abreu 8 50.ppt -...
TRANSCRIPT
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
2
Hypothesis: IBD arises from inappropriate handling of
intestinal bacteria
Defective innate immunityfollowed by exuberant adaptive
immune response
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
4
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
6
Etiologic Theories in Inflammatory Bowel Disease
Genetic predisposition
Mucosal or innate immune system
Environmental triggers
(luminal bacteria, infection)
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
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.
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
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)
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
9
Flagellated bacteria
Flagellin –Protein Component of Gram negative Bacterial Flagellum
~20,000 flagellin proteins per tail
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
11
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.
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
12
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
13
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
14
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
15
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
16
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)
Slide 31
T.1 animateMaria Abreu, 11/6/2009
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
17
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
18
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.
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
19
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.
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
20
DC
DC
DCLamina propria
MØ
TH17 cells
Gut lumen
Commensal bacteria
Citrobacter rodentium
Intestinal epithelial cells
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
Intestinal epithelial 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.
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
21
DC
Lamina propria
TH17 cells
Commensal bacteria
Microbial Pathogen
Intestinal epithelial cells
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)
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
22
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
23
Becker C et al. J Clin Invest. 2003;112:693.
Dendritic cell
Peyer’s patch
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
24
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
25
Dendritic cell
Peyer’s patch
NOD2
LRRSCARD CARD NOD2
IL-23
NF-κB
Nucleus
NF-κB
IL-12p40Defect in autophagy
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
IL-12p40Defect in autophagy
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
26
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
27
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.
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
29
Etiologic Theories in Inflammatory Bowel Disease
Genetic Predisposition
Mucosal or Innate Immune System
Environmental Triggers
(luminal bacteria, infection)
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
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
30
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.
Novel Insights Into IBD Pathogenesis: 2008 and Beyond Maria T. Abreu, MDIBD Fellows 2008
31
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
23