 phosphorylation

Watanabe et al., 2009

Lactobacillus casei YIT 9029

Macrophages NF-B Activation of NF-B Matsuguchi et al., 2003

Lactobacillus plantarum K8

Macrophages NF-B Inhibits IBα degradation

Kim et al., 2008

Lactobacillus plantarum K8

Macrophages MAPKs Decreases p38, JNK, ERK1/2 phosphorylation

Kim et al., 2008

Lactobacillus plantarum S1, DB22, & DS41

PBMCs apoptosis Increases TRAIL production and secretion

Horinaka et al., 2010

Bifidobacterium breve BbC50

Macrophages NF-B Decreases LPS binding to CD14

Menard et al., 2004

Streptococcus thermophilus St065

Macrophages NF-B Decreases LPS binding to CD14

Menard et al., 2004

Abbreviations: ERK, extracellular signal-regulated kinases; IEC, intestinal epithelial cell;

IBα, inhibitor of NF-B; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAPK,

mitogen-activated protein kinase; NF-B, nuclear factor-kappaB. Adopted from Thomas

and Versalovic, 2010.

22

1.10. Role of Probiotics in inflammatory bowel disease (IBD)

Pathogenesis of human intestinal inflammation is related to the imbalance of

pathogenic and beneficial microflora. Altered microbial composition and microbial

imbalance have been reported in patients suffering from IBD and other intestinal

disorders (Bjorksten et al.,1999; Kassinen et al., 2007; Sjogren et al., 2009; Yan et

al., 2011). An acute reduction of the intestinal microflora is observed in diseases

including IBD (Joossens et al., 2011; Manichanh et al., 2006; Packey et al., 2008;

Sartor, 2008). The effectiveness of probiotics reported through various studies,

have implicated probiotic bacteria as being beneficial in the prevention and

treatment of such gastrointestinal diseases (McFarland, 2006; McFarland et al.,

2008; Tong et al., 2007; Vanderpool et al., 2008).

Probiotic formulations are prepared with a sufficient amount of viable microflora to

improve host‟s microflora that are expected to produce beneficial effects by

providing protective barriers, enhancing immune responses and clearing

pathogens in the intestinal tract (Elmer, 2001; McFarland, 2000; Qamar et al.,

2001). There are reports on the effect of probiotic in the gastro-intestinal tract,

which showed modulation of immunoglobulins such as IgA, reduced inflammatory

cytokines and enhanced intestinal barrier function (Bai et al., 2006; O'Hara et al.,

2006a).

A number of clinical studies have been performed on the strains of Lactobacillus

and Bifidobacterium individually or in combination to prevent or treat gastro-

intestinal infections in which IBD is the most studied disease. The clinical studies

have evidenced a strong relation of bacteria in the pathogenesis of IBD and have

been reported to exert beneficial effect in the management of IBD through the

modulation of intestinal flora. For example, probotic Lactobacillus rhamnosus GG

(LGG) increased mucosal immunoglobulin A (IgA) levels (Malin et al., 1996),

improved intestinal permeability, and reduced disease activity when administered

to children with Crohn‟s disease (Gupta et al., 2000). Furthermore, VSL#3 has

been shown to be effective in the treatment of patients with ulcerative colitis,

Crohn‟s disease and pouchitis (Gionchetti et al., 2003; Gionchetti et al., 2000;

23

Guslandi et al., 2000), and improve the colonic permeability in interleukin-10 (IL10)

deficient mice model (Madsen et al., 2001).

In vitro studies have shown that probiotic bacteria reduce the production of pro-

inflammatory cytokines such as tumour necrosis factor-α (TNF-α) in macrophages

(Fioramonti et al., 2003; Menard et al., 2004). However, in contrast to the reports

discussed above, probiotics have also been reported to induce pro-inflammatory

mediators and cytokines such as, nitric oxide (NO) and TNF-α through the

modulation of various signalling pathways and are inducing the immune function.

The effects of probiotics have been shown to differ by different strains when they

are used individually and some of the strains have reported to produce no effects.

Intestinal function is largely controlled by constitutively produced NO. On the other

hand, iNOS-induced NO contributes to the inflammation and cell damage.

However, all these studies point to a beneficial effect of probiotics in regulating gut

inflammation.

1.11. Inflammation

Inflammation is a normal, protective response of the immune system of the host

tissue to injury caused by physical trauma, noxious chemicals, or invasion by

pathogenic microorganisms. The role of inflammation is vital in controlling and

ultimately eliminating the infectious agents, promoting wound healing and

restoration of tissue integrity. It is central to many disease conditions, such as

infection and immune reactions. Inflammation is also involved in the pathogenesis

of autoimmune chronic diseases such as, rheumatoid arthritis and cancer.

The immune cells of the host are capable of releasing a wide range of

inflammatory mediators such as nitric oxide (NO), prostaglandin E2, tumour

necrosis factor- (TNF-), interferon- (IFN-) etc., that promote the inflammatory

process in response to an infection, and the process is initiated mainly by the

resident macrophages, dendritic cells and mast cells. However, if the immune

system is inappropriately stimulated, the inflammatory response can cause injury

to the host tissues. At the onset of an infection or injury, the Toll-like receptors

24

(TLRs) present on the surface of the immune cells, cells become activated and

release inflammatory mediators. Thus initiate the set of responses referred as

inflammation against the pathogens and injury (Heinsbroeck et al., 2009; O'Hara et

al., 2006).

1.12. Role of Macrophages in Inflammation

Macrophages play a key role in the innate immune response and form a bridge

between innate and adaptive immune response. About 100 years ago, Elie

Metchnikoff first proposed the idea of an involvement of macrophages in

mammalian immunity (Mosser et al., 2009; Nathan, 2008). These cells are

produced in the bone marrow and spread to all the body tissues through blood,

and are an essential mediator of inflammatory reactions.

Activation of macrophages induces inflammatory processes such as migration of

macrophages, release of cytokines and pro-inflammatory mediators that includes

tumour necrosis factor- (TNF-), interferon- (IFN-), interleukin-1 (IL-1),

interleukin-6 (IL-6), prostaglandins (PGs) and nitric oxide (NO), and phagocytosis

(Celec, 2004; Tajima et al., 2008). Macrophage cells are the most potent

phagocytic cells in the immune system that produce NO through the expression of

inducible nitric oxide synthase (iNOS) as a killing mechanism in the host defence

reaction (Diefenbach et al., 1998; Schonhoff et al., 2003, Stamler et al., 2001)

(Figure 3).

In response to inflammatory stimuli, the activated macrophages produce and

release PGE2, through the expression of inducible cyclooxygenase-2 (COX-2).

The potent vasodilatory property of PGE2 increases blood flow around the

inflammatory sites to transfer and release immune cells in a highly specific order.

In the initial stages of inflammation neutrophils destroy most of the microbes. Next,

macrophages destroy the remaining microbes, remove the apoptotic bodies of

dead neutrophils and present these to T lymphocytes. Thus macrophages initiate

the mechanisms of acquired immunity which ends in the production of different

antibodies, cytokines and memory cells. These memory cells are the key element

25

for vaccines. This is how macrophages switch from being pro-inflammatory to anti-

inflammatory cells and remove all the tissue debris to achieve the healing of the

injured cells (Fujiwara & Kobayashi, 2005).

Figure 3: Role of NO in the killing of microbes by macrophages

Microbes are ingested by macrophage and peroxisomes are fused to the

phagosome, releasing reactive oxygen species. Then NO enters in the

phagosome and either forms peroxynitrite (ONOO-) by reaction with reactive

oxygen species or diffuses across the microbial cell wall to disrupt the microbial

DNA synthesis. Adopted from Schonhoff et al., 2003; Fernandez, 2013.

26

Macrophages are potently activated by bacterial lipopolysaccharide (LPS), a major

component of the outer membrane of gram-negative bacteria, induces a variety of

pathogenic responses and lead to the production of inflammatory mediators. The

binding of LPS with the Toll-like receptors (TLRs) present on the membrane of

macrophages leads to the activation of the intracellular signalling cascades (Figure

4).

LPS binds with LPS-Binding Protein (LBP), which transports LPS to the LPS

receptor CD14. CD14 receptors are surface proteins preferentially expressed on

immune cells and help initiating the immune response to LPS. Then extracellular

TLRs activate intracellular signalling cascade such as mitogen-activated protein

kinase (MAPK), Protein kinase C (PKC), Phosphatidylinositol 3-kinase (PI-3K)/

AKT etc. leading to the phosphorylation and activation of the immunological

transcription factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-

1). The signalling of TLRs occurs through two distinct pathways – MYD88-

dependent pathway and MYD88-independent pathway. Upon activation, TLRs

recruit the adaptor protein MYD88 and protein kinase IRAK. IRAK becomes

phosphorylated, leaves MYD88-TLR complex and associates with a protein

TRAF6. TRAF6 activates IB kinase (IKK) that phosphorylates IB leading to its

degradation through the ubiquitin-proteasome pathway (Deng et al., 2000; Sun et

al., 2004).

Activated transcription factors are then translocated into the nucleus and induce

the expression of pro-inflammatory genes such as, inducible nitric oxide synthase

(iNOS) and cyclooxygenase-2 (COX-2) that produces NO and PGE2 respectively.

A variety of other extra cellular stimulus such as heat, radiation etc. can also

activate different signalling pathways to initiate inflammation (Beutler et al., 2003;

Janssens et al., 2003; O‟Hara, et al., 2006).

27

Figure 4: The binding of LPS to the Toll-like receptor (TLR) present on the cell membrane leads to the activation of the intracellular signalling cascades. Adopted from Castrillo et al., 2001; Guha and Mackman, 2002, Guo

and Friedman, 2010; Madrid et al., 2000.

28

Macrophages produce both pro and anti-inflammatory actions during infection.

Pro-inflammatory activation of macrophages is induced by LPS or pro-

inflammatory cytokines such as IFN- and TNF-. On the other hand, anti-

inflammatory activation is initiated by the anti-inflammatory cytokines such as IL-4,

IL-10 or IL-13. The main difference between these two processes in macrophages

is the biochemical pathway used for the processing of the amino acid arginine.

LPS, IFN- or TNF- induce iNOS and produce NO in macrophage which is used

to kill the pathogens in the first phases of inflammation. In anti-inflammatory

response, arginase is induced in macrophages and produces proline and

polyamines, which catalyses the reconstitution of the damaged extracellular matrix

by inducing proliferation and collagen production, an event that occurs during the

final phases of inflammation (Classen et al., 2009; Forlenza et al., 2011).

1.13. Nitric oxide (NO) and its chemical properties

Nitric oxide (NO; nitrogen monoxide) is a colourless and gaseous 30 kDa diatomic

free radical, consisting of nitrogen and oxygen. Due to its gaseous nature it can

diffuse freely through membranes and is extremely short-lived in biological system

(Hickey et al., 2001).

1.13.1. Biological properties of nitric oxide (NO)

In biological system, nitric oxide (NO) acts as an important signalling chemical

messenger that regulates various physiological and pathophysiological responses

including vasodilatation, neurotransmission and immune system activation. In

1980, Furchgott et al. first demonstrated the involvement of NO as a substance in

endothelium dependent relaxation in smooth muscle cells (Furchgott et al., 1980).

Later, Palmer et al. identified NO as endothelium-derived relaxing factor (Palmer

et al., 1987). The half-life of NO in biological system is very short, ranging from 1

to 30 seconds. It is rapidly oxidized to nitrate and/or nitrite, and can diffuse across

cell membranes and interact with intracellular target molecule (Hickey et al., 2001).

29

A number of reports have shown NO with potent anti-inflammatory properties,

whereas other studies have shown it to promote inflammation-induced cell and

tissue dysfunction (Figure 5) (Grisham et al., 1999; Ibiza and Serrador, 2008). NO

interacts directly with a biological molecule under normal physiological conditions

when the rates of NO production are low and may serve regulatory or anti-

inflammatory functions. On the other hand, deleterious or pro-inflammatory effects

.

Figure 5: Regulatory, protective and deleterious biological effects of nitric oxide (NO). Adopted from Grisham et al., 1999; Ibiza and Serrador, 2008.

NO

Regulatory

Vascular Tone

Cellular adhesion

Vascular Permeability

Inhibits Platelet adhesion

Immunity

Protective

Antioxidant

Inhibits leukocyte adhesion

Protects cells against oxidant injury, pathogenic infection

Deleterious

Inhibits enzyme function

Promotes DNA damage

Induces lipid peroxidation

Depletes antioxidant stores

Increased susceptibility to: radiation, alkylating agents, toxic metals

30

are the reactions mediated by NO-derived intermediates such as reactive nitrogen

oxide species derived from the reaction of NO with oxygen or superoxide when

large amounts of NO are produced during inflammation (Grisham et al., 1999).

Formation of reactive nitrogen species is however not an unavoidable effect of the

NO production which is removed by the reaction with oxyhaemoglobin to form

nitrate. The simultaneous production of superoxide along with NO makes the

formation of peroxynitrite in the biological system (Hess et al., 2005; Pacher et al.,

2007).

As discussed further below, low levels of NO is an important regulator of

physiological processes but excessive production of NO is associated with various

carcinomas and inflammatory conditions such as Type-1 diabetes, arthritis,

inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). Most of its

pathological effects may be mediated through its interaction with superoxide

radicals to produce reactive nitrogen species (RNS) such as peroxynitrite (ONOO-

). This molecule can modify proteins through interaction with their transition metal

centres through a reaction with DNA structures (pyrimidine bases) of amino acids

in their peptide chains or prosthetic group (such as Heme) (Bogdan, 2001;

Nathan, 1992; Pacher et al., 2007).These unique properties of NO make it

different from any other neurotransmitters.

1.13.2. Biosynthesis of nitric oxide (NO)

Nitric oxide (NO) is synthesized by a family of enzymes called the nitric oxide

synthase (NOS), which converts the amino acid L-arginine into L-citruline and NO

(Korhonen et al., 2005; Kolios et al., 1998, HE et al., 2006). NOS catalyses the

oxidation of L-arginine which involves two separate reactions. In the first phase of

reaction L-arginine is converted to an intermediate N-hydroxy-L-arginine (NHLA).

This reaction requires nicotinamide adenine dinucleotide phosphate (NADPH) and

oxygen. In the second phase, N-hydroxy-L-arginine is oxidized to L-citruline and

NO which requires flavin adenine dinucleotide (FAD), flavin mononucleotide

(FMN), calmoduline (CAM) and tetrahydrobiopterin (BH4) as co-factors (Figure 6).

31

Figure 6: Schematic presentation of nitric oxide (NO) generation from L-arginine. Adopted from Aktan, 2004.

1.13.3. Nitric oxide synthases (NOS)

There are three different isoforms of nitric oxide synthase (NOS) named according

to their activity or the tissue type. Two are constitutively expressed and known as

neuronal NOS or nNOS or NOS1 located mostly in neurons and endothelial NOS

or eNOS or NOS3 expressed mostly in endothelial cells. The third isoform is the

inducible NOS known as iNOS or NOS2, which is not constitutively expressed, but

its expression is highly regulated by bacterial lipopolysaccharide (LPS) and

inflammatory cytokines like TNF-, IFN- etc. and is predominantly expressed in

areas of inflammation (Alderton et al., 2001; Hickey et al., 2001).

The constitutive expressions of eNOS and nNOS enzyme proteins in the cells are

regulated by Ca2+ fluxes and subsequent binding of calmodulin (CaM). But iNOS is

calcium-insensitive, due to its tight non-covalent interaction with calmodulin and

Ca2+ (MacMicking et al., 1997). Although these three isoforms catalyse the same

reaction to convert L-arginine and molecular oxygen to N-hydroxy-L-arginine and

further to citruline and NO, they differ in the regulation, the amplitude and duration

of the production of NO, and their cellular and tissue distribution (Bogdan, 2001;

MacMicking et al., 1997) (Table 3).

32

Table 3: Tissue distribution of different isoforms of nitric oxide synthase (NOS)

Name Size Tissue distribution Activators Expression

nNOS or

NOS1

161

kDa

Neurones Steroid hormones,

cytokines, NMDA

Constitutive,

Ca2+ sensitive

eNOS or

NOS3

133

kDa

Endothelial cells,

cardiac myocites,

epithelial cells,

reproductive organs

Shear stress,

histamine,

adenosine,

bradykinin,

thrombin, estradiol

Constitutive,

Ca2+ sensitive

iNOS or

NOS2

130kDa Macrophages, smooth

muscle cells,

endothelial cells

Cytokines,

endotoxins

Inducible, less

Ca2+ required

than nNOS

and eNOS

Adopted from Knowles et al., 1994; Franco et al., 1999; Alderton et al., 2001and

Aronoff et al., 2004.

Nitric oxide synthase has two domains in which an N-terminal oxygenase domain

containing binding sites for haem, BH4 and L-arginine, is linked by a CaM-

recognition site to a C-terminal reductase domain that contains binding sites for

FAD, FMN and NADPH (Wendy et al., 2001).

In the first phase of reaction during NO production, binding of BH4 and L-arginine

to the oxygenase domain increases haem redox potential forming a ferric haem.

On the reductase domain, electrons are donated from NADPH and flow through

FAD and FMN to the oxygenase domain, where they reduce the ferric haem to

ferrous haem. Binding of an oxygen molecule forms a ferrous-oxy complex which

equilibrates with a ferric-superoxide complex (considered to be the source of

superoxide formation by NOS due to dissociation). Simultaneous addition of an

33

electron and a proton from BH4 reduces the superoxide bound to hydroperoxide.

Further protonation induces the irreversible breaking of the oxygen-oxygen bond,

resulting in a ferryl iron with a protein-bound cation radical and H2O. This highly

oxidizing species rapidly oxygenates L-arginine to NHLA, and the enzyme resting

state is regenerated.

Throughout the second phase of reaction, a single electron from the reductase

domain reduces the ferric haem iron to ferrous-oxy complex allowing binding of

molecular oxygen and thus generating the ferrous-oxy complex. Finally, this

ferrous-oxy complex catalyses the oxidation of NHLA to NO, citrulline and water,

and regenerates the ferric haem protein again (Andrew et al., 1999).

1.14. Role of nitric oxide (NO) in immune response

During the past decades NO has been accepted as one of the most important

player in immune system as well as other organs because of its versatile property.

At the beginning NO was simply recognised as a product of macrophages

activated by cytokines and microbial compounds and is derived from the amino

acid L-arginine by the enzymatic activity of inducible nitric oxide synthase (iNOS)

to function as tumouricidal and antimicrobial molecule. But in recent years, NO

drew the attention of immunologists due to its protective and toxic effects and to

the fact that the other isoforms of NOS (eNOS and nNOS) are also involved in

activation of the immune system (Ibiza and Serrador, 2008; MacMicking et al.,

1997).

Constitutively produced low amount of NO preserves the cellular integrity and

mediate anti-inflammatory effects in the early phase of inflammation. But in the

advanced stage of inflammatory process, excess production of NO by iNOS cause

tissue injury (Korhonen et al., 2001). iNOS induced over production of NO

interacts with molecular oxygen and superoxide anion and forms reactive nitrogen

species peroxynitrite (ONOO-) and that‟s how immune cells kill pathogenic

microbes during phagocytosis (Figure 3).

34

Furthermore, the activity of NO is not limited to its production site. As an

uncharged gas, highly diffusible •NO radicals can be transferred to a distant site

through low molecular weight S-nitrosothiols (such as S-nitrosoglutathione), S-

nitrosylated protein and nitrosyl-metal complexes to the other immune cells such

as T and B lymphocytes, where iNOS-induced NO has been reported to regulate

the development, differentiation and function of these immune cells (Bogdan,

2011; Henson et al., 1999; Hess et al., 2005; Ibiza and Serrador, 2008; Kolb and

Kolb-Bachofen, 1998; Niedbala et al.,2006).

1.15. Role of Cyclooxygenase-2 and prostaglandin E2 in immune response

Cyclooxygenase (COX) is one of the key enzymes in the metabolic pathway. This

enzyme converts arachidonic acid into prostaglandins (PGs) that are well known

for their role in inflammation and immune response (Portanova et al., 1996). COX

has two isoforms. COX-1 is constitutively expressed in most tissues and is

involved in prostaglandin biosynthesis for cellular „housekeeping‟ functions and

gastric cytoprotection (Franco et al., 1999). On the other hand, COX-2 is the

inducible form which is selectively induced by pro-inflammatory cytokines and is

primarily expressed in immune cells (Table 4). COX-2 is responsible for the

production of inflammatory mediator PGE2 from arachidonic acid, which plays a

central role in the regulation of inflammation.

A substantial amount of PGE2 is produced by COX-2 at the site of inflammation

where it acts as a potent vasodilator and enhances vascular permeability to

increase the migration of leukocytes into the inflammatory sites (Aronoff et al.,

2004). It is usually undetectable in normal conditions but induced by a number of

mediators including cytokines, mitogens and bacterial endotoxin at sites of

inflammation and injury (Williams et al., 1996; Schwacha et al., 2002).

35

Table 4: Tissue distribution of different isoforms of cyclooxygenase (COX)

Name Size Tissue distribution Activators Expression

COX-1 70kDa Most tissues particularly in

platelet, stomach, kidney

Inflammatory

stimuli

Constitutive

COX-2 72 kDa Macrophages, monocytes,

synoviocytes, chondrocytes,

fibroblasts and endothelial

cells.

Cytokines,

endotoxins

Inducible

Adopted from Knowles et al., 1994; Franco et al, 1999; Alderton et al, 2001and

Aronoff et al., 2004.

1.16. Role of iNOS and COX-2 in inflammatory bowel disease (IBD)

Inducible nitric oxide synthase and cyclooxygenase-2 play key roles in the

inflammatory processes. Dysregulated expression of iNOS and/or COX-2 has

been reported to be associated with the pathophysiology of human inflammatory

disorders and cancers.

Several studies have evidenced the involvement of iNOS and iNOS-induced nitric

oxide (NO) in IBD (Boughton-Smith, 1994; Cross and Wilson, 2003; Middleton et

al., 1993). Studies have reported a significant increase of iNOS activity in the

colonic mucosa in patients with ulcerative colitis (UC) and Crohn's disease (CD),

compared to controls (Boughton-Smith et al., 1993; Dijikstra et al., 2002; Kolios et

al., 2004). Increased expression of COX-2 has been reported in both human IBD

and animal models of colitis (Hendel and Nielsen, 1997). The use of COX-2

inhibitors showed beneficial effects in patients with IBD (Mahadevan et al., 2002),

however, there are also reports that prostaglandins (PGs) produced by COX-2 are

important to improve the mucosal injury and to protect against bacterial invasion

(Reuter et al., 1996; Wallace, 2001).

36

Although the IBD-related cancer is still not clear, however, it has been generally

assumed that IBD-related cancer occurs as a result of chronic inflammation. The

association between inflammatory bowel disease and colorectal cancer has been

reported in many studies (Jess et al., 2006; Munkholm, 2003). The data suggest

that severity and duration of chronic inflammation in IBD is a significant risk factor

for intestinal cancer (Mantovani, 2005). Epidemiological data also suggest that

chronic colitis is the risk factor for colitis-associated colon cancer (Gupta et al.,

2007; Itzkowitz and Yio, 2004; Rutter et al., 2004).

It has been suggested that COX-2 mediated prostaglandin synthesis may be

involved in the process of intestinal cancer (Gupta et al., 2001; Kawai et al., 2002;

Otte et al., 2009). COX-2-induced PGE2 has been found to be increased in human

colorectal cancer tissue compared to normal colonic mucosa (Rigas et al., 1993;

Pugh and Thomas, 1994) and in experimental models of colon carcinoma cells

(Rao et al., 1996).

1.17. Role of Probiotics in cancer

A number of reports have demonstrated the role of probiotics in the prevention and

treatment of colorectal cancer. Most of these probiotics are from the strain of

Lactobacilli and Bifidobacteria (Azcárate-Peril et al., 2011; Fotiadis et al., 2008;

Liong, 2008; Marshal, 2008; Uccello et al., 2012). Pathogenesis of colorectal

cancer has been suggested to be linked with the inflammation induced by

pathogenic microbs which are also associated with the pathophysiology of IBD,

through their production of toxic metabolites in intestinal tract. These toxic

metabolites can bind to the cell surface receptors and lead to the mutation which

affects the intracellular signal transduction (Uronis et al., 2009).

Apart from the effects of probiotics on the prevention of the invasion of the

pathogens on the intestinal mucosa, probiotics scavenge toxic compound such as

carcinogens and mutagens from the body (Abdelali et al., 1995; Hosoda et al.,

37

1992; Renner and Munzner, 1991; Wollowski et al., 2001). They also modulate the

metabolic activity of the enzyme of pathogenic microflora such as nitroreductase

and β-glucuronidase which is reported to be associated with colon carcinoma

(Bouhnik et al., 1996; Goldin et al., 1992; Mital and Garg, 1995; Rowland, 1991).

Probiotics reduce the conversion of primary bile acids (from animal fat) such as

cholic acid (CA) and chenodeoxycholic acid (CDCA) to secondary bile acids

deoxycholic acid (DCA) and lithocholic acid (LCA), which are reported to have pro-

carcinogenic activity in intestinal epithelium (Bernstein et al., 2005; Narahara et al.,

2000).

Probiotic bacteria, specially Bifidobacteria ferment the non-digestible starch in the

large intestine and produce the short-chain fatty acids (SCFA) such as acetate,

propionate and butyrate, which have been reported as a strong anti-cancer agent

specially butyrate (Ewaschuk et al., 2006; Stein et al., 1996; Tong et al., 2004). In

addition to SCFA, probiotics can produce another group of fatty acids known as

conjugated linoleic acids (CLA), which have been reported to show anti-cancer

effects (Evans et al., 2010; Kelley et al., 2007). Besides, there is accumulating

evidence that demonstrated the ability of probiotics in the treatment and

prevention of intestinal cancer (de Moreno de LeBlanc et al., 2008; de Moreno de

LeBlanc and Perdigon, 2005; Fotiadis et al., 2008; Marteau et al., 1990;

Matsumoto et al., 2009; Perdigon et al., 2002; Tephly and Burchell, 1990).

38

1.18. Aims of the project

Lactic acid producing probiotic bacteria release active metabolites which have

been claimed to be responsible for their beneficial effect in the gastro-intestinal

tract (Menard et al., 2004). Despite claims of these beneficial effects in both

human and animal model, the exact mechanisms that mediate these actions have

not been clearly defined. Moreover, there are contradictions on the mechanism of

the protective effects of probiotics. Thus in this thesis experiments conducted were

aimed at establishing whether (i) on their own probiotics have any direct action on

the expression of potential inflammatory proteins including iNOS and COX-2, and

(ii) probiotics regulate the process of inflammation induced by inflammatory

agents. In addition studies were also aimed at unravelling the underlying

mechanisms that may mediate any direct effects of probiotics on the above

processes.

39

Chapter 2

Materials and methods

41

2.0. Materials and methods

2.1. Culture of probiotic bacteria

Commercially available Lactobacillus rhamnosus GG (LGG) (single strain probiotic

bacteria) and VSL#3 (combination of eight strains of probiotic bacteria) were

cultured in bacterial culture medium called de Man, Rogosa and Sharpe (MRS)

broth (Oxoid Limited, UK) (Table 5). There are different phases in bacterial growth

known as lag phase, log phase, stationary phase and death phase. Active

metabolites released by probiotic bacteria are believed to be the responsible factor

for their anti-inflammatory effect. So, the growth curves were constructed for these

specific bacteria to find out the most active phase of growth and compare the anti-

inflammatory effect in the different phases.

