the effects of polysaccharides secreted by c. aponinum

The effects of polysaccharides secreted by C. aponinum from the Blue Lagoon on immune responses of keratinocytes

in vitro

Vala Jónsdóttir

Thesis for the degree of Master of Science

University of Iceland

Faculty of Medicine

Department of Biomedical Science

School of Health Sciences

The effects of polysaccharides secreted by C. aponinum from the Blue Lagoon on immune

responses of keratinocytes in vitro

90 ECTS

Vala Jónsdóttir

Thesis for the degree of Master of Science

Supervisor:

Jóna Freysdóttir, Department of Immunology and Centre for Rheumatology

Research, Landspítali - the National University Hospital of Iceland and

Faculty of Medicine, University of Iceland

Advisors:

Ingibjörg Harðardóttir, Professor, Biochemistry and Molecular Biology,

Faculty of Medicine, University of Iceland and Department of Immunology

Landspítali - the National University Hospital of Iceland

Ása Bryndís Guðmundsdóttir, Ph.D. student and pharmacist, Department of

Immunology and Centre for Rheumatology Research, Landspítali - the

National University Hospital of Iceland and Faculty of Medicine, University of

Iceland

Faculty of Medicine

Department of Biomedical Sciences

School of Health Sciences

June 2017

Áhrif fjölsykra seyttum af C. aponinum úr Bláa Lóninu á ónæmissvör hyrnisfrumna in vitro

90 ECTS

Vala Jónsdóttir

Ritgerð til meistaragráðu í lífeindafræði

Umsjónakennari:

Jóna Freysdóttir, prófessor, Ónæmisfræðideild og Rannsóknastofu í

gigtsjúkdómum, Landspítala og Læknadeild Háskóla Íslands

Leiðbeinendur:

Ingibjörg Harðardóttir, prófessor, Lífefna- og sameindalíffræði, Læknadeild

Háskóla Íslands og Ónæmisfræðideild Landspítala

Ása Bryndís Guðmundsdóttir, doktorsnemi og lyfjafræðingur, Rannsóknastofu

í gigtsjúkdómum, Ónæmisfræðideild Landspítala og Læknadeild Háskóla

Íslands

Læknadeild

Námsbraut í lífeindafræði

Heilbrigðisvísindasvið Háskóla Íslands

Júní 2017

Thesis for a Magister Scientiae degree at the University of Iceland. All rights

reserved. No part of this publication may be reproduced in any form without

prior permission of the copyright holder.

© Vala Jónsdóttir 2017

ISBN (apply at http://www.landsbokasafn.is/)

Printing Nón

Reykjavik, Iceland 2017

http://www.landsbokasafn.is/

9

Abstract

Psoriasis is a chronic autoimmune disease that affects 2-3% of adults

worldwide. Psoriasis is characterized by red, scaly and well-demarked skin

lesions that arise through chronic interactions between hyper-proliferative

keratinocytes and activated residential immune cells. Bathing in the Blue

Lagoon in conjunction with UVB light treatment has been shown to be a

useful alternative treatment for psoriasis and to give better results than UVB

treatment alone. Previous results from the research group showed that when

dendritic cells (DCs) were matured in the presence of exopolysaccharides

from Cyanobacterium aponinum (EPS-Ca), one of the main organisms in the

Blue Lagoon, the DCs secreted more IL-10 than DCs matured in the absence

of EPS-Ca. Furthermore, DCs matured in the presence of EPS-Ca led to less

differentiation of ROR-γt+IL-17

+ T cells and increased differentiation of

FoxP3+IL-10

+ T cells. These results suggest that EPS-Ca has anti-

inflammatory effects on DCs and increases their potential to induce T

regulatory cells at the same time as it decreases their potential to induce a

Th17 type immune response. In the skin, keratinocytes interact with immune

cells and are important in psoriatic inflammation. The main aim of this study

was to determine the effects of EPS-Ca, and of DCs matured in the presence

of EPS-Ca and co-cultured with T cells, on activation of keratinocytes.

HaCaT cells (immortalized keratinocytes) were stimulated with either

TNF-α and IFN-γ (Th1 stimulation) or TNF-α and IL-17 (Th17 stimulation) in

the presence or absence of EPS-Ca. Unstimulated HaCaT cells were also

cultured with or without EPS-Ca. In addition, HaCaT cells were cultured with

DCs that had been matured in the presence or absence of EPS-Ca and co-

cultured with allogeneic CD4+ T cells. Furthermore, HaCaT cells were co-

cultured with immature DCs (imDCs) stimulated with either Th1 or Th17

stimulation and with mature DCs (mDCs) without being stimulated. The

concentration of cytokines and chemokines in supernatants was determined

by ELISA and surface molecule expression by flow cytometry.

When HaCaT cells were stimulated with Th1 stimulation, EPS-Ca

increased their secretion of IL-6, CCL3, CCL20 and CXCL8 and their

expression of CD29 (β1 integrin) but decreased their secretion of CXCL10.

When the HaCaT cells were stimulated with Th17 stimulation, EPS-Ca

increased their IL-6 and CCL3 secretion and their expression of ICAM-1.

10

HaCaT cells cultured with DCs matured in the presence of EPS-Ca and

cultured with T cells, secreted more CCL20 and CXCL8 than HaCaT cells co-

cultured with DCs matured in the absence of EPS-Ca and cultured with T

cells. EPS-Ca did not affect CXCL10 secretion of the co-culture.

Co-culturing imDCs and HaCaT cells and stimulating them with either Th1

or Th17 stimulation increased secretion of IL-6 and CCL20 (only for Th1

stimulation) compared with that when the HaCaT cells or imDCs were

cultured alone. When mDCs where co-cultured with unstimulated HaCaT

cells an increase in CCL20, CXCL8 and CXCL10 secretion was seen in the

co-culture compared with that when the cells were cultured alone. The effects

of EPS-Ca on the ability of imDCs and mDCs to induce chemokine secretion

could not be determined due to time-constraints.

Although the effects of EPS-Ca on DCs in prior studies appeared to be

anti-inflammatory, in the present study EPS-Ca had pro-inflammatory effects

on HaCaT cells stimulated in a Th1 or Th17 environment. In fact, EPS-Ca

even had a pro-inflammatory effect on HaCaT cells when they were

otherwise unstimulated. In addition, maturation of DCs in the presence of

EPS-Ca and co-culturing them with T cells led to their induction of pro-

inflammatory effects on HaCaT cells. This suggests that the EPS-Ca has pro-

inflammatory effects on HaCaT keratinocytes.

11

Ágrip

Psóríasis er krónískur bólgusjúkdómur sem hefur áhrif á 2-3% fullorðinna

einstaklinga á heimsvísu. Sjúkdómurinn einkennist af rauðum, vel

afmörkuðum skellum í húð sem myndast vegna samspils offjölgunar og

ófullkominnar sérhæfingar hyrnisfrumna og krónísks bólgusvars hvítfrumna í

húð. Böðun í Bláa Lóninu samhliða UVB ljósameðferð hefur reynst

áhrifaríkari meðferð við psóríasis en þegar einungis er stuðst við UVB

ljósameðferð. Fyrri niðurstöður rannsóknarhópsins leiddu í ljós að

angafrumur þroskaðar í návist utanfrumufjölsykra sem seytt er af

Cyanobacterium aponinum (EPS-Ca), annarri af aðallífverum Bláa Lónsins,

seyta meira af ónæmisbælandi boðefninu IL-10 en angafrumur þroskaðar án

EPS-Ca. Angafrumur þroskaðar í návist EPS-Ca ræstu einnig og sérhæfðu

ósamgena CD4+ T frumur í samrækt frekar í T bælifrumur (Treg) en í

sjúkdómshvetjandi Th17 frumur sem bendir til bólguhemjandi áhrifa EPS-Ca.

Hyrnisfrumur eru meginfrumugerð efsta húðlagsins. Hyrnisfrumur hafa

samskipti við frumur ónæmiskerfisins og gegna mikilvægu hlutverki í

bólgusvari húðar í psóríasis. Aðalmarkmið þessa verkefnis var að ákvarða

áhrif EPS-Ca og angafrumna þroskaðra í návist EPS-Ca og samræktuðum

með T frumum á örvun hyrnisfrumna.

HaCaT frumur (hyrnisfrumulína) voru ræstar annað hvort með TNF-α og

IFN-γ (Th1 boðefnaumhverfi) eða TNF-α og IL-17 (Th17 boðefnaumhverfi) og

ræktaðar með eða án EPS-Ca. Óræstar HaCaT frumur voru einnig ræktaðar

með eða án EPS-Ca. Þá voru HaCaT frumur einnig ræktaðar með

angafrumum sem höfðu verið þroskaðar í návist EPS-Ca og samræktaðar

með ósamgena CD4+

T frumum. Enn fremur voru HaCaT frumur

samræktaðar með óþroskuðum angafrumum annað hvort í Th1

boðefnaumhverfi eða Th17 boðefnaumhverfi og með þroskuðum

angafrumum án örvunar. Seytun boðefna og flakkboða var ákvörðuð með því

að mæla styrk þeirra í æti með ELISA aðferð og tjáning yfirborðssameinda

metin með frumuflæðisjá.

Þegar HaCaT frumur voru ræstar í Th1 boðefnaumhverfi jók EPS-Ca

seytingu þeirra á IL-6, CCL3, CCL20 og CXCL8, sem og tjáningu þeirra á

yfirborðssameindinni CD29 (β1 integrin) en minnkaði seytingu þeirra á

CXCL10. Þegar HaCaT frumur voru ræstar í Th17 boðefnaumhverfi jók EPS-

Ca seytingu þeirra á IL-6 og CCL3 og tjáningu á ICAM-1. EPS-Ca jók einnig

boðefna- og flakkboðaseytun óræstra HaCaT frumna.

12

HaCaT frumur ræktaðar með angafrumum þroskuðum í návist EPS-Ca og

samræktuðum með T frumum, seyttu meira af CCL20 og CXCL8 en HaCaT

frumur samræktaðar með angafrumum þroskuðum án EPS-Ca og

samræktuðum með T frumum. EPS-Ca hafði ekki áhrif á CXCL10 seytun

samræktarinnar.

Styrkur IL-6 var meiri í samrækt óþroskaðra angafrumna og HaCaT

frumna í Th1 og Th17 boðefnaumhverfi samanborið við í ræktun frumnanna

einna og sér og átti það einnig við um CCL20 í Th1 boðefnaumhverfi. Þegar

þroskaðar angafrumur og óræstar HaCaT frumur voru ræktaðar saman sást

aukin seytun á CCL20, CXCL8 og CXCL10 samanborið við ræktun

frumnanna sitt í hvoru lagi. Ekki vannst tími til að ákvarða áhrif EPS-Ca á

samrækt angafrumna og HaCaT frumna.

Þrátt fyrir að EPS-Ca hafi ýtt undir bælivirkni hjá angafrumum og T

frumum í fyrri rannsóknum, jók EPS-Ca ræsingu og bólguvirkni HaCaT

frumna sem voru örvaðar í Th1 eða Th17 boðefnaumhverfi, sem og þegar

HaCaT frumurnar voru óörvaðar. Auk þess jók EPS-Ca getu angafrumna til

að virkja HaCaT frumur. Þessar niðurstöður benda til þess að EPS-Ca hafi

bólguhvetjandi áhrif á HaCaT frumur.

13

Acknowledgements

First and foremost, I would like to thank my supervisors, Professor Jóna

Freysdóttir and Professor Ingibjörg Harðardóttir for their excellent guidance,

support and encouragement throughout the project, in both research and

writing periods. I would also like to give my thanks to Ása Bryndís

Guðmundsdóttir, who is a vital part of the project and has prepared the EPS-

Ca. Thank you all for giving me the opportunity to join the team, I am both

glad and proud to have been a member.

I would like to give my sincere thanks to both staff and fellow students at

the Department of Immunology and Centre for Rheumatology Research at

Landspítali - the National University Hospital of Iceland for their assistance,

discussions and support. I would like to give special thanks to Hildur

Sigurgrímsdóttir, PhD student and Jón Þórir Óskarsson, MSc for teaching me

most of the methods used in this project and to Fannar Theódórs, MSc

student, for support and encouragement throughout my studies.

Last but not least I would like to thank my family and friends for their

emotional support and encouragement. I especially want to thank my partner,

Andreas Egede, for always being there for me and designing the two

beautiful figures used in the introduction chapter in this thesis.

The Centre for Rheumatology Research and Department of Immunology

at Landspítali - the University Hospital of Iceland and the Faculty of Medicine

at the University of Iceland provided research facilities for this study.

This work was supported by the Technology Development Fund, Rannís

and the Landspítali - The National University Hospital Research Fund.

