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Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008

1

Maria T. Abreu, MD

Disclosures• Abbott• UCB• Salix• P&G• Prometheus


2

Hypothesis: IBD arises from inappropriate handling of

intestinal bacteria

Defective innate immunityfollowed by exuberant adaptive

immune response


3

Bacteria and the Intestinal Mucosa Have Adapted to Each Other

TLRs

APCs

••Nutrition/metabolism (30% Nutrition/metabolism (30% of calories)of calories)••Epithelial repair:Epithelial repair:

•• ProliferationProliferation•• RestitutionRestitution

••Phagocytosis/clearance of Phagocytosis/clearance of lamina propria bacterialamina propria bacteria

Sonnenburg, J et al. Science

Increase in IBD Incidence for Patients Exposed to Salmonella/Campylobacter

1.5

1

0.5

00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Per

cent

age

With

IBD

Years After Salmonella/Campylobacter Date

Gradel K et al. Gastroenterology. 2009;137:495.

ExposedUnexposed


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Mucosal or innate immune system

Etiologic Theories in Inflammatory Bowel Disease

Genetic predisposition

Environmental triggers

(luminal bacteria, infection)

IBD

Recognition of Bacteria through TLRs and NLRs

NLRs

Innate Immunity

CARD LRRsNOD

TLRs LRRs

TIR

bacterial product

bacterial product

Adaptive Immunity

PYDBIRs


5

Innate and Adaptive Immune Responses/ Acute and Chronic Inflammation

Innate Immunity Adaptive ImmunityRapid(within hours)

Slower onset(5–6 days)

Less specific Specific

No memory response Memory response

Innate and Adaptive Immunity Are Linked

“Threat”detection

Pathogen and damageassociated

molecular patterns

TLRs, NODs· Release of antimicrobials· Recruitment of cells· Localized inflammation

KILL PATHOGENSCLEAR INFECTION

Cytokines and

chemokines

0–6 hours

Immediateresponses

Innate immunityBroad action

PRIME

1–5 days

Longer-termmobilization

ACTIVATE

MODULATE

Adaptive immunityAntigen-specific

B cell or T cell

TLR

Ligand


6


Genetic predisposition

Mucosal or innate immune system

Environmental triggers


IBD

IFN-b

IRF3

TRIFTRIF

NF-kB

IRAK

TRAF6

IkB

NF-kB

TLR-5

Flagellin TLR-2

Lipopeptides TLR-1,6

Bacteriallipopeptide TLR-4

LPS

TLR-9

CpG DNA

TLR-3

dsRNA

TLR-11

UPEC/Profilin TLR-7,8

ssRNA

MyD88

MyD88

MyD88

MyD88

MyD88

MyD88

Antigen presenting cells:MacrophagesDendritic cells

T Cells

TLRs Recognize Pathogen-Associated Molecular Patterns


7

IL-1 Lipoproteins polyIC LPS CpG

The world of TLRs: all ligands available in gut flora! The world of TLRs: all ligands available in gut flora!

MyD88

MalTRIF

MyD88

MalTRAM

TRIF

MyD88

IL-1RI TLR2 TLR3 TLR4 TLR9

TBK-1

NF-kB IRF-3 NF-kB IRF7

+ ?

TNF IFNb TNF IFNa TNF IFNb TNF IFNa

MyD88

IRAK-1

sustained

IRAK4 and IRAK1 IRAK4 and IRAK1

NF-kB

IRAK4

TRAF6

IkB

TLRTLR--44

LPSLPS

NF-kB

MyD88MyD88 TRIF

TRAM

Innate signaling pathways implicated in genetics of IBD

NOD2/CARD15IL-23RIRGMATG16L1IL-12BTLR2, 3, 4, 5, 6, 9TNFS15STAT3

Van Limbergen et al. IBD 13:338; 2007; Barrett, JC et al. Nat Gen 40:955, 2008

MalMal

IRF3 IRF3P

IRF3 IRF3P IFN-b

One would predict that perturbations in innate One would predict that perturbations in innate immunity would disproportionately effect handling immunity would disproportionately effect handling of gut bacteria. of gut bacteria.


