Grup de fisiologia digestiva i adaptacions nutricionals

Institut de Nutrició i Seguretat Alimentaria

1

Outline

Weaning and the consequences of stress

Animal blood plasma SDP Spray-drying

SDP in a model of mild intestinal inflammation

SDP in a model of colitis

SDP in a model of acute lung inflammation

The mechanism(s) of action of SDP

Experimental design

SEB

Craig et al (2012) Nature 489: 231-41.

Izcue et al (2009) Annu Rev Immunol 27: 313-38.

Lallès et al (2009) Animal 3: 1625-42.

Piglets post-weaning

Remus et al 2013, Livestock Sci 155: 294-300

Van Dijk et al 2002, J Anim Sci 86:17-25

Niewold et al 2007, Vet. Microbiol. 124: 3629

Animal blood plasma

Experimental design

Inflammatory agent: Entorotoxin B from Staphyloccus aureus (SEB)

Intestinal inflammation

APC

T cell

Mucosal defensins

Luminal water contents

Diffuse GALT (LPL, IEL)

Organized GALT (PP, MLN)

• Recruitment of cytotoxic populations

Mucosal cytokines

Epithelial permeability

• Recruitment of cytotoxic populations

• Pro-inflammatory cytokine release

• Flux of dextran and HRP and tight junction proteins

• Cryptdin 4 and β-defensin 1 expression APC

T cell

Pérez-Bosque et al (2006) J Nutr 136:2838-43

Control SEB SEB-SDP

0

2

4

6

pm

ol

HR

P/c

m²·

h

0

40

80

120

160

pm

ol

FD

/cm

2·h

Control

SEB

SEB-SDP

* #

Effects of SEB administration

Pérez-Bosque & Moretó (2010) Proc Nutr Soc 69: 447-53.

0.6

0.8

1.0

1.2

1.4

0.4 0.6 0.8 1.0 1.2

TN

F-α

(n

g/g

tis

sue)

IL-10 (ng/g tissue)

Control

SEB

SEB-SDP

0.6

1.2

1.8

2.4

0.4 0.9 1.4 1.9

TN

F-α

(n

g/g

tis

sue)

IL-10 (ng/g tissue)

Control

SEB

SEB-SDP 50

100

150

200

250

50 100 150 200

TN

F-α

(p

g/m

L)

IL-10 (pg/mL)

Control

SEB

SEB-SDP

Mucosal defensins

Luminal water contents

Diffuse GALT (LPL, IEL)

Organized GALT (PP, MLN)

• Recruitment of cytotoxic populations

Mucosal cytokines

Epithelial permeability

• Recruitment of cytotoxic populations

• Pro-inflammatory cytokine release

• Flux of dextran and HRP and tight junction proteins

• Cryptdin 4 and β-defensin 1 expression APC

T cell

Mucosal interconnection

Gill et al (2010) Nat Immunol 11: 558-60.

Oral SDP supplements: • Increase performance of poults the first week after placement. • Reduced mortality in turkeys exposed to P. multocida.

CTL

Campbell et al (2004) J Appl Poult Res 13:388–93.

Su

rviv

al

(%)

Weaning (day 19)

C57BL/6 mice

LPS challenge 12.5 µg i.n. (day 33)

Sacrifice (6 h / 24 h after the challenge)

Inflammatory agent: Lipopolysaccharide from E. coli

ON DIET (14 days)

Control diet

Control diet + LPS

SDP* suppl. (8%) + LPS

*Supplied by

• Brochoalveolar lavage fluid (BALF) • Lung tissue • Blood

Adaptive immunity

Innate immunity

• Activated monocytes and neutrophiles, pro-inflammatory cytokines & chemokines.

Regulatory immunity

• Activated Th lymphocytes, pro-inflammatory cytokines.

• Treg lymphocytes, anti-inflammatory cytokines.

0

1

2

3

4

pg

/mg

pro

t

Control

LPS

LPS-SDP

IL-10 concentration

0

1,000

2,000

3,000

4,000

5,000

6 h 6 h 24h

pg

/mL B

ALF

050

100150

Non challenged Challenged

*

#

* #

TNF-α concentration

0

20

40

60

80

100

120

6h 6h 24h

pg

/mL B

ALF

Non challenged Challenged

IFN-γ concentration

* #

*

#

#

0

40

80

120

160

200

0.0 2.0 4.0T

NF-α

(p

g/m

L B

ALF)

IL-10 (pg/mg protein)

Control

LPS

LPS-SDP

Maijó et al (2012) J Nutr 142:264-70 Maijó et al (2012) Br J Nutr 107:867-75

Adaptive immunity

Innate immunity

• Activated monocytes and neutrophiles, pro-inflammatory cytokines & chemokines.

Regulatory immunity

• Activated Th lymphocytes, pro-inflammatory cytokines.

• Treg lymphocytes, anti-inflammatory cytokines.