15

Contents

Ágrip .............................................................................................................11

Abstract .......................................................................................................... 9

Acknowledgements .....................................................................................13

Contents .......................................................................................................15

List of figures ...............................................................................................19

Declaration of contribution ........................................................................21

List of abbreviations ...................................................................................23

1 Introduction ............................................................................................25

1.1 The immune system .........................................................................25

1.1.1 The innate immune system .......................................................25 1.1.2 The adaptive immune system ...................................................26

1.2 The skin ............................................................................................28

1.2.1 The skin and the immune system .............................................29

1.2.2 Keratinocytes ............................................................................30

1.2.2.1 HaCaT cells .......................................................................30

1.2.3 Surface molecule expression on keratinocytes ........................31

1.2.3.1 TLRs ..................................................................................31

1.2.3.2 Adhesion molecules ..........................................................31

1.2.3.3 Other molecules ................................................................32 1.3 Inflammation .....................................................................................32

1.3.1 Inflammation in the skin ............................................................32

1.3.1.1 Psoriasis ............................................................................33

1.3.2 Cytokine and chemokine production by keratinocytes in

psoriasis............ ........................................................................35

1.3.2.1 Cytokines ...........................................................................35

1.3.2.2 Chemokines ......................................................................36 1.4 The Blue lagoon................................................................................37

1.4.1 The Blue Lagoon treatment for psoriasis ..................................38

1.4.2 Cyanobacteria and polysaccharides .........................................38

2 Aims .........................................................................................................41

3 Materials and methods ..........................................................................43

16

3.1 Culture of Cyanobacteria and preparation of extracellular

polysaccharides ............................................................................ 43 3.2 HaCaT cell culture ........................................................................... 43 3.3 Activation of HaCaT cells................................................................. 43 3.4 Harvesting HaCaT cells ................................................................... 44 3.5 Flow cytometry on HaCaT cells ....................................................... 44

3.6 Isolation of peripheral blood mononuclear cells (PBMCs),

monocytes and CD4+ T cells ......................................................... 44

3.7 Differentiation of monocytes and maturation of DCs ....................... 45

3.8 Co-culture of DCs and HaCaT cells ................................................ 45 3.9 Co-culture of DCs and T cells .......................................................... 46 3.10 Co-culture of DCs, T cells and HaCaT .......................................... 46

3.11 ELISA ............................................................................................ 46 3.12 Statistical analysis ......................................................................... 47

4 Results .................................................................................................... 49

4.1 Effects of EPS-Ca on cytokine and chemokine secretion by

HaCaT cells ................................................................................... 49 4.2 Effects of EPS-Ca on expression of surface molecules on HaCaT

cells ............................................................................................... 53 4.3 Effects of DCs matured and activated in the presence of EPS-Ca

and co-cultured with T cells on chemokine secretion by HaCaT

cells ............................................................................................... 54 4.4 Effects of DCs on cytokine and chemokine secretion by HaCaT

cells ............................................................................................... 58

5 Discussion ............................................................................................. 62

6 Conclusions ........................................................................................... 67

References .................................................................................................. 69

7 Appendix A ............................................................................................. 79

7.1 The effects of different concentrations of HaCaT cells and

different times of stimulation on their cytokine and chemokine

secretion ........................................................................................ 79 7.2 The effects of different cytokine stimulations on cytokine and

chemokine secretion by HaCaT cells ............................................ 81 7.3 The effects of different concentrations of IL-17 and TNF-α on one

hand and IFN-γ and TNF-α on the other hand for stimulation of

cytokine and chemokine secretion by HaCaT cells ...................... 85

8 Appendix B ............................................................................................. 87

17

8.1 The effects of EPS-Ca on cytokine and chemokine secretion by

HaCaT cells following Th1 and Th17 stimulation not including

TNF-α .............................................................................................87 8.2 The effects of EPS-Ca on expression of surface molecules on

HaCaT cells ....................................................................................90

19

List of figures

Figure 1. The anatomical structure of the epidermis and dermis .................. 29

Figure 2. A simplefied overview of an active psoriatic plaque ...................... 34

Figure 3. The effects of EPS-Ca on secretion of cytokines and

chemokines by Th1 stimulated HaCaT cells .................................. 50


chemokines by Th17 stimulated HaCaT cells ................................ 51


chemokines by unstimulated HaCaT cells ..................................... 52

Figure 6. The effects of EPS-Ca on CD29 expression on Th1

stimulated HaCaT cells .................................................................. 53

Figure 7. The effects of EPS-Ca on ICAM-1 expression on Th17


Figure 8. The effects of EPS-Ca on chemokine concentration in the

supernatant of DC and T cell co-cultures ...................................... 55

Figure 9. The effects of supernatants from DCs matured and

stimulated in the absence or presence of EPS-Ca and then

co-cultured with T cells, on cytokine secretion by HaCaT

cells ................................................................................................ 56

Figure 10. The effects of supernatants from DCs matured and

stimulated in the absence or presence of EPS-Ca and then

co-cultured with T cells added along with the cells, on

cytokine secretion by HaCaT cells ................................................. 57

Figure 11. Secretion of cytokines and chemokines by Th1 stimulated

HaCaT cells and imDCs ................................................................. 59

Figure 12. Secretion of cytokines and chemokines by Th17 stimulated

HaCaT cells and imDCs ................................................................. 60

Figure 13. Secretion of cytokines and chemokines by co-cultures of

unstimulated HaCaT cells and mDCs ............................................ 61

Figure A1. The effects of different cell concentrations and stimulation

times on cytokine and chemokine secretion by HaCaT cells. ....... 80

Figure A2. Photograph of HaCaT cells in culture. ......................................... 80

20

Figure A3. The effects of different cytokine stimulations on cytokine

and chemokine secretion by HaCaT cells...................................... 82

Figure A4. The effects of different combinations of two cytokines on

the cytokine and chemokine secretion by HaCaT cells ................. 83

Figure A5. The effects of combinations of three different cytokines on

the cytokine and chemokine secretion by HaCaT cells ................. 84

Figure A6. The effects of different concentrations of TNF-α, IL-17 and

IFN-γ on cytokine and chemokine secretion by HaCaT cells. ....... 86

Figure B1. The effects of EPS-Ca on secretion of cytokines and

chemokines by Th1 stimulated HaCaT cells (without TNF-α) ..... 888

Figure B2. The effects of EPS-Ca on secretion of cytokines and

chemokines by Th17 stimulated HaCaT cells (without TNF-

α) .................................................................................................. 899

Figure B3. The effect of EPS-Ca on CD29 expression on Th1 or Th17


Figure B4. The effects of EPS-Ca on CD44 expression on Th1 or

Th17 stimulated HaCaT cells ......................................................... 91

Figure B5. The effects of EPS-Ca on ICAM-1 expression on Th1


Figure B6. The effects of EPS-Ca on CD183 expression on Th1 or


Figure B7. The effects of EPS-Ca on E-cadherin expression on Th1

or Th17 stimulated HaCaT cells. .................................................... 93

Figure B8. The effects of EPS-Ca on HLA-DR expression on Th1 or


Figure B 9. The effects of EPS-Ca on HLA-I expression on Th1 or


21

Declaration of contribution

Vala Jónsdóttir performed all experimental work included in this thesis. Ása

Bryndís Guðmundsdóttir Ph.D. student isolated and prepared the EPS-Ca

used in this study.

23

List of abbreviations

APCs Antigen-presenting cells

CCL CC-chemokine ligand

CCR CC-chemokine receptor

CLA Cutaneous lymphocyte-associated antigen

CLR C-type lectin receptor

CXCL CXC-chemokine ligand

CXCR CXC-chemokine receptor

DCs Dendritic cells

DMEM Dulbecco's Modified Eagle Medium

EDTA Ethylenediaminetetraacetic acid

EGF Epidermal growth factor

EPS-Ca Exopolysaccharide extract from C. aponinum

FBS Foetal bovine serum

GM-CSF Granulocyte colony-stimulating factor

HRP Horseradish peroxidase

ICAM Intercellular adhesion molecule

ID Inhibitor DNA binding

IFN Interferon

IL Interleukin

imDCs Immature dendritic cells

KGF Keratinocyte growth factor

LCs Langerhans cells

LFA Leukocyte function antigen

LPS Lipopolysacccharides

mDCs Mature dendritic cells

24

MFI Mean fluorescence intensity

MHC Major histocompatibility complex

NF- κB Nuclear-factor kappa B

NLR NOD-like receptor

PAMP Pathogen-associated molecular patterns

PBMCs Peripheral blood mononuclear cells

PBS Phosphate-buffered saline

pDCs Plasmacytoid DCs

PRRs Pattern recognition receptors

RPMI Roswell park memorial institute

RT Room temperature

RUNX Runt-related transcription factor

SEM Standard error of the mean

SI Secretion index

Tfh T follicular helper cells

TGF Transforming growth factor

Th T helper cell

TLR Toll-like receptor

TNF Tumour necrosis factor

Tregs T regulatory cells

VLA Very late antigen

25

1 Introduction

1.1 The immune system

The immune system is the body’s defence against infections. Through series

of steps called immune responses, the immune system attacks and

neutralizes microorganisms, such as parasites, fungi, bacteria and viruses

and foreign substances that invade our body. The immune system and its

responses in humans can be divided into two categories, the innate immune

system and the adaptive immune system. The innate immune system

responds to all potential pathogens and is always ready for combat, but

innate immune responses are non-specific and do not lead to lasting

immunity. If the innate immune system is unsuccessful in destroying a

pathogen, the adaptive immune system is activated. The adaptive immune

response adapts specifically to an infection from a specific pathogen and the

responses are developed during the lifetime of an individual. Adaptive

immune responses create immunological memory and sometimes lifelong

protective immunity (1).

1.1.1 The innate immune system

The innate immune system includes a variety of cells with different functions.

The cells of the innate immune system are equipped with pattern recognition

receptors (PRRs) on their surface, which recognize pathogen-associated

molecular patterns (PAMPs) on microbes invading the host. There are

several PRR families, such as the toll-like receptor (TLR) family, the NOD-like

receptor (NLR) family and the C-type lectin receptor (CLRs) family (2). The

innate immune system also includes a large variety of molecules such as the

ones belonging to the complement system and the antimicrobial peptides, all

of which are available to control or destroy pathogens (1).

Macrophages, neutrophils and dendritic cells (DCs) are phagocytes that

belong to the innate immune system. Macrophages are relatively long lived

phagocytes that differentiate from monocytes. They reside in tissues where

they protect the body by engulfing extracellular pathogens and other

substances that display molecules on their surface that do not belong to

healthy cells of the body (1). Neutrophils are relatively short lived compared

with macrophages, but they are the most numerous cells in innate immune

responses. Under normal conditions, neutrophils circulate in the blood, but

when an infection occurs, neutrophils migrate to the site of infection and are

usually the first responders to infection and inflammation (1).

26

Like macrophages, DCs are phagocytes that originate from precursor

cells in the bone marrow (3,4). The most important role of DCs is to activate

lymphocytes by displaying antigens on their cell surface and initiating

adaptive immune response (1,4). Immature DCs (imDCs) reside in tissues

and are actively phagocytic and constantly sample their surrounding

environment for pathogens. ImDCs have a low potential to activate T cells as

their expression of major histocompatibility complex (MHC) class II and T cell

co-stimulatory molecules is low. Once imDCs sense a danger in their

surroundings, they differentiate into mature DCs (mDCs). During the

maturation process, the cells upregulate MHC class II expression and

necessary co-stimulatory molecules to activate naïve T cells, while

simultaneously losing the ability to take up antigens. During maturation the

DCs also upregulate molecules to enable migration to neighbouring lymph

nodes where the mDCs can interact with T cells and B cells (5). DCs thus act

as messengers between the innate and the adaptive immune systems (6).

DCs that reside in the skin are called Langerhans cells (LCs). They are

radiation-resistant and can self-renew independently of precursor cells from

the blood and bone marrow, although circulating monocytes have also been

shown to replenish the LC population during inflammation (7).

Plasmacytoid DCs (pDCs) are a unique DC subset that is mostly

developed from DC progenitors in the bone marrow. Their primary function is

the secretion of interferon (IFN)-α and IFN-β (type I interferons) in response

to viruses, thus promoting antiviral responses by other immune cells,

although they also secrete other cytokines and chemokines (8). pDCs are

also able to induce tolerogenic immune responses (9).

1.1.2 The adaptive immune system

The adaptive immune system consists of two kinds of effector cells, B cells

and T cells, collectively known as lymphocytes. Lymphocytes have highly

variable antigen receptors on their surface and each lymphocyte bears a

single unique variant, making it specific to a single antigen. Activation of

lymphocytes can generate long-lasting immunological memory and

subsequent exposure to the same pathogen enables an immediate and

stronger response, giving the individual a protective immunity against the

pathogen (1).

B cells mainly function in the humoral immunity component by secreting

antibodies. Each B cell produces antibodies of a single variety and specificity.

Antibodies bind specifically to molecules from the pathogen that initiated an

immune response and they have two main functions: to neutralize the

27

pathogen or toxins that they bind to and to direct the pathogens to other cells

of the immune system that kill and dispose of the pathogen (1).

In contrast to B cells, which interact with pathogens in the extracellular

space of the body, T cells only recognize foreign antigens that are displayed

on the surface of the body’s own cells, in a special glycoprotein called MHC.