Table: 5. Composition of MRS broth

Typical Formula gm/litre

Peptone 10.0

`Lab-Lemco‟ powder 8.0

Yeast extract 4.0

Glucose 20.0

Sorbitan mono-oleate 1 ml

Dipotassium hydrogen phosphate 2.0

Sodium acetate 3H2O 5.0

Triammonium citrate 2.0

Magnesium sulphate 7H2O 0.2

Manganese sulphate 4H2O 0.05

pH 6.2 ± 0.2 @ 25°C

http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0359&org=82&c=UK&lang=EN

42

2.2. VSL#3

Commercially available probiotic bacterial formulation VSL#3 (Actial Farmaceutica

Lda, Italy) was purchased from a local chemist shop. Each packet contained viable

lyophilized 450 billion live gram+ve lactic acid bacteria and bifidobacteria, including

4 species of lactobacilli, 3 species of Bifidobacteria and 1 species of

Streptococcus salivarius subsp. Thermophilus.

The eight strains of VSL#3 were:

Streptococcus thermophilus

Bifidobacterium breve

Bifidobacterium longum

Bifidobacterium infantis

Lactobacillus acidophilus

Lactobacillus plantarum

Lactobacillus paracasei

Lactobacillusdelbrueckii subsp. bulgaricus

2.2.1. Construction of bacterial growth curve

The bacteria was grown in MRS broth, and sample of bacterial culture was

collected to construct the bacterial growth curve using the modified method

described in Pena et al., 2003 and Yan et al., 2007. To become accustomed with

the growing temperature, sample of frozen bacteria was inoculated in 20 ml MRS

broth and incubated overnight at 37oC with shaking. An aliquot of 500 µl overnight

culture was added to 150 ml autoclaved and, pre-warmed MRS broth, and

incubated at 37oC, with shaking. A sample was collected from the culture every

hour for the determination of turbidity, optical density measured at 600 nm

wavelength to get the concentration of bacteria and pH determined. The curves for

bacterial growth and pH were constructed by plotting the absorbance and pH

against time (shown in chapter 4, figure 22).

43

2.3. Preparation of Sample

2.3.1. Freeze-dried probiotic culture supernatant

As described in section 2.2.1, 100 µl of bacteria (thawed from frozen stock) was

inoculated in 20 ml autoclaved MRS broth and incubated overnight at 37oC, with

shaking. An aliquot of 500 µl overnight culture was added in 150 ml autoclaved,

pre-warmed MRS broth and incubated at 37oC, with shaking. Samples were

collected from log and stationary phases of the bacterial growth. The bacteria free

supernatants were collected by centrifugation at 500 g for 10 min, filtered through

0.2 micron (µ) filter and freeze-dried. Dry samples were stored at –80oC until use.

2.3.2. Probiotic-conditioned cell culture medium

Probiotic-conditioned cell culture medium was prepared using the modified method

described in Yan et al., 2007 and Menard et al, 2004. To prepare the conditioned

medium, 100 µl of bacteria (thawed from frozen stock) was inoculated in 50 ml

autoclaved MRS broth / Fresh freeze-dried bacteria from one sachet was grown in

100 ml autoclaved MRS broth and incubated overnight at 37oC with shaking. The

culture was centrifuged at 500 g for 10 min, supernatant discarded and the pellet

washed twice with autoclaved phosphate buffer saline (PBS). Bacterial pellet was

then re-suspended in a sterile 50 ml centrifuge tube containing 30 ml of cell culture

medium Dulbecco‟s modified Eagle medium (DMEM) with 20% foetal bovine

serum (FBS), and incubated at 37oC for another 24 hrs, with shaking. The

bacteria-containing cell culture medium was centrifuged at 500 g for 10 min,

supernatant collected and filtered through 0.2 micron filter. Samples were stored at

–80oC until use.

2.3.3. Sonicated probiotic in cell culture medium

As described in paragraph 2.3.2, the culture of probiotic was centrifuged, washed

with autoclaved PBS and re-suspended in 30 ml cell culture medium (DMEM) with

10% FBS and 1% Pen/Strep. Instead of incubation, the suspension was sonicated

for 10 min, with a 30 sec rest, and centrifuged for 10 min at 3000 g. Samples were

stored at –80oC until use.

44

2.4. Cell culture

2.4.1. Culture and sub-culture of J774

The murine macrophage cell line J774 was obtained from European Collection of

Animal Cell Cultures and grown in T-75 tissue-culture flask in complete culture

medium (Table 6) in a humidified tissue culture incubator at 37oC in 5% CO2. For

sub-culture, the spent culture medium was replaced by fresh culture medium.

Adherent cells were gently scraped by using a sterile cell scraper and re-

suspended at a ratio of 1:10 in new T-75 flasks. Cells were then grown in a

humidified tissue culture incubator at 37oC in 5% CO2. The medium was changed

and cells sub-cultured every 2-3 days to sustain the growth of cells. The number of

times cells have been sub-cultured or passed into new flasks, is generally termed

as passage number.

Table 6: Composition of the complete culture medium

Name Percentage (%)

Dulbecco‟s Modified Eagle Medium (DMEM, low glucose- ie

1000 mg/ml, without sodium pyruvate)

89%

Foetal Bovine Serum (FBS) 10%

100 units ml-1 Penicillin + 100 units ml-1 Streptomycin 1%

2.4.2. Culture and Sub-culture of epithelial cells

Epithelial cell lines Caco-2, HT-29 and SW-620 and uterine endometrial cell line

Hela cells were cultured in T-75 tissue-culture flask in the complete culture

medium (Table-6). For sub-culture, the spent culture medium was removed from

the flask, cell monolayers were harvest by trypsinisation (Li et al., 2007) and re-

suspended at a ratio of 1:10 in a new T-75 flask.

45

2.4.2.1. Trypsinisation of the cell monolayer

Trypsinisation is the process of dissociation of adherent cell monolayers from the

flask by using trypsin. Trypsin is a proteolytic enzyme which breaks down the

adhesion proteins to dissociate the cells from the culture flask and helps making

single cell suspension (Olsen et al., 2004; Shibeshi et al., 2008). In trypsinisation,

trypsin-EDTA is used as a combined method to enhance the effect of trypsin.

EDTA (Ethylenediaminetetraacetic acid) is a calcium chelating agent which

chelates the calcium and helps the cells to release from the culture flask (Senoo et

al., 2000).

To trypsinise the cells, the cell monolayers were washed twice with sterile

phosphate buffer saline (PBS) and 500 µl trypsin-EDTA was added to the flask

and incubated for 5-10 min. Cells were not exposed to trypsin-EDTA for long to

avoid the reduction of the cell viability (Farea et al., 2013; Sutradhar et al, 2010).

When the cells were detached from the flask, 5 ml fresh complete culture medium

was added to the flask to neutralise trypsin. The clusters of cells were aspirated

with a sterile pasteur pipette and re-suspended at a ratio of 1:10 in a new T-75

flask. The cells were then grown in a humidified tissue culture incubator at 37oC in

5% CO2. The medium was changed and cells sub-cultured every 2-3 days to

sustain the growth of cells.

2.5. Plating of cells for experimentation

Confluent monolayer of the cells was scraped / trypsinised and re-suspended in

fresh cell culture medium. After the determination of number, cells were plated in

micro-well plate at the required seeding density and incubated overnight to allow

the attachment to the plate. The cells were then treated with bacterial

lipopolysaccharide (LPS) for activation and incubated for 24 hrs at 37oC in 5%

CO2. To confirm activation, total nitrite was measured in spent cell culture medium

using Griess assay and iNOS expression was detected by Western Blot assay.

46

2.6. Determination of cell number

Confluent monolayer of cells in the culture flask was scraped or trypsinized and re-

suspended in 10 ml of fresh culture medium. An aliquot of 50 µl cell suspension

was added to 50 µl trypan blue to make 1:1 dilution. It was then added to the

chamber of either side of the haemocytometer (Neubauer type). The

haemocytometer was placed onto a microscope and cells in the middle large

square of each chamber (with an area of 1mm2 and a depth of 0.1 mm) were

counted and averaged. The average cell number was multiplied by 2 to adjust for

the dilution with trypan blue. Since the large square is equivalent to 10-4 cm3

(equivalent to 10-4 ml), the cell number of cells was again multiplied the by 104 to

give the total number of cells per ml.

Number of cells/ml = number of cells counted X 104

The cells suspension was diluted to get the required number of cells according to

the seeding density in a given volume of the culture medium.

2.7. Optimisation of the activation of J774 cells

J774 cells were plated into sterile 12 well plates at a seeding density of 106 cells

per well and incubated overnight for the attachment of the cells on the plate

(Figure 7). The cell culture medium was replaced with a serial dilution (0, 0.01,

0.03, 0.1, 0.3, 1, 3 and 10 μg/ml) of LPS and incubated for 24 hours. To confirm

the activation, the spent culture medium from each well was transferred to

individual eppendorf tubes to measure nitrite content. After the transfer of the

spent culture medium, the cell monolayers were washed twice with ice-cold

phosphate buffer saline (PBS). To get the lysate, 100 μl lysis buffer (100 mM Tris-

HCl pH 7.4, 1% SDS, 1.5 M NaCl) pre-heated at 95oC was added to each well and

the plate left on ice. The cells were scraped, cell lysates transferred from the wells

into individual eppendorf tubes and sonicated for 30 sec three times with 30 sec

rest to reduce the viscosity. The sonicated protein was centrifuged at 15000 g and

supernatant was taken in another set of eppendorf tubes and heated at 95oC for 5

min. The amount of total protein was determined in the cell lysates of each well,

using bicinchoninic acid (BCA) protein assay and the samples were stored in -

47

20oC until analysed. The expression of iNOS was detected in the extracted protein

by Western Blot assay.

Figure 7: Monolayer of macrophages J774 cells prior to treatment with drug

2.8. Measurement of total nitrite using Griess assay

Nitrite is the stable end product of NO, which is very short-lived in biological

system. It is readily oxidized by cellular oxygen and almost completely converted

to nitrite. In biological sample total nitrite is measured in cell culture medium by the

Griess assay as an indicator of NO production. The assay is based on the

diazotization reaction where nitrite reacts with the Griess reagents (1:1 ratio of 1%

sulfanilamide in 10% orthophosphoric acid – Reagent I and 0.2% N-

napthylethylenedianmine - Reagent II) to form an azo dye, which absorbs light at

540 nm (Green et al., 1982) (Figure 8).

48

Figure 8: Chemistry of the Griess Reaction (adopted from Held, 2001)

49

2.8.1. Preparation of Nitrite standard curve using Sodium Nitrite (NaNO2)

NaNO2 stock solution was prepared by dissolving 0.0345 g NaNO2 in 5 ml DMEM

to give a 100 mM stock. From 100 mM NaNO2 stock, a 1 mM solution was

prepared by adding 40 µl of 100 mM NaNO2 to 3960 µl DMEM. A serial dilution of

NaNO2 solution was prepared using 1 mM solution to construct the standard curve

(Table 7).

Table 7: Preparation of serial dilution of NaNO2

Concentration of NaNO2 (mM)

Volume of 1mM NaNO2 in µl

Volume of DMEM in µl

Final concentration of NaNO2

(nmol/100 µl)

0 0 1000 0

0.01 10 990 1

0.02 20 980 2

0.03 30 970 3

0.04 40 960 4

0.05 50 950 5

0.10 100 900 10

50

2.8.2. Procedure

A serial dilution of sodium nitrite solution (1, 2, 3, 4, 5 and 10 nmols/100 μl) in cell

culture medium was prepared to use as standards. To construct the standard

curve, 100 μl of standards ranging from 0 to 10 nmols/100 μl was added in

triplicate, to the wells of a non-sterile 96 well plate. An aliquot of 100 μl

supernatant from each well of LPS-treated cells was transferred to the other wells

of the plate in triplicate. The Griess reagent (1:1 mixture of Reagent I and Reagent

II) was prepared and 100 μl added into each well. The plate was incubated for 15

minutes at room temperature and the absorbance was measured at 540 nm on a

micro-well plate reader. A standard curve of sodium nitrite was constructed (Figure

9) using the absorbance of serial dilutions of sodium nitrite, and the amount of total

nitrite in LPS-treated cell culture supernatant was determined from the standard

curve.

Figure 9: A representative standard curve of Sodium Nitrite

0.00

0.20

0.40

0.60

0.80

1.00

0 2 4 6 8 10 12

Abso

rban

ce (5

40 n

m)

Nitrite (nmols)

51

2.9. Interference of bacterial culture medium (MRS broth) on the Griess assay

A serial dilution of MRS broth was prepared at the dilution of 1:1000, 1:500, 1:100,

1:50 and 1:10 in cell culture medium. 1 ml of each solution was transferred to the

wells of a 24-well plate and incubated in a humidified tissue culture incubator at

37oC in 5% CO2 for 24 hrs. A serial dilution of sodium nitrite solution of 1, 2, 3, 4, 5

and 10 nmol / ml in DMEM was prepared and 100 μl of each dilution was added to

the wells of two 96-well plates to construct six (6) sets of nitrite standard curves as

described in Section 2.8.2. Living the 1st set of standard curve, aliquot of 50 μl

solution from each of five dilutions of MRS broth were added to the wells of rest of

the five sets of standard curves. 100 μl Griess reagent was added to the wells of

first set of standard curve and 150 μl Griess reagent was added to the wells of last

five sets of standard curves containing the dilution of MRS broth. The plate was

incubated at room temp for 15 min and absorbance measured at 540 nm on a

micro-well plate reader. Standard curves were constructed by plotting the

absorbance against nitrite concentrations (data is shown in chapter 4, figure 26).

52

2.10. Measurement of total protein in cell lysates using bicinchoninic acid (BCA) protein assay

The bicinchoninic acid (BCA) protein assay is a detergent-compatible formulation

used for the colorimetric detection and quantification of total protein. The reaction

is based on the reduction of Cu2+ to Cu1+ by protein in alkaline medium and the

formation of a purple coloured water soluble complex measurable by

spectrophotometer. BCA is highly sensitive and selective for cuprous cation (Cu1+).

The reaction occurs in two steps. In the first step of the reaction known as biuret

reaction, the chelation of copper with protein in alkaline environment containing

sodium potassium tartrate form a light blue coloured chelate complex with cupric

ions. In the second step, BCA reacts with the reduced cation (Cu1+) that was

formed in the first step and a purple coloured reaction product results from the

chelation of two molecules of BCA with one cuprous ion (Cu1+) (Smith et al., 1985).

The macromolecular structure of protein, the number of peptide bonds and the

presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine)

are responsible for colour formation with BCA (Wiechelman, 1988) (figure 10).

Figure 10: Reaction of BCA associated with the detection of protein concentration in the cell lysates. Adopted from (adopted from Smith et al.,

1985).

53

2.10.1. Preparation of protein standard curve using bovine serum albumin (BSA)

A stock solution (10 mg/ml) was prepared by dissolving 0.02g endotoxin-free

bovine serum albumin (BSA) in 2 ml lysis buffer (1x). A serial dilution of BSA

solution was prepared using a 10 mg/ml solution to construct the standard curve

(Table 8).

Table 8: Preparation of serial dilution of BSA

Concentration of protein (µg/µl)

Volume of 10mg/ml BSA in

µl

Volume of 1X lysis buffer taken in µl

Final concentration of protein (µg/10µl)

0 0 100 0

0.1 10 990 1

0.2 20 980 2

0.3 30 970 3

0.5 50 950 5

1.0 100 900 10

1.5 150 850 15

2.0 200 800 20

2.5 250 750 25

3.0 300 700 30

54

2.10.2. Procedure

The amount of total protein in each well of the treated cells was determined using

bicinchoninic acid (BCA) protein assay. To construct the standard curve, 10 µl of

each standard ranging from 0 to 30 μg per 10 µl was added in triplicates to the

wells of a non-sterile 96-well plate. An aliquot of 10 μl cell lysate from each well

was added in triplicate to the other wells of the 96-well plate. BCA reagent (1:50

ratio of BCA reagent B and BCA reagent A) was prepared and 100 μl of BCA

reagent added into the wells and incubated for 45 min at room temperature. The

absorbance was measured at 620 nm using the micro-well plate reader. A

standard curve of BSA was constructed by plotting the absorbance of standards

and the amount of total protein in the samples was determined from the standard

curve (Figure 11).

Figure 11: A representative standard curve of protein (BSA)

0.000

0.040

0.080

0.120

0.160

0 1 2 3 4

Abso

rban

ce (6

20 n

m)

Protein (μg)

55

2.11. Detection of protein expression by Western Blot assay

Western blot is an analytical technique which is used to detect the change of the

expression of a specific protein from a mixture of proteins under different

conditions. Western blotting, also called immunoblotting is performed by

separating the proteins of interest in cell lysates based on molecular weight by

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and

immobilized onto a membrane. The Western blotting analysis is performed in six

steps: (1) extraction and quantification of protein samples; (2) separation of the

protein in SDS-PAGE denaturing gel electrophoresis; (3) transfer of the separated

protein to a membrane; (4) blocking nonspecific binding sites on the membrane;

(5) incubation the membrane with antibodies and (6) detection of signal (Blancher

and Jones, 2001).

2.11.1. Preparation of gel

An 8% sodium dodecyl sulfate-polyacrylamide gel called resolving gel (4.68 ml

ultra pure water, 2.67 ml 30% acrylamide/0.8% bisacrylamide solution, 2.5 ml Tris-

HCl (1.5 M; pH 8.8), 100 μl 10% SDS, 50 μl 10% ammonium persulfate and 10 μl

N,N,N‟,N‟-Tetramethylethylenediamine, TEMED) /10 ml solution was prepared and

3.75 ml of resolving gel carefully applied between two glass plates. A thin layer of

n-butanol was added to the top of the gel to remove the air bubbles and allowed to

set for 45 min. After polymerisation, the layer of n-butanol was carefully washed

with water without touching the gel. A layer of 5% acrylamid stacking gel (2.84 ml

ultra pure water, 0.833 ml 30% acrylamid/0.8% bisacrylamide solution, 1.25 ml

Tris-HCl (0.5 M, pH 6.8), 50 μl 10% SDS, 25 μl 10% ammonium persulfate and 5

μl TEMED) was added on top of the resolving gel in between the two glass plates.

A 0.75 mm Teflon comb was inserted into the stacking gel between the glass

plates to form wells and allowed to set for 30 min. After polymerization of the

stacking gel, the teflon comb was removed and the wells washed with Tank Buffer

(0.025 M Tris-HCl, 0.192 M Glycine and 0.1% SDS).

56

2.11.2. Separation of protein

Aliquots of cell lysates were mixed with equal amount of loading buffer (4% SDS,

10% glycerol, 0.006% bromophenol blue, 2% β-Mercaptoethanol and 250 mM

Tris-HCl pH 6.8). The mixture was heated at 95oC for 5 min. The samples

containing 15 μg protein were loaded in the wells onto the 0.75 mm discontinuous

vertical slab mini-gel in an electrophoresis cell (Mini Protean II, Bio-Rad). To

identify the expected band 5 μl molecular weight marker was loaded in the first

well. Electrophoresis was run at 200 volts for the separation of iNOS and 100 volts

for COX-2 protein. Electrophoresis was terminated when the tracking dye had

moved to the end.

2.11.3. Transfer of protein from gel to membrane

The protein was then transferred from the gel on to polyvinylidene difluoride

(PVDF) membrane in transfer buffer (39 mM Glycine, 48 mM Tris-HCl, 0.0375%

SDS and 20% methanol) by applying current. The gel was removed from the

electrophoresis cell and placed on to PVDF membrane in between two filter

papers on a semi-dry transfer cell (Trans-Blot SD, Bio-Rad). The transfer was

carried out at 0.8 mA cm-2 current using transfer cell. For iNOS the transfer was

carried out for 2.5 hrs and for COX-2 it was 2 hrs.

2.11.4. Immunoblotting and enhanced chemiluminescence detection of proteins

The PVDF membrane was incubated in blocking buffer (10 mM Tri-HCl pH7.5, 100

mM NaCl, 0.1% Tween-20 and 5% w/v non fat dried milk) overnight at 4oC to

prevent the non-specific binding of immunochemicals with the proteins. The

membrane was incubated with primary antibody (anti-iNOS or anti-COX-2 specific

antibody) for 2 hrs at room temperature with shaking on a rotary shaker. The

membrane was then washed three times with wash buffer (10 mM Tri-HCl pH7.5,

100 mM NaCl and 0.1% Tween-20) with shaking to wash off the unbound protein.

The wash buffer was replaced with fresh solution after every 10 min. The

membrane was again incubated with the secondary antibody, anti-β-actin antibody

(for β-actin) and anti-biotin HRP-linked antibody (for molecular weight marker) for

57

1 hour at room temperature with shaking on a rotary shaker. It was again washed

as described above. The membrane was placed on a clean cling film. Enhanced

chemiluminescence (ECL) detection solution was prepared (reagents 1 and 2, at

1:1 ratio) as described by the manufacturer and applied to the membrane for 1 min

at room temperature. Excess solution was drained and the membrane was

carefully wrapped with cling film avoiding any trapped air bubbles. The membrane

was exposed to ECL film for 5-10 min in the dark room under red light. The film

was immediately developed using Kodak DEKTOLTM developer and fixed with

Kodak UNIDIXTM fixer.

2.11.5. Role of β-actin in Western blot assay

In Western blot analysis multiple steps are involved that include sample

preparation, sample loading, electrophoresis, protein transfer, antibody incubation

and signal detection. Hence, during Western blot experiment, a loading control is

used in order to interpret the result. Beta-actin (-actin), also known as

„housekeeping protein‟ is one of the most commonly used loading controls in

Western blotting. It is one of six different actin isoforms which are highly conserved

proteins and are involved in cell motility, structure and integrity; and are crucial for

tissue development (Roper et al., 2005; Sheterline et al., 1999). -actin is

constitutively expressed at high levels in almost all tissues. Therefore, it is used as

a loading control in Western blot. However, the protein used as a loading control

should be of different molecular weight than the protein of interest to avoid the

overlapping of bands. A loading control such as -actin ensures that (1) the same

amount of protein sample is loaded in each lane; (2) proteins are transferred from

the gel to membrane with equal efficiency among different lanes and (3) antibody

incubation and signal detection are uniform across different lanes.

2.11.6. Quantification of western blots by scanning densitometry

Bands that developed on the film were quantified using ImageJ, an image

processing and analysing programme. Each band was scanned and its intensity

noted. The values obtained for probiotic treated samples were compared to those

of their respective controls.

58

2.12. Treatment of J774 cells with probiotics and measurement of nitric oxide (NO) production followed by the detection of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression

Confluent monolayer of J774 cells were scraped and plated as described in

Section 2.5. The cell culture medium was replaced by serial dilutions of probiotic-

conditioned medium or freeze-dried probiotic cultured supernatant in

presence/absence of LPS and incubated for 24 hrs. The samples were prepared in

LPS-free and LPS-containing (1 μg/ml) complete cell culture medium (Table 5)

and filtered through 0.2 micron filter prior to use. Total nitrite in the spent culture

medium of each well was measured by the Griess assay as described in chapter

2, section 2.8. Cell protein from each well was extracted and protein content was

measured using the BCA protein assay described in chapter 2, section 2.10. The

proteins from cell lysates were separated by SDS-PAGE. The proteins were then

transferred onto the PVDF membrane, blocked by 5% fat free milk and incubated

with specific antibody. To detect the expression of iNOS and COX-2 the

membrane was incubated with mouse anti-iNOS antibody (mouse origine; 1:2500)

(BD Transduction Laboratories) and mouse anti-COX-2 antibody (mouse origine;

1:5000) (BD Transduction Laboratories) respectively for 2 hours. To detect the

expression of β-actin, anti-β-actin monoclonal antibody (mouse origine; 1:5000)

(Sigma-Aldrich) was added along with the anti-iNOS antibody / anti-COX-2

antibody. The membrane was washed three times and again incubated with the

goat anti-mouse secondary antibody (1:5000) (BD Transduction Laboratories)

along with anti-biotin HRP-linked antibody (1:5000) (cell signalling) for molecular

weight marker. The antibodies were diluted in blocking buffer prior to use. The

protein band was visualised by using enhanced chemiluminescence (ECL)

detection solution (GE Healthcare / Amersham).

59

2.13. Detection of Nitrite in human intestinal epithelial cell culture medium

Nitric oxide (NO) degrades rapidly to nitrate and nitrite in aqueous solution and

only the nitrite can be measured using the Griess assay. Conversion of nitrate to

nitrite in biological fluids and tissue culture medium vary considerably. In animal

cells such as murine macrophage J774 cells, the total nitrite can be measured

easily by the Griess assay as NO is metabolised to nitrate and nitrite at 1:1 ratio

(Iyengar et al., 1987). In human cells, on the other hand, nitrate is the predominant

metabolite to nitrite (6:1) of NO metabolism (Wennmalm et al., 1992). Due to the

low quantity, in human cell culture medium nitrite was not detectable by Griess

assay. To facilitate this, the samples were treated with cadmium to catalyse the

conversion of nitrate to nitrite (Schmidt et al., 1995; Sun et al., 2003). After the

conversion, the nitrite content in culture medium was measured using the Griess

assay.

2.13.1. Conversion of nitrate to nitrite

A serial dilution of sodium nitrate (1, 2, 3, 4, 5 and 10 nmols/100 μl) in cell culture

medium was prepared in eppendorf tubes. Aliquots of 50 μl of samples from the

sodium nitrate solutions and LPS-treated human cell culture medium were diluted

using 140 μl water and deproteinized by adding 10 μl 30% zinc sulphate (ZnSO4)

solution. After mixing with ZnSO4, the solutions were incubated for 15 min at room

temp and centrifugation at 2000 g. The supernatant of sodium nitrate solutions and

supernatant of human cell culture medium were transferred to two sets of

eppendorf tubes containing equal number (5 beads/tube) of cadmium beads,

previously washed twice with 1 ml of water, 1 ml of 0.1 M HCl and finally 1 ml of

0.1 M NH4OH. The beads were soaked in 5 M HCl overnight before use. A third

set of the supernatant of sodium nitrate solutions was transferred to another set of

eppendorf tubes without cadmium beads. The solutions with and without cadmium

beads were incubated overnight on a shaker at room temp. The solutions were

centrifuged for 15 min at 3000 g and total nitrite in the supernatant was measured

by the Griess assay. A serial dilution of sodium nitrite was prepared and used

during Griess assay to construct the standard curve. Two standard curves were

constructed using the absorbances obtained from the set of sodium nitrite and

60

cadmium-converted sodium nitrate solutions and the concentration of nitrite in

human cell culture medium was calculated using the sodium nitrite standard curve

(shown in chapter 3, figure 18 - 21).

2.14. Determination of cell viability and cytotoxicity by the MTT assay

Cell viability was determined by the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-

diphenyltetrazolium bromide (MTT) assay, a colorimetric assay used to measure

the mitochondrial activity of the viable cells that metabolise MTT to purple

formazan (Mosmann, 1983). In this assay, the water soluble tetrazolium salt is

converted to insoluble purple compound formazan by the cleavage of the

tetrazolium ring by mitochondrial reductase in viable cells (Figure 12).

To determine the cell viability and cytotoxicity, confluent monolayers of cells in 96-

well plate were treated with drug for 24 hours at 37oC in a humidified CO2

incubator. After the treatment, the culture medium was replaced with 200 µl freshly

prepared 0.5 mg/ml MTT solution, and incubated for another 4 hours at 37oC in a

humidified CO2 incubator. The MTT solution was removed, 200 µl isopropanol

added to the wells and incubated for 30 min at room temperature on a shaker to

dissolve the water insoluble formazan crystals formed. The plate was read on a

multi-well plate reader at 540 nm. The viability of the drug treated cells was

calculated as a percentage of the control (untreated cells), considering the viability

of control as 100% using the formula below.

