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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