There are two kinds of MHC proteins, MHC class I and MHC class II. MHC

class I is expressed on all nucleated cells of the body and these molecules

display peptides from degraded cytosolic proteins, displaying intracellular

protein to the T cells. MHC class II molecules are only expressed on antigen-

presenting cells (APCs), such as DCs, macrophages and B cells and some

epithelial cells, including keratinocytes. MHC class II molecules display

extracellular proteins that the presenting cell has engulfed (1). T cells are

divided into subsets after which co-receptor they express and type of MHC

molecules they bind to. Cytotoxic T cells are CD8 positive (CD8+) and they

bind to MHC class I molecules. These T cells mainly recognize antigens

found on cells infected with intracellular pathogens, such as viruses or

tumour cells. When a CD8+

T cell is activated by an antigen, the CD8+

T cell

kills the target cell by forcing it into apoptosis (1). CD4 positive (CD4+) T cells

have multiple roles as they help, suppress or regulate immune responses by

secreting cytokines. Their role include helping with activation and growth of

CD8+ T cells, they maximize bactericidal activity of phagocytes and they are

essential in B cell activation and differentiation (1,10). CD4+ T cells bind to

MHC class II molecules displayed on APCs, most commonly in secondary

lymphoid organs. The cytokines secreted by the APCs upon activation of

naïve T cells polarize the CD4+ T cells to differentiate into a number of

different subsets of effector cells, including T helper (Th)1, Th2, Th17, T

follicular helper (Tfh) cells, T regulatory cells (Tregs) and memory T cells

(11,12). Different types of pathogens induce APCs to polarize T cells to

different subsets, however, T cell populations that are specific to the same

pathogen can be of different subsets (1). The Th cells are defined by the

cytokines they secrete and the transcription factors they express (10).

Th1 cells are characterized by their production of IFN-γ and their main

role is to eradicate infections by intracellular pathogens (12). IFN-γ activates

macrophages and enables them to successfully kill intracellular bacteria (10).

Th2 cells are characterized by their production of interleukin (IL)-4, IL-5 and

IL-13 and their main role is to respond to extracellular parasites, such as

worms (12). Th2 cytokines, particularly IL-4, promote B cells to produce IgE,

which primary role is to fight parasitic infections, but IgE is also a key player

in allergy. Th2 cytokines also activate eosinophils and mast cells (1,10). Th17

28

cells are characterized by their production of IL-17, mainly IL-17A and IL-17F,

but Th17 cells can also secrete IL-22 which induces proliferation of and

increases antimicrobial peptide secretion by epithelial cells (10,13). The main

role of Th17 cells is to respond to extracellular pathogens such as bacteria or

fungi by recruiting neutrophils to the site of inflammation. IL-17 produced by

Th17 cells is considered a key mediator of autoimmunity by maintaining a

positive feedback loop where it triggers a release of pro-inflammatory

cytokines from innate cells (such as IL-1β and tumour necrosis factor (TNF)-

α, creating and sustaining a pro-inflammatory environment that further drives

the release of IL-17 (10,13).

Activated APCs can induce T cells to home to specific tissues. APCs do

this by inducing expression of certain homing receptors and chemokine

receptors on the T cells, such as cutaneous lymphocyte-associated antigen

(CLA), CC-chemokine receptor (CCR)4 or CCR10, which are all skin-homing

receptors that direct T cells to the skin (10).

1.2 The skin

The skin is the largest organ of the body and it acts as a physical and

chemical barrier between the internal organs of the body and the

environment (1). In humans, the skin can be divided into two main anatomical

compartments (figure 1): the epidermis (the outermost compartment), which

is divided into four layers, the stratum basale (at the bottom), the stratum

spinosum, stratum granulosum and the stratum corneum (the outermost

layer) and the dermis, which is further divided into three layers (14).

The epidermis consists mostly of multiple layers of keratinocytes that form

a protection barrier against environmental damage. The stratum basale

contains a single row of undifferentiated basal keratinocytes, which divide

frequently and renew the cells of the epidermis. After the cells divide, some of

them begin to move to the next layer, the stratum spinosum, where they

begin their maturation process, but some cells replenish the basal layer (15).

Keratinocytes mature as they move through the layers and when they reach

the outermost layer their differentiation is complete. The stratum corneum

contains terminally differentiated keratinocytes known as corneocytes, which

are devoid of nucleus and most organelles (16).

The dermis consists mostly of loose connective tissue, which is rich in

extracellular-matrix-containing stromal cells, but it also contains immune

cells, along with different kinds of glands, blood vessels, lymphatic vessels,

nerve endings and sensory receptors (figure 1) (15).

29

Both the epidermis and dermis contain various immune cells, which will be

discussed in chapter 1.2.1.

Figure 1. The anatomical structure of the epidermis and dermis and the key

immunological cells in each compartment.

1.2.1 The skin and the immune system

The skin is both large and constantly exposed to various stimuli; therefore,

the immunosurveillance of the skin represents a major challenge for the

immune system. If an immune response is excessive, it will lead to chronic

inflammation and a possible development of autoimmunity and if an immune

response is inadequate overwhelming infections can develop (16).

Keratinocytes can function as instigators of cutaneous inflammation and

they are the major contributors of cytokine production in the epidermis.

Keratinocytes produce cytokines either constitutively or upon induction by

stimuli. The cytokines affect proliferation and differentiation of local cells,

induce migration of inflammatory cells to the skin, along with having a

systemic effect on the immune system (1,16).

A number of immune cells are found in the epidermis (figure 1). LCs are

imDCs found in the epidermis and they are most prominent in the stratum

spinosum. LCs require the transcription factors inhibitor DNA binding 2 (ID2)

and Runt-related transcription factor 3 (RUNX3) for their development and

they have a unique developmental pathway (17). In the presence of infection,

LCs take up antigens locally in the skin, after which they start to migrate to

the lymph nodes where they differentiate into mDCs and present their antigen

30

to antigen-specific T cells (1). In humans, the main T cells in the epidermis

are CD8+ T cells, which are found in large numbers in the stratum basale and

stratum spinosum (16,18).

Immune cells in the dermis include DCs, CD4+ T cells, NK cells, mast

cells and macrophages (16,18). The dermis constitutively contains several

populations of APCs, including at least three major DC subsets that can

migrate into skin draining lymph nodes (17).

1.2.2 Keratinocytes

Keratinocytes are epithelial cells and the dominant cell type of the epidermis

(15). They have numerous immunological functions and are able to sense

pathogens and discriminate between harmless organisms and harmful

pathogens through their PRRs (16).

Keratinocytes express MHC class II molecules when stimulated with IFN-

γ, enabling them to act as non-professional APCs for T cells. Keratinocytes

can also provide requisite signals for T cell proliferation (16,19).

Keratinocytes express various adhesion molecules, such as E-cadherin and

intercellular adhesion molecule (ICAM)-1, and a large variety of receptors,

both for cytokines, such as IL-1, IL-22, IL-17, TNF-α and IFN-γ, and

chemokines, such as CCR1, CCR3, CCR4 and CCR5 (1,20,21).

Keratinocytes also secrete a number of cytokines, both pro-inflammatory,

such as IL-1β, IL-6, IL-12, IL-18, IL-19 and TNF-α, and anti-inflammatory,

such as IL-10 (16,18). They also secrete a number of chemokines, including

CXC-chemokine ligand (CXCL)8, CXCL9, CXCL10, CXCL11, CC-chemokine

ligand (CCL)20 and CCL27 (18) and can produce a variety of antimicrobial

peptides, such as LL-37, β-defensins and S100A7 (22).

1.2.2.1 HaCaT cells

The HaCaT cell line is derived from skin keratinocytes from an adult human

male. The cell line was immortalized by spontaneous development of the cell

line, but HaCaT cells are neither tumorigenic nor invasive in vivo (23). HaCaT

cells were the first human keratinocyte cell line which mostly maintained the

ability to form a well-structured epidermis after transplantation in vivo and it is

able to differentiate in vitro with normal epidermal differentiation products

(23).

31

1.2.3 Surface molecule expression on keratinocytes

Keratinocytes express a large variety of surface molecules, including

activation and adhesion molecules. The main molecules measured in the

present project will be described below.

1.2.3.1 TLRs

TLRs are a family of receptors that recognize various evolutionary conserved

microbial components or PAMPs and play a key role in the innate immune

system, as mentioned before (16). Humans express TLRs 1-10 and each

TLR is specific for a certain group of foreign molecules (1). Primary human

keratinocytes in vitro constitutively express mRNA for TLR1, 2, 3, 4, 5, 6, 9

and 10, enabling them to respond to various bacterial molecules, such as

lipopeptides, lipopolysacccharides (LPS), flagellin, double-stranded DNA and

unmethylated CpG motifs (1,24). Primary keratinocytes respond to

stimulation through TLR1/TLR2 heterodimers, TLR6/TLR2 heterodimers,

TLR3, 4, 5 and 9, suggesting that the receptors are fully functional (24–26).

HaCaT cells respond to stimuli for TLR1-6 and TLR9 (27).

1.2.3.2 Adhesion molecules

ICAM-1, also known as CD54, is primarily expressed on activated endothelial

cells and cells of the immune system but also on keratinocytes and HaCaT

cells (20,28,29). In keratinocytes and HaCaT cells the expression of ICAM-1

is increased by IFN-γ stimulation (20,28) and with LPS stimulation in HaCaT

cells (29). ICAM-1 binds to members of the integrin family, especially

leukocyte function antigen (LFA)-1, which is found on various immune cells

(1).

E-cadherin is an adhesion molecule, primarily expressed on epithelia, and

E-cadherin expression has been reported both in primary keratinocytes (1)

and in HaCat cells (30). In the skin, E-cadherin mediates adherence junctions

that attach cells to each other, connecting both cells that are side by side in

the same layer and cells in different layers (31).

CD29 is the β1 integrin of the very late antigen (VLA) compounds and is

expressed on the surface of primary keratinocytes and HaCaT cells (30). On

keratinocytes, CD29 mediates the attachment of basal keratinocytes to the

basement membrane but it can also mediate intercellular adhesion, survival

and migration (32,33). As keratinocytes differentiate they lose their

adhesiveness to the basal membrane and move from the basal layer towards

the upper part of the epidermis and CD29 expression is lost (32).

32

1.2.3.3 Other molecules

As mentioned in chapter 1.1.2, keratinocytes express both MHC class I and

MHC class II molecules (1). IFN-γ is a strong inducer of MHC class I and II

expression in primary human keratinocytes (34). MHC class I and class II

expression has also been confirmed on HaCaT cell surface (35).

CD44 is a surface receptor that binds to hyaluronan, which is a major

component of extracellular matrix and other glycosaminoglycans (11). In

addition to its adhesion role, CD44 also plays a role in epidermal

differentiation, lipid synthesis and skin barrier function (11,36). In vivo, CD44

is expressed in both the basal and suprabasal layers of the human epidermis

(37). CD44 is also expressed on HaCaT cells (38).

Primary human keratinocytes express CCR1, CCR3, CCR4, CCR6,

CCR10, CXC-chemokine receptor (CXCR)1, CXCR2, CXCR3 and CXCR4

(39). They can secrete CCR1, CCR10, CXCR1, CXCR2 and CXCR3 during

wound healing and during wound healing they also secrete the corresponding

ligands of those receptors, which stimulate keratinocyte migration and/or

proliferation. Primary human keratinocytes do not express CCR2, CCR5,

CCR7 or CCR8 (39).

1.3 Inflammation

Inflammation is a response to injury or infection. It is a localized condition

characterized by red, swollen, warm area that is tender or painful (15).

Inflammation occurs as a response to inflammatory mediators, released as a

consequence of the recognition of pathogens by macrophages or other cells

of the immune system, or as a response to tissue damage (1).

Inflammation has three essential roles in host defence: to deliver

additional effector molecules and cells to the site of infection, to induce local

blood clotting to hinder spread of the invading pathogen and to promote

repair of the injured tissue (1).

1.3.1 Inflammation in the skin

If microorganisms breach the skin layer, they are met by cells and molecules

in the skin tissue that mount an immediate onset of acute inflammation.

Inflammation has several beneficial effects in infection, such as recruiting

innate phagocytes and APCs from the blood stream to the infected tissue and

increasing the flow of lymphocytes to the lymph nodes where they react with

APCs (1). During inflammation in the skin, human keratinocytes secrete a

number of cytokines and chemokines (see section 1.2.1) (16). The cytokines

33

initiate an inflammatory response and activate immune cells already resident

in the tissue, whereas the chemokines attract neutrophils, macrophages and

T cells to the dermis, and their cytokine production becomes an additional

source for inflammatory mediators (1).

1.3.1.1 Psoriasis

Psoriasis is a common chronic autoimmune disease that affects around 2-3%

of adults worldwide. Psoriasis is characterized by red, scaly and well-

demarked skin lesions that arise through chronic interactions between hyper-

proliferative keratinocytes and activated residential immune cells (40,41).

Histological changes (figure 2) that distinguish psoriatic plaques include

dramatic hyperplasia in the epidermis, increased vascularity in the dermis,

incomplete keratinocyte differentiation, and infiltration of effector T cells, DCs,

neutrophils and macrophages (42,43). Genetic and epigenetic factors are

strongly involved in psoriasis pathogenesis, but many environmental factors,

such as trauma, chemical irritants or microbial infections, also play an

important role (43).

The initial event that triggers the first psoriatic lesion is unknown but

external factors, such as stress, physical trauma, drugs, alcohol, smoking

and certain infections can trigger the initial psoriatic episode in individuals

with genetic predisposition (22,42). The precise mechanism of the onset of

psoriasis has yet to be determined, despite extensive research on the matter.