% Cell viability = x 100%

A540 of Drug Treated Cells

A540 of Control Cells

61

Figure 12: Metabolism of MTT to purple formazan salt by the cleavage of the tetrazolium ring by mitochondrial reductase in viable cells (adopted from

Ebada et al., 2008)

2.15. Treatment of cells with the inhibitors of various signalling pathways

To determine the involvement of the signalling pathways in the induction of iNOS

and NO, cells were treated with the inhibitors of the respective signalling

pathways. To get an idea of the non-cytotoxic concentration range of inhibitors, the

cell viability test was performed using the MTT assay, before setting up the

experiment. Confluent monolayers of cells were treated for 30 minutes with

different concentrations of inhibitor alone prior to the addition of probiotic-

conditioned medium. Cells were then incubated with culture medium (Control) and

VSL#3-CM (1:10 dilution) without and with different concentrations of inhibitor for

24 hrs. The nitrite content in the cell culture medium was measured by the Griess

assay and the expression of iNOS in the cell measured using the Western blot

analysis.

2.16. Statistical analysis

Statistical analysis of data was carried out using Graph-Pad Prism (Graph-Pad

Software). The one-way analysis of variance (ANOVA) test was carried out using

Dunnett's multiple comparison test. The difference between the treatments and

controls was considered significant at the level of p<0.05. Results were expressed

as mean ± standard error of mean (M±SEM).

63

Chapter 3

Results

65

Development of in vitro inflammatory model

using human intestinal epithelial

cell lines

67

3.1. Introduction

Intestinal epithelium acts as a physical barrier in innate and adaptive immune

system and interacts directly with pathogenic and non-pathogenic (probiotic)

microflora. Nitric oxide (NO), a signalling molecule involved in the pathogenesis of

intestinal inflammation, has been reported to be the main mediator in the

pathogenesis of inflammatory bowel disease (IBD) and is related to the tissue

damage in IBD patients (Dijkstra et al., 1998). Moreover, there is evidence that

the intestinal epithelium produce NO which is highly regulated by pro-inflammatory

cytokines (Kolios et al., 1995; Kolios et al., 1998). In this regard, a number of

human colon carcinoma cell lines such as HT-29, Caco2, SW-620, and human

embryonic kidney epithelial cell line HEK 293 have been treated with inflammatory

mediators for use as an inflammatory model. A uterine endometrial carcinoma cell

line such as Hela cells have also been exploited for such studies.

The excessive production of inducible nitric oxide synthase (iNOS) by the

epithelium may be a target for the beneficial actions of probiotics. Thus it was the

intention to explore whether probiotics could regulate this process. To investigate

this, initial studies were conducted to develop epithelial cell culture models that

could be induced to express iNOS and thus NO production. Different cytokines

and/or LPS were used at different concentrations and at different combinations to

induce iNOS expression in HT-29, Caco2, SW-620 and Hela cells. Induction was

monitored by measuring nitrite accumulation using the Griess assay.

68

3.2. Methods

3.2.1. Activation of cells with individual and combinations of different cytokines

Various cell lines were exposed to increasing concentrations of LPS (1-500 g/ml)

or cytokines (IFN-: 10-1000 U/ml, IL-1U/ml and TNF-: 10-300 U/ml) alone and

in combination (LPS+INF- , LPS+IL-1, LPS+TNF-; INF- +IL-1, INF- +TNF-

; INF- +IL-1+TNF-) for 24 hours. Induction of iNOS under these conditions

was monitored by western blotting. In parallel experiments, J774 macrophages

were induced with LPS (1 g/ml) or IFN- (10 U/ml) and used as a positive control

for the expression of iNOS and NO production. The accumulation of nitrite in the

culture medium was measured by the Griess assay as described in section 2.8. In

some studies this was carried out after conversion of nitrate to nitrite to ensure any

NO produced could be detected.

3.2.2. Detection of total NO production in cadmium treated culture medium

The Griess assay only detects nitrite in solution. However, it is reported that a

significant proportion of the NO resealed by human cells is converted to nitrate. As

a result it is possible that the amount of NO released by activated human cells

may be underestimated if the Griess assay is conducted without conversion of the

entire nitrate to nitrite prior to the assay. Thus parallel studies were carried out

where culture media from cells were treated with cadmium to convert nitrate to

nitrite as described in section 2.8. The data below describes the results obtained.

3.2.2.1. Standardisation of the cadmium catalysed conversion of nitrate to nitrite.

To ensure optimal conversion of nitrate to nitrite the experimental conditions had

to be optimised by varying the number of metallic cadmium beads and incubating

the beads in HCl.

A set of sodium nitrate solution (1, 2, 3, 4, 5 and 10 nmols/100μl) in cell culture

medium was prepared. Aliquots of 50μl sodium nitrate solutions or LPS-treated

69

human cell culture medium were diluted using water (140 l) and deproteinised by

adding 30% ZnSO4 solution (10 l). Following the incubation at room temperature

for 15 min and centrifugation at 2000 g supernatants were transferred to two sets

of eppendorf tubes containing equal number of cadmium beads (3-5 beads). The

beads were soaked in 5 M HCl overnight and washed with water, 0.1M HCl and

finally with 0.1M NH4OH before use. A third of the supernatant of sodium nitrate

solutions was transferred to another set of eppendorf tubes without cadmium

beads to compare the change in nitrite production. The solutions with and without

cadmium beads were incubated overnight on a shaker at room temp. Following

centrifugation for 15 min at 3000 g total nitrite in the supernatants was measured

by the Griess assay. A standard curve of sodium nitrite was constructed using the

absorbance of serial dilutions of sodium nitrite, and the nitrite in sodium nitrate

solution or LPS-treated cell culture supernatant was determined from the standard

curve.

70

3.3. Results

3.3.1. Activation of HT-29 cells with lipopolysaccharide (LPS), interferon-

(IFN-) interleukin-1 (IL-1) and tumour necrosis factor- (TNF-) alone and

in combinations

Table 9 shows the data obtained for nitrite production in HT-29 treated with either

LPS, IFN- or TNF-. As shown in the table none of the stimuli used induced NO

production. On the other hand, LPS and IFN- individually induced NO production

in J774 cell. Similarly, combinations of these stimuli and in addition IL-1 at

optimum concentrations that were not cytotoxic also failed to induce NO synthesis

by the HT-29 cells (Table 10). Western blot analysis of lysates revealed that iNOS

was not induced in HT-29 cells under the conditions above. In contrast, iNOS was

strongly expressed in J774 macrophages exposed to either LPS or IFN- (Figures

13 and 14).

71

Table 9: Effects of LPS, IFN- and TNF-on NO production in HT-29 and J774 cells

Conditions Nitrite (nmol)/µg protein

(Mean; n=3) SD SEM

Control 0.002 0.001 0.001

LPS (1 g/ml) 0.002 0.001 0.001

LPS (10 g/ml) 0.002 0.002 0.001

LPS (100 g/ml) 0.002 0.003 0.001

IFN- (10 U/ml) 0.003 0.003 0.002

IFN- (100 U/ml) 0.002 0.001 0.001

IFN- (1000 U/ml) 0.003 0.005 0.003

TNF- (10 U/ml) 0.002 0.001 0.001

TNF- (25 U/ml) 0.002 0.002 0.001

TNF- (50 U/ml) 0.002 0.001 0.001

TNF- (75 U/ml) 0.003 0.002 0.001

TNF- (100 U/ml) 0.002 0.001 0.001

LPS (g/ml)- J774 0.058 0.033 0.019

IFN- (10 U/ml)-J774 0.034 0.029 0.016

Confluent monolayers of Cells were treated with different concentrations of LPS,

IFN- or TNF- and J774 cells were treated with LPS or IFN- alone for 24 hrs. After

incubation, the nitrite in the spent culture medium was measured by the Griess

assay as described in the methods (chapter 2, section 2.8). Results are presented

as mean ± SEM of three different experiments.

72

Figure 13: Induction of iNOS in HT-29 cells and J774 macrophages

Confluent monolayers of HT-29 cells were treated with different concentrations of

LPS, IFN- or TNF- and J774 cells were treated with LPS or IFN- alone for 24

hrs. Cell protein was extracted and iNOS expression in the lysates detected by

western blot analysis as described in the methods (chapter 2, section 10.0 to

11.0). The blot is representative of at least three experiments.

iNOS

HT-29 cells

Control + - - - - - - - - - - - + - -

LPS (g/ml) - 1 10 100 - - - - - - - - - 1 -

IFN-(U/ml) - - - - 10 100 1000 - - - - - - - 10

TNF-(U/ml) - - - - - - - 10 25 50 75 100 - - -

J774 cells

73

Table 10: NO production in HT-29 cells after treatment with different

combinations of LPS, IFN- IL-1 and TNF-

Conditions Nitrite (nmol)/µg

protein (Mean; n=3)

SD SEM

Control 0.002 0.001 0.001

LPS (1μg/ml)+IFN- (10U/ml) 0.004 0.005 0.003

LPS (1μg/ml)+IFN- (100U/ml) 0.005 0.005 0.003

LPS (10μg/ml)+IFN- (10U/ml) 0.004 0.004 0.002

LPS (10μg/ml)+IFN- (100U/ml) 0.005 0.004 0.002

LPS (100μg/ml)+IFN- (100U/ml) 0.003 0.004 0.002

LPS (100g/ml)+IFN- (300U/ml) 0.001 0.002 0.001

LPS (100g/ml)+IL-1 (300U/ml) 0.001 0.002 0.001

LPS (100g/ml)+TNF- (300U/ml) 0.001 0.000 0.001

IFN- (300U/ml)+IL-1 (300U/ml) 0.001 0.001 0.002

IFN-(300U/ml)+TNF- (300U/ml) 0.002 0.001 0.001

TNF- (300U/ml)+IL-1 (300U/ml) 0.002 0.001 0.002

IFN- (300U/ml)+IL-1 (300U/ml)+ TNF- (300U/ml)

0.002 0.001 0.002

Confluent monolayers of cells were treated with combinations of different

concentrations of LPS, IFN-, IL-1 and TNF-for 24 hrs. After incubation, the

nitrite in the spent culture medium was measured by the Griess assay as

described in the methods (chapter 2, section 2.8). Results are presented as mean

± SEM of three different experiments.

74

Figure 14: Cytokine stimulated induction of iNOS in HT-29 cells

Confluent monolayers of cells were treated with combinations of different

concentrations of LPS, IFN- IL-1 and TNF- for 24 hrs. Cell protein was

extracted and iNOS expression in the lysates detected by western blot analysis as

described in the methods (chapter 2, section 10.0 to 11.0). The blot is

representative of at least three experiments.

iNOS

Control +ve + - - - - - - - - - - - -

LPS (g/ml) - - 1 1 10 10 100 100 100 100 - - - -

IFN- (U/ml) - - 10 100 10 100 100 300 - - 300 300 - 300

IL-1 (U/ml) - - - - - - - - 300 - 300 - 300 300

TNF- (U/ml) - - - - - - - - 300 - 300 300 300 -

-

-

-

75

3.3.2. Activation of Caco-2 cells with lipopolysaccharide (LPS) alone and in

combination with tumour necrosis factor- (TNF-

Table 11 shows nitrite production in Caco-2 cells treated with LPS alone and in

combination with TNF- The table showed that neither LPS alone or in

combination with TNF- induced NO production in these cells. Western blot

analysis of lysates from the treated cells showed that iNOS was not induced in

Caco-2 cells on exposure to LPS and TNF- (Figure 15).

Table 11: Effects of LPS and TNF-on NO production in CaCo-2 cells


protein (Mean; n=3)

SD SEM

Control 0.000 0.001 0.001

LPS (10 g/ml) 0.002 0.002 0.001

LPS (10 g/ml)+TNF- (100 U/ml) 0.002 0.001 0.001

Confluent monolayers of cells were treated with LPS alone and in combination

with TNF-α for 24 hrs. After incubation, the nitrite in the spent culture medium was

measured by the Griess assay as described in the methods (chapter 2, section

2.8). Results are presented as mean ± SEM of three different experiments.

76

Figure 15: LPS and TNF- induced iNOS expression in Caco-2 cells

Confluent monolayers of cells were treated with LPS alone and in combination

with TNF-α for 24 hrs. Cell protein was extracted and iNOS expression in the

lysates detected by western blot analysis as described in the methods (chapter 2,

section 10.0 to 11.0). The blot is representative of at least three experiments

iNOS

Control

LPS (g/ml)

TNF-(U/ml)

+ve + - -

- - - 10 10

- - - - 100

-

-

-

77

3.3.3. Activation of SW-620 cells with lipopolysaccharide (LPS) alone and in

combinations with tumour necrosis factor- (TNF-

Table 12 shows the data obtained for nitrite production in SW-620 cells exposed to

either LPS alone or in combination with TNF-As shown in the epithelial cells

above none of the concentrations of LPS or LPS-TNF- combinations induced NO

synthesis in SW-620 cells. Western blot analysis of lysates also showed that iNOS

was not induced in SW-620 cells under the conditions above (Figures 16).

Table 12: Effects of LPS and LPS-TNF-α on NO production in SW-620 cells


protein (Mean; n=3)

SD SEM

Control 0.000 0.001 0.000

LPS 1.0 g/ml 0.001 0.001 0.001

LPS 3.0 g/ml 0.001 0.001 0.001

LPS 10 g/ml 0.001 0.001 0.001

LPS (10 g/ml + TNF- 100 U/ml) 0.003 0.001 0.001

LPS (25 g/ml + TNF- 100 U/ml 0.001 0.001 0.001

LPS (50 g/ml + TNF- 100 U/ml) 0.003 0.001 0.001

LPS (125 g/ml + TNF- 100 U/ml) 0.002 0.001 0.001

LPS 250 (g/ml + TNF- 100 U/ml) 0.002 0.001 0.001

LPS 500 (g/ml + TNF- 100 U/ml) 0.003 0.001 0.001

78

Confluent monolayers of cells were treated with different concentrations of LPS

alone and in combination with a fixed concentration of TNF- for 24 hrs. After




Figure 16: LPS and TNF-α induced iNOS expression SW-620 cells


alone and in combination with a fixed concentration of TNF- for 24 hrs. Cell

protein was extracted and iNOS expression in the lysates detected by western blot

analysis as described in the methods (chapter 2, section 10.0 to 11.0). The blot is


iNOS

Control +ve - - - - - - - - - -

LPS (g/ml - - 1 3 10 10 25 50 125 250 500

TNF-(U/ml) - - - - - 100 100 100 100 100 100

-

-

79

3.3.4. Activation of Hela cells with different concentrations of lipopolysaccharide (LPS) alone and in combinations with tumour necrosis

factor- (TNF-

Table 13 shows the data obtained for nitrite production in uterine endometrial cell

line Hela cells exposed to either LPS alone or in combination with TNF-As

shown in the Table 13 none of the concentrations of LPS or LPS-TNF-

combinations induced NO synthesis in Hela cells. Western blot analysis of lysates

also revealed that iNOS was not induced in Hela cells under the conditions above

(Figures 17).

Table 13: Effects of LPS and TNF-on NO production in Hela cells


protein (Mean; n=3)

SD SEM

Control 0.001 0.001 0.001

LPS (10 g/ml) 0.001 0.002 0.001

LPS (25 g/ml) 0.001 0.001 0.001

LPS (50 g/ml) 0.002 0.001 0.001

LPS (125 g/ml) 0.003 0.002 0.001

LPS (250 g/ml) 0.002 0.002 0.001

LPS (10 g/ml + TNF- 100 U/ml) 0.002 0.001 0.001

LPS (25 g/ml + TNF- 100 U/ml 0.001 0.002 0.001

LPS (50 g/ml + TNF- 100 U/ml) 0.001 0.001 0.001

LPS (125 g/ml + TNF- 100 U/ml) 0.003 0.001 0.001

LPS 250 (g/ml + TNF- 100 U/ml) 0.003 0.002 0.001

LPS 500 (g/ml + TNF- 100 U/ml) 0.003 0.002 0.001

80


alone and in combination with a fixed concentration of TNF- for 24 hrs. After




Figure 17: LPS and LPS-TNF- induced iNOS expression in Hela cells


alone and in combination with a fixed concentration of TNF- for 24 hrs. Cell

protein was extracted and iNOS expression in the lysates detected by western blot

analysis as described in the methods (chapter 2, section 10.0 to 11.0). The blot is


.

iNOS

Control +ve - - - - - - - - - - - -

LPS (g/ml) - - 10 25 50 125 250 10 25 50 125 250 500

TNF-(U/ml) - - - - - - - 100 100 100 100 100 100

-

-

81

3.3.5. Conversion of nitrate to nitrite in cadmium treated sodium nitrate solution

Figure 18 shows the conversion of nitrate to nitrite in the cell culture medium after

the treatment with cadmium. As shown in the figure the conversion of nitrate was

gradually increased with the change of the conditions. In the first 2 graphs (A, B),

a little conversion of nitrate to nitrite occurred due to less cadmium being added.

However, the conversion was increased with the increase of the number of

cadmium beads (C, D). Almost complete conversion of nitrate to nitrite occured

when the beads were soaked in 5M HCl overnight before use (E).

82

A B

C D

E.

Figure 18: Development of cadmium catalysed conversion of nitrate to nitrite.

83

Sodium nitrate solution in culture medium was diluted with water and

deproteinized with 30% ZnSO4 as described in the methods (Chapter 2, Section

2.13). Following the incubation and centrifugation supernatants were exposed to

cadmium beads previously washed with water, 0.1M HCl and finally with 0.1M

NH4OH. The solutions were incubated overnight on a shaker at room temp. A

second set of the supernatant was also incubated without cadmium beads to

compare the change in nitrite production. Following centrifugation total nitrite in the

supernatants was measured by the Griess assay as described in the methods

(Chapter 2, Section 2.8). The nitrite curves were constructed using the absorbance

obtained from the solutions by the Griess assay. Medium was incubated with 3 (A,

B) or 5 cadmium beads (C, D). In other experiments the 5 cadmium beads were

soaked in 5M HCl overnight (E) before being used.

84

3.3.6. Effects of LPS and TNF- on nitrite production in SW-620 cells

Figure 19 shows the detection of nitrite in medium from SW-620 cells after the

cadmium-catalysed conversion of nitrate. The graph shows that the combination of

TNF- (100 U/ml) and LPS (10-500 g/ml) stimulated SW620 cells and produced

NO that was gradually increased with the concentration of LPS. However, the

detected amount of nitrite converted from nitrate was in the pico-molar range when

compared to the concentrations of nitrite produced by LPS in J774 cells.

Figure 19: Effects of LPS and TNF- on nitrite production by SW-620 human

intestinal epithelial cells.

Confluent monolayers of cells were treated with combinations LPS and TNF- for

24 hrs. The culture medium was diluted with water and deproteinized with 30%

ZnSO4 as described in the methods (Chapter 2, Section 2.13). Following

incubation and centrifugation supernatants were exposed to cadmium beads

previously washed with water, 0.1M HCl and finally with 0.1M NH4OH and

incubated overnight on a shaker at room temp. After centrifugation nitrite in the


(Chapter 2, Section 2.8). Results are presented as mean ± SEM of three different

experiments. The left Y-axis shows the nitrite production in SW620 cells (ρmols)

and the right Y-axis shows the nitrite production in J774 cells (ηmols).

Control

g/ml)

LPS (10 g/ml)

LPS (25 g/ml)

LPS (50 g/ml)

LPS (125 g/ml)

LPS (250 g/ml)

LPS (500 g/ml)

LPS (1

0.00

2.50

5.00

7.50

10.00

0.00

0.25

0.50

0.75

1.00

SW620 cellsTNF- (100 U/ml)

J774 cells

Nitr

ite p

rodu

ctio

n(

mol

s/

g pr

otei

n)

Nitr

ite p

rodu

ctio

n(

mol

s/

g pr

otei

n

85

3.3.7. Effects of LPS on nitrite production in Hela cells

Figure 20 shows the production of nitrite in LPS-treated Hela cells after the

cadmium-catalysed conversion of nitrate. The graph shows that the cells

stimulated with LPS resulted in the production of NO. However, the amount of

nitrite detected after the conversion was not consistent with the concentration of

LPS and was low in the pico-molar range when compared to the nanomoles levels

produced by LPS in activated J774 cells.

Figure 20: Effects of LPS on nitrite production in Hela uterine endometrial cells

Confluent monolayers of cells were treated with different concentrations of LPS for








experiments. The left Y-axis shows the nitrite production in Hela cells (ρmols) and

the right Y-axis shows the nitrite production in J774 cells (ηmols).

Control

g/ml)

LPS (10 g/ml)

LPS (50 g/ml)

LPS (125 g/ml)

LPS (250 g/ml)

LPS (

0.0

2.5

5.0

7.5

10.0

0.00

0.25

0.50

0.75

1.00

Hela cell J774 cell

Nitr

ite p

rodu

ctio

n(

mol

s/

g pr

otei

n)

Nitr

ite p

rodu

ctio

n(

mol

s/

g pr

otei

n

86

3.3.8. Effects of LPS and TNF- on nitrite production in Hela cells

Figure 21 shows the production of nitrite in LPS and TNF- -induced Hela cells

after the cadmium-catalysed conversion of nitrate. The graph shows that the

combination of TNF- (100 U/ml) and LPS (10-500 g/ml) stimulated Hela cells

with the production of NO which showed a gradual increase as the concentration

of LPS increased up to 250 g/ml but decreased at 500 g/ml which might be due

to toxicity to the cells. The increases observed were however not statistically

different to controls. Moreover, the amount of nitrite detected after conversion of

nitrate was again in the low pico-molar range.

Figure 21: Effects of LPS and TNF- on nitrite production in Hela uterine

endometrial cells

Confluent monolayers of cells were treated with combinations LPS and TNF- for








experiments. The left Y-axis shows the nitrite production in Hela cells (ρmols) and

the right Y-axis shows the nitrite production in J774 cells (ηmols).

Control

g/ml)

LPS (10 g/ml)

LPS (25 g/ml)

LPS (50 g/ml)

LPS (125 g/ml)

LPS (250 g/ml)

LPS (500 g/ml)

LPS (1

0.00

2.50

5.00

7.50

10.00

0.00

0.25

0.50

0.75

1.00

Hela cellTNF- (100 U/ml)

J774 cell

Nitr

ite p

rodu

ctio

n(

mol

s/

g pr

otei

n)

Nitr

ite p

rodu

ctio

n(

mol

s/

g pr

otei

n

87

3.4. Discussion

NO, and its cytotoxic metabolites peroxynitrite have been implicated as the main

mediator in the pathogenesis of IBD. A pronounced increase in iNOS expression

and NO production in the inflamed mucosa have been reported in patients with

ulcerative colitis (Boughton-Smith et al., 1993; Lundberg et al., 1994; Middleton et

al., 1993). Over expression of iNOS protein, increased iNOS activity or NO

production and increased iNOS mRNA levels have also been reported both in

ulcerative colitis and Crohn‟s disease (Kimura et al., 1998; McLaughlan et al.,

1997; Singer et al., 1996). The increasing evidence support the notion that

excessive production of NO, presumably from the epithelium, may be responsible

for the pathogenesis of these disease states (Cavicchi et al., 2000; Ikeda et al.,

1997; Kolios et al., 1998; Rachmilewitz et al., 1995).

In many cell types, NO is induced by various cytokines and microbial endotoxin,

and iNOS is the major isoform expressed by intestinal epithelial cells (Salzman et

al., 1996; Witthoft et al., 1998). As a result several human colon epithelial

carcinoma cell lines have been widely used as in vitro experimental model of

intestinal epithelial cells (Bruno et al., 2005; Huerta et al., 2009; Kim et al., 2012;

Kolios et al., 1995; Vignoli et al., 2001). These cell lines have been characterized

and used to investigate epithelial cell function and barrier regulation. Although,

derived from colon carcinoma, when cultured under specific condition, these cells

morphologically and functionally resemble the enterocytes lining the intestine

(Ferrec et al., 2001; Futschik et al., 2002; Lee et al., 2007; Stadelmann et al.,

2012; Stragand et al., 1981). Thus as part of this thesis attempts were made to

develop an in vitro epithelial culture model of gut inflammation that could be used

to identify the actions of probiotics and investigate mechanisms associated with

the effects observed. Initially, experiments were focused on the human colon

carcinoma cell line HT-29 but subsequently extended to using both Caco2 and

SW-620 cells. In each case, the cells were treated with endotoxin, inflammatory

cytokines and in combination over a wide concentration range selected from other

studies in the literature.

88

Despite the reports in the literature and the extensive approach we took, none of

the cells selected could be induced to express iNOS or produce NO. This was

unexpected especially as the HT-29 cell line has been reported to induce iNOS

expression and NO production in response to a combined treatment with IL-1 (10

ηg/ml) and IFN-(300 U/ml) that was potentiated threefold when treated with the

combination of IL-1, IFN- and TNF- (100 ηg/ml). Similarly, iNOS expression

was greatly enhanced when cells were treated with a combination of IL-1, IFN-

and TNF- (Kolios G et al., 1998; Kolios G et al., 2004). The concentrations used

in these reported studies were included in the experiments for this thesis but with

no significant effect and it is currently not clear why no induction was achieved in

our laboratory. It is worth noting however that at least one other report has shown

that while HT-29 cells responded to TNF- by expressing iNOS and producing NO

they failed to be stimulated by IFN- (Kim et al., 2012). Thus it is likely that

different cytokines exert different effects in these and other cells and the response

may be cytokine dependent. This does not however explain the lack of response

observed in the studies reported in this thesis especially as various combinations

and concentrations of inflammatory mediators were used.

Caco-2 is the other extensively used colon carcinoma cell line (Marion-Letellier et

al., 2008; Megias et al., 2007; Resta-Lener et al., 2005), has been reported to be

stimulated following treatment with the combination of cytokines IL-1β, TNF- and

IFN-. A significant induction of NO and iNOS mRNA has been shown in response

to these cytokines (Marion et al., 2003). A separate article has reported the

stimulation of Caco2 cells only with IFN-. However, the induction of NO and iNOS

was much higher when the cells were treated in combination with IL-1β, TNF-

and IFN- (Chavez et al., 1999).

The human colon carcinoma cell line SW620 has been reported to constitutively

express iNOS and NO (Jenkins et al., 1994) and can also be stimulated by LPS

and the cytokines IL-1β, TNF- and IFN- Schuerer-Maly et al., 1994). In the

present studies, SW620 and the other two cell lines did not respond to the

89

treatment with any of the inflammatory mediators used. To be able to progress

with the project alternative cell lines were considered including Hela cells which

are a uterine endometrial carcinoma cell line and also the murine J774

macrophages. Similar to other cell lines, no nitrite or iNOS expression was

detected in Hela cells treated under identical conditions to those described above.

Initially the detection of NO was carried out by determining nitrite accumulation in

the culture medium at the end of each experiment. However, in aqueous solution,

NO degrades rapidly to nitrate and nitrite and only the nitrite can be measured

using Griess assay. Thus, the entire nitrate remains undetected. This can

sometimes result in underestimation or the lack of detection of NO especially

where the ratio of nitrate to nitrite is biased more towards nitrate production. This

is in fact the reported case in human cell systems (Wennmalm et al., 1992). In

animal cells, such as murine macrophage J774 cells, nitrite is detectable and the

total nitrite can be measured easily by Griess assay because the ratio of nitrate to

nitrite is reported to be 1:1 (Iyengar et al., 1987). Moreover, J774 cells produce

significantly more NO than most other cell lines and thus making it easy to detect

its production by measuring nitrite alone.