8

Inflammatory response is required for pathogen killing

But….it can cause tissue damage and death

Crohn’s disease= defective innate immunity

Ulcerative colitis= exuberant innate immunity

Host Defense: a double-edged sword

Questions that remain unanswered about TLRs in the intestine

• Small intestine versus colonic expression– Differences in bacterial load--cause and effect

• Which cell types: IEC, lamina propria lymphocytes, DC’s, mf

• Polarization• Homeostasis versus injury• Acute versus chronic inflammation• Can this pathway be manipulated for clinical

benefit (infection versus chronic inflammation)


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Flagellated bacteria

Flagellin –Protein Component of Gram negative Bacterial Flagellum

~20,000 flagellin proteins per tail


10

Serologic Expression Cloning of Cecal Bacterial Antigens

Colonies Expressing Cecal Bacteria Antigens

Serum from colitis mouse

Antibody Reaction

Flagellins

Induction of Mucosal Inflammation

by Flagellin CBir1 Specific T cells

CBA-specificLodes et al, J Clin Investigation, 113(9):1296-1306, 2004


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Recombinant flagellin protects the gut from radiation induced damage

Burdelya LG, et al. Science 2008;320:226-30.

Allelic Variants of NOD2 Are Associated With Crohn’s Disease

• Carriage of NOD2 allelic variants1-6:– Crohn’s disease 27%–39%– Control population14%–16%– UC population 12%–14%5

• Carriage of 2 mutations is estimated to carry absolute risk of CD of 3%

CARD1 CARD2 NBD LRR

R702WR702WSNP8SNP8

G908RG908RSNP12SNP12

1007fs1007fsSNP13SNP13

1,21,2

P268SP268SSNP5SNP5

1. Hugot JP et al. Nature. 2001;411:599.2. Ogura Y et al. Nature. 2001;411:603.

3. Hampe J et al. Lancet. 2001;357:1925.4. Lesage S et al. Am J Hum Genet. 2002;70:845.

5. Cuthbert AP et al. Gastroenterology. 2002;122: 867.6. Ahmad T et al. Gastroenterology. 2002;122:854.


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NOD2 Role in Pathogenesis

Maeda S et al. Science. 2005;307:734.

NOD2 Phenotypes in Animal Models

NOD2-/-

Pauleau AL et al. Molecular & Cellular Biology. 2003;23:7531.

Commensal bacteria

NOD2-/-

Kobayashi KS et al. Science. 2005;307:731.

Control of pathogenic bacteria

NOD22939iC

Maeda S et al. Science. 2005;307:734.

DSS treatment


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van Heel DA et al. Lancet. 2005;365:1794.

Patients Carrying NOD2 Mutations Have a Decreased Response to Its Ligand MDP

121110

9876543210-1

Inte

rle

uk

in 8

(µg

/L)

1 10 100 1,000

MDP µg/L

wt/wt

702Trp/702Trp702Trp/1007fs908Arg/1007fs

1007fs/1007fs

Autophagy defends the cytoplasm

Functional autophagy Absence of autophagy


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Crohn’s Disease May Represent Decreased Ability to Destroy Intracellular Bacteria–“Autophagy”

• Breakdown of bacteria: E. coli, Salmonella, Legionella, M. tuberculosis

• ATG16L1 SNP: Ileal CD ORs 1.45 (95% CI: 1.21–1.74) for carrying allele G

• Immune-regulated guanosine triphosphatase

– impt M Tb (Chr 5q33.1)– OR 1.38 (1.15–1.66)

IRGM

ATG16L

1. Parkes M et al. Nat Genet. 2007;9:830.2. Yamazaki K et al. J Hum Genet. 2007;52:575.

3. Prescott NJ et al. Gastroenterology. 2007;132:1665.4. Rioux JD et al. Nat Genet. 2007;39:596.5. Hampe J et al. Nat Genet. 2007;39:207.