One theory suggests that following a trauma, stressed keratinocytes

release the antimicrobial peptide LL-37 that binds to self-DNA or RNA

released by damaged keratinocytes. The LL-37 complexes activate pDCs

that are usually absent in healthy skin, but infiltrate into developing psoriatic

lesions. pDCs release type-I IFN, which triggers activation and expansion of

autoimmune T cells present at the site, leading to the development of a

psoriatic lesion (44). Dermal DCs are activated by the massive amount of

cytokines released (called cytokine storm) and they start to mature and

migrate to the skin draining lymph nodes to present their antigen to naïve T

cells (44). The antigen presented by the DCs can be either of microbial or

self-origin. The DCs promote T cell differentiation into Th1 and/or Th17 cells

that express the skin homing receptor CLA that facilitates the targeting of T

cells to inflamed skin (44,45).

CCL20, CXCL9, CXCL10 and CXCL11 secreted by keratinocytes at the

psoriatic plaque attract Th1 and Th17 cells to migrate to the psoriatic plaque

where they secrete cytokines that furthers the inflammation (figure 2). Other

34

immune cells, such as macrophages, are drawn to the dermis and this leads

to the formation of a psoriatic plaque (40,42,44). The Th17 cells secrete IL-

17A, IL-17F and IL-22, which stimulate keratinocyte proliferation and their

release of TNF-α and antimicrobial peptides, such as β-defensins, S100A7,

S10A9, and chemokines, such as CXCL1, CXCL3, CXCL5 and CXCL8.

Inflammatory DCs produce IL-23, nitric oxide radicals and TNF-α (16). The

release of TNF-α, IL-1β and transforming growth factor (TGF)β from

keratinocytes promotes fibroblasts to release keratinocyte growth factor

(KGF) and epidermal growth factor (EGF), which in conjunction with TGFβ,

contribute to tissue reorganization and further allows the epidermis to

contribute to disease pathogenesis (16).

Figure 2. A simplefied overview of an active psoriatic plaque. In the plaque,

keratinocytes are activated and secrete pro-inflammatory cytokines and chemokines, attracting immune cells to the site of inflammation. The cytokines induce differentiation of imDCs to mDCs, which migrate to lymph nodes and activate T cells. Activated T cells then migrate to the psoriatic plaque and contribute to the cytokine storm in the plaque.

35

1.3.2 Cytokine and chemokine production by keratinocytes in psoriasis

As mentioned in section 1.2.2, keratinocytes are able to produce a vast

number of cytokines and chemokines. In psoriatic lesions, an increase in

TNF-α, INF-γ, IL-6, IL-12, IL-17, and IL-18 is observed along with a decrease

in IL-10 and IL-4 (46). Three of these, i.e. TNF-α, IL-17 and IFN-γ, appear to

be the key players in the development of psoriasis (46). An increase in CCL2,

CCL3, CCL5, CCL20, CCL26, CCL27, CXCL8, CXCL10 and CX3CL1 has

also been confirmed (46,47). Below is an overview of some of the cytokines

and chemokines produced by keratinocytes that are elevated in psoriasis and

were measured in this project.

1.3.2.1 Cytokines

Primary human keratinocytes and HaCaT cells secrete IL-6 (48,49). Its

secretion by primary human keratinocytes increases significantly when the

cells are stimulated with IL-17 and TNF-α (48), whereas HaCaT cells have

been shown to secrete IL-6 upon stimulation with IFN-γ (49). IL-6, along with

IL-1β (humans) or TGFβ (mice), induces differentiation of naïve CD4+ T cells

to Th17 cells, is chemotactic for T cells and can directly stimulate T cell

migration to the epidermis (50,51). IL-6 also has various effects on

keratinocytes. It stimulates their proliferation and induces retraction of the

cytokeratin network from the cytoplasmic periphery and its reorganization

around the cell nucleus, leading to the formation of thick keratin bundles and

granules. Keratinocyte cultures treated with IL-6 also form wider intracellular

spaces and present higher levels of β-cadherin (52). IL-6 has also been

shown to directly induce epidermal hyperplasia (42).

IL-19, a member of the IL-10 cytokine family, is secreted by primary

keratinocytes upon stimulation with IL-17A and either IL-1β or TNF-α (53,54)

and HaCaT cells upregulate mRNA production for IL-19 following IL-4

stimulation (55). IL-19 is the most strongly upregulated mediator in

keratinocytes in psoriasis, with mRNA expression in psoriatic skin being

several thousand times higher than in healthy skin (53,56). The mRNA

production is followed by protein production and is reflected in high systemic

amounts of IL-19, with blood concentrations correlating with disease severity

represented by the psoriasis area and severity index value (53). IL-19 binds

to a heterodimeric transmembrane receptor, composed of IL-20R1 and IL-

20R2 and keratinocytes express both IL-19 receptor components (56). IL-19

receptor component expression is low in keratinocytes in normal skin but

higher in keratinocytes in psoriatic lesions (56,57). IL-19 does not appear to

36

induce the characteristic changes associated with psoriasis in primary human

keratinocytes unless it is combined with IL-17A, but the combination

upregulates IL-1β, CXCL8 and CCL20 expression (53). HaCaT cells treated

with IL-19 show an increase in migration activity (58).

TNF-α is one of the key inflammatory mediators in psoriasis. It can be

found in high amounts around psoriatic skin lesions and monoclonal anti-

TNF-α antibodies, such as Infliximab, successfully treat psoriasis in some

patients (59). When primary human keratinocytes are stimulated with TNF-α

they show increased motility, activate nuclear-factor kappa B (NF-κB)

signalling and increase expression of IL-6, CXCL8, CCL20, CCL27 and TNF-

α, as well as surface expression of ICAM-1 (60,61). HaCaT cells also

upregulate expression of IL-1β, IL-6, CXCL8 and ICAM-1 following

stimulation with TNF-α (62). Psoriasis patients have a higher expression of

NF-κB compared with healthy controls and it has been proposed that TNF-α,

locally produced in psoriatic lesions, drives a positive feedback loop

increasing NF-κB activation and thereby increasing expression of pro-

inflammatory cytokines, including TNF-α. The extent to which keratinocytes

contribute to the TNF-α load seen in psoriatic skin lesions remains a matter

of some debate (60).

1.3.2.2 Chemokines

Primary human keratinocytes secrete CCL3 after stimulation with poly(I:C)

(structurally similar to double-stranded RNA) through TLR3. The secretion is

regulated by IFN-α and IFN-β (63). CCL3 plays an important role in

regulating T cell mediated immunity. It binds to the CCR1 and the CCR5

receptors and it is chemotactic for T cells, monocytes, imDCs, NK cells and

neutrophils (64). No reports were found on HaCaT cell secretion of CCL3.

CCL3 levels are elevated in serum in patients with psoriatic arthritis and has

thus recently become of interest in psoriasis development (47).

CCL20 is constitutively expressed at low levels by keratinocytes but

differentiated normal human keratinocytes produce more CCL20 than

undifferentiated ones (65). CCL20 expression is upregulated in psoriasis

(66,67). TNF-α and IL-1β as well as IFN-γ and IL-17 induce CCL20

expression in human keratinocytes (66,68) and HaCaT cells secrete CCL20

following stimulation with IL-1 (69). CCL20 binds to CCR6 and promotes LC

migration to the epidermis. CCR6 is primarily expressed on T cells,

particularly Th17 cells, one of the key players in psoriasis (67). Psoriatic skin-

homing CLA+ T cells show increased chemotactic responses to CCL20

gradient compared with cells from normal donors (68).

37

CCL27 is constitutively produced by normal primary human keratinocytes

and its expression is enhanced upon stimulation with TNF-α and IL-1β in both

primary human keratinocytes and HaCaT cells (70,71). CCL27 binds to the

CCR10 receptor and is chemotactic for CLA+

T cells but not CD4+ or CD8

+

naïve T cells (70). The majority of skin-resident T cells express CLA,

suggesting that CCL27 attracts memory T cells that have previously been

activated in the skin to cutaneous lesions (72,73). Overproduction of CCL27

by keratinocytes is associated with an increased influx of CCR10+ CD4

+ T

cells to the skin in patients with psoriasis (73).

Primary human keratinocytes secrete CXCL8 upon stimulation with TNF-α

and IL-17 (74). HaCaT cells express CXCL8 in response to synthetic double

stranded RNA analogue that signals trough the TLR3 receptor. HaCaT cells

express TLR3 receptors both on the cell membrane and within the

intracellular compartments (75). HaCaT cells have also been shown to

enhance their expression of CXCL8 when stimulated with IFN-γ (49). CXCL8

plays an important role in wound repair by recruiting T cells to the wound but

may also stimulate keratinocytes to proliferate at the wound edge, as they

express CXCR2 (the CXCL8 receptor) on their surface (76).

CXCL10 is constitutively produced by keratinocytes with their production

being enhanced in psoriasis (67,77). Th1 activation induces TNF-α and IFN-γ

production, which in turn stimulates secretion of CXCL10 by the lymphocytes,

but also other cells, such as keratinocytes, neutrophils and monocytes. IL-27,

which is involved in priming of Th1 cells, can stimulate keratinocytes to

secrete CXCL10 and CXCL11, while inhibiting TNF-α-mediated IL-1α and

CCL20 secretion (77). HaCaT cells have been shown to secrete CXCL10

following stimulation by IFN-γ (69). CXCL10 binds to CXCR3, which is

primarily expressed on T cells, particularly Th1 cells which are one of the key

players in psoriasis, but also on NK cells and B lymphocytes (67,77).

1.4 The Blue lagoon

The Blue Lagoon is a geothermal spa located in Southwestern Iceland. It

consists of a mixture of sea water and fresh water (65:35) and is

supersaturated with silica (78). The Blue Lagoon has an unusual ecosystem

with very low microbial diversity and the few dominant players exhibiting

seasonal fluctuations. The explanation for this low biodiversity is most likely

found in the salinity of the fluid, its unique chemical composition with high

concentration of silica and the source of the dilute geothermal sea water (78).

38

The main organisms in the Blue Lagoon are the Cyanobacterium aponinum

and the Silicabacter lacuscaerulensis (78).

1.4.1 The Blue Lagoon treatment for psoriasis

Bathing in the Blue Lagoon in conjunction with UVB light treatment has been

shown to be a useful alternative treatment for psoriasis and to give better

results than UVB treatment alone (79). These results were confirmed in two

recent clinical studies, a pilot study and a large cohort study (80,81). The

results from the pilot study indicate that the mechanism behind the beneficial

effects of bathing in the Blue Lagoon may be linked to down-regulation of

peripheral Th17 cells (80).

Although bathing in the Blue Lagoon has increased in popularity over

the years, little is known about the biological activities present in the Lagoon.

Extracts from the silica mud and the two microorganisms found in the Blue

Lagoon induced expression of involucrin, loricrin, transglutaminase-1 and

filaggrin in primary human epidermal keratinocytes, indicating that they may

improve skin barrier function, which may explain some of the beneficial

effects of bathing in the Blue Lagoon experienced by psoriasis patients (82).

1.4.2 Cyanobacteria and polysaccharides

Cyanobacteria, also known as blue green algae, are simple microorganisms

that have characteristics in common with both bacteria and algae. They

resemble bacteria in their prokaryotic cellular organization but resemble

algae and other higher plants in being photoautotrophic (83). Cyanobacteria

are larger than other bacteria and are mostly aquatic. All blue green algae

are unicellular, though many grow in colonies or filaments, often surrounded

by a gelatinous or mucilaginous sheath depending on environmental

conditions (83).

Many microorganisms secrete organic compounds and the blue green

algae are no exception as they have been shown to secrete polysaccharides.

Microbial polysaccharides are attracting escalating interest for their possible

applications in pharmaceutical industries as well as in the food and cosmetic

industry due to the industrial success of some microbial polysaccharides (84).

In a research project leading to the present one, the research group has

shown that monocyte-derived DCs matured in the presence of

exopolysaccharide extract from C. aponinum (EPS-Ca) from the Blue Lagoon

secreted high levels of the anti-inflammatory cytokine IL-10. In addition,

maturing DCs in the presence of extract from C. aponinum increased their

39

potential to differentiate CD4+ T cells into Treg cells but decreased CD4

+ T

cell differentiation into inflammatory Th17 cells (85).

41

2 Aims

The main aim of the study was to determine the effects of EPS-Ca and of

DCs matured in the presence of EPS-Ca and co-cultured with T cells, on

activation of keratinocytes.

The specific aims were:

1. To determine the effects of EPS-Ca on cytokine and chemokine

secretion by HaCaT cells and on their expression of surface

molecules linked with activation.

2. To determine whether DCs matured in the presence of EPS-Ca and

cultured with T cells affect cytokine and chemokine secretion by

HaCaT cells.

3. To determine whether DCs matured in the presence of EPS-Ca

affect the cytokine and chemokine secretion of HaCaT cells.

43

3 Materials and methods

3.1 Culture of Cyanobacteria and preparation of extracellular polysaccharides

The cyanobacteria C. aponinum was cultured by the Blue Lagoon staff and

supernatant collected. The supernatant was lyophilized, filtered and re-

lyophilized. The remains are a mixture of polysaccharides (EPS-Ca).