Since in human cells most of the NO is converted to nitrate other than to nitrite,

additional experiments were therefore carried out where the collected culture

medium at the end of each experiment was treated with cadmium to reduce all the

nitrate to nitrite and the latter then detected by the Griess assay. Despite this

approach, the levels of nitrite detected in media from the human cell lines did not

vary significantly from those in control non-activated cells. This was not due the

nitrate not been converted to nitrite, because the assay was optimised as

described in section 3.3.5 to ensure optimal conditions that facilitated the complete

conversion of nitrate to nitrite (figure 18) were used. The likely explanation is that

these cells did not respond to stimulation by the pro-inflammatory mediators and

as such could not express iNOS or produce NO. The direct evidence in support of

this was the lack of concurrent expression of iNOS under the treatment conditions.

90

On the other hand, the murine J774 macrophages responded readily to even just

LPS treatment alone and produced detectable amounts of NO and significant

induction of iNOS.

Although the exact reason(s) for the lack of response by the human cell lines were

not identified, there are reports that the passage number may have a critical

influence on the characteristics and function of cells in culture. Indeed several

reports have demonstrated that passage number and culture condition may affect

cell characteristics over time (Chang-Liu and Woloschak, 1997; Guo et al.,

2012; Hughes et al., 2007; Sambuy et al., 2005). Cell lines of high passage

number have been reported to show changes in morphology, growth rates, protein

expression, transfection efficiency and response to stimuli when compared to

lower passage cells (ATCC Bulletin number 7;Briske-Anderson et al., 1997;

Esquenet et al., 1997; Lipiec et al., 2012; Yu et al., 1997).

In this regard it should be noted that the HT- 29 cell line used in this study was

from passage 144. Unfortunately, there were no other records of passage

numbers for the rest of the human cell lines examined as the cells were inherited

from frozen stocks used previously. It is worth noting however that the passage

number of our HT-29 cells was much higher than that of the HT-29 cells (passages

20-45) used by Son et al. and Jijon et al. (Jijon et al., 2004; Son et al., 2005).

Similarly, in another report using Caco-2 cells the passage number was restricted

to between 15-60 passages (Gutierrez-Orozco et al., 2013; Son et al., 2005;

Wehkamp et al., 2004). There are, however, reports that the HT-29 cell line does

maintain its function and characteristics even at higher passage (Gutierrez-

Orozco et al., 2013). Thus, it is not clear whether the high passage of the cells

used in this thesis may have influenced the outcomes reported, but this factor is

worth being considered.

Although macrophages are not epithelial in nature, they play a critical role in

inflammation and in particular, in gut inflammation or pathologies, where they

91

appear to play a central role (Bassaganya-Riera et al., 2012). Therefore, J774

macrophages cell line was selected for use in all future studies.

93

Chapter 4 Results

95

The effect of probiotic culture supernatant and probiotic-conditioned medium on nitric oxide

production and iNOS/COX-2 expression in J774 macrophages

97

4.1. Introduction

Probiotic bacteria have been reported to produce beneficial effects through their

released metabolites. The studies in this chapter were therefore aimed at

investigating the effects of the supernatant obtained from the cultures of probiotics

and also of probiotic conditioned medium on nitric oxide (NO) production and on

inducible nitric oxide synthase (iNOS) or cyclooxygenase-2 (COX-2) expression in

J774 macrophages. In studies using the supernatant, bacterial cultures were

grown in MRS broth and subsequently centrifuged collecting the supernatant for

use in the experiments. In experiments with conditioned medium, the cultured

bacteria was centrifuged and bacterial cells were further cultured in normal cell

culture medium Dulbecco‟s Modified Eagle Medium (DMEM) for 24 hours and the

media collected after centrifuging and discarding the bacterial cells. The collected

medium referred to as conditioned medium was used in the studies. The aim of

these studies was to establish whether the bacteria produced mediators exert

effects on the pathways (iNOS and COX-2) involved in the production of

inflammatory mediators.

To investigate whether the bacterial culture medium (MRS broth) itself has any

effect on NO production in J774 cells, parallel experiments were also carried out

using only the MRS broth. A cell viability study of MRS broth was also carried out

using the MTT assay.

This chapter contains data showing the bacterial growth curves, optimisation of

macrophage activation and the effects of freeze-dried culture supernatant of

Lactobacillus rhamnosus GG (LGG), LGG-CM and VSL#3-CM on nitric oxide (NO)

production.

98

4.2. Methods

4.2.1. Construction of the bacterial growth curve

The bacteria were inoculated in MRS broth and grown at 37oC for 24 hours with

shaking. MRS broth without bacteria was also incubated to use as control.

Samples were collected from the culture every hour up to 6th hour and again from

17th up to 24th hour. The turbidity of the culture was determined by taking the

absorbance at 600 nm wavelength (Pena et al., 2003 and Yan et al., 2007) and pH

was measured from the same sample. The curves for bacterial growth and pH

were constructed by plotting the absorbance and pH against time.

4.2.2. Preparation of Sample

4.2.2.1. Freeze-dried LGG culture supernatant

LGG was cultured in MRS broth at 37oC for 24 hours with shaking. Samples were

collected from log (7th hour) and stationary phases of the bacterial growth. The

bacteria free supernatants were collected by centrifugation at 500 g for 10 min,

filtered through 0.2 micron (µ) filter and freeze-dried. Dry samples were stored at –

80oC until use.

4.2.2.2. Probiotic conditioned cell culture medium

Bacteria were cultured in MRS broth at 37oC for 24 hours with shaking. Bacterial

pellet was collected after centrifuging the culture at 500 g for 10 min and washed

with autoclaved phosphate buffer saline (PBS). The pellet was then re-suspended

in a sterile 50 ml centrifuge tube containing 30 ml of cell culture medium with 20%

FBS and incubated at 37oC for another 24 hrs, with shaking. After centrifugation at

500 g for 10 min, the culture medium was collected and filtered through 0.2 micron

filter. Samples were stored at –80oC until use.

4.2.3. MTT assay

Cell viability was determined by MTT assay to measure the mitochondrial activity

of the viable cells that metabolise the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-

99

diphenyltetrazolium bromide (MTT) to purple formazan. Cells monolayers in 96-

well plate were exposed to different dilutions of MRS broth for 24hours. The

culture medium was replaced with 200 µl freshly prepared 0.5mg/ml MTT solution,

and incubated for another 4 hours at 37oC in a humidified CO2 incubator. The MTT

solution was removed and the formazan crystals formed were dissolved by adding

200 µl isopropanol by incubating for 30 min at room temperature with shaking. The

plate was read at 540 nm and viability of the drug treated cells was calculated as a

percentage of the control (untreated cells), considering the viability of control as

100%.

4.2.4. Activation of macrophages J774 by LPS

J774 cells were treated with different concentrations of LPS (0.01-10 g/ml) for 24

hours. The nitrite, accumulated in the culture medium was measured by the Griess

assay and iNOS induction was detected by western blotting as described in

chapter 2, section 10.0 to 11.0. Bands that developed on the film were quantified

using the image processing and analysing programme ImageJ. The values

obtained for probiotic-treated samples were compared to those of their respective

controls.

4.2.5. Effects of MRS broth on the sensitivity of the Griess assay

A serial dilution of sodium nitrite solution (1, 2, 3, 4, 5 and 10 nmol / ml) in DMEM

were prepared and used to construct a nitrite standard curve. Another five

standard curves were set up on the same plate and 50 μl solution of diluted MRS

broth (1:1000 -1:10) added to the wells of the standard curves. 100 μl Griess

reagent was added to the wells of first set of standard curve and 150 μl Griess

reagent added to the wells of last five sets of standard curves. The plate was

incubated at room temp for 15 min and absorbance measured at 540 nm on a

micro well plate reader. Standard curves were constructed by plotting the

absorbance against nitrite concentrations as described in the methods (Chapter 2;

Section 2.8)

100

4.3. Results

4.3.1. Growth phases of bacteria and change in pH

Figure 22 shows the growth of the bacteria Lactobacillus rhamnosus GG (LGG)

and bacteria present in VSL#3; and changes of pH of the cultures with the

increase of culture time. As shown in Figure 22 (A), the growth of LGG and

bacteria present in VSL#3 was evident from the 2nd hour (starting of log phase) of

incubation. The growth continued until the 6th hour when the last optical density

was measured. The next optical density measured at 17th hour shows the end of

log phase and starting of the stationary phase. Figure 22 (B) shows the decrease

of pH that also started after the 2nd hour and correlated with the increase of the

growth of bacteria shown in figure 22 (A). However, the intensity of the decrease in

pH was lower in LGG culture compared to VSL#3, which may be the effect of the

presence of multiple strains of bacteria in VSL#3 culture. These bacteria are called

lactic acid producing bacteria and it is evident from the data that shows the

decrease of pH during their growth.

101

A.

B.

Figure 22: Bacterial growth curves and changes in pH with growth

0 2 4 6 8 10 12 14 16 18 20 22 24 26

0.00

0.50

1.00

1.50

2.00

2.50ControlVSL#3LGG

Time (hour)

Opt

ical

den

sity

(600

nm

)

0 2 4 6 8 10 12 14 16 18 20 22 24 26

3.50

4.00

4.50

5.00

5.50

6.00

ControlVSL#3LGG

Time (hour)

pH

102

Sample of frozen bacteria was inoculated in MRS broth and incubated overnight at

37oC with shaking. From the overnight culture, an aliquot of 500 µl was added in

150 ml autoclaved, pre-warmed MRS broth, and incubated at 37oC for 24 hours

with shaking. Samples were collected from the culture every hour, optical density

measured at 600 nm wavelength and pH determined. The curves for bacterial

growth and pH were constructed by plotting the absorbance and pH against the

time.

103

4.3.2. Effects of bacterial lipopolysaccharide (LPS) on nitrite production in J774 macrophages

Figure 23 shows nitrite production in J774 cells following exposure to different

concentrations (0.01-10 g/ml) of lipopolysaccharide (LPS). LPS caused a

concentration-dependent production of nitric oxide (NO) which appeared to be

highest at 1g/ml declining at 3 g/ml and above due potentially to cytotoxicity to

the cells (Figure 23A). Western blot analysis of lysates also showed the

concentration-dependent induction of inducible nitric oxide synthase (iNOS) in the

LPS-treated cells (Figure 23B) which correlated with the NO production in Figure

23A. The concentration of LPS that caused the highest production of NO (1 g/ml)

was chosen to activate the J774 macrophages throughout the study.

A.

B.

Figure 23: LPS-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774 macrophages

0 0 0.01 0.03 0.1 0.3 1.0 3.0 10.0

0

25

50

75

100

125

Log [LPS (g/ml)]

Nitri

te p

rodu

ctio

n (

mol

/ g

prot

ein)

% m

axim

al re

spon

se

iNOS 130 kD

LPS (g/ml) 0 0.01 0.03 0.1 0.3 1.0 3.0 10.0

104

Confluent monolayers of J774 cells were treated with different concentrations of

LPS for 24 hrs. The nitrite content in the spent culture medium was measured by

Griess assay as described in materials and method (Chapter 2; Section 2.8).

Results are presented as mean ± SEM of three different experiments.

Cell protein was extracted and iNOS expression in the lysates detected by western

blot analysis as described in the materials and method (chapter 2, section 10.0 to

11.0). The blot is representative of three experiments.

105

4.3.3. Effect of Freeze-dried MRS broth and Lactobacillus rhamnosus GG (LGG) culture supernatant (collected from the log phase) on basal and lipopolysaccharide (LPS)-induced nitric oxide (NO) production in J774 cells

Figure 24 illustrates the effects of increasing concentration of freeze-dried MRS

broth and LGG (0.01-1 mg/ml) on nitric oxide (NO) production in non-stimulated

and LPS (1 g/ml)-stimulated J774 cells which is evident from the nitrite

accumulation in the treated wells. The negative control MRS broth showed slight

increase in the basal NO production at higher concentration. LGG, however, had

no effect on basal NO production. In contrast, both MRS broth and LGG increased

the LPS-induced NO production which was higher at the lower concentration (0.01

mg/ml). Higher concentrations of either MRS or LGG caused inhibition of LPS-

induced NO production however, none of the effects showed statistical

significance when compared to the LPS controls.

The negative control MRS broth was not expected to produce any change on NO

production. However, as it produced effect on basal NO and LPS-induced NO

production in J774 cells, it was planned to clarify whether the MRS broth has any

effect on the sensitivity of the Griess assay that is used to measure NO in the

culture medium.

106

Figure 24: Effect of freeze-dried MRS broth and Lactobacillus rhamnosus GG (collected from the log phase) on basal and lipopolysaccharide (LPS)-induced NO production in J774 macrophage

Confluent monolayers of cells were treated for 24 hours with different

concentrations of freeze-dried MRS/LGG in the absence and presence of LPS.

The nitrite content in the spent culture medium was measured by Griess assay as

described in materials and method (Chapter 2, Section 2.8). Control represents

the cells treated with culture medium only. Results are presented as mean ± SEM

of four different experiments. Statistical significance was determined by one way

ANOVA using Dunnett‟s multiple comparison test.

Cont

rol

0.01

0.05 0.1

0.5

1.0

LPS

0.01

0.05 0.1

0.5

1.0

0

50

100

150

200MRSLGG

MRS/LGG (mg/ml) LPS (1g/ml) +MRS/LGG (mg/ml)

Nitr

ite p

rodu

ctio

n(%

of L

PS a

ctiv

ated

cel

ls)

107

4.3.4. Effect of Freeze-dried MRS broth and Lactobacillus rhamnosus GG (LGG) culture supernatant (collected from the stationary phase) on basal and lipopolysaccharide (LPS)-induced nitric oxide (NO) production in J774 cells

Freeze-dried MRS broth and LGG culture supernatant (0.01-1 mg/ml), collected

from the stationary phase of bacterial growth showed a slight induction of basal

NO in J774 cells (Figure 25). In contrast, both MRS and LGG inhibited the LPS-

induced NO production, that was statistically significant when compared to LPS

control (MRS: p<0.05 - 0.01; LGG: p<0.01)

As described in Figure 24, MRS broth was not expected to produce any effect. In

this data, the basal induction of NO and inhibition of LPS-induced NO production

by LGG may be the effect of the compound secreted by LGG that has been

accumulated in the culture media. However, as MRS broth produced similar

effects as LGG, the MRS-induced effect on the sensitivity of the Griess assay was

measured and decided not to use the bacteria culture supernatant to continue the

experiment.

108

Figure 25: Effect of freeze-dried MRS broth and Lactobacillus rhamnosus GG (collected from the stationary phase) on basal and lipopolysaccharide (LPS)-induced NO production in J774 macrophage

Confluent monolayers of cells were treated for 24 hours with different

concentrations of freeze-dried MRS/LGG and incubated for 24 hours in the

absence and presence of LPS (1 g/ml). The nitrite content in the spent culture

medium was measured by Griess assay as described in materials and method

(Chapter 2, Section 2.8). Control represents the cells treated with culture medium

only. Results are presented as mean ± SEM of four different experiments.

Statistical significance was determined by one way ANOVA using Dunnett‟s

multiple comparison test. In MRS-treated graph, ♦p<0.05 and ♦♦p<0.01 denote

significant difference from LPS (1 g/ml) treated cells. In the LGG-treated graph,

●●p<0.01 denotes significant difference from LPS (1 g/ml)-treated cells.

Control

0.010.05 0.1 0.5 1.0

LPS 0.01

0.05 0.1 0.5 1.00

50

100

150MRSLGG

MRS/LGG (mg/ml) LPS (1g/ml) +MRS/LGG (mg/ml)

Nitr

ite p

rodu

ctio

n(%

of L

PS a

ctiv

ated

cel

ls)

109

4.3.5. Effects of MRS broth on the sensitivity of the Griess assay

To ensure that none of the effects on nitric oxide production in J774 cells seen in

the results above (Figure 24 and Figure 25) were caused by the MRS broth in

which the bacteria Lactobacillus rhamnosus GG were cultured, experiments were

carried out investigating whether the broth alone (ie, broth that had not had

bacterial cultures) interfered with the Griess assay. Serial dilution of sodium nitrite

solution that is used to construct the standard curve for nitrite measurement was

incubated with Griess reagent in presence and absence of MRS broth (1:1000 -

1:10). The data shows that the curves constructed using the absorbance taken

from MRS containing wells was similar to the curve constructed with the

absorbance of sodium nitrite solution alone (Figure 26).

It is clear from the data that MRS broth had no interfering effect on the detection of

nitrite by Griess assay and that the induction of nitric oxide (NO) in mammalian

cells may be the effect of MRS broth that supports the culture and growth of

bacteria, which may not be appropriate for mammalian cells.

110

Figure 26: Effects of MRS broth on the sensitivity of the Griess assay

MRS broth was diluted (1:1000 - 1:10) in the cell culture medium. Each solution (1

ml) was transferred to the wells of 24-well plate and incubated in humidified tissue

culture incubator at 37oC in 5% CO2 for 24 hrs. A serial dilution of sodium nitrite

solution (1, 2, 3, 4, 5 and 10 nmol/ml) in DMEM was prepared and used to

construct a nitrite standard curve. Five more standard curves were set up on the

same plate and 50 μl solution of diluted MRS broth (1:1000 -1:10) added to the

last five sets of standards. 100 μl Griess reagent was added to the wells of first set

of standard curve and 150 μl Griess reagent added to the wells of last five sets of

standard curves. The plate was incubated at room temperature for 15 min and

absorbance measured at 540 nm on a micro-well plate reader. Standard curves

were constructed by plotting the absorbance against nitrite concentrations as

described in the materials and methods (Chapter 2, Section 2.9).

111

4.3.6. Effect of MRS broth on the viability of J774 macrophages

Figure 27 shows the effect of different dilutions of MRS broth (1:500 - 1:10) on the

viability of J774 cells. After the treatment for 24 hrs none of the dilutions of MRS

broth showed cytotoxicity to J774 cells. However, when MRS broth was used

simultaneously with lipopolysaccharide (LPS), cell viability was reduced to 40%

followed by an increase of up to 62% and 75% at the dilutions of 1:25 and 1:10.

The data shows that at lower concentration, while used with LPS, MRS broth was

toxic to the cells as LPS alone however; it has shown a protective effect on LPS-

treated cells at higher concentration, where the viability of the cells has increased.

Figure 27: Effect of MRS broth on viability of macrophage J774 cells

Confluent monolayers of J774 cells in 96-well plate were incubated with different

dilutions of MRS broth with or without LPS (1 µg/ml) for 24 hrs. The culture

medium in the treated wells was replaced with 200 μl fresh medium containing

0.05 mg/ml MTT solution and incubated for 4 hours at 37°C. As described in

materials and methods (Chapter 2, Section 2.14), the medium was removed, 200

μl Isopropanol added to the wells and incubated for 30 min at room temperature

on a shaker to dissolve the formazan crystals produced by the metabolism of MTT.

The plate was read on a micro-well plate reader at 540 nm. The viability of the

Control1:500

1:1001:50

1:251:10

LPS 1:500

1:1001:50

1:251:10

0

25

50

75

100

125

LPS (1g/ml) +MRS broth

MRS broth

***

% o

f Con

trol

112

MRS broth treated cells was calculated as a percentage of the control considering

the viability of the control (untreated cells) as 100%. The data are the mean SEM

of 3 independent experiments. Statistical significance was determined by one way

ANOVA using Dunnett‟s multiple comparison test. *** p<0.001 denotes significant

difference from Control.

113

4.3.7. Effects of LGG-CM on basal and lipopolysaccharide (LPS)-induced nitric oxide (NO) production in J774 macrophages

Figure 28 demonstrates the effect of different dilutions (1:500 - 1:10) of LGG-CM

on basal and lipopolysaccharide (LPS)-induced nitric oxide (NO) production in

J774. The results obtained showed that LGG-CM gradually increased the basal

NO level. However, the induction was not statistically significant. In contrast, it

inhibited the LPS (1 g/ml)-induced NO production where the inhibition was

statistically significant (p<0.01) at 1:10 dilution.

The data suggest that Lactobacillus rhamnosus GG (LGG) release metabolites in

the culture medium which has the ability to induce nitric oxide in normal cells and

play a role to improve the immune function. On the other hand, it has the ability to

suppress the pathogen-induced inflammatory mediator NO during infection.

.

Figure 28: Effects of LGG-CM on the basal and lipopolysaccharide (LPS)-induced nitric oxide (NO) production in J774 macrophage

Control

1:5001:100

1:501:25

1:10LPS

1:5001:100

1:501:25

1:100

20

40

60

80

100

120

LGG-CM LPS (1g/ml) +LGG-CM

Nitri

te p

rodu

ctio

n(%

of L

PS a

ctiv

ated

cel

ls)

114

Confluent monolayers of J774 cells were treated with a serial dilution of LGG-CM

in the absence and presence of LPS (1 g/ml). Following 24 hours incubation, the

accumulation of nitrite was measured by the Griess assay as described in

materials and methods (Chapter 2, Section 2.8). The results presented are the

mean ± SEM of five independent experiments. Statistical significance was

determined by one way ANOVA using Dunnett‟s multiple comparison test.

●●p<0.01 denote significant difference from LPS (1 g/ml)-treated cells.

115

4.3.8. Effects of LGG-CM medium on basal and lipopolysaccharide (LPS)-induced cyclooxynase-2 (COX-2) expression in J774 macrophages

Figure 29 shows the densitometric quantification of representative western blots of

cyclooxynase-2 (COX-2) expression detected in cell lysates after 24 hours

treatment with LGG-CM in J774 cells. LGG-CM caused an increase in the basal

COX-2 expression at the dilution of 1:25 and 1:10. On the other hand it did not

show any significant effects on lipopolysaccharide (LPS) (1 g/ml)-induced COX-2

expression.

The data suggest that the released metabolites by Lactobacillus rhamnosus GG

(LGG) induce the expression of COX-2 in normal, non-stimulated cells to improve

the immune function, which is a key enzyme in immune reaction and responsible

for the production of inflammatory mediator prostaglandin E2 (PGE2). However, it

may not have effect on the pathogen-induced COX-2 expression.

Figure 29: Effects of LGG-CM on basal and LPS-induced cyclooxynase-2 (COX-2) expression in J774 macrophage

Control

1:5001:100

1:501:25

1:10LPS

1:5001:100

1:501:25

1:100

50

100

150

200

LPS (1g/ml) +LGG-CM

LGG-CM

COX-

2 ex

pres

sion

(% o

f LPS

act

ivat

ed c

ells

)

116

Confluent monolayers of J774 cells were treated with a serial dilution of LGG-CM

in the absence and presence of LPS (1 g/ml). Following 24 hours incubation cell

lysates were collected and analysed by Western blot analysis to detect the

expression of COX-2 as described in materials and methods (Chapter 2, Section

2.10 - 2.11). The results presented are the mean ± SEM of five independent

experiments. Statistical significance was determined by one way ANOVA using

Dunnett‟s multiple comparison test.

117

4.3.9. Effect of VSL#3-CM medium on basal and lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774 macrophages

Figure 30 shows the effects of the different dilutions (1:500 – 1:10) of VSL#3-CM

on nitric oxide (NO) production in J774 cells. The results obtained show that

VSL#3-CM gradually increased basal NO level. The induction was statistically

significant at p<0.05 level at higher concentration. However, it did not produce any

effect on lipopolysaccharide (LPS) (1 g/ml)-induced NO production.

Representative western blot showed the gradual induction of the basal iNOS

expression in VSL#3-CM-treated cells that correlated with the NO production

shown in the same cells .

The data suggest that the bacterial strains present in VSL#3 release metabolites in

culture medium and these metabolites can induce the nitric oxide in normal cells

through the expression of inducible nitric oxide synthase (iNOS). On the other

hand, it did not seem to play any role on the regulation of pathogen-induced iNOS

expression and NO production.

118

Figure 30: Effects of VSL#3-CM on basal and LPS-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774 macrophages

Confluent monolayers of J774 cells were treated with a serial dilution of VSL#3-

CM in the absence and presence of LPS (1g/ml). Following the 24 hours

incubation, the accumulation of nitrite was measured by the Griess assay as

described in materials and methods (Chapter 2, Section 2.8). The results of nitrite

production presented are the mean ± SEM of five independent experiments.


multiple comparison test. *p<0.05 denotes significant difference from the control. Cell lysates were collected and analysed by Western blot analysis to detect the

expression of iNOS as described in materials and methods (chapter 2, section

2.10 - 2.11). Expression of -actin was used to determine the loading efficiency.

Control1:500

1:1001:50

1:251:10

LPS1:500

1:1001:50

1:251:10

0

20

40

60

80

100

120

VSL#3-CM LPS (1g/ml) +VSL#3-CM

Nitri

te pr

oduc

tion

(% of

LPS

activ

ated

cells

)

iNOS

β-actin

119

4.3.10. Effects of VSL#3-conditioned medium on basal and lipopolysaccharide (LPS)-induced cyclooxynase-2 (COX-2) expression

Figure 31 shows the densitometry quantification of representative Western blots of

cyclooxynase-2 (COX-2) expression in J774 cells detected in cell lysates after 24

hours treatment with VSL#3-CM. VSL#3-CM caused a gradual increase in basal

COX-2 expression. The expression was statistically significant at p<0.05 level. On

the other hand, it did not show any effect on LPS (1 g/ml)-induced COX-2

expression.

Similar to the effect of LGG shown in figure 29, this data also suggest that the

released metabolites by bacteria present in VSL#3 have the ability to induce the

expression of COX-2 in non-stimulated cells to improve the immune function.

However, it may not have any effect on the regulation of pathogen-induced COX-2

expression.

Figure 31: Effects of VSL#3-CM on basal and lipopolysaccharide (LPS)-induced cyclooxynase-2 (COX-2) expression in J774 macrophage

Control

1:5001:100

1:501:25

1:10LPS

1:5001:100

1:501:25

1:100

25

50

75

100

125

LPS (1g/ml) +VSL#3-CM

VSL#3-CM

CO

X-2

expr

essi

on(%

of L

PS a

ctiv

ated

cel

ls)

120


CM in the absence and presence of LPS (1 g/ml). Following the 24 hours

incubation, cell lysates were collected and analysed by Western blot analysis to

detect the expression of COX-2 as described in materials and methods (Chapter

2, Section 2.11). The results presented are the mean ± SEM of five independent


Dunnett‟s multiple comparison test. *p<0.05 denotes significant difference from the

control.

121

4.4. Discussion

Due to the incomplete activation of the human intestinal epithelial cell lines and

failure to detect nitrite after the cytokine treatment, the murine macrophages J774

cells were used as an in vitro inflammatory model.

In accordance with the study proposal, freeze-dried probiotic (Lactobacillus

rhamnosus GG; LGG) culture supernatant was prepared and used to investigate

its effects on bacterial lipopolysaccharide (LPS)-induced nitric oxide (NO)

production. Active metabolites released by the live probiotic bacteria are claimed

to produce anti-inflammatory effect and increase intestinal immunity (Bermudez-

Brito et al., 2012; Corthesy et al., 2007; kaci et al., 2011; O'Hara et al., 2006).

Therefore, cell-free extracts of LGG culture in MRS broth were used to determine

the role of active bacterial metabolite(s) in producing this response in cultured

cells.

To get the sample containing appropriate number of live bacteria, the bacteria

growth curves were constructed and the samples were collected from different

phases to compare the effects. The bacteria were grown in MRS broth in aerobic

condition and bacteria free supernatant of MRS broth was collected following the

method previously described. The number of bacteria was confirmed by

measuring the optical density of the culture at 600 nm (OD600). The optical density

of the culture 1.0 represented the number of bacteria of approximately 1x109

CFU/ml (Menard et al, 2004; Pena et al., 2003; Yan et al., 2007). The bacteria

culture medium MRS broth (freeze-dried) alone was also used in the experiment to

check its effect on J774 cells.

