Post on 28-May-2020
Gut Immunity
Shomron Ben-Horin
Laboratory of Gastro-Immunology
Gastroenterology Department
Sheba Medical Center, Tel-Aviv University, Israel
Disclosures
Dr. Shomron Ben-Horin received consultancy fees from:
Abbot Laboratories, Schering-Plough, Optimer Inc.
Immunology made easy
Thymocytes Naïve
CD4+ T cellTh17/Th
IL-17
IL-17F
IL-6
TNF-a
IL-23
IL-23R
IFN
-g
IL-4
IFN-g
IL-10
TGF-b1
IL-4
IL-5
IL-13STAT6
GATA3
Treg
Th2
IL-4R
CD25
FoxP3
Th1
IL-12R
STAT1
STAT4
T-bet
TGF-b1
IL-6
Human host response after exposure
to a typical immunology slide
Let’s start with a brief review of :
Gut immune system organization
Gut immune system functions
Traditional view -1
The epithelial functions consist of physical barrier and absorption apparatus
Traditional view 1: epithelium is only a barrier…
Traditional view -2
IBD is caused by exaggerated gut immune response (but CDAD is not….)
Traditional view 2: Crohn’s disease is an immune hyper-activity disorder…
Immune in-adequacy Immune hyper-reactivity
Traditional view -3
Immune memory is antigen specific
Traditional view 3: Immune memory is only antigen-specific….
Intestinal Mucosa Defense- Organization
Non-Immune
Immune
Two types of mechanisms to control pathogens,
normal flora (1014) & dietary molecules
Intestinal Mucosa Defense: Non-immune
Response type - Immune reaction:
Innate response
Adaptive response
Topographic - Immune compartmentalization:
Intestinal Mucosa Defense: Immune
Topographic Organization of GALT
Mesenteric lymph nodes
Peyer’s Patches
Cryptopatches
Solitary lymphoid follicles
Solitary immune cells:
Intraepithelial lymphocytes (IELs)
Lamina propria lymphocytes (LPLs)
Atkins D, Nature Rev Immunol 2009
Peyer’s patchDome-like protruding into lumen
Sub-epithelial dome (SED) -
3-4 Follicles (B cell germinal center), inter-follicular region
(T-cells), high endothelial venules (HEV) & lymphatics
Villous epithelium & Microfold cells (M cells)
M cells: No villi, thin glycocalyx, basally folded around immune cells. Role: Uptake of Antigens &Transcytosis
Neutra MR, Nature Immunol 2001
Peyer’s patchDome-like protruding into lumen
Sub-epithelial dome (SED) -
3-4 Follicles (B cell germinal center), inter-follicular region
(T-cells), high endothelial venules (HEV) & lymphatics
Villous epithelium & Microfold cells (M cells)
M cells: No villi, thin glycocalyx, basally folded around immune cells. Role: Uptake of Antigens &Transcytosis
M cell
Jang MH, PNAS 2004
Other inductive sites
Cryptopatches
Clusters of hematopoeitic progenitors and dendritic cells
Role: Extra-thymic development of T-cells
Solitary follicles
Clusters of lymphocytes - B cells and dendritic cells
Antigen sampling and presentation >> IgA production
Topographic Organization of GALT -
Effectors
Solitary
follicle
Peyer’s patch
Cryptopatch
Paneth Cell
Isolated Lymphocytes
Intra-epithelial lymphocytes (IELs) – CD8+ and gd T-cells
Lamina propria lymphocytes (LPLs) – CD4+ T cells & B-cells
Primed antigen-specific effector cells:
Excrete pro-inflammatory cytokines
Exert T-cell cytocytoxicity
Secrete Immunoglobulines (IgA)
Atkins D, Nature Rev Immunol 2009
Suzuki K, Trends Immunol 2008
Secretory IgA (& pentameric IgM) produced by LP
B-cells and actively secreted to gut lumen by epithelium
Topographic Organization of GALT
Mesenteric lymph nodes
Peyer’s Patches Inductive
Cryptopatches sites
Solitary lymphoid follicles
Solitary immune cells:
Intraepithelial lymphocytes (IELs) Effector
Lamina propria lymphocytes (LPLs) sites
Atkins D, Nature Rev Immunol 2009
Intestinal effector T & B cells – Education/migration
1. Naïve T & B cells presented with antigen by
macrophages/DCs in Peyer’s patches or mesenteric lymph
nodes >> effector cells
2. Exit through efferent lymph vessels into circulation/spleen
So how do they return to intestinal wall ?