3.2 HaCaT cell culture

The HaCaT cell line was cultured in Dulbecco's Modified Eagle Medium

(DMEM) with 10% foetal bovine serum (FBS) and 5% penicillin/streptomycin

(all from Gibco®, ThermoFisher Scientific), in 75 cm

2 flasks (Nunc,

Denmark)

at 37°C in 5% CO2 atmosphere and 95% humidity.

The cells where counted with CountessTM

Automated Cell Counter

(InvitrogenTM

), using disposable CountessTM

Cell Counting Chamber Slides.

The cells where stained with 0.4% Trypan Blue Stain (InvitrogenTM

) prior to

counting.

3.3 Activation of HaCaT cells

HaCaT cells where trypsinized from their culture flasks with 0.25% trypsin-

ethylenediaminetetraacetic acid (EDTA) solution (Gibco®,

ThermoFisher

Scientific) for 14 min at 37°C. The cells were counted (described in chapter

3.2) and diluted to 5x105 cells/ml in medium, of which 0.5 ml were seeded per

well in a 48-well plate (Nunc, ThermoFisher Scientific).

The cells were cultured for 24 h and then stimulated for 24 h with 40 ng/ml

TNF-α and 50 ng/ml IL-17A (both from R&D Systems), to mimic Th17 type

stimulation, or with 20 ng/ml TNF-α and 100 ng/ml IFN-γ (both from R&D

Systems) to mimic Th1 type stimulation. The cells were activated in the

presence or absence of EPS-Ca at concentration of 1, 10, 50 and 100 μg/ml.

Several different concentrations of cells, concentrations of the stimulants

as well as culture times were tested before the specific conditions described

above were chosen. Furthermore, all combinations were tested with or

without LPS being present. The results from these set-up experiments are

shown in appendix A.

44

3.4 Harvesting HaCaT cells

Following activation, the supernatant was removed from each well and spun

at 1400 rpm at 4°C for 3 min and stored at -80°C.

The HaCaT cells were washed twice with 300 µl phosphate-buffered

saline (PBS) and trypsinized with 100 µl of 0.25% Trypsin-EDTA solution in

each well for 14 min at 37°C. The cells were removed from the plate, placed

in DMEM and spun at 1400 rpm at 4°C for 3 min. The cells were then

resuspended in staining buffer (PBS containing 0.5% bovine serum albumin

(BSA; Millipore), 2 mM EDTA and 0.1% NaN3) in fluorescence-activated cell

sorting tubes (Falcon, BD Biosciences).

3.5 Flow cytometry on HaCaT cells

After harvesting, the cells were incubated with a 2% blocking solution (1:1

mixture of normal mouse serum and normal human serum) on ice for 10 min.

The cells where then stained with fluorochrome-labelled monoclonal

antibodies against CD29, CD54, CD62L, CD183 (CXCR3), E-cadherin, HLA-I

(all from BioLegend), CD44 (Bio-Rad) and HLA-DR (BD Biosciences).

After incubating with the antibodies for 20 min on ice, the cells where

washed with staining buffer and resuspended and fixed with 300 μl of 1%

paraformaldehyde in PBS.

Between 10,000 and 20,000 cells were collected on Navios Flow

Cytometer (Beckman Coulter) and the data analyzed with the Kaluza Flow

Cytometry Analyzes software (Beckman Coulter). Results are given as

percentage of positive cells and mean fluorescence intensity (MFI).

3.6 Isolation of peripheral blood mononuclear cells (PBMCs), monocytes and CD4+ T cells

PBMCs were isolated from buffy coats from the Icelandic Blood Bank

(National Bioethics Committee approval #:06-068). One part of buffy coat

was diluted with two parts of PBS and then the PBMCs were isolated with

density gradient cell separation medium (Histopaque-1077, Sigma) and

washed three times with magnetic-activated cells sorting buffer (PBS with

0.5% BSA and 2 mM EDTA) and counted.

MicroBead-labelled antibodies (Miltenyi Biotec) were then bound to the

cells on ice, CD14 MicroBeads for monocyte isolation and CD4 Microbeads

for CD4+ T cell isolation. After labelling the cells, they were placed on

LV+/VS

+ columns (Miltenyi Biotec) in a strong magnetic field. The unbound

45

cells were washed through the column. The column was removed from the

magnetic field and then the antibody-bound cells were washed through the

column. The collected cells were counted as described in chapter 3.2.

3.7 Differentiation of monocytes and maturation of DCs

MicroBead isolated CD14+ monocytes were cultured in Roswell Park

Memorial Institute (RPMI) 1640 L-glutamine medium, with 10% FBS and 5%

penicillin/streptomycin (all from Gibco®,

ThermoFisher Scientific). CD14+

monocytes were diluted to 5x105 cells/ml and 1 ml was seeded per well in a

48-well plate (Nunc) and the cells cultured for 7 days. IL-4 (12.5 ng) and

granulocyte colony-stimulating factor (GM-CSF) (25 ng) (both from R&D

Systems) were added to each well to induce differentiation of the monocytes

into imDCs. On day 3 or 4, 500 μl of medium containing 12.5 ng of IL-4 and

25 ng of GM-CSF was added to each well.

To mature and activate the imDCs into mDCs, 500 µl of 2.5 x105 cells/ml

solution in RPMI 1640 medium was seeded per well in a 48-well plate and

10 ng/ml of IL-1β, 50 ng/ml TNF-α (both from R&D Systems) and 0.5

μg/ml of LPS (Sigma) were added to each well, in the presence or

absence of 100 μg/ml EPS-Ca. The cells were cultured for 24 h.

3.8 Co-culture of DCs and HaCaT cells

HaCaT cells were trypsinized from their culture flasks and 0.5 ml of 5x105

cells/ml solution was seeded per well in a 48-well plate. The cells were

cultured for 24 h. DCs were harvested either as imDCs or mDCs, the medium

was removed from the HaCaT cells and 500 µl of 5x104 cells/ml solution of

DCs was added to each well.

When imDCs were harvested and co-cultured with HaCaT cells, the co-

culture was performed in DMEM and HaCaT stimulations used (see chapter

3.3), with the addition of LPS (0.1 µg/ml), which stimulates the DCs (86) and

not the HaCaT cells (see appendix A). The cells were co-cultured for 24 h.

When mDCs were harvested and co-cultured with HaCaT cells, the co-

culture was performed in RPMI 1640 medium without any HaCaT cell

stimulation. However, LPS (0.1 µg/ml) was added to maintain the activation

state of the mDCs. The cells were co-cultured for 24 h.

Co-culture of imDCs and HaCaT cells was set-up with the aim of

determining the effects of DCs matured in the presence of EPS-Ca on

activation of HaCaT cells but time did not allow completion of those studies.

46

3.9 Co-culture of DCs and T cells

When the CD14+ monocytes had been differentiated and matured into mDCs

(at day 8 of the DC culture, see chapter 3.7), CD4+ T cells were isolated as

described in chapter 3.6 and 100 µl of 2x106 cells/ml solution seeded in a 96-

well plate (Nunc, ThermoFisher Scientific) in RPMI 1640 medium. The mDCs

were harvested and 100 µl of 2x105 cells/ml solution was added to each well

containing T cells. The T cells and DCs were cultured together for 6 days

before harvesting, either as cells and supernatant or supernatant only.

3.10 Co-culture of DCs, T cells and HaCaT

HaCaT cells where trypsinized and removed from their culture flasks and 0.5

ml of 5x105 cells/ml solution seeded per well in a 48-well plate. The cells

were cultured for 24 h without any stimulation. The medium was removed

from the HaCaT culture and 500 µl of either supernatant from the DC and T

cell co-culture or supernatant and cells from the DC and T cell co-culture

added to the HaCaT cells. The cells were cultured for 24 h and then

harvested. For comparison, DCs and T cells were co-cultured for an

additional day without HaCaT cells being present.

3.11 ELISA

Concentrations of cytokines (IL-1β, IL-6, IL-10, IL-12p40, IL-19, IL-27 and IL-

33) and chemokines (CCL3, CCL20, CCL27, CXCL8 (IL-8) and CXCL10) in

supernatants collected from cell cultures (see chapters 3.4, 3.8 and 3.10)

were measured using DuoSet ELISA kits (R&D Systems). The DuoSet kits

were used according to the manufacture’s protocols. MaxiSorp plates (Nunc)

were coated with purified mouse anti-human cytokine or chemokine

antibodies overnight at room temperature (RT). The following day, unbound

binding sites were blocked with PBS containing 1% BSA, 5% sucrose and

0.05% NaN3 for 1 h at RT. The plates where washed 4 times with PBS

containing 0.05% Tween 20 (Sigma) and supernatants and standards added

to the wells and incubated for 2 h at RT. After incubation, the wells were

washed 4 times and biotinylated goat anti-human cytokine or chemokine

antibodies added to the wells and incubated for 2 h at RT. After the

incubation, the wells were washed 4 times and horseradish peroxidase

(HRP)-conjugated streptavidin added to the wells and incubated for 20 min at

RT in darkness. After the incubation, the wells where washed 4 times and

then TMB One substrate solution (Kementec) was added to the wells and

incubated in darkness. The reaction was stopped by addition of 0.18 M

sulphuric acid to the wells and the absorbance measured at 450 nm in a

47

microplate reader (Multiskan/EX from Thermo, Finland).

Each sample was run in duplicate. Assay diluent was used as blank.

Values (absolute) in pg/ml were calculated from seven-point standard curves

constructed by serial 2-fold dilutions of representative recombinant human

cytokines. The results are shown as secretion index (SI), which is calculated

by dividing the concentration of cytokines/chemokines in supernatants from

cells stimulated with EPS-Ca by the concentration of cytokines/chemokines in

supernatants from cells stimulated without EPS-Ca.

3.12 Statistical analysis

The data were presented as mean + standard error of the mean (SEM). Data

points determined by GraphPads outlier calculator to be outliers were

excluded from the data. Statistical significance was analysed using

SigmaStat 3.1 (Systat Software Inc) and results considered statistically

significant if p<0.05. When analyzing data from HaCaT cells, equal variance

tests and normality tests failed for most of the data sets and, therefore, one-

way ANOVA on ranks with Dunn's post-hoc test was used to do statistical

evaluations. As the co-culture data were, with only few exceptions, normally

distributed, one-way ANOVA with Bonferroni post-hoc test was used for

statistical evaluation between the control and treatment groups.

49

4 Results

4.1 Effects of EPS-Ca on cytokine and chemokine secretion by HaCaT cells

To analyse the effect of EPS-Ca on cytokine and chemokine secretion by

keratinocytes, HaCaT cells were stimulated with Th1 or Th17 stimulation for

24 h in the absence or presence of EPS-Ca at various concentrations.

Th1 stimulated HaCaT cells secreted more IL-6, IL-19, CCL3 and CXCL8

when they were activated in the presence of EPS-Ca than when they were

activated in the absence of EPS-Ca (figure 3). These effects of EPS-Ca were

dose dependent. EPS-Ca also increased secretion of CCL20, with 10 µg/ml

of EPS-Ca being the most effective concentration. EPS-Ca decreased

HaCaT cell secretion of CXCL10 at the highest concentration tested (figure

3).

Th17 stimulated HaCaT cells secreted more IL-6, IL-19 and CCL3 when

they were activated in the presence of EPS-Ca than when they were

activated in the absence of EPS-Ca (figure 4). The effects of EPS-Ca were

dose dependent. A trend towards increased secretion of CXCL8 by HaCaT

cells activated in the presence of EPS-Ca was observed, but the difference

was not statistically significant. The effect of EPS-Ca on Th17 stimulated

HaCaT cell secretion of CCL20 was similar to that for Th1 induced secretion.

However, the effect of EPS-Ca on Th17 induced CCL20 secretion was

observed as for the Th1-associated stimulation, where there seems to be a

peak in secretion with EPS-Ca at 10 µg/ml, but it was not statistically

significant. No CXCL10 secretion was detected in HaCaT cells following the

Th17-associated stimulation (data not shown).

50

Figure 3. The effects of EPS-Ca on secretion of cytokines and chemokines by Th1 stimulated HaCaT cells. HaCaT cells were cultured for 48 h and stimulated with

TNF-α at 20 ng/ml and IFN-γ at 100 ng/ml, mimicking Th1 microenvironment, for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The supernatant was collected and the concentration of cytokines and chemokines measured by ELISA. The absolute values were obtained in pg/ml and are shown for the control cells below each graph. The results are shown as mean secretion index (SI) which is the concentration of the cytokine/chemokine in supernatant from cells treated with EPS-Ca divided by its concentration in supernatant from cells not treated with EPS-Ca + SEM. *Different from control, p<0.05. N=14-17, except for IL-19, N=5-11.

51

Figure 4. The effects of EPS-Ca on secretion of cytokines and chemokines by Th17 stimulated HaCaT cells. HaCaT cells were cultured for 48 h and stimulated

with TNF-α at 40 ng/ml and IL-17 at 50 ng/ml, mimicking Th17-associated microenvironment, for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The supernatant was collected and the concentration of IL-6, IL-19, CCL3, CCL20, CXCL8 and CXCL10 was measured by ELISA. The absolute values are obtained in pg/ml and shown for the control cells. The results are shown as mean secretion index (SI) which is the concentration in supernatant from cells treated with or without EPS-Ca divided by the its concentration in supernatant from cells not treated with EPS-Ca + SEM. *Different from control p<0.05. N=14-17, except for IL-19, N=5-11.