Bacteria-free supernatant of LGG produced a slight induction of basal nitrite and

reduced LPS-induced NO production in J774 cells. LGG, collected from the

stationary phase of their growth, showed more pronounced effect than the culture

collected from the log phase. This might be due to some metabolite(s) released by

LGG that accumulated in the culture until it was collected in the stationary phase.

This finding is supported by the report of Pena et al., reporting that bacteria-free

122

culture supernatant collected from late log phase of LGG growth produced marked

reduction of pro-inflammatory mediator tumour necrosis factor- (TNF-) but most

potent inhibitory effect produced by LGG-culture harvested at 24th hour (Pena et

al, 2003).

MRS broth (freeze-dried) alone used as negative control during the assay also

produced a similar effect on basal and LPS-induced NO production in J774 cells.

Whether the effect of MRS broth on NO production was due to any interference

with nitrite detection by Griess assay was examined. However, No such effect of

MRS broth on nitrite detection was observed.

It has been reported that Lactobacillus plantarum (one of eight bacteria in VSL#3)-

free supernatant grown in MRS broth reduced the nitrite level significantly in

Salmonella typhimurium-infected macrophages (Rishi et al., 2011). However, there

is no report on the effect of MRS broth alone in macrophages, as a control. MRS

broth is the medium that supports the culture and growth of bacteria. On the other

hand, J774 is the mammalian cell line derived from mouse macrophage. It needs

different medium to maintain the growth and culture. Besides, the composition of

and inorganic compounds present in MRS broth are not appropriate for J774 cells

and, indeed, might be toxic to the mammalian macrophage J774 cells.

Interestingly, the MTT test of MRS broth has shown a protective effect on LPS-

treated cells at higher concentration, where it increased the percentage of viable

cells. These results suggested the combined effect of MRS broth and LGG

secreted compound(s) on the inhibition of LPS-induced nitrite production.

However, to overcome this problem, instead of freeze-dried bacterial culture

supernatant, bacteria-conditioned cell culture medium was used as probiotic

sample during the study period.

Previous studies have shown improved immunity as well as anti-inflammatory

effects of bacterial cell components in vitro (Jijon, et al., 2004; Grangette, et al.,

2005). LGG-derived soluble compounds have been reported to inhibit cytokine-

induced apoptosis in the intestine (Yan et al., 2002). Again, Yan et al. reported on

123

isolated LGG-derived soluble factors and their effect on the suppression of

cytokine-induced colon epithelial cell apoptosis (Yan et al., 2007), through the

activation of AKT in PI3K-dependant pathway in cultured cells. To date, the known

mechanism of the function of probiotics includes production of anti-inflammatory

and blocking of pro-inflammatory cytokines, antagonism to pathogenic bacteria

(Otte et al., 2004), increasing the secretory immunoglobulin A (IgA) production

(Macpherson et al., 2004) and maintaining the barrier function (Resta-Lenert et al.,

2003; Resta-Lenert et al., 2006).

In this study, both LGG and VSL#3-CM increased the basal NO production in J774

cells in a concentration-dependant manner. This result was supported by other

reports on the induction of inflammatory mediators by probiotics to induce

immunity. Korhonen et al. reported that LGG induced a low level NO production in

presence of interferon- (IFN- that was inhibited by NOS inhibitors cycloheximide

and by NF- inhibitor pyrrolidine dithiocarbamate (PDTC). LGG and IFN--

stimulation also increased iNOS mRNA and protein levels in J774 cells. It

produced NO in T84 human epithelial cells in the presence of interleukin-1 (IL-

1, TNF- and IFN- (Korhonen et al., 2001). Another report by Korhonen et al.

demonstrated LGG-induced NO production in J774 cells which was inhibited by

NOS inhibitors (L‟NMMA and 1400W), protein synthesis inhibitor (cycloheximide)

and by NF-B inhibitor pyrrolidine dithiocarbamate (PDTC) (Korhonen et al.,

2002). LGG-induced antiviral activity in animal and human intestinal cell line and in

macrophage has been reported by Maragkoudakis et al. through the increased NO

production. L. casei one of eight strains within VSL#3 has also been reported to

induce NO in the same cell lines (Maragkoudakis et al., 2010). Dai et al. has

suggested the induction of NO by VSL#3 in the treatment of irritable bowel

syndrome (IBS) (Dai et al., 2012). Corridoni et al. has reported on the VSL#3-CM-

induced TNF- production in the regulation of epithelial permeability in

experimental ileitis through the modulation of innate immune effect on intestinal

epithelium (Corridoni et al., 2012).

124

The effects of LGG and VSL#3 differed on LPS-induced NO production. LGG-CM

significantly inhibited LPS-induced NO production that supports the previous report

of Yan et al. (2007). On the other hand, VSL#3-CM did not show any effect on

LPS-induced NO production and iNOS expression. Pena et al. also reported on

the effect of LGG on the inhibition of LPS-induced TNF- production and

suggested that inhibition of LGG-induced TNF- in macrophages depends on the

concentration of LPS (Pena et al., 2003).

Both of LGG-CM and VSL#3-CM caused a gradual increase in basal COX-2

expression; however, none of them had effect on LPS-induced COX-2 expression.

Freeze-dried LGG has been reported to induce the COX-2 expression in human

T84 epithelial cells (Korhonen et al., 2004). LGG increased COX-2 expression but

reduced the iNOS expression in the experimental colitis in rat model (Holma et al.,

2001). In contrast to COX-2 expression both of LGG and VSL#3 have been

reported to reduce the iNOS activity and PGE2 generation in iodoacetamide-

induced experimental colitis in rat models (Shibolet et al., 2002). Hart et al.

reported on the anti-inflammatory effect of VSL#3 through the inhibition of LPS-

induced IL-12 production in human dendritic cells (Hart et al., 2004). Petrof et al.

also reported the attenuation of intestinal inflammation by VSL#3-CM through the

reduction of TNF- in mouse model (Petrof et al., 2004). Another study reported

anti-inflammatory effect of VSL#3 on intestinal inflammation in the interleukin (IL)-

10 gene-deficient mice through the reduction in mucosal TNF- and IFN-

secretion (Madsen et al., 2001).

In this phase of study, iNOS expression and PGE2 production was not detected in

LGG and VSL#3-condition medium-treated cells and culture medium. Therefore,

the data is not enough to come to a conclusion whether LGG and VSL#3-CM have

any role on the regulation of expression and function of iNOS and COX-2 in J774

macrophage cells.

125

Chapter 5

Results

127

Investigation of the signalling pathways involved in VSL#3-CM-induced NO production and

iNOS expression in J774 macrophages

129

5.1. Introduction

On the basis of the effects of VSL#3-CM on the expression of inducible nitric oxide

synthase (iNOS) and production of nitric oxide (NO) in macrophages J774 cells

presented in Chapter 4, experiments in this chapter were carried out to determine

whether VSL#3-CM is regulating the signalling pathways involved in NO

production and iNOS expression in J774 cells.

Macrophages are the key element in the inflammatory process and accelerate the

cellular reaction in the intestinal epithelial cells. In response to pathogens, resident

macrophages become activated and generate iNOS-induced NO. In

macrophages, the expression of iNOS is mainly governed by the transcription

factor nuclear factor kappa B (NF-B), which is expressed ubiquitously and

regulates the induction of inflammatory protein associated with immune and

inflammatory responses (Baeuerle et al., 1994; Connelly et al., 2001; Ghosh et al.,

1998; Kleinert et al., 2004). In non-stimulated cells, NF-κB remains in its inactive

form in cytoplasm as a complex with the inhibitor protein IκB and thus prevented

from nuclear localisation for signal transduction. After activation, NF-κB becomes

free of IκB and translocate to the nucleus where it binds to the DNA and induces

the expression of iNOS protein (Kim YH, 2009). In the macrophages, due to the

bacterial endotoxin or other external stimulation several intracellular signalling

molecules become activated which lead to the phosphorylation of IκB through the

activation of the enzyme IκB kinase (IKK) and make the NF-κB free from its bound

state.

Protein kinase C (PKC) is a family of serine/threonine kinase enzymes that play a

major role in the macrophage activation. Mitogen-activated protein kinase (MAPK)-

stimulated macrophage activation is dependent on PKC (Valledor et al., 2000).

Lipopolysaccharide (LPS)-induced NO production has been reported to be

reduced in PKC-deficient mouse macrophages (Larsen et al., 2000). LPS-

activated signaling pathways associated with IB, NF-B and MAP kinases,

including p38 are dependent on the PKC activation has been reported to be

reduced in PKC deficient mouse macrophage (Castrillo et al., 2001).

130

Phosphatidylinocitol 3-kinase (PI3K) is an important intracellular signalling enzyme

involved in the phosphorylation of phosphatidylinocitol lipids. PI3K family is divided

into three different classes: Class I, Class II and Class III. It is activated by G

protein-coupled receptors and tyrosine kinase receptors. It plays an important role

in biological system and immune response. Macrophages deficient in PI3K has

been reported to produce impaired response in LPS-induced NO production

(Koyasu, 2003; Sakai et al., 2006).

AKT, also known as protein kinase B (PKB) is a serine/threonine specific protein

kinase that can be stimulated with various stimuli including growth factors and

cytokines. AKT plays important roles in various cellular processes including NF-

B-dependent transcription and inflammatory cytokine production (Rajaram et al.,

2006).

p38 MAPK (p38α p38β, 38γ and p38δ) is another serine/threonine kinase enzyme

and is one of the major sub-groups of mitogen-activated protein kinase (MAPK)

family. p38 MAPK has been reported to be activated by a variety of inflammatory

stimulus which includes LPS and cytokines and play important role in macrophage

activation during inflammation (Blink e al., 2001).

Proteasome is a multi-subunit, ATP-dependant protease complex localised in the

cell cytoplasm that degrades unwanted proteins through ubiquitination and plays

important role in inflammatory response. It degrades the IB which makes the NF-

B free from IB to translocate to the nucleus (Kloetzel et al., 1999; Ortiz-Lazareno

et al., 2008; Qureshi et al., 2005).

In macrophages, the induction of iNOS and production of NO is dependent on the

activation of several kinase pathways. However, it is not clear whether these

pathways are regulated by VSL#3-CM either in blocking LPS-induced iNOS and

NO or in VSL#3-CM itself directly inducing iNOS and NO. Thus, the studies in this

http://en.wikipedia.org/wiki/Class_III_PI_3-kinase



131

chapter were conducted to identify the signalling pathway(s) may mediate the

actions of VSL#3-CM either in blocking or inducing iNOS and NO.

Nitric oxide plays important roles in the maintenance of vascular tone (Ignarro et

al., 1999; Ignarro and Napoli, 2004; Moncada and Higgs, 2006). Besides, the role

of iNOS-induced NO has drawn attention for its beneficial effects in

atherosclerosis (Kibbe et al., 1999; Kolyada et al., 2001; Shah, 2000; Shears et

al., 1997; Wu et al., 2011). Therefore, apart from J774 cells, experiments were

also carried out to investigate the effect of VSL#3-CM on the regulation of iNOS

and NO production in a second cellular model, rat aortic smooth muscle cells

(RASMCs).

This chapter includes the data showing the effect of VSL#3-CM on the expression

of iNOS and the production of iNOS and NO, and regulation of various signalling

pathways associated with the induction of iNOS protein.

132

5.2. Methods

5.2.1. Preparation of VSL#3-conditioned cell culture medium (VSL#3-CM)

Commercially available formulation of VSL#3 was grown in MRS broth. Aliquot of

100 l of bacterial culture was transferred in 100 ml autoclaved MRS broth and

incubated overnight at 37oC with shaking. The culture was centrifuged at 500 g for

10 min, supernatant discarded and the pellet washed twice with autoclaved

phosphate buffer saline (PBS). Bacteria pellet was then re-suspended in a 50 ml

sterile centrifuge tube containing 30 ml of cell culture medium (DMEM) with 20%

FBS, and incubated at 37oC for another 24 hrs, with shaking. The culture medium

containing bacteria was centrifuged at 500 g for 10 min, supernatant collected and

filtered through 0.2 micron filter. Samples were stored at –80oC until use.

5.2.2. Preparation of sonicated probiotic in cell culture medium

As described in paragraph 5.2.1, the overnight culture of VSL#3 was centrifuged,

washed with autoclaved PBS and re-suspended in 30 ml cell culture medium

(DMEM) with 10% FBS and 1% Pen/Strep. The suspension was sonicated for 10

min with a 30 sec rest, and centrifuged for 10 min at 3000 g. Supernatant samples

of sonicated bacteria were stored at –80oC until use.

5.2.3. Cell viability assay - MTT assay

Cell viability was determined by the MTT assay to measure the mitochondrial

activity of the viable cells that metabolise 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-

diphenyltetrazolium bromide (MTT) to purple formazan. Cell monolayers were

exposed to different dilutions of VSL#3-CM or inhibitors. Following 24 hours of

incubation, cells were treated with MTT at the concentration of 0.5 mg/ml and

incubated for a further 4 hours at 37oC in a humidified CO2 incubator. The MTT

solution was removed and the formazan crystals dissolved by adding 100µl

isopropanol by incubating at room temperature with shaking. The plate was read

at 540 nm and viability of the drug-treated cells was calculated as a percentage of

the control (untreated cells), considering the viability of control as 100%.

133

5.2.4. Treatment of J774 cells with probiotics and measurement of nitrite production followed by the detection of inducible nitric oxide synthase (iNOS) and cyclooxigenase-2 (COX-2) expression

Confluent monolayers of cells were exposed to different dilutions of VSL#3-CM in

the absence or presence of LPS (1 g/ml). Total nitrite in the spent culture medium

was measured by the Griess assay. Cell protein from each well was extracted and

protein content was measured using the BCA protein assay. The proteins from cell

lysates were separated by sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE). It was then transferred onto the polyvinylidene

difluoride (PVDF) membrane, blocked by 5% fat free milk and incubated with

specific antibody. To detect iNOS and COX-2 protein, the membranes were

incubated with mouse anti-iNOS monoclonal antibody (1:2500) (BD Transduction

Laboratories) and anti-COX-2 antibody (1:5000) (BD Transduction Laboratories)

followed by goat anti-mouse antibody (1:5000) (BD Transduction Laboratories).

The antibodies were diluted in blocking buffer prior to use. The protein band was

visualised by using enhanced chemiluminescence detection solution.

5.2.5. Treatment of cells with the antibiotic polymysxin B (PmB)

PmB (Sigma-Aldrich) is a natural peptide, and an endotoxin neutralising

compound. Cells were treated with VSL#3-CM (1:25 and 1:10), LPS (1 g/ml) and

PmB (10 M) individually and in combinations for 24 hours to determine any

endotoxin contamination during the preparation of VSL#3-CM. Total nitrite in the

spent culture medium was measured by the Griess assay.

5.2.6. Treatment of cells with the inhibitors of various signalling pathways

To determine the involvement of the signalling pathways in the VSL#3-CM-

induction of iNOS and NO, cells were treated with the inhibitors of the respective

signalling pathways. To get an idea of the non-cytotoxic concentration range of

inhibitors, the cell viability test was performed using the MTT assay, before setting

up the experiment. Confluent monolayers of cells were treated for 30 minutes with

different concentrations of inhibitor alone prior to the addition of probiotic-

134

conditioned medium. Cells were then incubated with culture medium (Control) and

VSL#3-CM (1:10 dilution) with or without different concentrations of inhibitor for 24

hrs. The nitrite content in the cell culture medium was measured by the Griess

assay and the expression of iNOS in the cell detected by the Western blot

analysis.

135

5.3. Results

5.3.1. Effects of VSL#3-CM on nitric oxide (NO) production in non-stimulated and lipopolysaccharide (LPS)-stimulated J774 macrophages

Figure 32 shows the effects of VSL#3-CM on basal and lipopolysaccharide (LPS)

(1 g/ml)-induced nitric oxide (NO) production in J774 cells. When treated with

different dilutions (1:500 – 1:10), VSL#3-CM induced the basal NO in a

concentration dependent manner. In contrast, it inhibited the LPS-induced NO

production, also in a concentration dependent manner. Both of the basal NO

induction and LPS-induced NO inhibition by VSL#3-CM were statistically

significant at p<0.05-0.01.

The data suggest that bacteria-released metabolites present in VSL#3 conditioned

medium improves the immune function through the production of NO that is used

in phagocytosis. On the other hand, it has the ability to suppress the pathogen-

induced inflammation through the inhibition of NO production as inflammatory

mediator.

Figure 32: Effects of VSL#3-CM on NO production in non-stimulated and LPS-stimulated J774 macrophages

Control1:500

1:1001:50

1:251:10

LPS 1:500

1:1001:50

1:251:10

0

25

50

75

100


Nitri

te p

rodu

ctio

n(%

of L

PS a

ctiv

ated

cel

ls)

136

Confluent monolayers of J774 cells were treated with different dilutions of VSL#3-

CM in the absence and presence of LPS (1g/ml). Following 24 hours incubation,

the accumulation of nitrite in the culture medium was measured by the Griess

assay as described in materials and methods (Chapter 2, Section 2.8). The results

presented are the mean ± SEM of ten independent experiments. Statistical

significance was determined by one way ANOVA using Dunnett‟s multiple

comparison test. *p<0.05 and **p<0.01 denote significant difference from control;

●p<0.05 and ●●p<0.01 denote significant difference from LPS (1g/ml) treated

cells.

137

5.3.2. Effects of VSL#3-CM on inducible nitric oxide synthase (iNOS) expression in basal and lipopolysaccharide (LPS)-stimulated J774 macrophages

The densitometric quantification of representative Western blots in Figure 33

showed concentration-dependent induction of inducible nitric oxide synthase

(iNOS) expression in cells treated with different dilutions of VSL#3-CM (1:500 –

1:10) alone. It correlated with the basal nitric oxide (NO) production shown in

figure 32 and was statistically significant (p<0.05-0.01), but showed little inhibition

of lipopolysaccharide (LPS) (1g/ml)-induced iNOS expression.

The data supports the data of Figure 32 that bacteria-released metabolites present

in VSL#3-conditioned medium produce NO through the induction of the NO-

producing enzyme iNOS. On the other hand, it may suppress the pathogen-

induced inflammation perhaps through the regulation of the function of iNOS, not

through the expression of iNOS.

Figure 33: Effects of VSL#3-CM on iNOS expression in basal and LPS-stimulated J774 macrophages

Control1:500

1:1001:50

1:251:10

LPS1:500

1:1001:50

1:251:10

0

25

50

75

100

125

LPS (1g/ml) +VSL#3-CM

VSL#3-CM

iNOS

exp

ress

ion

(% o

f LPS

act

ivat

ed c

ells

)

iNOS

β-actin

138

Confluent monolayers of J774 cells were treated with different dilutions of VSL#3-

CM in the absence and presence of LPS (1 g/ml). Following 24 hours incubation,

cell lysates were collected and analysed by Western blot analysis to detect the

expression of iNOS as described in materials and methods (Chapter 2, Section

2.11). Expression of -actin was used to determine the loading efficiency of proein.

The results presented are the mean ± SEM of ten independent experiments.


multiple comparison test. *p<0.05 and **p<0.01 denote significant difference from

the control.

139

5.3.3. Effects of VSL#3-CM on cyclooxigenase-2 (COX-2) expression in basal and lipopolysaccharide (LPS)-stimulated J774 macrophages

The densitometric quantification of representative Western blots from the lysates

of J774 cells treated with different dilutions of VSL#3-CM in the absence and

presence of lipopolysaccharide (LPS) (1 g/ml) showed increase in basal COX-2

expression by the higher concentration of VSL#3-CM and little inhibition on LPS

induced COX-2 expression. Both the basal induction and inhibition of LPS-induced

COX-2 expression by VSL#3-CM were statistically insignificant (Figure 34).

COX-2 plays important role in cellular immune reaction through the production of

prostaglandin E2 (PGE2). The data suggests that bacteria-released metabolites

present in VSL#3-conditioned medium have a role in the induction of basal COX-2

at least at higher concentration. On the other hand, it may not have the ability to

regulate the pathogen-induced COX-2 expression.

Figure 34: Effects of VSL#3-CM on COX-2 expression in basal and LPS-stimulated J774 macrophages

Control1:500

1:1001:50

1:251:10

LPS 1:500

1:1001:50

1:251:10

0

25

50

75

100

125


COX-

2 ex

pres

sion

(% o

f LPS

act

ivat

ed c

ells

)

COX-2

β-actin

140


CM in the absence and presence of LPS (1 g/ml). Following 24 hours incubation,

cell lysates were collected and analysed by Western blot analysis to detect the

expression of COX-2 as described in materials and methods (Chapter 2, Section

2.11). Expression of -actin was used to determine the loading efficiency of

protein. The results presented are the mean ± SEM of five independent


Dunnett‟s multiple comparison test.

141

5.3.4. Effect of Polymyxin B (PmB) on VSL#3-CM-induced nitric oxide (NO) production in J774 macrophages

J774 cells were treated with Polymyxin B (PmB) (10 g/ml), VSL#3-CM (1:25 –

1:10) and lipopolysaccharide (LPS) (1 g/ml) alone and in combination, to check

the possible contamination of endotoxin in VSL#3-CM that induced the basal nitric

oxide (NO) production. The results obtained, showed that VSL#3-CM induced

basal NO both in the presence and absence of PmB, while PmB itself did not

induce NO.

On the other hand, PmB inhibited the LPS-induced NO production but showed

little inhibition on NO production when cells were treated with PmB, VSL#3-CM

and LPS in combination (Figure 35).

The data suggests that the induction of basal NO in J774 macrophages was made

by the bacteria-released metabolites present in VSL#3-CM. NO production was

unaffected by the bacterial endotoxin neutralising compound PmB whereas, PmB

suppressed the NO production induced by bacterial endotoxin LPS.

Figure 35: Effect of Polymyxin B (PmB) on VSL#3-CM-induced NO production in J774 macrophages

0

50

100

150

Nitr

ite p

rodu

ctio

n(%

of L

PS a

ctiv

ated

cel

ls)

Control - - - - - - - - - - - - PmB (10g/ml) - + - - + + - - - + + +

VSL#3-CM (1:25) - - + - + - + - - - + - VSL#3-CM (1:10) - - - + - + - + - - - + LPS(1g/ml) - - - - - - + + + + + +

142

Confluent monolayers of J774 cells were treated with Polymyxin B, VSL#3-CM

and LPS alone and in combination. Following 24 hours incubation, the

accumulation of nitrite was measured using Griess assay as described in the

materials and methods (Chapter 2, Section 2.8). The results presented are the

mean ± SEM of five independent experiments. Statistical significance was


143

5.3.5. Effect of VSL#3-CM on the viability of J774 macrophages

Cell viability was studied to determine the toxic effect of VSL#3-CM in J774 cells.

Figure 36 shows that VSL#3-CM was not cytotoxic at concentrations of up to the

dilution of 1:25 (1:500 – 1:25). However, the viability of cells was reduced

significantly (p<0.01) to 60% at the dilution of 1:10 which showed similar toxicity as

lipopolysaccharide (LPS) alone.

Figure 36: Effect of VSL#3-CM on the viability of J774 macrophages

Confluent monolayers cells in 96-well plate were incubated with different dilutions

of VSL#3 with or without LPS (1µg/ml) for 24 hrs. The culture medium in the

treated wells was replaced with 200μl fresh medium containing 0.05 mg/ml MTT

solution and incubated for 4 hours at 37°C. As described in materials and methods

(Chapter 2, Section 2.14), the medium was removed, 100μl Isopropanol added to

the wells and incubated for 30 min at room temperature on a shaker to dissolve

the formazan crystals. The plate was read at 540nm. The viability of the VSL#3

treated cells was calculated as a percentage of the control considering the viability

of the control (untreated cells) as 100%. The data are the mean SEM of 3

independent experiments. Statistical significance was determined using one way

ANOVA followed by Dunnett‟s multiple comparison test. **p<0.01 denotes

significant difference from the control.

Control

1:5001:100

1:501:25

1:10LPS

1:5001:100

1:501:25

1:100

20

40

60

80

100

120


Cell

viab

ility

(% o

f con

trol

)

144

5.3.6. Comparison of the effects of VSL#3-CM and sonicated bacteria on basal nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774 macrophages

Two separate experiments were carried out in parallel using VSL#3-CM and

supernatant of sonicated bacteria. J774 cells were treated with similar dilutions of

VSL#3-CM and sonicated bacteria (1:500 – 1:10) individually to examine their

effect on normal cellular level of nitric oxide (NO) production and inducible nitric

oxide synthase (iNOS) expression. The results obtained showed that basal NO

level increased in VSL#3-CM-treated cells in a concentration dependent manner

that was statistically significant (p<0.05). In contrast, there was no induction of NO

when the cells were treated with the supernatant of sonicated bacteria (Figure

37A).

Similarly, The representative Western blots showed the gradual induction of iNOS

expression in cells treated with VSL#3-CM (Figure 37B), but did not show any

significant change in iNOS expression in sonicated bacteria treated cells (Figure

37C).

The data suggest that the whole bacteria or its component may not have the effect

rather, the bacteria-released metabolites has the ability to activate the

macrophages through the induction of iNOS and NO production.

145

A. Effects of VSL#3-CM and sonicated bacteria on basal NO production

B. VSL#3-CM-induced basal iNOS expression

C. Sonicated bacteria-induced basal iNOS expression

Figure 37: Effects of VSL#3-CM and sonicated bacteria on basal NO production and iNOS expression in J774 macrophages

Control

1:500

1:100 1:5

01:2

51:1

00.0

0.1

0.2

0.3 Sonicated bacteriaConditioned-medium

* *

VSL#3 (Dilution)

Nitr

ite p

rodu

ctio

n(

mol

s/

g pr

otei

n)

iNOS

β-actin

β-actin

iNOS

146

Confluent monolayers of cells were treated with different dilutions of VSL#3-CM

and sonicated bacteria. Following 24 hours incubation, the accumulation of nitrite

was measured by Griess assay as described in the method section (Chapter 2,

Section 2.8). The results presented are the mean ± SEM of three independent


Dunnett‟s multiple comparison test. *p<0.05 denotes significant difference from

control. The expression of iNOS was detected in cell lysates using Western blot

assay as described in materials and methods (Chapter 2, Section 2.11).

Expression of -actin was used to determine the loading efficiency of protein.

147

5.3.7. Effect of dexamethasone on VSL#3-CM-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774 macrophages

To determine whether VSL#3-CM-induced basal nitric oxide (NO) production is

susceptible to inhibition by glucocorticoids, cells were treated with VSL#3-CM and

lipopolysaccharide (LPS) (1 g/ml) in the absence and presence of

dexamethasone (Sigms-Aldrich). The concentration of dexamethasone was

selected after performing the MTT assay. Cells were also treated with the culture

medium and ethanol (EtOH; 0.05%) in the same experiment to determine the

effect on NO production as dexamethasone was dissolved in EtOH.

Dexamethasone inhibited both VSL#3-CM and LPS-induced NO production and

inducible nitric oxide synthase (iNOS) expression. In both cases, the inhibition of

NO production was statistically significant (VSL#3-CM - p<0.01, LPS - p<0.01).

EtOH did not show any effect on NO production or iNOS expression (Figure 38A

and 38B).

Dexamethasone inhibits the activation of transcription factor nuclear factor kappa

B (NF-B). The data shows that it has partially inhibited the induction of NO and

iNOS at non-cytotoxic concentration. Therefore, suggest that the activation of

macrophages by VSL#3-CM may not involve only the NF-B pathway.

148

A.

B.

