intestinal homeostasis and its breakdown in ibd … · intestinal homeostasis and its breakdown in...
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Intestinal homeostasis and its breakdown in IBD
Fiona Powrie
Translational Gastroenterology Unit
Experimental Medicine Division Nuffield Dept of Clinical Medicine
University of Oxford [email protected]
Fondation Merieux
10/6/13
Modified from Xavier and Podolsky 2007
** genes associated with IBD risk Innate immunity (NOD2, ATG16L1) Inflammation (IL-23R, STAT3)
Microbiota changes
Intestinal barrier function
Anti-microbial defense
Balance between effector and regulatory T cell responses
NOD2 ATG16L1
IL23R, STAT3 IL10
Adapted from Xavier Nature 2011
Maladaptations between the intestinal microbiota and innate and adaptive immune response promote IBD
Helicobacter hepaticus
C57BL6
Deficiencies in H hepaticus -induced IL-10 leads to IL-23-mediated colitis
Hh
CD11c
DAPI
Kullberg et al, JEM 2003; Hue et al., JEM 2006
Anti-IL10R
IL-23 dependent colitis IL-17-independent
Contro
l mLN
H. Hepatic
us mLN
Control c
aecum
H. hepatic
us caecu
m
Control c
olon
H. hepatic
us colo
n
0
1
2
3
**
**
%Fo
xp3
- IL1
0+ a
mo
ng C
D4
+ T
ce
lls
Control m
LN
H. Hepatic
us mLN
Control c
aecum
H. hepatic
us caecu
m
Control c
olon
H. hepatic
us colo
n
0
10
20
30
40
50
**
**
% IL
10
+ a
mo
ng C
D4
+Fo
xp3
+ T
ce
lls
IL-10+Foxp3+ IL-10+Foxp3-
The IL-23/Th17 Pathway-distinct mediators may promote particular tissue responses
IL-1, IL-6
TGF-β, IL-23
Naïve T-cell
Th17 cell (CD8, unconv T cells, ILC)
IL-17A IL-17F
IL-21
IL-22
IFN-γ TNF-α
GM-CSF
IL23R
Myeloid Cells
APCs
Stromal Cells
RORgt
Inflammation GWAS: AS, CD, UC, Psoriasis Models; colitis, joint, skin, uveitis, CNS
Host defence AMP’s Barrier function, repair
IL-23 acts directly on T cells to promote colitis
WT
23R-/-
RAG-/-
IL23R-/- CD4+
CD45RBhi
Ahern et al., Immunity 2010
IL-1
7
IFN-g
23R-/-
Foxp3 +
WT 23R-/- 0
1
2
3
4
5 p<0.05
% F
oxp
3 a
mo
ng
CD
4+
Colitis is characterised by high numbers of mature and myeloid progenitor cells-role of GM-CSF?
CD11b
GR1
neutrophils Inflamm monos
CD4+ CD45RBhi
RAG-/-
control colitis
0 102
103
104
105
0
102
103
104
105
Specimen_001_colon 2RR.fcs…lymphocytes
<PE-A>: GMCSF
<A
PC
-A>
: IL
-17A
7.04 8.72
2855.8
IL-1
7A
GM-CSF
Colonic CD4+
T Griseri et al., Immunity 2012
0
5
10
15
20
p=0.0007
control colitic
N0 colonic GMP
x 1
02 c
ell
s
CFU-GM control colitic
Marginal CFU activity in the colon ~10X increased during colitis
GM-CSF promotes chronic intestinal inflammation
colitis
0
2
4
6
8
10
12
RBhi +isotype control RBhi + anti-GM-CSF
p=0.009
p=0.05
(x1
04 c
ell
s)
0
10
20
30
colonic neutrophils
N0 CD11b+Gr1hi cells
Griseri et al., Immunity 2012
0
1
2
3
4 p=0.02
N0 GMP
(x10
3 c
ells)
p=0.05
anti-
GM-CSF isotype
Rosenberg HF Nat Rev Imm 2013
Eosinophils and the gastrointestinal tract
• Eosinophils are abundant throughout the GI tract
• Present in germ free mice
• Contributors to the immune response
• Can mediate tissue damge
• Increased in acute colitis
• Increased activation in IBD
HSC
GMP MEP
CLP
BONE
MARROW
Stem cells
Lymphoid
progenitors Myeloid
progenitors Erythroid
progenitors
Haematopoiesis Chronic Intestinal Inflammation
IFN-γ
GM-CSF Blo
od
GMP
EMH
microbiota
Lamina
propria
damage
Neutrophils
Inflammatory
monocytes
GM-CSF
Th1/
Th17
DC
IFN-γ
IL-17
GM-CSF promotes dysregulated myelopoiesis: Colitogenic role for tissue toxic eosinophils
Eosinophils
IL-23
IL-23-driven innate colitis-genetically controlled response to Hh with hallmarks of type 17 response
a-IL-23 a-IL-17A a-IFNg
Hue et al., JEM 2006; Buonocore et al., Nature 2010; Boulard JEM 2012; Szabady
unpublished
Colitis and colon cancer in 129SvEv C57Bl6 resistant (mapped to Chr3)
Colon
129Rag 129RagIL220
2
4
6
8
10**
His
top
ath
olo
gy S
co
reRAG RAG/IL-22KO
IL23-responsive innate lymphoid cells
• IL23 induces Th17/Th1 cytokines by a novel population of innate lymphoid cells
Thy1
SCA-1
Innate lymphoid cell
IL-23R RORγt
IL-23
IL17A IL22 IFNγ
Buonocore et al., Nature 2010
IL-17+
Lin-ILC Colon
0
2
4
6
8
10
12
*
Th
y1
hi S
CA