52

To determine whether EPS-Ca by itself induced cytokine/chemokine

secretion by the HaCaT cells, the cells where cultured for 48 h without any

stimulation but in the presence of EPS-Ca for the last 24 h. Unstimulated

HaCaT cells secreted very low levels of IL-6, CCL3 and CCL20 compared

with that secreted by Th1 or Th17 stimulated cells. HaCaT cells cultured in

the presence of EPS-Ca secreted more IL-6, CCL3 and CCL20 than HaCaT

cells cultured in the absence of EPS-Ca (figure 5). As the experiment was

only performed twice evaluation of whether the differences were statistically

significant could not be performed.

Figure 5. The effects of EPS-Ca on secretion of cytokines and chemokines by unstimulated HaCaT cells. HaCaT cells were cultured for 48 h either in the absence

(C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml for the last 24 h. The supernatant was collected and the concentration of cytokines and chemokines measured by ELISA. The absolute values were obtained in pg/ml and are shown for the control cells below each graph. The results are shown as mean secretion index (SI) which is the concentration of cytokine/chemokine in supernatant from cells treated with EPS-Ca divided by its concentration in supernatant from cells not treated with EPS-Ca + SEM. N=2.

HaCaT cells stimulated with either Th1 or Th17 stimulation, with or without

EPS-Ca being present, showed little or no secretion of IL-1β, IL-10, IL-12p40,

IL-27 or IL-33 (data not shown).

53

4.2 Effects of EPS-Ca on expression of surface molecules on HaCaT cells

To analyse whether EPS-Ca affected expression of surface molecules on

HaCaT cells stimulated with Th1 or Th17 stimulation for 24 h in the absence

or presence of EPS-Ca at various concentrations. Expression of various

molecules that are expressed on normal skin was analysed by flow

cytometry.

Of all the molecues measured, EPS-Ca only affected the expression of

CD29 (integrin-β1) and ICAM-1 (CD54). The highest concentration of EPS-

Ca increased the MFI of CD29 on Th1 stimulated HaCaT cells (figure 6).

EPS-Ca increased the proportion of Th17 stimulated HaCaT cells expressing

ICAM-1 and also increased the mean expression levels of ICAM-1 on Th17

stimulated HaCaT cells (figure 7).

Figure 6. The effects of EPS-Ca on CD29 expression on Th1 stimulated HaCaT cells. HaCaT cells were cultured for 48 h and stimulated with TNF-α at 20 ng/ml and

IFN-γ at 100 ng/ml, mimicking Th1 microenvironment, for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The HaCaT cells were harvested and stained with fluorochrome-labelled monoclonal antibodies against CD29 analyzed by flow cytometry. The results are shown as mean fluorescence intensity (MFI). *Different from control p<0.05. N=11-12.

54

Figure 7. The effects of EPS-Ca on ICAM-1 expression on Th17 stimulated HaCaT cells. HaCaT cells were cultured for 48 h and stimulated with TNF-α at 40

ng/ml and IL-17 at 50 ng/ml, mimicking Th17 microenvironment, for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The HaCaT cells were harvested and stained with fluorochrome-labelled monoclonal antibodies against ICAM-1 and analyzed by flow cytometry. The results are shown as percentage of positive cells and as mean fluorescence intensity (MFI). *Different from control p<0.05. N=9-11.

EPS-Ca did not affect the expression of E-cadherin, HLA-I, HLA-DR,

CD44 or CD183 on the surface of HaCaT cells stimulated with either Th1 or

Th17 stimulation (appendix B). No expression of CD62L, TLR2, TLR4 or

TLR6 was detected on the surface of HaCaT cells (data not shown).

4.3 Effects of DCs matured and activated in the presence of EPS-Ca and co-cultured with T cells on chemokine secretion by HaCaT cells

Next the effects of DCs matured and stimulated in the absence or presence

of EPS-Ca and then co-cultured with T cells on cytokine secretion by HaCaT

cells were examined. DCs matured in the presence or absence of EPS-Ca

were co-cultured with allogeneic CD4+ T cells for 6 days before being added

to unstimulated HaCaT cells for 24 h and then either the co-cultured DCs and

T cells were added along with the culture supernatant or the supernatant was

added without the cells. DC and T cell co-culture control where cultured for 7

days.

When the co-cultured DCs and T cells were cultured without HaCaT cells

for 24 h, co-cultures in which the DCs had been matured and stimulated in

the presence of EPS-Ca had higher levels of both CCL20 and CXCL8 in the

supernatant (figure 8). No CXCL10 secretion was detected (data not shown).

When the DC and T cell co-culture supernatant was added to the HaCaT

cells the same effect of the EPS-CA was observed for the CCL20 and CXCL8

55

secretion, i.e. higher levels of these cytokines in supernatants from co-

cultures in which the DCs had been matured and stimulated in the presence

of EPS-Ca (figure 9). The DC and T cell co-culture supernatant induced

CXCL10 secretion and the levels were unaffected by whether the DCs had

been matured and stimulated in the presence of EPS-Ca (figure 10).

Finally, when both cells and supernatant from the DC and T cell co-

culture were added to the HaCaT cells all three chemokines were detected.

Cells and supernatant from the DC and T cell co-culture in which the DCs

were matured and stimulated in the presence of EPS-Ca increased the

secretion of CCL20 but did not have an effect on CXCL8 or CXCL10

secretion (figure 10).

Figure 8. The effects of EPS-Ca on chemokine concentration in the supernatant of DC and T cell co-cultures. DCs were matured and stimulated in the absence (C)

or presence of 100 µg/ml EPS-Ca for 24 h, and then co-cultured with allogeneic CD4+

T cells for 7 days. The supernatant was collected and the concentration of chemokines measured by ELISA. The absolute values were obtained in pg/ml and shown for each chemokine for the control cells. The results are shown as mean secretion index (SI) which is the concentration of chemokines in supernatant from co-cultures of DCs treated with EPS-Ca and T cells divided by its concentration in supernatant from co-cultures of DCs not treated with EPS-Ca and T cells. *Different from control. p<0.05. N=4.

56

Figure 9. The effects of supernatants from DCs matured and stimulated in the absence or presence of EPS-Ca and then co-cultured with T cells, on cytokine secretion by HaCaT cells. DCs where matured and stimulated in the absence (C) or

presence of 100 µg/ml EPS-Ca for 24 h, and then co-cultured for 6 days with allogeneic CD4

+ T cells. HaCaT cells were cultured for 24 h before supernatant from

the DC and T cell co-culture was added. The supernatant was collected and the concentration of chemokines was measured by ELISA. The absolute values were obtained in pg/ml and shown for each chemokine in the control cells. The results are shown as secretion index (SI) which is the concentration of chemokine in supernatant from co-cultures of DCs treated with EPS-Ca and T cells and HaCaT cells divided by its concentration in supernatant from co-cultures of DCs not treated with EPS-Ca and T cells and EPS-Ca. *Different from control. p<0.05. N=11-12.

57

Figure 10. The effects of supernatants from DCs matured and stimulated in the absence or presence of EPS-Ca and then co-cultured with T cells added along with the cells, on cytokine secretion by HaCaT cells. DCs were matured and

stimulated in the absence (C) or presence of 100 µg/ml EPS-Ca for 24 h, and then co-cultured for 6 days with allogeneic CD4

+ T cells. HaCaT cells were cultured for 24 h

before the cells and supernatants from the DC and T cell co-culture was added. The supernatant was collected and the concentration of chemokines was measured by ELISA. The absolute values were obtained in pg/ml and shown for each chemokine in the control cells. The results are shown as secretion index (SI) which is the concentration of chemokine in supernatant from co-cultures of DCs treated with EPS-Ca and T cells and HaCaT cells divided by its concentration in supernatant from co-cultures of DCs not treated with EPS-Ca and T cells and HaCaT cells. *Different from control. p<0.05. N=11-12.

58

4.4 Effects of DCs on cytokine and chemokine secretion by HaCaT cells

To determine the effects of DCs on Th1 or Th17 stimulated HaCaT cells,

imDCs were added to HaCaT cells and the co-cultured cells stimulated with

Th1 or Th17 stimulation and LPS.

HaCaT cells were cultured for 24 h and then stimulated with Th1-

associated stimulation for another 24 h. At the time of stimulation, imDCs and

LPS were added to the HaCaT cells (figure 11). Both TNF-α and LPS are

known to stimulate imDCs (87,88).The co-culture secreted IL-6, CCL3,

CCL20, CXCL8 and CXCL10. Co-cultures of HaCaT cells and imDCs in Th1-

associated stimulation secreted more IL-6 and CCL20 than either cell type

alone and more than that secreted by the two cell types combined (figure 11).

Co-cultures of HaCaT cells and imDCs in secreted more CCL3 and CXCL8

than either cell type alone but not more than that secreted by the two cell

types combined (figure 11). The LPS had previously been observed not to

stimulate the HaCaT cells (Appendix A). Th1 stimulated HaCat cells secreted

IL-6, CCL3, CCL20, CXCL8 and CXCL10. ImDCs stimulated with the Th1

stimulation and LPS secreted IL-6, CCL3 and CXCL8, but negligible levels of

CCL20 and CXCL10.

59

Figure 11. Secretion of cytokines and chemokines by Th1 stimulated HaCaT cells and imDCs. HaCaT cells were cultured for 24 h, then imDCs were added and

the co-cultured cells stimulated for 24 h with TNF-α at 20 ng/ml, IFN-γ at 100 ng/ml and LPS at 0.1 µg/ml, mimicking Th1 microenvironment for the HaCaT cells and inducing maturation for the imDCs. HaCaT cells cultured alone and stimulated with TNF-α at 20 ng/ml, IFN-γ at 100 ng/ml and LPS at 0.1 µg/ml are shown in comparison as are DCs cultured alone with the same stimulation. The supernatant was collected and the concentration of cytokines and chemokines measured by ELISA. The results are shown as absolute values in pg/ml. N=3 for imDCs and HaCaT cells, N=9 for co-cultures of HaCaT cells + imDCs.

60

Co-culturing HaCaT cells and imDCs and stimulating them with Th17

stimulation and LPS led to more IL-6 secretion than secreted by either cell

type alone and more than that secreted by the two cell types combined

(figure 12). Levels of CCL3, CCL20 and CXCL8 in the co-cultures did not

exceed the levels of these cytokines in cultures of either HaCaT cells or

imDCs stimulated alone (figure 12).

CXCL10 was not detected in cultures of cells stimulated with Th17

stimulation (data not shown).

Figure 12. Secretion of cytokines and chemokines by Th17 stimulated HaCaT cells and imDCs. HaCaT cells were cultured for 24 h, then imDCs were added and

the co-cultured cells stimulated with TNF-α at 40 ng/ml, IL-17 at 50 ng/ml and LPS at 0.1 µg/ml, mimicking Th17 microenvironment for the HaCaT cells and inducing maturation for the imDCs, for 24 h. The supernatant was collected and the concentration of cytokines and chemokines measured by ELISA. The results are shown as absolute values in pg/ml. N=3 for imDCs and HaCaT cells, N=9 for co-cultures of HaCaT cells + imDCs.

In order to analyse the effect of co-culturing unstimulated HaCaT cells

with mDCs, HaCaT cells were cultured for 24 h before mDCs were added

and the cells co-cultured for another 24 h in the presence of LPS. LPS had

previously been observed not to stimulate the HaCaT cells (Appendix A). Co-

culturing HaCaT cells and mDCs led to higher levels of CCL20, CXCL8 and

61

CXCL10 than for the cells were cultured individually and higher levels than

what would be expected if the levels in the two cultures were added together

(figure 13).

Figure 13. Secretion of cytokines and chemokines by co-cultures of unstimulated HaCaT cells and mDCs. HaCaT cells were cultured for 24 h, then

mDCs and LPS at 0.1 µg/ml were added and cultured for 24 h. The supernatant was collected and the concentration of cytokines and chemokines was measured by ELISA. The results are shown as absolute values in pg/ml. N=3 for mDCs and HaCaT cells, N=9 for co-cultures of HaCaT cells + mDCs.

62

5 Discussion

The main findings of this project are the overall increase in cytokine and

chemokine secretion of the HaCaT cells when cultured or stimulated in the

presence of EPS-Ca, indicating a pro-inflammatory effect of EPS-Ca on the

HaCaT cells. The pro-inflammatory effect of EPS-Ca on the HaCaT cells was

unexpected as previous studies on the immunomodulatory effects of EPS-Ca

had shown it to increase secretion of the anti-inflammatory cytokine IL-10 by

DCs without affecting secretion of the pro-inflammatory cytokine IL-12p40 as

well as increasing their potential to induce Treg cells but decreasing their

potential to induce a Th17 type immune response (85).