Figure 38: Effect of dexamethasone on VSL#3-CM-induced NO production and iNOS expression in J774 macrophages

Confluent monolayers of cells were pre-conditioned with 10 M dexamethasone

for 30 minutes prior to the addition of VSL#3-CM. Cells were then incubated with

culture medium (Control), VSL#3-CM (1:10 dilution) or with LPS (1 g/ml) alone

and with dexamethasone for 24 hrs. Cells were also treated with ethanol (EtOH) in

the same experiment. Following 24 hours incubation, the accumulated nitrite in the

culture medium was quantified by the Griess assay as described in the method

section (Chapter 2, Section 2.8). The results presented are the mean ± SEM of

four independent experiments. Statistical significance was determined by one way

ANOVA using Dunnett‟s multiple comparison test. **p<0.01 denotes significant

difference from Control; ●●p<0.01 denote significant difference from VSL#3-CM

and ♦♦p<0.01 denotes significant difference from LPS. The expression of iNOS

was detected in cell lysates by Western blot analysis as described in materials and

methods (Chapter 2, Section 2.11). Expression of -actin was used to determine

the loading efficiency of protein. The representative Western blot is one of four

separate experiments.

Control Dexa (10 M) 1:10 Dexa (10 M) LPS Dexa (10 M) EtOH0.00

0.25

0.50

0.75

Control VSL#3-CM (1:10) LPS (1M)

Nitr

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n(

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g pr

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n)

iNOS

-actin

149

5.3.8. Effect of dexamethasone on the cell viability

Cells were treated with different concentrations of dexamethasone (1-20 M).

Viability study showed that the compound was not cytotoxic at concentrations up

to 10 M (Figure 39). The cell viability was reduced significantly (p<0.05-0.01)

when dexamethasone was used at the concentrations of 15 µM and 20 µM.

Figure 39: Effect of dexamethasone on the cell viability

Confluent monolayers of cells in 96-well plate were exposed to different

concentrations of dexamethasone. As described in materials and methods

(Chapter 2, Section 2.8), after 24 hours incubation, the culture medium in treated

wells was replaced with 200μl fresh medium containing 0.05 mg/ml MTT solution,

and incubated for 3-4 hours at 37°C. The medium was removed and 200μl

isopropanol added to the wells and incubated at room temperature on a shaker for

30 min to dissolve the formazan crystals produced from the metabolism of MTT.

The plate was read on at 540nm. The viability of the dexamethasone treated cells

was calculated as a percentage of the control (untreated cell) considering the

viability as 100%. The results presented are mean ± SEM of 3 independent

experiments. Statistical significance was determined using one way ANOVA

followed by Dunnett‟s multiple comparison test. *p<0.05 and **p<0.01 denote

significant difference from Control.

Control 1 5 10 15 200

25

50

75

100

125

Dexamethasone (M)

Cell

viab

ility

(% o

f con

trol

)

150

5.3.9. Role of the enzyme protein kinase C (PKC) on VSL#3-CM-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774 macrophages

Figure 40 shows the role of protein kinase C (PKC) enzyme on nitric oxide (NO)

production and inducible nitric oxide synthase (iNOS) expression. The cells were

treated with a PKC inhibitor bisindolylmaleimide (BIM) (Calbiochem) at

concentrations ranging from 1 to 30 M which was dissolved in DMSO. The

concentration was selected after performing the MTT assay. Therefore, the cells

were also treated with the culture medium only as control and dimethylsulfoxide

(DMSO) (0.1 %) in the same experiment to determine whether DMSO itself has

any effect on NO production. To compare the role of BIM on VSL#3-CM, cells

were also treated with BIM in presence of lipopolysaccharide (LPS).

The results obtained showed that BIM did not produce any effect on NO

production in control cells (Figures 40A and 40B). However, there was slight

induction of iNOS which was inhibited by BIM at 30 M. VSL#3-CM itself induced

NO and iNOS that was statistically significant (p<0.01). BIM has partially but

significantly inhibited VSL#3-CM-induced NO production and iNOS expression in a

concentration dependent manner (p<0.05 - p<0.01) (Figures 40C and 40D). It has

produced similar effect in LPS-activated cells (Figures 40E and 40F). DMSO had

no effect on NO production and iNOS expression (Figures 40C and 40D).

PKC is involved in the activation of macrophages that acts through the NF-B

pathway. However, the inhibition of VSL#3-CM-induced NO and iNOS suggest

that PKC pathway is only partially involved in the induction of iNOS and NO. This

data support the data of Figure 38 which showed Dexamethasone that inhibits the

NF-B activation, only partially inhibited VSL#3-CM-induced NO and iNOS

expression.

151

A.

B.

C.

D.

Control 1M 3 M 10 M 30 M0.0

0.1

0.2

0.3

0.4

Control

BIM (M)

Nitr

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n(

mol

s/

g pr

otei

n)

Control 1:10 1M 3 M 10 M 30 M DMSO0.0

0.1

0.2

0.3

0.4

VSL#3-CM (1:10)

BIM (M)

Nitri

te p

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ctio

n(

mol

s/

g pr

otein

)

β-actin

iNOS

iNOS

β-actin

152

E.

F.

Figure 40: Role of protein kinase C (PKC) on VSL#3-CM-induced NO production and iNOS expression in J774 macrophages

Confluent monolayers of cells were treated with different concentrations of BIM

ranging from 1 to 30µM for 30 minutes prior to the addition of VSL#3-CM. Cells

were then treated with complete culture medium (control) and VSL#3-CM (1:10

dilution) alone and in combination with different concentrations of BIM (1 to 30µM).

Cells were also treated with culture medium and dimethylsulfoxide (DMSO) in the

same experiment to determine its effect on NO production as BIM was dissolved

DMSO. Following 24 hours incubation, accumulated nitrite in the culture medium

was quantified by the Griess assay as described in the method section (Chapter 2,

Section 2.8). The results presented are mean ± SEM of four independent


Dunnett‟s multiple comparison test. *p<0.05 and **p<0.01 denote significant

difference from Control; ●p<0.05 and ●●p<0.01 denote significant difference from

VSL#3-CM and, ♦p<0.05 and ♦♦p<0.01 denote significant difference from LPS.

LPS 1M 3 M 10 M 30 M0.0

0.2

0.4

0.6

LPS (M)

BIM (M)

Nitr

ite p

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n(

mol

s/

g pr

otei

n)

iNOS

β-actin

153

The expression of iNOS was detected in cell lysates using Western blot analysis

as described in materials and methods (Chapter 2, Section 2.11). Expression of -

actin was used to determine the loading efficiency of protein. The representative

Western blot is one of four separate experiments.

154

5.3.10. Effect of protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM) on the cell viability

As showed in Figure 41, J774 cells were treated with different concentrations of

BIM (1-30 M). Viability study of BIM showed that the compound was nontoxic to

the cells up to 10 M. The cell viability was reduced significantly (p<0.05) when

BIM was used at the concentration of 30 M.

Figure 41: Effect of protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM) on the cell viability

Confluent monolayers of cells in 96-well plate were exposed to different

concentrations of bisindolylmaleimide (BIM). As described in materials and

methods (Chapter 2, Section 2.8), after 24 hours incubation, the culture medium in

the treated wells was replaced with 200μl fresh medium containing 0.05 mg/ml

MTT solution and incubated for 3-4 hours at 37°C. The medium was removed,

200μl isopropanol added to the wells, and incubated at room temperature for 30

min on a shaker to dissolve the formazan crystals produced by metabolism of

MTT. The plate was read on at 540nm. The viability of the BIM treated cells was

calculated as a percentage of the viability of the control (untreated cells). The

results presented are mean ± SEM of 3 independent experiments. Statistical

significance was determined using one way ANOVA followed by Dunnett‟s multiple

comparison test. *p<0.05 denotes significant difference from Control.

Control 1 3 7 10 300

25

50

75

100

125

BIM (M)

Cel

l via

bilit

y(%

of c

ontr

ol)

155

5.3.11. Role of phosphatidylinocitol 3-kinase (PI3K) on VSL#3-CM-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774 macrophages

To determine the role of phosphatidylinocitol 3-kinase (PI3K) on the nitric oxide

(NO) production and inducible nitric oxide synthase (iNOS) expression, cells were

treated with VSL#3-CM in the absence and presence of a PI3K inhibitor,

LY294002 (Calbiochem) at concentrations ranging from 1 to 30 M. The

concentration of LY294002 was selected after performing the MTT assay. To

compare the role of LY294002 on VSL#3-CM, cells were also treated with

LY294002 in presence of lipopolysaccharide (LPS).

The results obtained showed that LY294002 did not produce any change in control

cells (Figures 42A and 42B). VSL#3-CM itself induced NO that was statistically

significant (p<0.05). LY294002 inhibited VSL#3-CM-induced NO production and

iNOS expression in a concentration dependant manner. The inhibition of NO

production by LY294002 was statistically significant at the concentration of 30µM

(p<0.05) (Figures 42C and 42D). LY294002 also inhibited LPS-induced NO

production and iNOS expression at both 10 M (p<0.01) and 30 M (p<0.01)

concentrations (Figures 42E and 42F).

PI3K plays an important role in immune response and involved in NO production

through the expression of iNOS. However, LY294002 at non-cytotoxic

concentration (3 M) made only slight inhibition on NO and iNOS production that

was not statistically significant. The data therefore is not conclusive to say whether

VSL#3-CM has any role on the regulation of PI3K pathway.

156

A.

B.

C.

D.

Control 1M 3 M 10 M 30 M0.00

0.05

0.10

0.15

0.20

Control

LY294002 (M)

Nitr

ite p

rodu

ctio

n(

mol

s/

g pr

otei

n)

Control 1:10 1M 3 M 10 M 30 M0.00

0.05

0.10

0.15

0.20

VSL#3-CM (1:10)

LY294002 (M)

Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

β-actin

iNOS

iNOS

β-actin

157

E.

F.

Figure 42: Role of phosphatidylinocitol 3-kinase (PI3K) on VSL#3-CM-induced NO production and iNOS expression in J774 macrophages


LY294002 ranging from 1 to 30 µM for 30 minutes before VSL#3-CM was

introduced. Cells were then treated with complete culture medium (Control) and

VSL#3-CM (1:10 dilution) alone and in combination with different concentrations of

LY294002 (1 to 30µM). Following 24 hours incubation, accumulated nitrite in the

culture medium was quantified by the Griess assay as described in the method

section (Chapter 2, Section 2.8). The results presented are mean ± SEM of four

independent experiments. Statistical significance was determined by one way

ANOVA using Dunnett‟s multiple comparison test. *p<0.05 denotes significant

difference from Control; ●p<0.05 denotes significant difference from VSL#3-CM

and ♦♦p<0.01 denotes significant difference from LPS. The expression of iNOS

was detected in cell lysates using Western blot assay as described in materials

and methods (Chapter 2, Section 2.11). Expression of -actin was used to

determine the loading efficiency of protein. The representative Western blot is one

of four separate experiments.

LPS 1M 3 M 10 M 30 M0.0

0.1

0.2

0.3

LPS (M)

LY294002 (M)

Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

iNOS

β-actin

158

5.3.12. Effect of phosphatidylinocitol 3-kinase (PI3K) inhibitor LY294002 on the cell viability

To determine the cytotoxic effect of the inhibitor LY294002, cells were treated with

different concentrations of LY294002 (1-30 µM). Viability study of LY294002

showed that the compound was not cytotoxic at concentrations up to 7µM shown

in figure 43. The cell viability was reduced significantly (p<.0.5-0.01) when the

concentrations of LY294002 were increased to 10µM and 30µM.

Figure 43: Effect of phosphatidylinocitol 3-kinase (PI3K) inhibitor LY294002 on the cell viability

Confluent monolayers of J774 cells in 96-well plate were exposed to different

concentrations of LY294002. As described in the materials and methods section

(Chapter 2, Sction 2.8), after 24 hours incubation, the culture medium in treated

wells was replaced with 200μl fresh medium containing 0.05 mg/ml MTT solution

and incubated for 3-4 hours at 37°C. The medium was removed and 200μl



The plate was read on a Multiscan II plate reader at 540nm. The viability of the

LY294002 treated cells was calculated as a percentage of the viability of the

control (untreated cells). The results presented are mean ± SEM of 3 independent

experiments. Statistical significance was determined using one way ANOVA

followed by Dunnett‟s multiple comparison tests. *p<0.05 and **p<0.01 denote

significant differences from Control.

Control 1 3 7 10 300

25

50

75

100

125

LY294002 (M)

Cel

l via

bilit

y(%

of c

ontr

ol)

159

5.3.13. Effect of protein kinase B (AKT) inhibitor on VSL#3-CM induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774 cells

To determine the role of AKT (also known as protein kinase B) on nitric oxide (NO)

production and inducible nitric oxide synthase (iNOS) expression, cells were

treated with control and VSL#3-CM in the absence and presence of the AKT

inhibitor XIII (Calbiochem), an AKT inhibitor, at concentrations ranging from 0.1 to

10 M. The concentration of AKT inhibitor XIII was selected based on toxicity test

by MTT assay. To compare the role of AKT inhibitor XIII on VSL#3-CM, cells were

also treated with AKT inhibitor XIII in presence of lipopolysaccharide (LPS).

The results obtained showed that AKT inhibitor XIII did not produce any effect in

control cells (Figures 44A and 44B). VSL#3-CM itself induced NO that was

statistically significant (p<0.01). AKT inhibitor XIII inhibited VSL#3-CM-induced NO

and iNOS expression at higher concentrations that was statistically significant

(p<0.01) at the concentration of 10 M (Figures 44C and 44D). AKT inhibitor XIII

completely inhibited LPS induced NO production and iNOS expression at 10 M

(p<0.01) concentrations (Figures 44E and 44F).

The suppression of NO by AKT inhibitor occurred only at higher and toxic

concentration and no change was made upto the concentrations from 0.1 to 3 M

that was non-toxic to cells. Therefore the data suggest that AKT may not have any

role on NO production in response to VSL#3-CM.

160

A.

B.

C.

D.

Control 0.1 M 1 M 3 M 7 M 10 M0.00

0.05

0.10

0.15

0.20

Control

AKT inhibitor XIII (M)

Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

Control 1:10 0.1 M 1 M 3 M 7 M 10 M0.00

0.05

0.10

0.15

0.20

VSL#3-CM (1:10)


Nitri

te pr

oduc

tion

(m

ols/

g pro

tein)

β-actin

iNOS

iNOS

β-actin

161

E.

F.

Figure 44: Effect of protein kinase B (AKT) inhibitor on VSL#3-CM induced NO production and iNOS expression in J774 cells


AKT inhibitor XIII, ranging from 0.1 to 10 M, for 30 minutes prior to addition of

VSL#3-CM. Cells were then treated with complete culture medium (control) and

VSL#3-CM (1:10 dilution) alone, and in combination with different concentrations

of AKT inhibitor XIII (0.1 to 10µM). Following 24 hours incubation, accumulated

nitrite in the culture medium was quantified by Griess assay as described in the

materials and methods section (Chapter 2, Sction 2.8). The results presented are

mean ± SEM of three independent experiments. Statistical significance was


**p<0.01 denotes significant difference from Control; ●●p<0.01 denotes significant

difference from VSL#3-CM and ♦♦p<0.01 denotes significant difference from LPS.


as described in materials and methods (Chapter 2, Section 2.11). Expression of -

actin was used to determine the loading efficiency of protein. The representative

Western blot is one of three separate experiments.

LPS 0.1 M 1 M 3 M 7 M 10 M0.0

0.1

0.2

0.3

0.4

0.5

LPS (1M)


Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

iNOS

β-actin

162

5.3.14. Effect of AKT inhibitor XIII on the cell viability

Figure 45 shows the viability of J774 cells in response to the AKT inhibitor XIII. To

determine the cytotoxic effect of the AKT inhibitor XIII, cells were treated with

different concentrations of AKT inhibitor XIII (0.1-10 M). The compound was not

cytotoxic at concentrations up to 3µM. But the compound found highly toxic at the

concentration of 7 and 10 M where the cell viability was reduced significantly

(p<0.01).

Figure 45: Effect of AKT inhibitor XIII on the cell viability


concentrations of AKT inhibitor XIII. As described in materials and methods

(chapter 2, section 2.8), after 24 hours incubation, the culture medium in treated

wells was replaced with 200μl fresh medium containing 0.05 mg/ml MTT solution

and incubated for 3-4 hours at 37°C. The medium was removed, 200μl



The plate was read on at 540nm. The viability of the AKT inhibitor XIII treated

cells was calculated as a percentage of viability of the control (untreated cells).

The results presented are mean ± SEM of 3 independent experiments. Statistical

significance was determined using one way ANOVA followed by Dunnett‟s multiple

comparison tests. **p<0.01 denotes significant difference from Control.

Control 0.1 1 3 7 100

25

50

75

100

125


Cell

viab

ility

(% o

f con

trol

)

163

5.3.15. Role of mitogen-activated protein kinase enzyme p38 MAPK on VSL#3-CM-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774 macrophages

To determine the role of p38 MAPK in VSL#3-induced nitric oxide (NO) production

and inducible nitric oxide synthase (iNOS) expression, cells were treated with

VSL#3-CM in the absence and presence of SB203580 (Calbiochem), inhibitor of

p38 MAPK at concentrations ranging from 1 to 30 M. The concentration of

SB203580 was selected according to its toxicity determined by MTT. To compare

the role of SB203580 on VSL#3-CM, cells were also treated with SB203580 in

presence of lipopolysaccharide (LPS).

The results obtained, showed that SB203580 did not produce any significant

change in control cells (Figures 46A and 46B). VSL#3-CM itself induced NO that

was statistically significant (p<0.01). SB20380 inhibited VSL#3-CM-induced NO

production and iNOS expression in a concentration dependant manner. The

inhibition was statistically significant at concentrations of 3 M, 10 M and 30 M

(p<0.01) (Figures 46C and 46D). Similar to VSL#3-CM, SB203580 inhibited LPS

induced NO production and iNOS expression at 1 M (p<0.01) and 30 M

(p<0.01) concentrations (Figures 46E and 46F).

In macrophages, p38 MAPK play a major role in the immune and inflammatory

response. The inhibitor of p38 MAPK, SB203580 has markedly inhibited the

VSL#3-CM-induced NO and iNOS even at 3 M concentration that was non-

cytotoxic to the cells. The data clearly shows that p38 MAPK is activated by

VSL#3-CM and induced the production of NO and iNOS in J774 cells.

164

A.

B.

C.

D.

Control 1 M 3 M 10 M 30 M0.00

0.01

0.02

0.03

0.04

0.05

SB203580 (M)

Control

Nitr

ite p

rodu

ctio

n(

mol

s/

g pr

otei

n)

Control 1:10 1 M 3 M 10 M 30 M0.00

0.01

0.02

0.03

0.04

0.05

SB203580 (M)

VSL#3-CM (1:10)

Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

β-actin

iNOS

iNOS

β-actin

165

E.

F.

Figure 46: Role of p38 MAPK inhibitor SB203580 on VSL#3-CM-induced NO production and iNOS expression in J774 macrophages


SB20380 ranging from 1 to 30 M for 30 minutes prior to the addition of VSL#3-

CM. Cells were then treated with complete culture medium (Control) and VSL#3-

CM (1:10 dilution) alone and in combination with different concentrations of

SB20380 (1 to 30 M). Following 24 hours incubation, accumulated nitrite in the

culture medium was quantified by the Griess assay as described in the materials

and methods section (Chapter 2, Sction 2.8). The results presented are mean ±

SEM of three independent experiments. Statistical significance was determined by

one way ANOVA using Dunnett‟s multiple comparison test. **p<0.01 denotes

significant difference from Control; ●●p<0.01 denotes significant difference from

VSL#3-CM and ♦♦p<0.01 denotes significant difference from LPS. The expression

of iNOS was detected in cell lysates using Western blot analysis as described in

materials and methods (Chapter 2, Section 2.11). Expression of -actin was used

to determine the loading efficiency of protein. The representative Western blot is

one of three separate experiments.

LPS 1 M 3 M 10 M 30 M0.0

0.2

0.4

0.6

SB203580 (M)

LPS (1M)

Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

iNOS

β-actin

166

5.3.16. Effect of P38 inhibitor SB203580 on the cell viability

To determine the cytotoxic effect of the inhibitor SB203580, cells were treated with

different concentrations of SB203580 (0.1-10 M). Viability study of SB203580

showed that the compound was not cytotoxic at concentrations up to 3 µM, shown

in Figure 47. The cell viability was reduced significantly (p<0.01), when SB203580

concentration above 3 µM was used.

Figure 47: Effect of P38 inhibitor SB203580 on the cell viability


concentrations of SB203580. As described in materials and methods (Chapter 2,

Section 2.8), after 24 hours incubation, the culture medium in the treated wells

was replaced with 200 l fresh medium containing 0.05 mg/ml MTT solution and

incubated for 3-4 hours at 37°C. The medium was removed, 200 l isopropanol

added to the wells and incubated at room temperature on a shaker for 30 min to

dissolve the formazan crystals produced from the metabolism of MTT. The plate

was read on at 540 nm. The viability of the SB203580 treated cells was calculated

as a percentage of viability of the control (untreated cells). The results presented

are mean ± SEM of 3 independent experiments. Statistical significance was

determined using one way ANOVA followed by Dunnett‟s multiple comparison

tests. **p<0.01 denotes significant difference from Control.

Control 0.1 1 3 7 100

25

50

75

100

125

SB203580 (M)

Cel

l via

bilit

y(%

of c

ontr

ol)

167

5.3.17. Role of Proteasome on VSL#3-CM-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774 macrophages

To determine the role of proteasome on nitric oxide (NO) production and inducible

nitric oxide synthase (iNOS) expression, cells were treated with VSL#3-CM and

LPS (1 g/ml) individually in the absence and presence of an inhibitor of

proteasome, MG132 (Calbiochem), at concentrations ranging from 0.01 to 0.5

M. The concentration of MG132 was selected on the basis of MTT assay. To

compare the role of MG132 on VSL#3-CM, cells were also treated with MG132 in

presence of lipopolysaccharide (LPS).

The results obtained showed that MG132 did not have any effect on control cells

(Figures 48A and 48B). VSL#3-CM itself induced NO that was statistically

significant at the level of p<0.01. MG132 significantly inhibited (p<0.05) VSL#3-

CM-induced NO production and iNOS expression at 0.5 M concentration (Figures

48C and 48D). LPS-induced NO production and iNOS expression was inhibited by

MG132 at 0.25 M and 0.5 M concentrations. Inhibition was statistically

significant at 0.5 M at p<0.01 level (Figures 48E and 48F).

MG132 blocks the activation of NF-B by preventing the proteasomal degradation

of NF-B inhibitor protein IB. However, the data shows that MG132 did not inhibit

the production of NO and iNOS at non-cytotoxic concentration suggest that

proteasomal pathway may not be involved in VSL#3-CM-induced NO and iNOS

expression.

168

A.

B.

C.

D.

Control 0.01 M 0.1 M 0.25 M 0.5 M0.0

0.1

0.2

0.3

0.4

0.5

Control

MG132 (M)

Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

Control 1:10 0.01 M 0.1 M 0.25 M 0.5 M0.0

0.1

0.2

0.3

0.4

0.5

VSL#3-CM (1:10)

MG132 (M)

Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

β-actin

iNOS

iNOS

β-actin

169

E.

F.

Figure 48: Effect of Proteasome inhibitor MG132 on VSL#3-CM-induced NO production and iNOS expression in J774 macrophages


MG132 ranging from 0.01 to 0.5 M for 30 minutes prior to addition of VSL#3-CM.

Cells were then treated with complete culture medium (Control), VSL#3-CM (1:10

dilution) and LPS alone and in combination with different concentrations of MG132

(0.01 to 0.5 M). Following 24 hours incubation, accumulated nitrite in the culture

medium was quantified by the Griess assay as described in the materials and

methods section (Chapter 2, Sction 2.8). The results presented are mean ± SEM

of four independent experiments. Statistical significance was determined by one

way ANOVA using Dunnett‟s multiple comparison test. **p<0.01 denotes

significant difference from Control; ●p<0.05 denotes significant difference from

VSL#3-CM and ♦♦p<0.01 denotes significant difference from LPS. The expression

of iNOS was detected in cell lysates using Western blot analysis as described in

methods section (Chapter 2, Section 2.11). Expression of -actin was used to

determine the loading efficiency of protein. The representative Western blot is one

of four separate experiments.

LPS 0.01 M 0.1 M 0.25 M 0.5 M0.0

0.1

0.2

0.3

0.4

0.5

LPS (M)

MG132 (M)

Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

β-actin

iNOS

170

5.3.18. Effect of Proteasome inhibitor MG132 on the cell viability

Cells were treated with different concentrations of MG132 (0.01-1 M). Viability

study of MG132 showed that the compound was not cytotoxic at concentrations up

to 0.25 M (Figure 49). But the concentration of MG132 above 0.25 M was toxic

to the cells. The cell viability reduced significantly (p<0.01) at concentrations of 0.5

M and 1 M.

Figure 49: Effect of Proteasome inhibitor MG132 on the cell viability


concentrations of MG132. As described in materials and methods (chapter 2,

section 2.8), after 24 hours incubation, the culture medium in the treated wells was

replaced with 200 l fresh medium containing 0.05 mg/ml MTT solution, and

incubated for 3-4 hours at 37°C. The medium was removed, 200 l isopropanol



was read at 540nm. The viability of MG132 treated cells was calculated as a

percentage of the viability of the control (untreated cells). The results presented

are mean ± SEM of 3 independent experiments. Statistical significance was

determined using one way ANOVA followed by Dunnett‟s multiple comparison

tests. **p<0.01 denotes significant difference from Control.

Control0.01 0.1 0.25 0.5 1

0

25

50

75

100

125

MG132 (M)

Cel

l via

bilit

y(%

of c

ontr

ol)

171

5.3.19. Role of nuclear factor kappa B (NFκB) signalling pathway on VSL#3-CM-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774 macrophages

To determine the involvement of transcription factor NF-B in the VSL#3-CM-

induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS)

expression, cells were treated with VSL#3-CM and LPS (1 g/ml) individually in

the absence and presence of an NF-B activation inhibitor CAY10470 (Cambridge

Bioscience) at concentrations ranging from 0.1 to 100 ηM. The concentration of

CAY10470 was selected according to its toxicity detected by MTT assay. To

compare the role of CAY10470 on VSL#3-CM, cells were also treated with

CAY10470 in presence of lipopolysaccharide (LPS).

The results obtained showed that CAY10470 did not produce any effect on control

cells (Figures 50A and 50B). VSL#3-CM induced the NO and iNOS that was

statistically significantly at p<0.01 level. CAY10470 inhibited the NO at 10 ηM and

100 ηM concentrations. The inhibition was statistically significant (p<0.01) at the

concentration of 100 ηM (Figures 50C and 50D). Similar to VSL#3-CM, CAY10470

inhibited LPS-induced NO production and iNOS expression at 10 ηM and 100 ηM

concentrations. The inhibition was statistically significant at p<0.05 level at the

concentration of 100ηM. The inhibition was 20% at 10ηM and 34% at 100ηM

(Figures 50E and 50F).

In macrophages, activation of transcription factor NF-B is crucial for NO

production and iNOS expression to produce immune response. The slight

inhibition of VSL#3-CM-induced NO production by CAY10470 suggests that the

NF-B pathway may only be partially involved in the activation of J774 cells. This

data also support the effect of Dexamethasone in figure 38 where Dexamethasone

partially inhibited the VSL#3-CM-induced NO and iNOS production.

172

A.

B.

C.

D.

Control 0.1 M 1 M 10 M 100 M0.00

0.05

0.10

0.15

0.20

0.25

0.30

Control

CAY10470 (M)

Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

Control 1:10 0.1 M 1 M 10 M 100 M0.00

0.05

0.10

0.15

0.20

0.25

0.30

VSL#3-CM (1:10)

CAY10470 (M)

Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

iNOS

β-actin

β-actin

iNOS

173

E.