-1+
freq
uen
cy (
%)
Control Hh
Hh RAG-/- Hh RAG-/- +anti-Thy-1
IL-23
ILC
IL-23
IL-17
IFN-g
IL-22
IL-23 ILC
Neutrophils
Mf
IL-23 drives innate colitis through promoting IFN-g
and IL-17 by innate lymphoid cells
Innate cell involved in the tissue
inflammatory response
Respond to early microbially-
induced IL-23
RORgt-dependent cytokine
module
May be involved in early
amplification of the inflammatory
response
Increased Type 17 ILC in CD
lesions (Geremia et al., JEM 2011)
INFLAMMATION
CD127
RORγT
C-kit
CD4
RORγT
CD127
Thy-1
NKp46
Type 3 ILC
Required for lymphoid organogenesis Dependent on RORgt Lti-like cells produce IL-17 and IL-22 in response to IL-23-host protective in CR infection
C-kit
IL-22 producing ILC Bears NK markers Mediates anti-microbial immunity in intestine
Mebius NRI 2003; Eberl G et al., Nat Immunol 2004 ;Takatori et al., JEM 2009; Cella et al.,
Nature 2009; Buonocore et al., Nature 2010, Sonnenberg et al., Immunity 2011
RORγT
CD127
Thy-1
IL-17, IL-22 and IFN-gamma producing ILC Promote intestinal inflammation
H. Hepaticus-induced invasive colorectal cancer
5 m analysis
6w AOM
Highest tumor grade
19%
(n=3 )
64%
(n=9 )
7%
(n=1 )
6%
(n=1 )
75%
(n=1 2 )
29%
(n=4 )
0%
20%
40%
60%
80%
100%
129SvEv C3B
No CRC Low-grade dysplasia
High-grade dysplasia CRC
129 CRC C3B normal
Boulard et al., JEM 2012
E-cadherin RORg DAPI IL-7Ra DAPI
Changes in the ILC compartment during H.hepaticus induced cancer
Hh+AOM
Anti-Thy-1 tx reverses established inflammation and cancerous changes
129 RAG-/- AOM
ip
after 19w
Analysis: Colitis? Cancer ?
H. hepaticus
Start of experiment
0w 6-10w
12w-18w
Anti-Thy1
12w:inflammation cancer
Anti-Thy1
31% n=4
91%
n=10
62%
n=8
0%
20%
40%
60%
80%
100%
iso anti-Thy1
CRC
dysplasia
No CRC
IL-22R
IL-22 as a pro-tumorigenic factor?
• Survival and growth factor produced by Th22, Nk-22, ILC, LTi
• IL-22R expression restricted to epithelium
• Signals through Stat3, which is involved in other CAC models
• Polymorphisms in IL-22 associated with a 1.46-fold increased risk for development of CRC (Thompson et al. Cancer Causes Control 2010)
Stat3P
IL-22R1
IL-2
2
IL-10R2
IL-2
2
live, CD45+, lin-
CD
4
Nkp46 Thy1.2
IL-2
2
IL-17
live, CD45+,lin-,Thy1hi
ILC produced IL-22 is acting on the epithelium to promote proliferation
Hh+AOM
pStat3 Y705
+ anti- IL-22 + iso
25mm 25mm
25mm 25mm
CRC
6-10w
AOM
1-3d 20w
Hh
Ab tx 1w
+ anti- IL-17 + anti- IL-6
Cyclin D1
25mm 25mm
+ anti- IL-22 + iso
IL-22 sustains colon cancer
Start of experiment
0w
129 RAG-/- AOM
ip
after 19w
Analysis: Colitis? Cancer ?
H. hepaticus
6-10w
12w-18w
Anti-IL-22
0%
20%
40%
60%
80%
100%
CRC
dysplasia
no CRC
Kirchberger et al., JEM 2013
ILC promote colitis and sustain colon cancer through IL-22 production
Genetic control of bacteria-driven colon cancer (1.5Mb region on Chr 3-also acts in other models) Functions in haematopietic cells to control early innate response to H. Hepaticus DN 17+22+ Thy1+ ILC accumulate in CRC and mediate a functional role in perpetuation of the disease IL-22 is required for pStat3 in IEC Neutralisation of IL-22 ameliorates established cancer-cell extrinsic control of neoplastic cells IL-22+ T and non-T cells in human CRC-novel therapeutic target (Flavell, Nature 2012;Karin Nature 2012)
Gr
CRC
H.hepaticus
DC
IL-23
cILC
IL-23R+
Thy1hi
Sca1hi
pStat3
IL-22 IL-17
Dysbiosis?
ROS inflammatory
recruitment
AOM
proliferation
cILC
IL-22
Inflammatory cytokines contribute to the initiation and perpetuation of bacteria-driven colon cancer
• Is there an IL-22 signature assoc with spontaneous CRC? • Mutations in CAC? • Is dysbiosia of the microbiota a contributor to Hh-driven CAC?
Collaborators: D.Littman (NYU) Dan Cua (Merck) Brent Mckenzie (CSL) M. Oukka (Benaroya Inst) Paul Crocker (Dundee)
CRUK
Powrie Lab Chris Scheiring Matt Shale Scott Steward Tharpe Stephanie Kirchberger Olivier Boulard Claire Pearson Allesandra Geremia Carolina Arancibia Tibo Griseri Thomas Krausgruber Philip Ahern Sophia Buonocore Kevin Maloy (SWDSP) Margherita Coccia Simon Travis (TGU) Holm Uhlig (TGU) Daniel Royston (Pathologist)