One of the first things that came to mind, when searching for an

explanation for the different effects of EPS-Ca on DCs on one hand and

keratinocytes on the other, was whether the stimulation of the HaCaT cells

was so extensive or so powerful in pushing the HaCaT cells to a pro-

inflammatory state that the EPS-Ca could not reverse it. It should be noted,

however, that the Th1 and Th17 stimulations used in the present study are

comparable with that used by others in previously published papers on

HaCaT cells (89–91). However, when testing whether EPS-Ca had different

effects on HaCaT cells when they were either less stimulated (omitting TNF-α

from the stimulation) or not stimulated with anything but EPS-Ca, EPS-Ca still

had a pro-inflammatory effect on cytokine and chemokine secretion by the

HaCaT cells. It was, therefore, concluded that the pro-inflammatory effects of

EPS-Ca were not due to extensive stimulation of the HaCaT cells and that

EPS-Ca itself can act as an independent stimulant of HaCaT cells inducing a

pro-inflammatory effect.

As mentioned above, the pro-inflammatory effect of EPS-Ca on the

HaCaT cells was unexpected as previous studies on the immunomodulatory

effects of EPS-Ca had shown it to have anti-inflammatory effects on DCs

(85). DCs and keratinocytes are very different cell types and play different

roles in the body. Keratinocytes are epithelial cells that develop from

epidermal stem cells and their primary role is to form a barrier that isolates

and protects the body from its environment (92). Although keratinocytes have

immunological functions and participate in immune responses, they are not

specialized cells of the immune system (1,92). DCs are, on the other hand,

phagocytic cells that originate mostly from monocytes and DC progenitors in

the bone marrow. The main role of DCs is to activate the adaptive immune

system and to direct lymphocyte responses to a suitable pathway, for

example by promoting Th1, Th17 or Treg responses (1,93). Keratinocytes

63

and DCs express many of the same surface molecules, such as TLRs, MHC

class I and II (1) and co-stimulatory molecules necessary to stimulate T cells,

but they do express them at various levels and in different environmental

circumstances (1,94–96). DCs and keratinocytes produce different types of

cytokines and chemokines to the same stimuli, DCs for example produced IL-

12p40 following stimulation with LPS (97), whereas HaCaT cells did not

(Appendix A). Different cell types also have different intracellular signalling

pathways. This may explain why the cells react differently to EPS-Ca.

In the search for an explanation of the different effects of EPS-Ca on DCs

(85) and HaCaT cells (this study), it was explored whether DCs matured in

the presence of EPS-Ca and then co-cultured with HaCaT cells could

mediate anti-inflammatory effects on the HaCaT cells. To simulate inflamed

skin where, in addition to DCs, T cells are also present, T cells were included

in the co-culture. Although EPS-Ca had previously been shown to have an

anti-inflammatory effect on DCs (85), DCs matured and stimulated in the

presence of EPS-Ca and co-cultured with T cells had pro-inflammatory

effects on HaCaT cells increasing their secretion of the chemokines CCL20

and CXCL8. Thus, the indirect route, i.e. via DCs, appeared to have the

same pro-inflammatory effect on the HaCaT cells as EPS-Ca had directly on

the HaCaT cells. The CCL20 present in the culture medium was most likely

secreted by the HaCaT cells (and the increased secretion induced by the co-

culture of DCs matured and activated in the presence of EPS-Ca and T

cells), since very little CCL20 was detected in the supernatant from the DC

and T cell co-culture. However, as all three cell types are capable of

secreting CXCL8, it cannot be confirmed from the ELISA measurements in

the present study which cell(s) contributed to the increase in CXCL8

(49,98,99). Intracellular staining and flow cytometry could be used to

determine which cell is most likely responsible for the increase in CXCL8

secretion. Results from these co-culture experiments, furthermore, revealed

that soluble molecules in the supernatant of the DCs treated with EPS-Ca

and co-cultured with T cells were responsible for the increased levels of

CCL20 as adding supernatant only from the DC and T cell co-culture or

supernatant and cells had the same effect on CCL20 secretion by the HaCaT

cells.

As the focus in the present study was primarily on investigating the

effects of EPS-Ca on keratinocytes, the cytokines and chemokines measured

are the ones known to be secreted by keratinocytes. Although some

cytokines and chemokines are secreted by both keratinocytes and DCs,

some are not, and in this study different cytokines and chemokines were

64

measured than in the study leading to this one (85). This may partly explain

the difference in the results obtained in the previous study and the present

study. In the study by Gudmundsdottir et al, IL-10 was the only cytokine

measured in the DC and T cell co-culture (85), so it is possible that pro-

inflammatory cytokines were secreted at an increased level as well as the

anti-inflammatory cytokine IL-10. As the previous study on the effects of EPS-

Ca on maturation and activation of DCs had shown no effect on IL-12p40

secretion, the only pro-inflammatory cytokine measured (85), we wanted to

explore the effects of DCs matured in the presence of EPS-Ca on HaCaT

cells, without the presence of T cells.

Unfortunately, there was not enough time to perform DC and HaCaT cell

co-culture experiments with EPS-Ca. However, results from co-cultures of

both imDCs and mDCs with HaCaT cells revealed that they increased

secretion of IL-6 and CCL20 (imDCs) or CCL20, CXCL8 and CXCL10

(mDCs), compared with that when the cells were cultured alone. The

increased IL-6 and CXCL8 secretion could be due to increased secretion by

the DCs, HaCaT cells or both (49,100), but the increase in CCL20 or

CXCL10 secretion is most likely due to increased secretion by the HaCaT

cells, since the DCs did not secrete any CCL20 or CXCL10 when cultured

alone. As CCL20 is a chemokine that attracts T cells to the skin (68), it would

have been interesting to determine the effects of having EPS-Ca present

during the imDC (or mDC) and HaCaT cell co-culture.

The HaCaT cells secreted many different cytokines and chemokines when

stimulated with either Th1 or Th17 stimulations and previous studies have

shown their secretion of almost all of them (48,49,69). However, to our

knowledge HaCaT cells have not previously been reported to secrete CCL3,

although CCL3 secretion has been reported for primary human keratinocytes

(63). On the other hand, in the present study there was very little or no

secretion of IL-1β, but HaCaT cells and primary keratinocytes are known to

secrete IL-1β (48,101). In a previous study, Cho et al. using a stimulation

identical to one of the stimulations used for setting up the culture conditions

in the present study showed IL-1β being secreted by the HaCaT cells (101).

Although Cho et al. used the same medium and percentage of FBS as used

in the present study, they may have grown the cells in different culture plates

or at different concentration than in the present study but this was not

specified in their publication (101).

In the present study, the HaCaT cells were grown in a monolayer, which

could be representative of the stratum basale of the epidermis. This omits the

65

other three layers of the epidermis. It is important to keep in mind that the

stratum basale is the innermost layer of the epidermis and of the four layers

of the epidermis this layer has the least contact to the outer world (15). In

psoriasis, the incomplete keratinocyte differentiation results in an incomplete

barrier function, so in many ways, the psoriatic plaque resembles an open

wound, where outside molecules are able to penetrate the skin barrier and

come into contact with other skin cells, for example DCs, which are found in

abundance in the psoriatic plaque (102). In creating the model used in this

study, the goal was to simulate the environment in a psoriatic plaque and,

therefore, both DCs and T cells, that are key players in psoriasis, were added

to cultures of the HaCaT cells. However, other cells, not included in the

present study, could also play an important role in psoriasis. In addition, there

are many cytokines and chemokines involved in psoriasis, and although the

cytokines used to stimulate the HaCaT cells in the present study are key

players in psoriasis, they do not include all cytokines involved in the psoriatic

cytokine storm. In order to overcome some of these limitations, the HaCaT

cells could be cultured three dimensionally in the presence of both T cells

and DCs.

The only previous study investigating the effects of EPS-Ca investigated

its immunomodulatory effects on DCs (85). The effects of algae extracts

containing C. aponinum from the Blue Lagoon on keratinocytes have been

studied and showed that topical application of the extract on healthy skin for

4 weeks reduced IL-1 and IL-6 secretion induced by UVB irradiation in

irradiated areas compared with untreated skin areas (82). However, the

extract was made from the C. aponinum algae mass and since EPS-Ca is an

exopolysaccharide secreted by C. aponinum it is unlikely that the extract of

the algae mass contained any EPS-Ca.

Although HaCaT cells are able to differentiate to form a well-structured

epidermis in vivo, it is important to remember that this is a cell line and,

therefore, there is a difference between HaCaT cells and primary

keratinocytes. Very little can be found in the literature about differences in

cytokine and chemokine secretions between HaCaT cells and primary

keratinocytes. This could be because there is very little or no difference or

because it has yet to be studied. There are, however, some reports of HaCaT

cells responding to stimulations from various molecules in a different manner

than primary human keratinocytes. Liu et al. showed that HaCaT cells are

less resistant to hydrogen peroxide-induced oxidative stress than normal

human epidermal keratinocytes and that HaCaT cells have a more

senescence phenotype (103). Seo et al. showed that HaCaT cells have a

66

different gene transcriptional profile in epidermal differentiation genes (such

as filaggrin, loricrin and involucrin) than primary keratinocytes following

stimulation with IFN-γ, IL-4, IL-17A or IL-22 (104). HaCaT cells, however, had

the same gene transcriptional profile of human β2-defensin in response to

IFN-γ, IL-4 or IL-17A (104). It is, therefore, unlikely that this model represents

fully the response of human keratinocytes in vivo. The HaCaT cell line has,

however, been widely used in scientific research through the years,

especially in psoriasis research (105–107). The use of primary keratinocytes

instead of HaCaT cells would overcome some of the limitations of the model

used in the present study. Primary keratinocytes are, however, difficult to

obtain upon request and they do not live long in cell cultures. They can,

however, be bought, although import to Iceland is problematic. Since a large

number of keratinocytes had to be used for the creation of this model, it was

concluded that it would be best to use the HaCaT cell line while setting up the

model and once the model had been established and tested, the main

experiments would be repeated using primary keratinocytes. Therefore, the

next step will be to study the effects of EPS-Ca on primary human

keratinocytes using the knowledge gained in this study for determining the

culture and stimulation conditions.

67

6 Conclusions

Although previous reports on the effects of EPS-Ca on DCs and T cells

indicated an anti-inflammatory effect, the effect of EPS-Ca on HaCaT

keratinocytes seems to be pro-inflammatory. Supernatant from co-cultures of

DCs matured in the presence of EPS-Ca and T cells also had a pro-

inflammatory effect on the HaCaT cells as did EPS-Ca by itself. Thus, it is

concluded that EPS-Ca has pro-inflammatory effects on keratinocytes and

that its effects on keratinocytes does not explain the beneficial effects of

bathing in the Blue Lagoon.

69

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7 Appendix A

7.1 The effects of different concentrations of HaCaT cells and different times of stimulation on their cytokine and chemokine secretion

To determine the concentration of HaCaT cells to be used in further

experiments, 3 different concentrations of HaCaT cells were seeded in a 48-

well plate prior to stimulation. To determine whether the HaCaT cells should

be allowed to adjust/reach confluency in the wells prior to the stimulation

being added, the stimulants were added at 0, 24 or 48 h after the cells were

added to the plate. Higher concentrations of HaCaT cells seeded in each well

resulted in higher concentrations of IL-6, CCL20, CXCL8 and CXCL10 (figure

A1). Allowing the HaCaT cells to adjust in the wells for either 24 or 48 h prior

to stimulation appeared to increase concentrations of CCL20 and CXCL8 at

all cell concentrations and CXCL10 at lower cell concentrations (figure A1).

No IL-1β, IL-10, IL-27 or IL-33 was detected in any of the cell cultures (data

not shown). It was concluded that optimal stimulation was reached when cells

were seeded at 5x105 cells/ml and the stimulation added 24 h after seeding.

To confirm that seeding of 5x105 cells/ml and culturing for 24 h before

stimulation was a suitable condition, the cells were monitored and they were

observed to be confluent but not overpopulated (figure A2).

80

Figure A1. The effects of different cell concentrations and stimulation times on cytokine and chemokine secretion by HaCaT cells. HaCaT cells were seeded at 3

different concentrations, 1x105 cells/ml, 2.5x10

5 cells/ml and 5x10

5 cells/ml in 48-well

tissue culture plates. The cells were stimulated with a mixture of 100 ng/ml IL-17, 100 ng/ml IFN-γ and 40 ng/ml TNF-α, either as soon as cells were seeded (0 h), or 24 h or 48 h later. The cells were stimulated for 24 h befere the supernatant was collected and the concentration of cytokines and chemokines measured by ELISA. The absolute values were obtained in pg/ml. N=1.

Figure A2. Photograph of HaCaT cells in culture. Five x105 cells/ml were cultured

for 23 h and then viewd and photographed in a light microscope at 10x magnification.

81

7.2 The effects of different cytokine stimulations on cytokine and chemokine secretion by HaCaT cells

To determine what cytokines to use for HaCaT stimulation, the cells were

initially stimulated with a single cytokine using a concentration used by others

in previous publications (figure A3). IL-1β was tested at two different

concentrations, whereas IL-17, IL-22, IFN-γ and TNF-α were tested at one

concentration. All the cytokine stimulations were performed in the presence

or absence of 100 ng/ml LPS. No IL-1β was detected when the HaCaT cells

were stimulated with IL-17, IL-22, IFN-γ or TNF-α in the presence or absence

of LPS (data not shown). IL-22 alone induced little or no cytokine secretion

(figure A3). For IL-6 secretion, stimulation with TNF-α and IFN-γ led to higher

secretion of IL-6 than that observed using other cytokines as stimulants. For

CCL20, stimulation with TNF-α or IL-1β at 10 ng/ml led to higher secretion

than that observed using the other cytokines. For CXCL8, stimulation with

TNF-α induced high secretion, although stimulation with IL-1β and IL-17 also

triggered substantial secretion. Stimulation with IL-1β, IFN-γ and IL-17 with

LPS were the main inducers of CXCL10 secretion. LPS did not have a major

effect on cytokine secretion by the HaCaT cells, with few exceptions (figure

A3).