100

Surv

ival

(%

)

80

60

40

20

00 1 2 3 4 5 6 7 8 9 10 (day)

5% DSS Water

Atg16L1+/+ (n=5)∆/∆ (n=5)

Defective Autophagy Increases Inflammatory Response and Decreases Paneth Cell Function

ATG16L1-/- die with DSS

Cadwell K et al. Nature. 2008;456:259.Saitoh T et al. Nature. 2008;456:264.

Van Limbergen J, et al Inflamm Bowel Dis. 2008 Mar;14(3):338-46.Amre, DK et al. Inflamm Bowel Dis. 2009 Apr;15(4):501-7.

ATG16L1 associated with ileal involvment


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Possible role of autophagy genes in CD

Th1/Th2/Th17 Paradigm

Therefore, IL-12 vs IL-23 determine fate of Th1 or Th17 cells.

T “naive”

Th1 IFN-gCrohn’s disease?

IL-12

Th2 IL-4, IL-5, IL-13, IL-25Ulcerative colitisIL-4

IL-17Crohn’s disease?

Autoimmunity+IL-6, TGFb

IL-23IL-23

Th17IL23RIL23R


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Differentiation of Effector T Cells

Miossec P et al. N Engl J Med. 2009;361:888.

NaïveCD4+T cells

Interleukin-23Interleukin-21

TGF-β+

Interleukin-6+

Interleukin-1

Interleukin-17

Th17

Interleukin-23R

ROR-gt

++

Interleukin-12Interleukin-18

Interferon-g

Th1

Interleukin-12RInterferon-g

T-bet

Interleukin-12Interferon-g

+

+

Interleukin-4

Interleukin-4

Interleukin-4

Interleukin-4GATA3

Th2

Interleukin-4R

+

Crohn’s

UC

T.1

Absence of IL-23 Protects Against Colitis in IL-10-/- Mice

Yen D et al. J Clin Invest. 2006;116:1310.

IL-10-/-KO IL-10-/- x p19-/- (IL-23) IL-10-/- x p35-/- (IL-12)

T.1 animateMaria Abreu, 11/6/2009


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IL-12 and IL-23 Blockade in IBD

• IL-12 p40 neutralizing antibodies are therapeutic in mouse colitis models and in human CD

• Genomewide association study: 3 SNPs identified (2 were Nod2)• Uncommon variant of IL-23R: arginine 381 ® glutamine• Carriage of Arg381Gln is protective of Crohn’s disease: OR 0.26 (0.15–

0.43)• Other SNPs in IL-23R confer risk in both CD and UC (latest GWAS)

Duerr RH et al. Science. 2006;314:1461.Parkes M. Nat Genetics. 2007;39:830.

Brant S et al. IBD Journal 2008

IL-12Rb1 IL-12Rb2

IL-12

p35

IL-12Rb1 IL-23R

Anti-p40 Ab Anti-p40 AbIL-23

p19p40 p40

Genes in the IL-23/IL-17 Pathway Interact to Increase Crohn’s Disease Susceptibility

Taylor KD et al. Inflamm Bowel Dis. 2008;14:1185.

4

3

2

1

Od

ds

Ra

tio

for

CD

Number of “Risk” Haplotypes Present0 1 2 3

1.3

4.0

4

2.5

pMH<0.0001

IL-23R, IL-17A, IL-17RA, and IL-12RB1


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Composition of intestinal microbiota regulates the intestinal Th17:Treg balance

J

sIgA

DENDRITIC CELL

IEL

TLRs

Segmented filamentous

bacteria

GOBLETCELL

Th17 cells T-reg cellsIvanov II et al. Cell 139, 485–498, October 30, 2009

Which innate immune signals triggersimultaneous synthesis of TGF-β and IL-6?

IL-6 + TGF-β

CD4+ T cell

TH17RORgt

IL-17AIL-17FIL-22IL-10

Naïve

Torchinsky MB et al. Nature. 2009;458:78.