Migration of Cells into Tissues
Selectin ligands,L-SelectinCCR 9
E, P Selectins
Mucosa
ACTIVATION ARRESTROLLING
Integrins
a4b7Adhesion MoleculeMADCAM-1
TRANSMIGRATION
Cytokines &
chemokines
(TECK - CCR9
ligand)
Oral immunization with S.typhy Ty21a induce antigen-responsive cells expressing a4b7 integrin, whereas
parenteral vaccination does not
Traditional view 1: Immune memory is only antigen-specific….
Kantele A, J Immunol 1999
0
20
40
60
80
100
Pa
tie
nts
in
re
mis
sio
n
(%)
L-sel
ectin
a4b7
inte
grin
Oral immunization
Parenteral
Un-vaccinated
P=<0.001 P=0.01
% A
ntigen-r
esponsiv
e c
ells
Intestinal dendritic cells in Peyer patches & mesenteric LN prime memory T-cells to express gut-homing molecules:
a4b7 integrin and CCR9 chemokine receptor
Traditional view 1: Immune memory is only antigen-specific….
Mora JR, Nature 2003
Campbell DJ, J Exp Med 2002
Johansson-Lindbom B, J Exp Med 2003
CD4+ cells primed by dendritic cell in the mesenteric lymph nodes preferentially migrate back to the GALT
Traditional view 1: Immune memory is only antigen-specific….
Iwata M, Immunity 2004
CD4+ cells primed by MLN DC (labeled with green CFSE)
or by PLN DC (red – TRITC labeled)
After injection into mice
>>
T cells primed by MLN DC(Green labeled) are
Much more able to migrate back to Peyer Patch
Media
Gliadin-TTG
TT
0.9%
5.1%
2.6%
3.8%
7.1%
0.8%
b7integrin
CFSE
Ben-Horin S et al, J Leuk biol 2006
Gliadin-TTG specific CD4+ memory T-cells, but not Tetanus
specific cells, preferentially express the gut-homing b7 integrin
Traditional view 1: Immune memory is only antigen-specific….
Media
Gliadin-TTG
TT
0.9%
5.1%
2.6%
3.8%
7.1%
0.8%
b7integrin
CFSE
Ben-Horin S et al, J Leuk biol 2006
Gliadin-TTG specific CD4+ memory T-cells, but not Tetanus
specific cells, preferentially express the gut-homing b7 integrin
NEW view 1: Immune memory is antigen-specific….AND tissue-specific
Feagan B, N Engl J Med 2005
Anti a4b7 integrin mAb (Vedolizumab) induces remission
in moderately active ulcerative colitis
Traditional view 1: Immune memory is only antigen-specific….
0
10
20
30
40
Pa
tie
nts
in
rem
iss
ion
(%
)
Vedol
izum
ab 2
mg/
kg
Vedol
izum
ab 0
.5m
g/kg
Place
bo
P=0.02P=0.03
Intestinal epithelial cells (m-IC) induce expression of gut homing
phenotype by T-cells in the absence of intestinal dendritic cells
Edele F, J Immunol 2008
Traditional view 2: epithelium is only a barrier…
CD8+ cells only
CD8+ cells &
bone marrow DC
CD8+ cells &
bone marrow DC &
gut epithelial cells
CD8+ cells &
bone marrow DC &
gut epithelial cells (transwell)
Topographic Organization of GALT
Mesenteric lymph nodes
Peyer’s Patches Inductive
Cryptopatches sites
Solitary lymphoid follicles
Solitary immune cells:
Intraepithelial lymphocytes (IELs) Effector
Lamina propria lymphocytes (LPLs) sites
Epithelium ??
Intestinal Epithelium expresses
bacterial sensing molecules
Traditional view 2: epithelium is only a barrier…
Giraradin SE, Science 2003
Intestinal Epithelium expresses
bacterial sensing molecules
Traditional view 2: epithelium is only a barrier…
NOD1 – Recognition of Gram-
bacterial wall tri-peptide
NOD2- Recognition of Gram-/+
bacterial wall MDP
>> Activation of MAPK-NFkB pathways
& Secretion of chemokines/peptides
Giraradin SE, Science 2003
Peptidoglycan (G+)LipoproteinLipoarabinomannan(Mycobacterial)LPS (Leotosopia)LPS (Porphyromonas)GPI (Trypanosoma cruzi)Zymosan Yeast)
LPS (G-)Lipoteichoic acids (G+) RSV F Protein
dsRNA Flagellin Unmethylated CpG DNA
TLR9TLR5TLR3TLR4CD14TLR1TLR2TLR6
MD-2
Toll-Like Receptors: Recognition of Pathogen
associated molecular patterns PAMP)
Artis D, Nature Rev Immunol 2008
Su
rviv
al
(%)
Deficient TLR signaling increases
mortality after DSS colitis
Depletion of intestinal flora
increases mortality after DSS colitis,
and LPS restores protection
Rakoff-Nahoum S, Cell 2004
Day 0 3 6 9 12 15 18 21
100
80
60
40
20
0
A
WT
MyD88-/-
TLR4-/-
TLR2-/-
B
Commensal triggering of TLR maintains epithelial
homeostasis and protects against DSS colitis
Traditional view 2: epithelium is only a barrier…
Selective NFkB ablation in intestinal epithelium of mice by knocking out NEMO (IKK-g) leads to innate and later adaptive
immune cell inflitrate and intestinal inflammation
Traditional view 2: epithelium is only a barrier…
Nenci A, Nature 2007
Murine models validity for human studies
Arch Gynecol - Links
The effect of intraperitoneal colchicine on the formation of peritoneal adhesions in the rat
Shapiro I, Granat M, Sharf M.