F.

Figure 50: Effect of NFκB inhibitor CAY10470 on VSL#3-CM-induced NO production and iNOS expression in J774 macrophages


CAY10470 ranging from 0.1 to 100 ηM for 30 minutes before VSL#3-CM was

added. Cells were then treated with complete culture medium (Control), VSL#3-

CM (1:10 dilution) and LPS alone and in combination with different concentrations

of CAY10470 (0.1 to 100 ηM). Following 24 hours incubation, accumulated nitrite

in the culture medium was quantified by the Griess assay as described in the

materials and methods section (Chapter 2, Sction 2.8). The results presented are

mean ± SEM of four independent experiments. Statistical significance was


**p<0.01 denotes significant difference from Control; ●●p<0.01 denotes significant

difference from VSL#3-CM and ♦p<0.05 significant difference from LPS (1µg/ml).


as described in methods (chapter 2, section 2.11). Expression of -actin was used

to determine the loading efficiency of protein. The representative Western blot is

one of four separate experiments.

LPS 0.1 M 1 M 10 M 100 M0.00

0.05

0.10

0.15

0.20

0.25

0.30

LPS (1M)

CAY10470 (M)

Nitri

te p

rodu

ctio

n(

mol

s/

g pr

otei

n)

iNOS

β-actin

174

5.3.20. Effect of NF-κB inhibitor CAY10470 on the cell viability

The viability assay of CAY10470, performed with different concentrations of

CAY10470 (0.01-100 ηM) shown in Figure 51. The compound CAY10470 was

nontoxic to cells up to 10 ηM. The cell viability was reduced significantly (p<0.01)

when CAY10470 concentrations used above 10 ηM.

Figure 51: Effect of NF-κB inhibitor CAY10470 on the cell viability


concentrations of CAY10470. As described in materials and methods (chapter 2,

section 2.8), after 24 hours incubation, the culture medium in the treated wells was

replaced with 200μl fresh medium containing 0.05 mg/ml MTT solution and

incubated for 3-4 hours at 37°C. The medium was removed, 200μl isopropanol



was read at 540nm. The viability of the CAY10470 treated cells was calculated as

a percentage of the viability of control (untreated cells). The results presented are

mean ± SEM of 3 independent experiments. Statistical significance was

determined using one way ANOVA followed by Dunnett‟s multiple comparison test.

**p<0.01 denotes significant difference from Control.

Control .01 .1 1 10 1000

25

50

75

100

125

CAY10470 (M)

Cel

l via

bilit

y(%

of c

ontr

ol)

175

5.3.21. Effect VSL#3-CM on basal and lipopolysaccharide (LPS) induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in rat aortic smooth muscle cells (RASMC)

RASMCs were treated with different dilutions of VSL#3-CM in the absence and

presence of lipopolysaccharide (LPS) (100 g/ml) to see the effect on basal and

LPS induced nitric oxide (NO) production and inducible nitric oxide synthase

(iNOS) expression. The accumulation of nitrite in cell culture medium was used as

an indicator of NO production. The results obtained showed that there was no

effect of VSL#3-CM on the normal cellular level as well as LPS-induced NO

production in rat aortic smooth muscle cells (Figure 52A).

The representative Western blots showed little inhibition of VSL#3-CM on LPS-

induced iNOS expression (Figure 52B).

The data suggest that the effect of the metabolites present in VSL#3-CM may be

cell specific that may not have the ability to regulate the NO and iNOS expression

in non-stimulated aortic smooth muscle cells however, further study is required to

come to a conclusion.

176

A.

B.

Figure 52: Effect VSL#3-CM on basal and lipopolysaccharide (LPS) (100 µg/ml) induced NO production and inducible nitric oxide synthase (iNOS) expression in rat aortic smooth muscle cells (RASMC)

Confluent monolayers of RASMC cells were treated with a serial dilution of VSL#3-

CM alone and simultaneously with LPS (100 µg/ml). Following 24 hours

incubation, the accumulation of nitrite was measured using Griess assay as

described in materials and methods (Chapter 2, Section 2.8). The results

presented are mean ± SEM of four independent experiments. Statistical

significance was determined by one way ANOVA using Dunnett‟s multiple

comparison test. The expression of iNOS was detected in cell lysates using

Western blot analysis as described in materials and methods (chapter 2, section

2.11). Expression of -actin was used to determine the loading efficiency of

protein. The representative Western blot is one of four separate experiments.

Control1:500

1:1001:50

1:251:10

LPS1:500

1:1001:50

1:251:10

0

50

100

150

200


Nitri

te p

rodu

ctio

n(%

of L

PS a

ctiv

ated

cel

ls)

β-actin

iNOS

177

5.4. Discussion

Nitric oxide (NO) and reactive nitrogen species peroxynitrite (ONOO־) are the

important mediators in the pathogenesis of inflammatory bowel disease (IBD). The

mucous membrane of patients suffering from IBD had been reported to produce

large amount of NO through the expression of inducible nitric oxide synthase

(iNOS) (Gupta et al., 1998). Impairment in the immune responses plays the key

role in the pathogenesis of IBD. So, there should be a balance in the host immune

modulation in response to the pathogens. Intestinal epithelial cells play important

roles in the immune modulation by interacting with the immune cells to induce

appropriate immune response during infection. However, excess immune

expression might be injurious to the epithelial tissue.

The production of iNOS-mediated NO in response to the pro-inflammatory

cytokines and bacterial lipipolysaccharide (LPS) was reported in various cell types

such as, macrophages (Jones et al., 2007), vascular smooth muscle cells (Zhang

et al., 2001) and messenglial cells (Lui et al., 2004). The induction of iNOS is

regulated by the activation of transcription factor nuclear factor kappa B (NF-B)

and post-transcriptional modification of iNOS gene such as mRNA stabilization.

NF-B becomes activated through the degradation of the inhibitor protein IB by

IκB kinase and translocate to the nucleus from the cytoplasm followed by the

binding to DNA via the B site (Kleinert et al., 2004). It has been claimed that the

non-pathogenic and probiotic bacteria suppress the inflammatory response

produced by the pathogenic bacteria through the activation of NF-B (Ng et al.,

2009; Petrof et al., 2004).

In the present study J774 macrophage cells were used to characterise the VSL#3-

CM-induced NO production and iNOS expression as well as to investigate the

effects of the inhibitors of various signalling molecules that are involved in the

process of iNOS expression and NO production. Due to the limitation of time the

investigation was focussed primarily on the VSL#3-CM-induced NO production

and iNOS expression.

178

In this study, VSL#3-CM has been shown to induce the basal iNOS expression

and NO production in J774 cells on a concentration dependent manner. The

stimulation of J774 cells by VSL#3-CM at higher concentration was to the same

degree as with LPS. On the other hand, it has inhibited the LPS-induced NO

production on a concentration dependent manner without affecting the LPS-

induced iNOS expression. These findings would suggest that VSL#3-CM was able

to regulate the LPS-induced NO production not by blocking iNOS expression but

presumably by blocking its ability to generate NO from L-arginine. This however

remains to be confirmed.

Although NO is produced from L-arginine through the expression of iNOS protein,

the enzymatic activity of iNOS is regulated by many factors and L-arginine

(Alderton et al., 2001). To perform its enzymatic activity, dimerization is essential

for iNOS, and this dimerization requires multiple cofactors such as heme,

calmoduline, flavin adenine dinucleatide (FAD), nicotinamide adenine dinucleotide

phosphate (NADPH), Flavin mononucleotide (FMN) and tetrahydrobiopterin (BH4).

However, among these cofactors, BH4 is rate-limiting for NO production (Milstien

et al., 1993; Muhl et al., 1994). The reduction of BH4 and L-arginine has been

reported to lead to the production of superoxide (Lirk et al., 2002; Pekarova et al.,

2011). Xia et al. (1997) has reported that due to the reduced availability of L-

arginine, the iNOS in macrophage generates both superoxide and NO that reacts

to form potent oxidant peroxynitrite (Lirk et al., 2002; Xia et al., 1996; Xia et al.,

1997; Xia et al., 1998). Sakai et al. has reported on the increased production of

nitrate from the reaction of iNOS-derived superoxide and NO due to the reduction

of BH4 and L-arginine (Lewis et al., 1995; Sakai et al., 2006). As BH4 or nitrate

content of the culture medium was not detected, it is hard to come to a conclusion

on this. However it can be suggested that VSL#3-CM, in the presence of LPS,

may have an effect on BH4 or any other factors responsible for the activity of

iNOS.

Polymyxin B, a natural peptide, derived from the bacterium Bacillus polymyxa, is a

potent antibiotic with bactericidal action, against almost all gram-negative bacterial

179

infection. In addition to its antibiotic function, PmB is used to neutralize the

contamination of endotoxin in reagents and cell culture medium (Cardoso et al.,

2007). It binds with the negatively charged site in the lipopolysaccharide layer.

Polymyxin B, the cyclic cataionic decapeptide contains lipophilic and hydrophilic

group that binds to lipid A, the major component of the endotoxin LPS and

neutralize it (Cardoso et al., 2007; Cooperstock, 1974; Ferrari et al., 2004). Gao

and co-workers reported on the inhibition of LPS-induced pro-inflammatory

cytokine TNF- by PmB (Gao et al., 2003a; Gao et al., 2003b).

The production of LPS-induced TNF- and other cytokines in various cells

including murine macrophages has been reported to be neutralized by PmB (Gao

et al., 2003a; Hogasen et al., 1995; van der Kleij et al., 2004). To exclude the

possibility of the endotoxin contamination in VSL#3-CM during its preparation,

experiments were carried out in J774 cells using VSL#3-CM and LPS in the

presence and absence of PmB. Polymyxin B did not affect the VSL#3-CM-induced

NO production, whereas, it inhibited the LPS-induced NO production confirming

that the production of NO in J774 cells induced by VSL#3-CM was not endotoxin

mediated.

VSL#3-CM caused the induction of basal COX-2 independently that was much

lower than the expression iNOS at the same dilution of (1:10), which suggest that

VSL#3-CM possibly modulate the regulation of iNOS and COX-2 in a different

way. COX-2-mediated PGE2 production has been reported to inhibit the NO and

IL-12 production by macrophages (Harbrecht et al., 1997; van der Pouw Kraan et

al., 1995; Yamashita et al., 2007). A substantial amount of PGE2 is produced by

the COX-2 at the site of inflammation where it acts as a potent vasodilator and

increase the vascular permeability to accelerate the migration of leukocytes into

the inflammatory sites and this is how COX-2 plays crucial roles in inflammatory

responses (Aronoff et al., 2004; Schwacha et al., 2002; Williams et al., 1996).

However, due to the time limitations, PGE2 was not measured and it is therefore

difficult to comment on the status of COX-2 expression in response to VSL#3-CM.

180

To support the proposition that VSL#3-CM-induced NO production and iNOS

expression was caused by the mediators released by probiotics. Cells were

treated separately with VSL#3-CM and the supernatant from the sonicated whole

bacteria in the cell culture medium (DMEM). It is evident from the results that the

induction of basal NO production and iNOS expression is attributed to the factors

released by the bacteria, because the bacterial sonicate had no effect on either

basal NO production or iNOS expression. In contrast, VSL#3-CM induced the

basal NO production and iNOS expression on a concentration dependent manner.

The induction of the iNOS expression and NO production by LPS is regulated by

the key transcription factor NF-B (Baldwin, 1996; Jaulmes et al., 2005; Karin et

al., 2000; Kim et al., 2009; Li et al., 2002). In murine macrophages J774 cells, the

dexamethasone has been reported to inhibit the induction of iNOS but showed no

effect on the degradation of the IB and activation of NF-B (Korhonen et al.,

2002) pointing to the post-transcriptional regulation of iNOS such as, iNOS mRNA

stabilisation. Korhonen et al. also reported that dexamethasone inhibited the

induction of iNOS and iNOS-induced NO production by destabilizing the iNOS

mRNA in LPS-treated cells. However, dexamethasone did not produce any

response on the iNOS mRNA and NO production in the cells treated with the

combination of LPS and IFN- (Korhonen et al., 2002). Shinoda et al. also

reported on the involvement of post-transcriptional regulation of iNOS induction by

dexamethasone in LPS-treated glioma cells (Newton et al., 1998; Shinoda et al.,

2003). This suggests that the mechanism of action of dexamethasone on the

expression of iNOS may be related to the type and stimulus used.

In this study, dexamethasone partially inhibited the VSL#3-CM-induced iNOS

expression and NO production. A similar effect was produced by the

dexamethasone on the LPS-induced iNOS expression and NO production.

However, the inhibition in VSL#3-CM-treated cells was lower than in LPS-treated

cells. These results indicate that VSL#3-CM possibly stimulates J774 cells to

express iNOS and produce NO through the pathways that may only be partially

181

dependent on NF-B and that the pathway may be different to that activated by

LPS.

Among the other signalling pathways which may be modulated by VSL#3-CM,

protein kinase C (PKC) was investigated to see whether it has involvement in

VSL#3-CM-induced iNOS expression and NO production. Protein kinase C plays a

major role in the macrophage activation and is divided into three classes based on

their structure and ability to bind with the cofactors (Newton, 2001). Protein kinase

C enzymes are involved in the regulation of cellular responses through the

phosphorylation of hydroxyl groups of amino acids serine and threonine residues.

These enzymes have been reported to expresse in many cell types, including

macrophages (Salonen et al., 2006; Wardsworth et al., 2002; Wen et al., 2011;

Wey et al., 2000), and its implication was considered as an important mediator of

cellular activation (Bhatt et al., 2010; Nakai et al., 1998).

Protein kinase C pathway represents a major signal transduction system in

inflammation (Spitaler et al., 2004). Specific PKC isoforms have been reported to

induce the LPS-stimulated iNOS in murine microglial cells. Wen et al showed that

the expression of iNOS in murine microglia occurs through the signalling pathways

mediated by cPKC and nPKC that involves phosphorylation of MAPKs and

activation of NF-B (Wen et al., 2011).

Bisindolylmaleimide (BIM) is a potent and highly selective PKC inhibitor that

inhibits the PKC phosphorylation by competitively inhibiting the ATP binding site

and interacting with the catalytic subunit (Coultrap et al., 1999; Hofmann, 1997).

LPS-stimulated PKC activation in murine macrophages, and individual PKC

isoenzymes-regulated cell signalling had been shown to differ among cell types

(Paul et al., 1997b; Paul et al., 1995). The inhibition of cPKC suppressed the

LPS-induced iNOS expression and NO production in the activated murine

macrophages J774 cells that has been suggested to be mediated by the inhibition

of transcription factor STAT 1 (Salonen et al., 2006). VSL#3-CM-induced NO

production and iNOS expression was only partially inhibited by BIM at the non-

182

toxic concentration. However, it caused the total inhibition of both NO and iNOS at

the concentration that was found toxic to the cells by MTT assay. The results

suggest that the VSL#3-CM-induced iNOS and NO involves the PKC dependent

pathway. Salonen et al (2006) reported the involvement of cPKC β in LPS

stimulated J774, however, as BIM is a general PKC inhibitor it is not possible to

comment on the specific PKC isoenzyme involvement in the VSL#3-CM-stimulated

J774 cells.

Phosphatidylinocitol 3-kinases (PI3K) is another important intracellular signalling

enzyme involved in the phosphorylation of Phosphatidylinocitol lipids. The role of

PI3K has been implicated in a wide variety of cellular functions including gene

expression (Cantley, 2002; El-Kholy et al., 2003). Phosphatidylinocitol 3-kinases

has been reported to produce the NO through the expression of iNOS in murine

macrophages (Chen et al., 2012; Kao et al., 2005).

LY2904002 is a compound derived from the naturally occurring bioflavonoid

quercetin that inhibits the PI3K activity through the competitive inhibition of an ATP

et al., 1994). LY2904002 has been

reported to suppress IKK/IB/NF-B mediated-iNOS expression by inhibiting PI3K

(Qi et al., 2012; Tsai et al., 2012). The role of PI3K on NO production through the

expression of iNOS has been characterised in murine macrophages (Chen et al.,

2012; Kao et al., 2005). A possible role for PI3K in the VSL#3-CM-induced NO

production and iNOS expression was investigated. When cells were treated with

the inhibitor of PI3K LY2904002, which is also an AKT activation inhibitor (Lai et

al.2010), both the VSL#3-CM-induced NO production and iNOS expression were

partially inhibited at higher concentration of LY2904002, that was toxic to the cells.

Slight inhibition was shown at lower non-cytotoxic concentration which suggests

that PI3K may be regulated by VSL#3-CM that requires more investigations.

However, the inhibition at higher concentration might be due to the suppression of

other signalling pathways in addition to PI3K.

183

AKT or protein kinase B (PKB) plays a key role in different cellular processes

including the signal transduction. LPS-induced iNOS expression and NO

production in mouse macrophages mediated by the phosphorylation of AKT have

been reported by Oh et al. (Oh et al., 2008).

AKT inhibitor XIII is a quinoxaline compound and an inhibitor of AKT1 and AKT2,

blocks the basal and stimulated phosphorylation of AKT signalling pathway.

Further studies were undertaken to examine the possible involvement of AKT in

the VSL#3-CM-induced NO production and iNOS expression. AKT is a

downstream signalling molecule of PI3K (Franke et al., 1997; Kao et al., 2005) and

has been reported to induce the iNOS expression and NO production in the

murine macrophage through the activation of PI3K/Akt pathway in response to

gram+ve bacterial cell membrane compound lipotechoic acid (LTA) (Kao et al.,

2005). However, AKT can be activated also in a PI3K independent pathway

(Brami-Cherrier et al., 2002; Sable et al., 1997; Widenmaier et al., 2009). LPS-

induced iNOS expression and NO production in mouse macrophages through the

phosphorylation of AKT were reported by Oh et al. (Oh et al., 2008). AKT inhibitor

XIII inhibited the VSL#3-CM-induced NO and iNOS, but only at a higher

concentration that was toxic to the J774 cells. The non-cytotoxic concentration of

the AKT inhibitor did not show any effect on the NO production and iNOS

expression. These results suggest that AKT may not be involved in VSL#3-CM-

stimulated NO production and iNOS expression in J774 cells.

p38 MAPK, one of the major subgroups of mitogen-activated protein kinase

(MAPK) family is activated by a variety of cellular stresses including the

inflammatory cytokines, oxidative stress and LPS which regulate the activity of

several factors including the NF-B (An et al., 2002; Papachristou et al., 2008;

Pomerantz et al., 2002; Tergaonkar, 2006). However, the differential regulation of

p38 MAPK on the iNOS expression has been reported by several authors.

Previous studies have shown that p38 MAPK pathway is cell type and stimulus

specific and either up-regulate (Chen et al., 1999a; Chen et al., 1999b; Fang et al.,

2011) or down-regulate the iNOS expression in the murine macrophage cells

184

(Chen et al., 1999; Chen and Wang, 1999; Guan et al., 1997; Lahti et al., 2006).

SB203580 is a pyridinyl imidazole compound and highly specific p38 MAPK

inhibitor. It inhibits the activity of p38 MAPK by blocking its catalytic activity

through the competitive inhibition with ATP molecule (Kumar et al., 1999). To

investigate the possible involvement of p38 MAPK in VSL#3-CM-induced iNOS

expression and NO production, cells were treated with p38 MAPK inhibitor

SB203580. SB203580 markedly inhibited the VSL#3-CM-induced NO production

and iNOS expression even at its non-toxic concentration suggesting the

involvement of p38 MAPK signalling pathway in VSL#3-CM-stimulated J774 cells.

Protein degradation is essential to the cells as it supplies the amino acids for fresh

protein synthesis and deactivates the enzymes and transcription factors as a way

of regulating the physiological processes. Proteasome is a multi-subunit, ATP

dependant protease complex localised in the cell cytoplasm that degrades the

unwanted proteins through ubiquitination, where particular proteins are tagged

with a small protein called ubiquitin for degradation (Kloetzel et al., 1999; Ortiz-

Lazareno et al., 2008; Qureshi et al., 2005). One of the important parts of

proteasome activity is the regulation of the transcription factors such as NF-B that

induces the expression of a large number of genes related to the immune

response and inflammatory process in response to the various stimuli (Adams,

2002; Karin et al., 2005; Miller et al., 2010). MG132 is a specific proteasome

inhibitor that blocks the degradation of ubiquitin conjugated proteins in the cells.

This keeps the NF-B bound to IB, leading to the inhibition of the activation of the

NF-B (Vabulas et al., 2005).

The proteasome-dependent protein degradation pathway was implicated in the

signalling pathways responsible for the regulation of NF-B activation (Wu et al.,

2004). As indicated above MG132 suppresses the NF-B activation by preventing

the IB degradation. To investigate the role of proteasome on NO production, cells

were treated with MG132. The result in this study showed that MG132 did not

have any effect on either VSL#3-induced NO production or iNOS expression at its

non-cytotoxic concentration. However, it has inhibited the production of NO and

185

expression of iNOS partially at 500 ηM concentration at which MG132 itself was

found to be toxic to the cells. MG132 partially inhibited LPS-induced NO at 250 ηM

and 500 ηM concentration. These findings suggest that VSL#3-CM-induced NO

production and iNOS expression may be independent of NF-B. However,

additional studies using a less cytotoxic inhibitor of the pathway are required.

NF-B is a ubiquitous transcription factor that enhances the transcription of pro-

inflammatory cytokines and is responsible for the induction of iNOS gene

expression and NO production (Kleinert et al., 2004). CAY10470 is a quinazoline

(QNZ) derivative and a potent inhibitor of transcriptional activation of NF-B. It acts

by inhibiting the phosphorylation of IB kinase (IKK) and inhibit the

phosphorylation of IB in IB-NF-B complex that keeps the NF-B bound to IB.

Thus, it prevents the activation of NF-B required to translocate to the nucleus and

reduces the synthesis of iNOS mRNA at the transcriptional level (Tobe et al.,

2003b; Wen et al., 2011; Wilczynska et al., 2006).

CAY10470-inhibited NF-B activation has been reported in different cell lines such

as mouse splenocytes and human Jurkat (Tobe et al., 2003b) cells. The

CAY10470-inhibition of LPS-induced iNOS expression in BV-2 cells through the

NF-B activation was also reported (Wen et al., 2011). In the present study, it

showed slight inhibition on the VSL#3-CM-induced NO and iNOS expression at

lower, non-cytotoxic concentration. At higher concentration (100 ηM), it partially

inhibited both NO production and iNOS expression. But the concentration of

CAY10470 exhibited cellular toxicity detected by MTT assay in J774 cells. These

results suggest that the VSL#3-CM-induced NO production and iNOS expression

may not implicate NF-B activation through the IB kinase pathway.

Previous studies have reported on the differential regulation of iNOS gene

expression in different species (Perrella et al., 1996) and cell types (Spink et al.,

1996; Xie et al., 1994) and also by different stimulus (Chen et al., 2000; Huang et

al., 2002; Kolyada et al., 2001; Zhang et al., 2001). Though VSL#3-CM induced

186

the basal iNOS and NO and inhibited the LPS-induced NO in murine macrophage

J774 cells, it showed no effect on either basal or LPS-induced iNOS expression

and NO production in RASMC. The data suggests that iNOS expression or NO

production may not be regulated by VSL#3-CM in RASMC. It can be said that

VSL#3-CM may be cell specific and may not have any effect on the regulation of

NO production and iNOS expression in RAVSM cells. However, the data provided

on the effect of VSL#3-CM on RASMC is from preliminary experiment. More study

is required to come to a valid conclusion.

The expression of VSL#3-CM-induced iNOS and production of NO have been

partially suppressed by the inhibitors of different signalling molecules which play

active roles in macrophage activation during inflammation. These data suggest

that the compound released by the bacteria present in VSL#3-CM regulate the

different signalling pathways associated with the expression of iNOS and

production NO in J774 macrophages.

187

Chapter 6 General discussion

189

6.0. General discussion

Probiotic bacteria have been reported to play important roles in improving

immunity (Lavasani et al., 2010; Sheil et al., 2007) and be effective in the

treatment and prevention of clinical conditions like IBD, IBS and other

gastrointestinal disorders. In the last decades, the awareness of the beneficial

effects of probiotics has been increased markedly (Benmark, 2007; Mach, 2006;

Rousseaux, et al., 2007). In recent years, probiotics have received extensive

attention in clinical research on inflammatory bowel disease. Several studies in

humans and animals have shown that a single probiotic strain or combination of

strains modulated the intestinal function and improved the disease condition

(Luyer et al., 2005; Preidis et al., 2009; Verna and Lucak, 2010). However, other

studies have shown that some probiotics have either minimal or no such effect

(Mileti et al., 2009; Prantera et al., 2002).

Probiotic-induced effects in the intestinal environment may be mediated by the cell

wall component (Bhatt et al., 2011; Dziarski and Gupta, 2005; Gupta et al., 1996;

Cox et al., 2007; Schwandner et al., 1999) or structurally diverse secreted

molecules (Sanchez et al., 2010; Seth et al., 2008; Thomas and Versalovic, 2010).

Several studies have suggested MAPK (Dai et al., 2012; Kim et al., 2006; Schlee

et al., 2008; Segawa et al., 2011), PI3K/Akt (Dai et al., 2013), NF-B (Dai et al.,

2013; Kim et al., 2006) and STAT (Jandu et al., 2009; Lee et al., 2010) signalling

as the target for the key biological signalling pathways of probiotics or their

products. These pathways can be modified by the individual strains, even from the

same probiotic species. For example, one strain of Lactobacillus species, L reuteri

strain, ATCCPTA 6475, inhibited the LPS-induced TNF- production in myeloid

cells by suppressing the activator protein (AP-1) pathway, whereas, L reuteri

strain DSM 17938, did not show any effect on the LPS induced TNF- production

(Lin et al., 2008).

The active metabolites released by these bacteria are believed to be the

responsible factor for their anti-inflammatory effect in intestinal inflammation and

other gastrointestinal disorders (Ewaschuk et al., 2006; Gionchetti, et al., 2006;

190

Limdi et al., 2006; O‟ Hara et al., 2006). Since, probiotic bacteria can survive the

low gastric pH and bile effects, and colonise the intestine by adhering to the

intestinal epithelium, these bacteria have become the potential candidates for the

treatment of these clinical conditions (Caselli et al., 2011; Corthesy et al., 2007;

Reid et al., 2003; Salminen et al., 1996; Van Immerseel et al., 2010). Although, the

mechanisms by which probiotics exert their effects in vivo have not yet been fully

clarified, the intestinal microflora appears to play a major role in improving the

inflammation in chronic inflammatory bowel diseases in animal and human

models. For example, colitis is associated with the altered colonic microflora in the

interleukin-10 (IL-10) gene-deficient mouse, and may be modulated either by

antibiotics or Lactobacillus subsp. treatment (Madsen et al., 2000). So far, the

most common probiotics that have been studied are Lactobacillus and

Bifidobacterium. Lactobacillus rhamnosus GG is one of the first Lactobacillus

reported to be therapeutically beneficial, administered to treat the acute rotavirus

diarrhoea (Kaila et al., 1992). Later, a combination of the lactic acid bacteria and

bifidobacteria such as VSL#3, have been used in the management of

gastrointestinal inflammation.

The present study was initially planned with an aim to look at the in vitro anti-

inflammatory effect of probiotic LGG and VSL#3 in the intestinal epithelial cell and

macrophage cell lines. NO is the important signalling molecule in the intestinal

epithelium and involved in the regulation of various epithelial function. Excess

production of NO has been reported in intestinal mucosal epithelium in IBD.

Human colon carcinoma cell lines have been widely used as in vitro experimental

model of intestinal epithelial cells (Bruno et al., 2005; Huerta et al., 2009; Kolios et

al., 1995; Vignoli et al., 2001).