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Regulatory T cellFOXP3+

TCRCD4

TH17 cellRORgt+

TCR CD4

Mature DC

Apoptotic cell

TLR signaling

Immature DC

Infected apoptotic cell

TGF-β IL-6IL-23?

TGF-β

Hypothesis

Torchinsky MB et al. Nature. 2009;458:78.

DC

DC

DCLamina propria

MØ

TH17 cells

Gut lumen

Commensal bacteria

Citrobacter rodentium

Intestinal epithelial cells

Infection of the Intestinal Epithelium With Citrobacter rodentium

Mangan PR et al. Nature. 2006;441:231.Zheng Y et al. Nature Med. 2008;14:282.


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DC

DC

DCLamina propria

MØ

TH17 cells

Gut lumen

Commensal bacteria

Citrobacter rodentium


Citrobacter rodentium Causes Apoptosis of Intestinal Epithelial Cells

Crane JK et al. Cell Microbiol. 2001;3:197.Nagai T et al. J Biol Chem. 2005;280:2998.

Vallance BA et al. Infect Immun. 2003;71:3443.Courtesy of J.M. Blander

DC

Lamina propria

MØ

TH17 cells


DC

DMapDMapDMap

EPEC-Secreted Protein FMitochondrial-Associated Protein

DEspFDEspF DEspF

?

Infection With a Citrobacter Mutant Unable to Induce Apoptosis

Deng W et al. Proc Natl Acad Sci U S A. 2004;101:3597.


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DC

Lamina propria

TH17 cells

Commensal bacteria

Microbial Pathogen


Novel Role for Apoptosis Along Surface Epithelia

DC

Infected apoptotic cells constitute an important innate immune signal

for TH17 cell differentiation.

Torchinsky MB et al. Nature. 2009;458:78.Courtesy of J.M. Blander

Absence of IL-23 Protects Against Colitis in IL-10-/- Mice

Yen D et al. J Clin Invest. 2006;116:1310.

IL-10-/-KO IL-10-/- x p19-/- (IL-23) IL-10-/- x p35-/- (IL-12)


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A Polymorphism in the IL-23R Gene Protects Against Crohn’s Disease

• Genomewide association study: 3 SNPs identified (2 were Nod2)• Uncommon variant of IL-23R: arginine 381 ® glutamine• Carriage of Arg381Gln is protective of Crohn’s disease: OR 0.26

(0.15–0.43)• Functional signaling consequences unknown

Duerr RH et al. Science. 2006;314:1461.Parkes M. Nat Genetics. 2007;39:830.

IL-23 IL-12Rb1IL-23R

IL-23

Naïve CD4+ T cell

+TGF-b

IL-6

IL-12Rb1IL-23R

IL-17-producing TH cell

IL-23

IL-17

Arg381GlnArg381Gln


23

Becker C et al. J Clin Invest. 2003;112:693.

Dendritic cell

Peyer’s patch


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Dendritic cell

Peyer’s patch

NOD2

PAMPsTLRs

Dendritic cell

Peyer’s patch

NOD2

LRRSCARD CARD NOD2

NF-κB

Nucleus

NF-κB

IL-12p40Defect in autophagy

PAMPsTLRs


25

Dendritic cell

Peyer’s patch

NOD2

LRRSCARD CARD NOD2

IL-23

NF-κB

Nucleus

NF-κB


PAMPsTLRs

Dendritic cell

Peyer’s patch

PAMPTLR

NOD2

LRRS

IL-23IL-23

IL-10IL-10

Increasedproduction

Decreased production

Watanabe T et al. Nat Immnuol. 2004;5:800.Netea MG et al. Eur J Immunol. 2004;34:2052

Cadwell K et al. Nature. 2008;456:259..

CARD CARD NOD2

IL-23

NF-κB

Nucleus

NF-κB



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IBD Specific Serologic Immune Markers

AntibodyAntibody AntigenAntigenNonNon--

IBD (%)IBD (%) CD (%)CD (%) UC (%)UC (%)

DNase DNase Sensitive Sensitive pANCApANCA

Histone HHistone H11, bacterial , bacterial antigen?antigen?