A double blind study was carried out to determine the effect of
colchicine on the formation of intraperitoneal adhesions in the rat. Adhesions were produced by a standard method in 92 rats.
The animals were than divided at random into two groups. One group (47 rats) was treated with intraperitoneal colchicine, whilst the other, the control group (45 rats) was treated with intraperitoneal normal saline in the same volume, frequency and duration.
Two kinds of adhesions were found. 1. "Surface adhesion" - consisting of two serosal surfaces attached together; their area was significantly smaller (p less than 0.00003), and their number was lower (p less than 0.03) in the colchicine group than in the control group. 2. "Filamentous adhesions" - thin, elastic cords connecting omentum or pelvic fat bodies to other viscera. We concluded that post-operative treatment with colchicine reduces the formation of intraperitoneal adhesions in the rat
Murine models validity for human studies
Arch Gynecol - Links
The effect of intraperitoneal colchicine on the formation of peritoneal adhesions in the rat
Shapiro I, Granat M, Sharf M.
A double blind study was carried out to determine the effect of
colchicine on the formation of intraperitoneal adhesions in the rat. Adhesions were produced by a standard method in 92 rats.
The animals were than divided at random into two groups. One group (47 rats) was treated with intraperitoneal colchicine, whilst the other, the control group (45 rats) was treated with intraperitoneal normal saline in the same volume, frequency and duration.
Two kinds of adhesions were found. 1. "Surface adhesion" - consisting of two serosal surfaces attached together; their area was significantly smaller (p less than 0.00003), and their number was lower (p less than 0.03) in the colchicine group than in the control group. 2. "Filamentous adhesions" - thin, elastic cords connecting omentum or pelvic fat bodies to other viscera. We concluded that post-operative treatment with colchicine reduces the formation of intraperitoneal adhesions in the rat
Paneth cell
Specialized epithelial cells at base of crypts
Respond to certain patterns of bacteria
Secrete anti-bacterial peptides (Defensins)
Traditional view 2: epithelium is only a barrier…
NOD2 Knockout mice have defective expression of
Defensins after Listeria infection
Does the Nod2 defect induce loss of function and
reduces innate immunity exposure to bacterial
infections mainly in the TI region?
Kobayashi KS, Science 2005
Rela
tive e
xp
ressio
n
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
infection- +- +
WT Nod2-/-
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
infection- +- +
WT Nod2-/-
60
50
40
30
20
10
0
infection - +- +WT Nod2-/-
Rela
tive e
xp
ressio
n
Rela
tive e
xp
ressio
n
Defcr5Defcr-rs10Defcr4
Traditional view 2: epithelium is only a barrier…
Paneth cell
NOD2 mutations found in 15% Crohn’s patients
and associated with diminished mucosal
a-defensin expression
>> reduced control of luminal bacteria ??