Intestinal epithelium acts as a physical barrier to the entry of pathogenic

microorganisms and toxins (Shu et al., 2001; Shu et al., 2002). Disruption of this

function results in the systemic infection through the production of pro-

inflammatory cytokines. The loss of intestinal barrier integrity is a consequence of

the mucosal inflammation due to the epithelial synthesis of NO by iNOS (Kolios et

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al., 1995). The human colon epithelial carcinoma cell lines have been

characterized and used as a model to investigate the epithelial cell function and

barrier regulation. Although, they were derived from the colon carcinoma, under

specific conditions, when cultured, these cells resemble, morphologically and

functionally, the enterocytes lining the intestine (Ferrec et al., 2001; Futschik et al.,

2002; Lee et al., 2007; Stragand et al., 1981).

In this study, activation of a number of human colon carcinoma cell lines such as

HT-29, Caco2, SW-620, and human embryonic kidney epithelial cell line such as

HEK 293 was tried for use as inflammatory model. A uterine endometrial

carcinoma cell line, Hela cells, was also tested for the activation with pro-

inflammatory cytokines and LPS. Nitrite production by the human epithelial

carcinoma cells has been reported in the studies performed by others. However, in

this thesis, neither nitrite nor iNOS expression was detectable in the cells activated

with various inflammatory mediators. On the other hand, murine macrophage J774

cells were readily activated by LPS that was evident from the nitrite measured in

the spent culture medium and from the expression of the iNOS detected by

Western blot analysis. For the detection of nitrite in the epithelial cell culture

medium, the culture medium was treated with cadmium beads to convert the

nitrate to nitrite (as the conversion of NO to nitrate is predominant than nitrite in

human epithelial cells). However, even this treatment did not cause significant

changes in nitrite levels above basal suggesting that the cells were not induced.

Therefore, murine macrophage J774 cells were used throughout the study.

In accordance with the study proposal, freeze-dried probiotic (LGG and VSL#3)

culture supernatant was prepared to look at its effect on the LPS-induced NO

production. The bacteria were grown in aerobic condition. Some of the strains

such as Bifidobacteria present in VSL#3 are anaerobic. Therefore, the effects

produced by VSL#3 might be produced by the aerobic bacteria only. However,

initially VSL#3-CM was prepared both in aerobic and anaerobic condition and

showed similar effect on the basal NO production. Hence in the rest of the study,

VSL#3 was grown in aerobic condition. Though the bacteria-free culture

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supernatant of LGG showed the inhibitory effects on LPS-induced NO production

however, due to the similar effect produced by only bacteria culture medium MRS

broth, the study was carried out using the bacteria-conditioned medium produced

in cell culture medium DMEM.

At this stage, the study was repeated using a commercially available formulation of

VSL#3. On the basis of the effect of VSL#3-CM on the basal NO production and

iNOS expression in J774 cells, this part of the study was carried out to investigate

the involvement of the signalling pathways regulated by VSL#3-CM.

VSL#3-CM has significantly increased the NO production in non-stimulated J774

cells through the expression of iNOS. The stimulation of J774 cells by VSL#3-CM

at higher concentration was 75% as compared with LPS-stimulation. However, the

basal induction of NO and iNOS was due to the products secreted by the bacteria

in VSL#3. This was confirmed by the fact that effects were seen using conditioned

media but not supernatant obtained from whole bacterial sonicate, which showed

no basal induction of NO and iNOS. Previous study has reported that heat-killed

whole Bifidobacterium and Lactobacillus sp. present in VSL#3, and their cell wall

component as well as cytoplasmic fractions stimulated the murine macrophages

and produced NO (Tejada-Simon and Pestka, 1999). Korhonen et al. (2001)

reported on the iNOS-induced NO production in J774 cells by a different strain of

probiotic LGG through the activation of NF-B. Another study has reported on the

Gram-positive bacterial cell wall component LTA-induced NO production and iNOS

expression in murine macrophages which was not by LPS as was unaffected by

PmB (Kao et al., 2005).

In contrast to the induction of basal NO production; VSL#3-CM has significantly

inhibited the LPS-induced NO production but caused slight inhibition on the LPS-

induced iNOS expression in J774 cells. This might be the effect of VSL#3-CM on

the inhibition of enzymatic activity of iNOS that restricted the production of NO.

There are several reports that showed the suppression of LPS-induced NO

production through the inhibition of iNOS expression or iNOS activity in murine

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macrophages including J774 cells (Lahti et al., 2000; Paul et al., 1995). Copious

amount of NO produced by iNOS, generate peroxinitrite by the reaction with

superoxide, has been reported to reduce the NO bioavailability (Buttery et al.

1996; Ischiropoulos and Beckman, 1992; Muijsers et al., 2000; Pacher et al.,

2007). In addition, peroxynitrite can oxidise BH4 which is required during iNOS-

induced NO production (Vasquez-Vivar et al. 1998; Kohnen et al. 2001; Kuzkaya

et al. 2003; Alp and Channon 2004; Forstermann 2006; Forstermann and Munzel

2006; Upmacis et al. 2007). NO production by iNOS depends on the multiple

levels of regulations which include transcriptional, translational and post-

translational regulation (Aktan, 2004; Rao, 2000). Besides, for the iNOS activity,

homodimerization is essential and dimerization requires co-factors such as,

calmodulin, FAD, FMN, NADPH and heme which is further stabilized by binding of

the tetrahydrobiopterin (BH4) and the substrate arginine (Baek et al., 1993; Sakai

et al., 2006). Among these cofactors, BH4 is the rate-limiting for NO production

(Milstien et al., 1993; Muhl et al., 1994).

In LPS-treated PI3K deficient macrophage cells, a reduced NO production has

been reported by Sakai et al. (2006) where the induction of iNOS protein was

unchanged. In addition, he reported on the unaltered LPS-induced iNOS mRNA

and the activation of NF-B which are required to induce iNOS were also

unaffected. The possible cause for this might be the formation of dimers of iNOS

protein, which he found was extremely impaired, and a decreased amount of BH4

(Sakai et al, 2006). Furthermore, he reported a significant reduction in the

expression of GTP cyclohydrolase 1 (GCH1), the rate limiting enzyme for BH4

synthesis (Sakai et al., 2006). However, it is not detected whether VSL#3-CM had

any role on the synthesis or functions of BH4 and requires more study to come to a

valid conclusion.

Moreover, BH4 is not the only cause which might limit the iNOS activity. There are

several other factors which can regulate the iNOS activity and reduce NO

generation. One of these factors is the substrate availability. In activated

macrophage L-arginine is utilized by both iNOS and arginase to produce NO and

194

urea which are upregulated by LPS-stimulation (Baydoun and Morgan, 1998;

Corraliza et al., 1995; Morris et al., 1998). Increased arginase activity thus might

prevent the NO production by sequestering the substrate (Chang et al., 1998).

However, neither L-arginine content nor arginase activity was measured in this

study. Hence it is not possible to comment whether VSL#3-CM has any role on L-

arginine availability.

In addition, sustained production of NO by iNOS in activated macrophage is

dependent on the availability of cytosolic L-arginine, and the influx of L-arginine

into the cell is mediated by several transport systems. The most common of which

is the cationic amino acid transporters (CATs) (Baydoun et al., 1993; Closs et al.,

2000). Nicholson et al. (2001) demonstrated that the transport of L-arginine across

the cell membrane via CATs is important and NO production by iNOS can be

affected by the reduction of cytosolic L-arginine which requires the cationic amino

acid transporter 2 (CAT2) to influx into the cell. CAT2 plays important role in the

regulation of iNOS activity. Previous reports have shown the reduced NO

production even with adequate iNOS expression in LPS- treated CAT2 negative

mice macrophages (Detmers et al., 2000; Guix et al., 2005; Moncada and Higgs,

1995; Nicholson et al., 2001; Thompson et al., 2008; Yeramian et al., 2006).

VSL#3-CM may regulate the production of NO through the suppression of CAT2.

However, this requires more studies to come to a conclusion.

Similar to iNOS, VSL#3-CM also increased the basal COX2 expression in J774

cells that was only at higher concentration. The induction of COX-2 by VSL#3-CM

was however lower compared to iNOS suggeats VSL#3-CM may not regulate the

COX-2 expression in a similar way like iNOS in non-stimulated cells. Like iNOS,

VSL#3-CM showed slight reduction on LPS-induced COX-2 expression in J774

cells. There are reports on the inhibition of COX-2 activity in the LPS-stimulated

cell lines. Jin et al. (2006) reported that a chinese traditional medicine

cryptotanshinone used as anti-inflammatory agent worked through the inhibition of

the enzymatic activity of COX-2, not by affecting the transcriptional or translational

195

level of COX-2 protein expression in mouse macrophage RAW 264.7 cells (Jin et

al., 2006).

However, opposing this report Jeon et al. (2008) showed that cryptotanshinone

down-regulated COX-2 by inhibiting the NF-B activation (Jeon et al., 2008). On

the other hand Jeon et al. (2008) reported on another chinese traditional anti-

inflammatory medicine Tanshinone I (Isolated from dried roots of Salvia

miltiorrhiza) in his study, which inhibited the COX-2 mediated PGE2 production

without affecting COX-2 protein expression in mouse macrophage cell line. He

suggested that tanshinone I might work through the inhibition of phospholipase A2

that releases the arachidonic acid which is the primary precursor of prostaglandin

biosynthesis. Previously Kim et al. (2002) suggested that tanshinone I inhibited

PGE2 production in LPS-induced RAW macrophage through the inhibition of

arachidonich acid metabolism and did not affect the COX-2 activity or COX-2

expression (Kim et al. (2002). However, as none of COX-2 activity or COX-2

induced inflammatory mediator PGE2 was measured in the VSL#3-CM-treated

culture medium in this study, it is hard to make any comment on the effect of

VSL#3-CM on the basal and LPS-induced COX-2 function.

In this study, the lower inhibition of VSL#3-CM-induced NO by dexamethasone

than LPS-induced NO suggests the possibility that VSL#3-CM stimulates J774

cells to express iNOS and produce NO through the pathways that may only be

partially dependent on NF-B. CAY10470, the NF-κB inhibitor, which works

through the inhibition of IB phosphorylation showed no inhibition on VSL#3-CM-

induced NO production and iNOS expression at its non-cytotoxic concentration

suggests the non-involvement of NF-κB. However, Korhonen et al. (2002) reported

on the different responses of dexamethasone on iNOS-induced NO production. In

his study, dexamethasone inhibited the iNOS expression and NO production

through the inhibition of iNOS mRNA but not through the inhibition of IB

degradation and NF-B activation in LPS-treated murine macrophages J774 cells.

In contrast, dexamethasone did not produce any response on iNOS mRNA and

NO production in the same cells treated with the combination of LPS and IFN-.

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These observations suggest that the effect of dexamethasone may be stimulus

specific (Korhonen et al., 2002) and its response on the VSL#3-CM-induced iNOS

expression needs to be confirmed by the detection of iNOS mRNA.

Involvement of PKC has been reported in the regulation of cellular responses in

different cell types including macrophage which is a major signalling pathway in

iNOS-induced NO production (Salonen et al., 2006; Wardsworth et al., 2002; Wen

et al., 2011; Way et al., 2000; Bhatt et al., 2010; Spitaler et al., 2004). Partial but

significant inhibition of VSL#3-CM-induced NO production and iNOS expression by

BIM at non-cytotoxic concentration suggests the activation of the PKC pathway by

VSL#3-CM. Salonen et al. (2006), in his report, has suggested that the iNOS

expression and NO production in macrophages may be up-regulated through the

activation of STAT 1 that does not involve NF-B and is mediated by PKC.

However, the complete inhibition of NO and iNOS at the concentration which was

toxic to the cells may be due to the suppression of other signalling pathways by

BIM.

The PI3K inhibitor, LY2904002, which is also an AKT inhibitor has been widely

used to study the role of PI3K in the regulation of various cellular pathways

including macrophages (Gharbi et al., 2007; Lai et al., 2010), showed slight

inhibition on the VSL#3-CM-induced NO production and iNOS expression at lower

non-cytotoxic concentration suggests that this pathway may be partly modulated

by VSL#3-CM. Also, the inhibition of neither NO nor iNOS by the Akt inhibitor XIII,

an AKT pathway inhibitor, at lower non-cytotoxic concentration suggests the non-

involvement of AKT pathway in the VSL#3-CM-induced macrophage stimulation.

However, the partial inhibition shown at higher concentration may be due to the

suppression of other pathways as it was shown toxic to the cells detected by the

MTT assay.

Kao et al. (2005) has reported on the gram +ve bacterial cell wall component LTA-

induced iNOS expression and NO production, which occurred through the

activation of tyrosine kinase in murine macrophages, and was inhibited by

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LY2904002. Kengatharan et al., (1996) also has reported on the similar effect of

LTA on the iNOS expression and NO production in murine macrophages J774.2

which occurred through the activation of phosphatidylcholine-phospholipase C

(PC-PLC), phosphorylation of tyrosine kinase, and NF-B.

The marked inhibition of VSL#3-CM-induced NO production and iNOS expression

by the p38 MAPK inhibitor SB203580, was statistically significant even at 3 µM

which was not cytotoxic. This implies involvement of p38 MAPK signalling pathway

in VSL#3-CM-induced NO production and iNOS expression in J774 cells. The

activation of p38 MAPK by LTA and LPS has been reported in murine

macrophages RAW 264.7 and J774 cells (Chen et al., 1999; Kao et al., 2005).

However, the inhibition may be occurred due to the suppression of other pathways

that include extracellular signal-regulated kinase (ERK) and c-Jun N-terminal

kinase (JNK). The activation of p38 MAPK and two other MAP kinases, ERK and

JNK has been reported to be induced by LPS in murine macrophages (Chan and

Riches, 2001; Hsu and Wen, 2002; Hwang et al., 1997). VSL#3 has been reported

to protect the epithelial barrier and increase the tight junction protein expression in

vivo and in vitro by activating the p38 MAPK signalling pathway (Dai et al., 2012).

On the other hand, DNA from bacteria in VSL#3 has been reported to suppress

the p38 MAPK phosphorylation without affecting the NF-B activation in IEC (Jijon

et al., 2004) suggest the characteristics of differential regulation of MAPK

signalling by VSL#3.

Macrophage proteasome plays a key role in LPS-induced macrophage activation

(Qureshi et al., 2005; Reis et al., 2011). Upon stimulation, the phosphorylated IB

is ubiquitinated and degraded by proteasome allowing translocation of NF-B into

the nucleus where it goes through the transcriptional process to express iNOS

gene. MG132 suppress the NF-B activation by preventing the IB degradation

and thereby preventing the nuclear localization of NF-B. However, in this study

MG132 did not show any effect on either VSL#3-induced NO production or iNOS

expression at its non-cytotoxic concentration suggests the non-involvement of

proteasome-regulated NF-B activation pathway.

198

In the present study, the effect of CAY10470 on VSL#3-CM-induced NO and iNOS

expression at non-cytotoxic concentration suggests that VSL#3-CM may not have

direct effect on the IB phosphorylation and NF-B activation for iNOS expression

and NO production. It only inhibited at higher concentration (100 nM) which was

toxic to the cells shown by MTT assay. In BV-2 cells, CAY10470 at 20 nM inhibited

the LPS-induced iNOS expression through the inhibition of NF-B activation (Wen

et al., 2011) which suggests that CAY10470 might respond differently in BV-2 and

J774 cells. However, the concentration of CAY10470 used to treat BV-2 cells was

twice than the concentration used to treat J774 cells in this study. Again,

CAY10470 at non-cytotoxic concentration (10 nM) showed similar effect on the

inhibition of LPS-induced NO production and iNOS expression as on VSL#3-CM-

induced NO and iNOS expression. In J774 cells the LPS-induced iNOS and NO

production occurs through the IB-NF-B activation and CAY10470 inhibits the

NF-B activation by blocking the IB phosphorylation. Therefore, it is not clear

whether the concentration of CAY10470 was too low to produce any effect on

either VSL#3-CM or LPS-induced iNOS expression and NO production in J774

cells and an increased concentration of CAY10470 in a non-cytotoxic range is

required to see its effect on iNOS and NO induction.

In macrophages, iNOS expression is not regulated only by NF-B, it is also

regulated by other transcription factors such as, activator protein-1 (AP-1)

(Bogdan, 2001; Hulme et al., 2012; Su et al., 2011) and signal transducer and

activator of transcription 1 (STAT 1) (Chung et al., 2010; Darnell et al., 1994;

Durbin et al., 1996; Meraz et al., 1996; Ramana et al., 2002; Xie et al., 1993). In

this study the effect of VSL#3-CM on the activation of AP-1 and STAT1 has not

been investigated. Hence it is not possible to comment whether these pathways

were regulated by VSL#3-CM which might induce NO production and iNOS

expression.

VSL#3 is a mixture of multiple types of bacteria and it can be expected that

different strains within this bacterial mixture may act through different mechanisms

in diverse situations (Hart et al., 2004; Sood et al., 2009). VSL#3 bacteria-secreted

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products has been reported to induce the expression of heat shock proteins in

colon epithelial cells which protects the cells against oxidative stress-induced

injury (Petrof et al., 2004). Another report has shown that VSL#3 mediated the

prevention of the onset of intestinal inflammation in mouse model through the

induction of TNF-and activation NF-B which improved the intestinal epithelial

barrier function (Pagnini et al., 2010). VSL#3 contained bacterial DNA has been

reported to exert TLR9-mediated activation of NF-B and JNK in bone marrow-

derived mouse macrophages (Rachmilewitz et al., 2004).

Individual strains from probiotic VSL#3 has been reported to modulate various

signalling pathways in macrophages and intestinal epithelial cells. Bifidobacteria

up-regulated the IL-10 production significantly by human dendritic cell (DC). In

contrast, Lactobacilli and Streptococcal strains have been reported to either

reduce or show no effect on IL-10 production in the same cells (Hart et al., 2004).

B. bifidum, B. breve, and B. infantis have been reported to induce significantly

higher IL-10 compared to the B. adolescentis in murine macrophages J774.1 cells

(He et al., 2002). L. paracsei, one of the strains of VSL#3 has been reported to

reduce pro-inflammatory cytokines in plasma and lymphocyte in patients with UC

(Federico et al., 2009). Another report has shown DNA from L. delbrueckii subsp.

bulgaricus stimulated the epithelial IL-8 secretion, whereas, Bifidobacterium

strains inhibited the basal IL-8 secretion (Jijon et al., 2004). Therefore, VSL#3-CM

may act through different mechanisms to modulate the immunity through the

induction of iNOS and NO in immune cells macrophages. However, the work that

has been done in this study is not enough to come to a conclusion on the effect of

VSL#3-CM both on basal and LPS-induced iNOS expression and NO production

and more work is required to make valid conclusion on it.

Nitric oxide plays important roles in vascular biology (Ignarro et al., 1999;

Moncada and Higgs, 2006). Constitutively expressed eNOS-derived NO protects

vascular layer in different pathological conditions including atherosclerosis

(Furchgott, 1999; Ignarro and Napoli, 2004; Napoli and Ignarro, 2001). But excess

production of NO by iNOS contributes to the tissue injury through the production of

200

peroxynitrite in atherosclerosis (Buttery et al. 1996; Detmers et al., 2000).

Therefore, to establish whether the effects of VSL#3-CM were specific to the J774

macrophages or common to other cell systems expressing iNOS, studies were

carried out on a second cellular model, cultured vascular smooth muscle cells

(VSMCs). The smooth muscle cells (SMCs) also express iNOS and produce NO in

response to the pro-inflammatory mediators and it is through the signalling

mechanisms similar to those reported in J774 cells. The pro-inflammatory

mediators such as LPS, IFN- and TNF- regulate the induction of iNOS through

the activation of different signalling pathways that include PKC, PI3K/AKT and p38

MAPK in SMCs. In murine macrophage J774 cells, the expression of iNOS and

production of NO occurs through the activation of the similar pathways.

However, as VSL#3-CM induced the expression of basal iNOS and NO and

reduced the LPS-induced production of NO in macrophages, rat aortic smooth

muscle cells (RASMC) were used to explore the effect of VSL#3-CM in the

absence and presence of LPS on the induction of iNOS and NO production. A

number of reports have shown the iNOS-induced NO production in RASMC in

response to LPS, IFN- and IL-1 through different mechanisms (Fries et al.,

2003; Teng et al., 2002). However, in contrast to the effect on J774 cells, VSL#3-

CM showed no effect on either basal or LPS-induced iNOS expression and NO

production in RASMCs.

The regulation of the iNOS gene expression differs in different species (Perrella et

al., 1996) and cell types (Spink et al., 1996; Xie et al., 1994) and also regulated

differently by various stimulus (Chen et al., 2000; Huang et al., 2002; Kolyada et

al., 2001; Zhang et al., 2001). Therefore, it can be suggested that VSL#3-CM may

not have any effect on the regulation of NO production and iNOS expression in

RASMCs as shown in macrophage. However, the work carried out in RASMCs is

somewhat limited and requires further extension before any valid conclusion may

be drawn.

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6.1. Summary and conclusion

From the in vitro and in vivo studies, probiotic formulation VSL#3 was reported to

be effective in the treatment and prevention of IBD. The aim of the present study

was to look at the anti-inflammatory effect of VSL#3-secreted compounds that had

been claimed to be the responsible factor for producing its beneficial effects.

Macrophages play important roles in the host immunity and inflammatory

responses. To maintain the homeostasis in the gastrointestinal tract a careful

balance of host response to the intestinal luminal content is required. The

pathogenesis of IBD is thus strongly related to the intestinal microbial imbalance.

The normal intestinal micro flora has been reported to be dominated by the

pathogens in IBD. The intestinal macrophages are situated at the interface of the

host and intestinal luminal environment to appropriately respond to the pathogens

and ingested stimuli. Macrophages also play important roles in the resolution of

inflammation and repair of intestinal mucosa during disease remission (Kamada et

al., 2008; Mahida, 2000; Schenk et al., 2007). However, intrinsic defects in

macrophages and inappropriate regulation in the signalling also contribute to the

pathogenesis of IBD. The mucosal macrophages have been reported to be

increased in both ulcerative colitis and Crohn‟s disease and these macrophages

secrete TNF- and pro-inflammatory cytokines which play major roles in the

inflammation.

The production of NO by the macrophages mediates the killing of pathogenic

bacteria that is initiated through the activation of various signalling pathways. In

this study, probiotics have been documented to modulate the different signalling

pathways in macrophages which were shown to be activated by the compound

secreted by the probiotic microflora present in the formulation of VSL#3.

Although, the individual strain of bacteria in VSL#3 had been reported to regulate

the signalling pathways differentially, in this study, the data so far gathered,

showed that the secreted soluble products in VSL#3-CM have both the

immunostimulatory and immunomodulatory effects. Becaus, immunostimulants

202

induce the enhancement of the body‟s defense mechanism and immunomodulants

influence the host‟s immune system either by enhancing or suppressing the

immune response (Koo et al., 2006). The immunostimulatory and

immunomodulatory effects of VSL#3 have been reported by several other

researchers (Bibiloni et al., 2005; Hart et al., 2004; Mastrangeli et al., 2009;

Pagnini et al., 2009; Sheil et al., 2007; Thomas and Versalovic, 2010). The data

from the present study revealed that VSL#3-CM up-regulated the pro-inflammatory

gene iNOS and induced the NO production in macrophages. On the other hand, it

has suppressed the bacterial lipopolysaccharide (LPS)-induced NO production,

without producing significant change on the iNOS expression. This may be due to

the interruption of the functional response in LPS-induced iNOS by VSL#3-CM

through the modulation of the co-factor BH4 and/or L-arginine availability.

VSL#3-CM induced the basal COX-2 expression but caused the slight inhibition of

COX-2 expression mediated by LPS. Like iNOS and iNOS-derived NO, COX-2

and COX-2-derived PGE2 are important regulators in immune response. However,

as the PGE2 was not detected, the regulation of the COX-2 expression by VSL#3-

CM remains unclear.

The studies carried out to determine the involvement of signalling pathways,

showed that VSL#3-CM stimulated the J774 macrophages through the activation

of more than one signalling pathway. The data gathered from the study showed

that the induction of iNOS and NO production by VSL#3-CM occurs through the

phosphorylation of protein kinase C (PKC), p38 MAPK and Phosphatidylinocitol 3-

kinases (PI3K).

Activation of the macrophages involves the other transcription factors such as

activator protein-1 (AP-1) and signal transducer and activator of transcription

(STAT) in macrophages. AP-1 play important role in immune response and induce

the inflammatory protein which is regulated by the activation of another member of

MAPK c-Jun N-terminal kinase (JNK). Recent reports have shown the iNOS

expression and NO production in the activated murine macrophages through the

203

AP-1 pathway (Hulme et al., 2012; Su et al., 2011). Various studies have

demonstrated the role of STAT-1 in inflammatory and immune response (Chung et

al., 2010; Meraz et al., 1996; Salonen et al., 2006). Salonen et al. (2006) reported

the involvement of the STAT 1 in LPS treated J774 cells. Chung et al. (2010) has

shown the IFN--induced macrophage activation through the STAT-1 activation.

Therefore, NF-B may not be the only transcription factor rather; in addition, other

transcription factors such as, AP-1 and / or STAT-1 which are not detected in this

study may be activated in response to the VSL#3-CM suggesting the possible

involvement of more than one signalling pathway in the process.

The suppression of LPS-induced NO by VSL#3-CM in the J774 macrophages

suggesting that its proposed anti-inflammatory actions are partly mediated through

the suppression of iNOS activity and thus NO production in vivo. However, VSL#3-

CM also induced the basal iNOS and NO through the activation of PKC, P38

MAPK and PI3K pathways. A balanced expression of iNOS and NO production is

necessary for the regulation of normal cellular functions. Therefore, it remains

unclear whether this is of any benefits of using the VSL#3-CM as it also induces

iNOS and NO production itself through the activation of the PKC, p38 MAPK and

PI3K pathways. Therefore, further studies are required to rationalize the use of

VSL#3 in the treatment of IBD.

205

6.2. Future work

Due to constraint of time, it was not possible to extend the experimental work

required to fully explain the results obtained with the conditioned media of the

probiotic formulation VSL#3. To produce the NO from L-arginine, the enzymatic

activity of iNOS depends on the cofactors. One of these cofactors is

tetrahydrobiopterin (BH4) that is the rate limiting factor in NO production. Due to

the unavailability of this cofactor iNOS produces superoxide instead of NO. It is

necessary to carry out the further study to confirm whether VSL#3-CM modulated

the BH4 availability or any other factor. Besides, the MTT assay showed higher cell

death in response to VSL#3-CM. Therefore, the determination of the superoxide

content of the culture medium would be required to test this mechanism.

The inhibitor of the NF-κB, CAY10470 showed no effect on the VSL#3-CM-

induced basal NO production and iNOS expression. Apart from the NF-B,

activator protein-1 (AP-1) and signal transducer and activator of transcription-1

(STAT-1) are the other transcription factors that are also involved in iNOS gene

expression in macrophages. Besides, it was not clear whether VSL#3-CM has any

influence on the transcriptional level of iNOS expression. Because,

dexamethasone works in the transcriptional level of iNOS expression and it only

partially inhibited the VSL#3-CM-induced iNOS expression and NO production.

Therefore to find out the involvement of AP-1 and STAT-1 pathway and also the

modulation in the transcriptional level of the iNOS expression, further studies are

necessary using the inhibitors of these transcription factors and iNOS mRNA. That

will confirm the involvement of the relevant signalling pathways which may be

responsible for the induction of basal iNOS expression and NO production in J774

macrophages.

207

Chapter 7 References

209

7.0. References

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