<5%<5% 1010––25%25% 5050––65%65%

ASCAASCASaccharomyces cerevisiae Saccharomyces cerevisiae (oligomannans)(oligomannans)

5%5% 5555––65%65% 5%5%

OmpCOmpC E. ColiE. Coli <5%<5% 3838––50%50% 2%2%

AntiAnti--II22 Pseudomonas fluorescensPseudomonas fluorescens <5%<5% 54%54% 2%2%

AntiAnti--FlagellinFlagellin CBir 1 AntigenCBir 1 Antigen 88--14%14% 50%50% 6%6%

Anti-ALCA IgG

laminaribiosideGlc(β1,3)Glc(β)

2%2% 27%27% 9%9%

Anti-ACCAIgA

Chitobioside GlcNAc(β1,4)GlcNAc(β)

12%12% 25%25% 25%25%

The risk of Crohn’s disease increases with anti-microbial Ab positivity

Joosens, M. et al. Inflammatory bowel disease journal 2009


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0.00

0.25

0.50

0.75

1.00

Time to surgery (months)

0 25 50 75 100 125 150 175

ab_sum=0

ab_sum=1

ab_sum=2

ab_sum=3

Pro

babi

lity

of n

on-p

rogr

essi

ve C

D

P<0.0001

N= 174

N= 47

N= 189

N= 243

Dubinsky MC et al CGH 2008;6:1105

Antibody Sum and Surgery

Adaptive Immune Responses to Microbial Products Increases With Decreased NOD2 Function

9.72

10.46

11.26

N=499 N=194 N=398.5

9.5

10.0

10.5

11.5

0 1 2

9.0

11.0P trend = .002

Le

vel o

f Ant

imic

rob

ial A

bs

Me

an Q

uart

ile S

um

NOD2/CARD15 Variant Status

Devlin SM et al. Gastroenterology. 2007;132:576.


28

Defective innate immunity is compensated by adaptive immune responses to intestinal

microbiota

Emma Slack, et al. Science 325, (2009) 617

MyD88-/-/Ticam -/-

F1 control

Patients with Crohn’s disease have antibodies against GM-CSF which impair

neutrophil function

Han X, et al. Gastroenterology. 2009 Apr;136(4):1261-71


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Genetic Predisposition

Mucosal or Innate Immune System

Environmental Triggers


IBD

Luminal Bacteria Stimulate Immune-Mediated Colitis

MiceIL-2-/-

IL-10-/-

TCRa-/-

CD3E26TGSAMP1/yit

DSSCD45RBhi®SCID

BacteriaNo bacteria

No colitis

TH1

TNFaIFNg

Colitis

IL-1bTNFaRats

B27/b2M TGIndomethacin

Nonhuman primateCottontop tamarin

No immune activation


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Host–Microbe Interaction Determines Severity and Location of Colitis

Kim SC et al. Gastroenterology. 2005:128:891.

Germ-freeIL-10-/-

Commensal bacteriaIL-10-/-

E. coli NC101IL-10-/-

E. coli K12/LF82IL-10-/-

Specific intestinal bacteria induce Th17 cells

• Same genetic strains of mice have different percentages of IL-17-producing T cells in SI LP depending on facility from

• Differentiation of Th17 cells correlates presence of cytophaga-flavobacter-bacteroidetes (CFB) bacteria in the intestine

• Absence of Th17 cell-inducing bacteria was accompanied by increase in Foxp3+ regulatory T cells (Treg) in the LP

• Composition of intestinal microbiota regulates the Th17:Treg balance in the LP

Ivanov II, et al (Littman DR) Cell Host Microbe. 2008 Oct 16;4(4):337-49.


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CD4CD4

IL-12IL-18

bacteria Lumen

activation

antigenpresenting

cell

IFNg

TNF

IL-10

CD4

regulatory T cell

apoptosis

NOD2

TLR4

AR

RTC

inflammationT cell modulation““innate” immune response

NOD2Tlr4

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