% o
f N
on
-dis
ease C
on
tro
l
HD5 Peptidegg/mg extract
100
80
60
40
20
0CD CD
(NOD2-wt) (NOD2-mut)Wehkamp J, PNAS 2005
Traditional view 2: epithelium is only a barrier…
WT bone marrow transfer does not restore function >> Epithelial dependency
NOD2- mice develop H. hepaticus granulomatous ileitis in
Peyer patches, reversed in transgenic human defensin mice
Biswas A, PNAS 2010
Traditional view 2: epithelium is only a barrier…
The a-defensins impact the microbial composition of the gut
Salzmann NH, Nature Immunol 2010
Traditional view 2: epithelium is only a barrier…
Defensin Knock-outs Transgenic defensin mice
Pre-incubation of intestinal epithelium with a-defensins ameliorates C.difficile Toxin B injury
Traditional view 2: epithelium is only a barrier…
Giesmann T, Gastroenterology 2008
Pre-incubation of intestinal epithelium with a-defensins ameliorates C.difficile Toxin B injury
NEW view 2: epithelium is a barrier…but also an immune organ
Giesmann T, Gastroenterology 2008
NEW view 2: epithelium is a barrier…but also an immune organ
Intestinal enterocytes induce expression of FoxP3 in activated
CD4+ cells and expansion of Treg population
Dotan I, Am J Gastrointest Physiol, 2008
Intestinal enterocytes up-regulate MICA expression and IL-15
secretion leading to Intra-epthelial CD8+ T-cell activation and
epithelial damage after exposure to gliadin in celiac
Kagnoff MF, J Clin Invest 2007
Ben-Horin S et al, Submitted
CFSE
CD
45R
O
Media GTG GTG-Trypsin
3%
3% 5%3%
4%3%
Index patient
4%
3%
19%
3%
17%
3%CD
45R
O
Celiac patient
In a bone-marrow transplanted celiac :No immune memory to celiac antigens
NEW view 2: epithelium is a barrier…but also an immune organ
Ben-Horin S et al, Submitted
In a bone-marrow transplanted celiac :Intestinal epithelial-lymphocyte chimerism
NEW view 2: epithelium is a barrier…but also an immune organ
11
10
9
8
7
6
5
4
3
2
1
0
-1
1 10 100 1000
wt/wt
702Trp/702Trp
702Trp/1007fs
908Arg/1007fs
1007fs/1007fs
MDPmg/L
IL8 n
g/m
l
Mutated NOD2 Monocytes Mount Defective Immune
Responses: Innate system dysfunction in Crohn’s disease?
Van heel D, Lancet 2005
Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…
PBMCs’ response to MDP and peptidoglycans is reduced in Crohn’s patients homozygous for the NOD2 mutation (Nod2fs) compared to controls (CTR) & Crohn’s patients
without defects in Nod2 (Crohn)
Netea MG, J Biol Chem 2005
Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…
Seidelin A, PLoS One 2009
Diminished response to MDP is also present in monocytes
from Crohn’s patients without NOD2/CARD15 mutations
Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…
NOD2 activating mutations cause constitutive NFkB
activation and early-onset sarcoidosis (Blau syn)
Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…
Okafuji I, Arthritis Rheum 2009
Van Beelen AJ, Immunity 2007
TLR2 & NOD2 signaling cause dendritic cells to induce IL-17
expression by memory T-cells
Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…
Yen D, J Clin Invest 2006
Th17 memory T-cells are induced by IL-23 and mediate
intestinal inflammation in IL-10 KO mice model
Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…
Secretion of IL17 by lamina propria mononuclear cells (LPMCs) is increased in patients with Crohn’s disease (CD) & ulcerative colitis (UC) compared to non-IBD patients patients
and healthy controls (HC)
Rovedatti L , Gut 2009
Cadwell K, Nature 2008
Defective Autophagy in ATG16L1 mutated mice & Crohn’s
patients results in disordered paneth cells and reduced
lysozyme secretion
Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…
Wild type ATG knock-out
Crohn’s
without ATG16L1
mutation
Crohn’s
with ATG16L1
mutation
Kuballa P, PLoS One 2009
siRNA silencing of ATG16L1 results in reduced Salmonella
Typhimurium internalization into autophagosome
Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…
Crohn’s patients in remission have decreased recruitment of PMN & reduced clearance of E.coli
injected into forearm
Smith AM, J Exp Med 2009
Traditional view 3: Crohn’s disease is an immune hyper-activity disorder…
Deficient Innate Immunity in Crohn’s disease ?
mucosa
tissue damageDeficient initial innate immunity: aggressive chronic adaptive response
GM-CSF Confers Therapeutic Advantage in
Crohn’s Disease
Day Day Day Follow-up15 29 57 day 30
60
50
40
30
20
10
0
Perc
en
tag
e o
f P
ati
en
ts
Primary End Point
(70-p0int decrease in CDAI)
Secondary End Point
(100-p0int decrease in CDAI)
Remission
(CDAI score 150 points)
P=0.08P=0.006 P=0.28
P=0.03
P=0.02
P=0.002
P=0.01
P=0.02
P=0.73
P=0.01
P=0.02
P=0.01
Sargramostim
Placebo
Day Day Day Follow-up15 29 57 day 30
Day Day Day Follow-up15 29 57 day 30
Korzenik JR, N Engl J Med 2005
NEW view 3: Crohn’s disease is an (innate) immune deficiency disorder…
TcdA & TcdB trigger IL-1b secretion by NLRP intra-cellular inflammasome, and disease is
reversed by IL-1b blockade
Ng J, Gastroenterology 2010
Traditional view 3: Crohn’s is immune hyper-activity….But CDAD is not….. ?
Transforming normal colon to inflammation
Immune in-adequacy Immune hyper-reactivity
Thank you for your attention