82

Figure A3. The effects of different cytokine stimulations on cytokine and chemokine secretion by HaCaT cells. HaCaT cells were cultured for 48 h and

stimulated for the last 24 h with a single cytokine; IL-1β at 1 and 10 ng/ml, IL-17, IL-22 and IFN-γ at 100 ng/ml or TNF-α at 40 ng/ml, in the absence or presence of 100 ng/ml of LPS. The supernatant was collected and the concentration of cytokines and chemokines measured by ELISA. N=1

83

Next, different combinations of two cytokines were tested for their ability to

induce cytokine and chemokine secretion by the HaCaT cells using IL-1β, IL-

17, IL-22, IFN-γ and TNF-α, in the presence or absence of 100 ng/ml LPS.

For IL-6, CCL20 and CXCL8 secretion, stimulation with a mixture of IL-17

and TNF-α or a mixture of IFN-γ and TNF-α induced the most secretion

(figure A4). For CXCL10 secretion, stimulation with a mixture of IFN-γ and IL-

17 or a mixture of IFN-γ and TNF-α induced the most secretion. The addition

of LPS during the cytokine stimulation did not increase the cytokine and

chemokine secretion by the HaCaT cells. As when the HaCaT cells were

stimulated with only one cytokine at a time, no IL-1β was detected when the

cells were stimulated with two cytokines (data not shown).

Figure A4. The effects of different combinations of two cytokines on the cytokine and chemokine secretion by HaCaT cells. HaCaT cells were cultured for

48 h and stimulated for the last 24 h with a combination of two different cytokines; IL-1β at 10 ng/ml, IL-17, IL-22 and IFN-γ at 100 ng/ml or TNF-α at 40 ng/ml, in the absence or presence of 100 ng/ml of LPS. The supernatant was collected and the concentration of cytokines and chemokines measured by ELISA. N=1.

84

As Th17 cells are expressed in psoriatic lesions and they secrete both IL-

17 and IL-22, stimulation of the HaCaT cells with a combination of these two

cytokines and either IFN-γ or TNF-α, in the presence or absence of 100

ng/ml LPS, was tested (figure A5). The mixture of IL-17, IL-22 and TNF-α

stimulation without LPS was more effective in inducing IL-6, CCL20 and

CXCL8 secretion, but the IL-17, IL-22 and IFN-γ stimulation was more

effective in inducing CXCL10 secretion. Addition of IL-22 to the other

cytokines for stimulation of the HaCaT cells did not increase their cytokine

secretion compared to that secreted without using IL-22 and hence IL-22 was

not used in subsequent experiments.

Figure A5. The effects of combinations of three different cytokines on the cytokine and chemokine secretion by HaCaT cells. HaCaT cells were cultured for

48 h and stimulated for the last 24 h with a combination of IL-17 and IL-22 at 100 ng/ml and either TNF-α at 40 ng/ml of IFN-γ at 100 ng/ml, in the absence or presence of 100 ng/ml of LPS. The supernatant was collected and the concentration of cytokines and chemokines measured by ELISA. N=1.

85

7.3 The effects of different concentrations of IL-17 and TNF-α on one hand and IFN-γ and TNF-α on the other hand for stimulation of cytokine and chemokine secretion by HaCaT cells

Based on the results from the previous experiments, it was decided to

stimulate the HaCaT cells with two different stimulations, with IFN-γ and TNF-

α, mimicking Th1 stimulation and with IL-17 and TNF-α mimicking Th17

stimulation. As LPS had little effect on cytokine and chemokines secretion by

the HaCaT cells, it was not included in the stimulation panel.

To determine what cytokine concentration was best suited to stimulate the

HaCaT cells, the cytokines were tested together at 3 different concentrations,

with TNF-α at 20, 40 or 80 ng/ml and with IL-17 or IFN-γ at 50, 100 or 200

ng/ml using IL-6, CCL20, CXCL8 and CXCL10 secretion by the HaCaT cells

for readout. Stimulating the HaCaT cells with either TNF-α at 40 ng/ml and

IFN-γ at 50 ng/ml or TNF-α at 20 ng/ml and IL-17 at 100 ng/ml led to good

but not maximum secretion of the cytokines and chemokines chosen and

were, therefore, used for further investigation of the effects of EPS-Ca on

activation of HaCaT cells.

When the final stimulation conditions had been determined, secretion of

other cytokines, such as IL-1β, IL-10, IL-12p40, IL-27 and IL-33 was tested.

Of these, only IL-10 and IL-33 were detectable in the supernatant, and only at

a concentration similar to the lowest point of the standard curves and hence

none of these were measured in further experiments performed (data not

shown).

86

Figure A6. The effects of different concentrations of TNF-α, IL-17 and IFN-γ on cytokine and chemokine secretion by HaCaT cells. HaCaT cells were cultured for

48 h and stimulated with different combinations of two of these cytokines in different concentrations for the last 24 h. The supernatant was collected and the concentration of cytokines and chemokines measured by ELISA. N=1.

87

8 Appendix B

8.1 The effects of EPS-Ca on cytokine and chemokine secretion by HaCaT cells following Th1 and Th17 stimulation not including TNF-α

The effects of EPS-Ca on cytokine and chemokine secretion by HaCaT cells

stimulated with Th1 or Th17 stimulation without TNF-α, was examined to

determine whether EPS-Ca had different effects on less stimulated HaCaT

cells than that described in chapter 3.

Secretion of IL-6, CCL3 and CCL20 by HaCaT cells stimulated with Th1

stimulation without TNF-α was 3-10% of what it was when the HaCaT cells

were stimulated with Th1 stimulation with TNF-α. EPS-Ca was much more

powerful in increasing secretion of IL-6 and CCL20 when the cells were

stimulated without TNF-α than when they were stimulated with TNF-α, but the

final concentration of these cytokines in cultures from HaCaT cells stimulated

without TNF-α were still less than that when they were stimulated with TNF-α

(2628 pg/ml versus 4201 pg/ml for IL-6 and 506 pg/ml versus 1467 pg/ml for

CCL20 without and with TNF included in the stimulation) (figure B1). EPS-Ca

increased CCL3 secretion by HaCaT cells stimulated without TNF-α but less

so than when they were stimulated with TNF-α (figure B1).

88

Figure B1. The effects of EPS-Ca on secretion of cytokines and chemokines by Th1 stimulated HaCaT cells (without TNF-α). HaCaT cells were cultured for 48 h

and stimulated with IFN-γ at 100 ng/ml for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The supernatant was collected and the concentration of cytokines and chemokines measured by ELISA. The absolute values were obtained in pg/ml and are shown for the control cells below each graph. The results are shown as mean secretion index (SI) which is the concentration of the cytokine/chemokine in supernatant from cells treated with EPS-Ca divided by its concentration in supernatant from cells not treated with EPS-Ca. N=2.

89

Secretion of CCL20 by HaCaT cells stimulated with Th17 stimulation

without TNF-α was 1% of what it was when the HaCaT cells were stimulated

with Th17 stimulation with TNF-α, but for IL-6 and CCL3 it was 36 and 57%

(figure B2). EPS-Ca was equally as effective in increasing IL-6 secretion

when the HaCaT cells were stimulated without TNF-α as it was when they

were stimulated with TNF-α, less effective in increasing CCL3 secretion and

much more effective in increasing HaCaT cell secretion of CCL20, increasing

its concentration by over 30 fold with 50 g/ml of EPS-Ca (figure B2).

Figure B2. The effects of EPS-Ca on secretion of cytokines and chemokines by Th17 stimulated HaCaT cells (without TNF-α). HaCaT cells were cultured for 48 h

and stimulated with IL-17 at 50 ng/ml for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The supernatant was collected and the concentration of cytokines and chemokines measured by ELISA. The absolute values were obtained in pg/ml and are shown for the control cells below each graph. The results are shown as mean secretion index (SI) which is the concentration of cytokine/chemokine in supernatant from cells treated with EPS-Ca divided by its concentration in supernatant from cells not treated with EPS-Ca. N=2

90

8.2 The effects of EPS-Ca on expression of surface molecules on HaCaT cells

EPS-Ca did not affect HaCaT cell expression of most of the surface

molecules examined. The ones that were affected are shown in chapter 3,

the others are shown below (figures B3-B9).

Figure B3. The effect of EPS-Ca on CD29 expression on Th1 or Th17 stimulated HaCaT cells. HaCaT cells were cultured for 48 h and stimulated with TNF-α at 20

ng/ml and IFN-γ at 100 ng/ml, mimicking Th1 microenvironment (left), or TNF-α at 40 ng/ml and IL-17 at 50 ng/ml mimicking Th17 microenvironment (right), for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The HaCaT cells were harvested and stained with fluorochrome-labelled monoclonal antibodies against CD29 and analyzed by flow cytometey. The results are shown as percentage of positive cells (top row) or as MFI (bottom row). N=11-12.

91

Figure B4. The effects of EPS-Ca on CD44 expression on Th1 or Th17 stimulated HaCaT cells. HaCaT cells were cultured for 48 h and stimulated with

TNF-α at 20 ng/ml and IFN-γ at 100 ng/ml, mimicking Th1 microenvironment (left), or TNF-α at 40 ng/ml and IL-17 at 50 ng/ml mimicking Th17 microenvironment (right), for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The HaCaT cells were harvested and stained with fluorochrome-labelled monoclonal antibodies against CD44 and analyzed by flow cytometey. The results are shown as percentage of positive cells (top row) or as MFI (bottom row). N=5-6.

Figure B5. The effects of EPS-Ca on ICAM-1 expression on Th1 stimulated HaCaT cells. HaCaT cells were cultured for 48 h and stimulated with TNF-α at 20

ng/ml and IFN-γ at 100 ng/ml, mimicking Th1 microenvironment for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The HaCaT cells were harvested and stained with fluorochrome-labelled monoclonal antibodies against ICAM-1 and analyzed by flow cytometey. The results are shown as percentage of positive cells (right) or as MFI (left). N=9-11.

92

Figure B6. The effects of EPS-Ca on CD183 expression on Th1 or Th17 stimulated HaCaT cells. HaCaT cells were cultured for 48 h and stimulated with

TNF-α at 20 ng/ml and IFN-γ at 100 ng/ml, mimicking Th1 microenvironment (left), or TNF-α at 40 ng/ml and IL-17 at 50 ng/ml mimicking Th17 microenvironment (right), for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The HaCaT cells were harvested and stained with fluorochrome-labelled monoclonal antibodies against CD183 and analyzed by flow cytometey. The results are shown as percentage of positive cells (top row) or as MFI (bottom row). N=8-9.

93

Figure B7. The effects of EPS-Ca on E-cadherin expression on Th1 or Th17 stimulated HaCaT cells. HaCaT cells were cultured for 48 h and stimulated with

TNF-α at 20 ng/ml and IFN-γ at 100 ng/ml, mimicking Th1 microenvironment (left), or TNF-α at 40 ng/ml and IL-17 at 50 ng/ml mimicking Th17 microenvironment (right), for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The HaCaT cells were harvested and stained with fluorochrome-labelled monoclonal antibodies against E-cadherin and analyzed by flow cytometey. The results are shown as percentage of positive cells (top row) or as MFI (bottom row). N=11-12.

94

Figure B8. The effects of EPS-Ca on HLA-DR expression on Th1 or Th17 stimulated HaCaT cells. HaCaT cells were cultured for 48 h and stimulated with

TNF-α at 20 ng/ml and IFN-γ at 100 ng/ml, mimicking Th1 microenvironment (left), or TNF-α at 40 ng/ml and IL-17 at 50 ng/ml mimicking Th17 microenvironment (right), for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The HaCaT cells were harvested and stained with fluorochrome-labelled monoclonal antibodies against HLA-DR and analyzed by flow cytometey. The results are shown as percentage of positive cells (top row) or as MFI (bottom row). N=8-9.

95

Figure B9. The effects of EPS-Ca on HLA-I expression on Th1 or Th17 stimulated HaCaT cells. HaCaT cells were cultured for 48 h and stimulated with

TNF-α at 20 ng/ml and IFN-γ at 100 ng/ml, mimicking Th1 microenvironment (left), or TNF-α at 40 ng/ml and IL-17 at 50 ng/ml mimicking Th17 microenvironment (right), for the last 24 h, either in the absence (C) or presence of EPS-Ca at 1, 10, 50 or 100 µg/ml. The HaCaT cells were harvested and stained with fluorochrome-labelled monoclonal antibodies against HLA-I and analyzed by flow cytometey. The results are shown as percentage of positive cells (top row) or as MFI (bottom row). N=8-9.

the effects of polysaccharides secreted by c. aponinum

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