Outline

Weaning and the consequences of stress

Animal blood plasma SDP Spray-drying

SDP in a model of mild intestinal inflammation

SDP in a model of colitis

SDP in a model of acute lung inflammation

The mechanism(s) of action of SDP

Experimental design

Craig et al (2012) Nature 489: 231-41.

mdr1a -/- mouse

Experimental design

On diet (5 wk)

• WT-CTL: Control diet • KO-CTL: Control diet • KO-SDP: SDP suppl. diet (8%)*

*Supplied by

Weaning Birth Sacrifice

Week

SPF area (4 wk) Conventional area (4 wk)

The model

P-glycoprotein

mdr1a -/- mouse

Experimental design

On diet (5 wk)

• WT-CTL: Control diet • KO-CTL: Control diet • KO-SDP: SDP suppl. diet (8%)*

*Supplied by

Weaning Birth Sacrifice

Week

SPF area (4 wk) Conventional area (4 wk)

The model

0

5

10

15

Sco

re

WT

KO

KO-SDP

*

#

WT-CTL KO-CTL KO - SDP

Infiltration

1 mm

100 µm

1 mm 1 mm

100 µm 100 µm

Pérez-Bosque et al (2010) Proc Nutr Soc 69:E262.

0

2

4

6

8

% o

f T

h l

ym

ph

ocy

tes

* #

0

5

10

15

20

% o

f T

h l

ym

ph

ocy

tes

* #

Moretó et al 2010 Dig Dis Week Meeting, New Orleans, LA.

0.0

0.5

1.0

1.5

2.0

% o

f T

h l

ym

ph

ocy

tes

#

0

2

4

6 *

#

0

1

2

3

4*

#

0

2

4

6

8

10%

of

Th

lym

ph

ocy

tes

#

*

0

5

10

15

20

25

30

Fo

ld c

han

ge

0

20

40

60

80

100

Fo

ld c

han

ge

0.0

0.5

1.0

1.5

pg

/mg

pro

tein

0

10

20

30

40

pg

/mg

pro

tein

0

20

40

60

80

pg

/mg

pro

tein

0

1

2

3

4

pg

/mg

pro

tein

*

#

* #

* #

*

#

*

#

*

#

Results

0.0

0.2

0.4

0.6

pg

/mg

pro

tein

WT

KO

KO-SDP

#

*

0

5

10

15

20

25

30

0.0 0.5 1.0

WT

KO

KO-SDP

Moretó et al 2010 Dig Dis Week Meeting, New Orleans, LA.

Slope = Permeability index (min-1)

FITC-Dextran (4 kDa)

t (min)

Rati

o

0 6 12

Crypt

lumen Pericryptal

space

0.000

0.002

0.004

0.006

0.008

0.010

0.012

min

-1

WT

KO

SDP-KO

*

#

6 min

12 min

0 min

0.0

0.5

1.0

1.5

β-Catenin E-Cadherin

Arb

itra

ry u

nit

s

WT

KO

KO-SDP

*

#

*

#

β-Catenin E-Cadherin

FITC-Dextran

WT

KO

Moretó et al (2009) Dig Dis Week Meeting, Chicago, IL.

0.0

0.5

1.0

1.5

Fo

ld c

han

ge

* #

Results

Moretó et al (2013) IUPS Meeting, Birmingham, UK.

0.0

0.5

1.0

1.5Fo

ld c

han

ge

WT

KO

KO-SDP

*

#

0

5

10

15

Go

ble

t ce

lls/

10

0 µ

m

* #

Apical junctional complex

Mucosal mucins

Diffuse GALT (LPL, IEL)

Organized GALT (MLN)

• Recruitment of cytotoxic populations

Mucosal cytokines

Epithelial crypt permeability

• Recruitment of cytotoxic populations

• Pro-inflammatory cytokine release

• Expression of β-catenin and E-cadherin

• Flux of dextran

• Expression of MUC2 & TFF3

mdr1a -/- mouse

Histopathological index

Apical junctional complex

Mucosal mucins

Diffuse GALT (LPL, IEL)

Organized GALT (MLN)

• Recruitment of cytotoxic populations

Mucosal cytokines

Epithelial crypt permeability

• Recruitment of cytotoxic populations

• Pro-inflammatory cytokine release

• Expression of β-catenin and E-cadherin

• Flux of dextran

• Expression of MUC2 & TFF3

mdr1a -/- mouse

Histopathological index

SEB mdr1a-/-

LPS

CTL

+SDP

+SDP

T activated / T regulatory lymphocytes

Pro / Anti inflammatory cytokines

Outline

Weaning and the consequences of stress

Animal blood plasma SDP Spray-drying

SDP in a model of mild intestinal inflammation

SDP in a model of colitis

SDP in a model of acute lung inflammation

The mechanism(s) of action of SDP

LPL DC

M = = = = = = = = = = = = = =

Naive