the multifactorial pathogenesis of inflammatory bowel …€¦ · the multifactorial pathogenesis...
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The multifactorial pathogenesis ofThe multifactorial pathogenesis ofinflammatory bowel diseaseinflammatory bowel disease
Claudio FiocchiClaudio Fiocchi
Department of Pathobiology, Lerner Research Institute Department of Pathobiology, Lerner Research Institute Department of Gastroenterology & HepatologyDepartment of Gastroenterology & Hepatology
The Cleveland Clinic FoundationThe Cleveland Clinic FoundationCleveland, Ohio, USACleveland, Ohio, USA
The clinical spectrum ofinflammatory bowel diseases (IBD)
Indeterminate, microscopic,lymphocytic, collagenous colitis
Ulcerative colitisUlcerative colitis(UC)(UC)
CrohnCrohn’’s diseases disease(CD)(CD)
Hypothetical stages in the pathogenesis of IBDHypothetical stages in the pathogenesis of IBD
Conception:Conception:GeneticGeneticsusceptibilitysusceptibility
Birth: ExposureBirth: Exposureto environmentto environment
ImmunoregulatoryImmunoregulatoryimbalanceimbalance
AbnormalAbnormalpermeabilitypermeability
MicroscopicMicroscopicdiseasedisease
ClinicalClinicaldiseasedisease
Priming eventPriming event
Triggering eventTriggering event
DiagnosisDiagnosis
Seidman E. (MB Cohen et al., IBD, 1998)
Distribution of IBD: pre-World War II
Distribution of IBD: 1950-1970
Distribution of IBD: 1970-1990
Distribution of IBD: 1990-present
Distribution of IBD: 1990-present
2,8313,518
4,0984,856
5,7156,609
7,702
8,862
9,980
11,337
12,645
13,997
15,440
16,891
18,036
19,651
21,061
0
5,000
10,000
15,000
20,000
25,000
85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01
11,60213,613
16,03718,072
20,81323,200
26,603
29,66133,114
36,979
41,243
46,215
51,477
57,078
60,631
66,714
72,672
0
10,000
20,000
30,000
40,000
50,000
60,000
70,000
80,000
85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01
Cumulative number of IBD patients registered in JapanUlcerative colitis Crohn’s disease
Courtesy of Dr. H. Ogata
Increasing incidence of IBD in Korea
Seoul
1990 20001995
Courtesy of Dr. Won Ho Kim
Seoul
Ulcerative colitis Crohn’s disease
1990 1995 2000
0
50
100
150
200
250
300
350
400
450
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
Hospitalized
Screened
Numbers of Chinese UC patients hospitalized and screened by endoscopy from 1990-2003
Courtesy of Dr. Qin Ouyang
Courtesy of Dr. K.L. Goh
Increasing incidence of IBD in Malaysia
Kitahora et al., 1995
Appleyard et al. Inflam Bowel Dis 10:106, 2004
Increasing incidence of IBD in Puerto Rico
0
5
10
15
20
Age
at d
iagn
osis
Age
at d
iagn
osis
19701970’’ 19801980’’0
5
10
15
20
% E
ntire
CD
pop
ulat
ion
% E
ntire
CD
pop
ulat
ion
19701970’’ 19801980’’
Changes in frequency and age of pediatric IBD
Crohn’s disease
Ulcerative colitis
Indeterminate colitis
Distribution of new onset pediatricIBD cases in Wisconsin
from 2000 to 2001
129
10
60
Kugathasan et al. J Pediatr 143:525, 2003
New IBD diagnosis
87 %6 %4 %2 %
0.5 %
General population
Asian
Other
Hispanic
African-American
Caucasian
Distribution among various racial groups andfamilial incidence of new onset pediatric IBD in Wisconsin
1st degree
2nd degree
No family history
IBD families
86 %6 %4 %2 %2 %
89 %
8 %
3 %
Kugathasan et al. J Pediatr 143:525, 2003
The incidence of The incidence of infectiousinfectious diseases has diseases has decreaseddecreasedand of and of immuneimmune disorders has disorders has increasedincreased
over the last four decades over the last four decades
IDDM
Multiple sclerosis
Crohn's disease
Asthma
1955 75 85 95
100
50
Infectious diseases
%
100
200
Immune disorders
%300
065
Hepatitis A
Rheumatic fever
Measles
Tuberculosis
1955 75 85 9565
temporarily
Bach J-F. N Engl J Med 2002; 347:911
Liver diseaseLiver disease
Heart diseaseHeart disease
19001900
DiphteriaDiphteria
BronchitisBronchitis
CancerCancer
StrokeStroke
InjuriesInjuries
DiarrheaDiarrhea
PneumoniaPneumonia
TuberculosisTuberculosis
20002000
Chronic liver diseaseChronic liver disease
Chronic kidney diseaseChronic kidney disease
SuicideSuicide
DiabetesDiabetes
Pneumonia/influenzaPneumonia/influenza
InjuriesInjuries
Chronic lung diseaseChronic lung disease
StrokeStroke
CancerCancer
Heart diseaseHeart disease
The ten leading causes of deathThe ten leading causes of deathin the United States during the last centuryin the United States during the last century
Cohen, M.L. Nature 2000, 406: 762-767
Increase inautoimmune diseases
Increase inautoimmune diseases
Increase inhost susceptibility
Restricted immunesystem stimulation
Selectivenutrition
Shelteredhousing
Lack ofparasites
Hygiene &sanitation
Clean food& water
New antigenexposure
Cohen, M.L. Nature 2000, 406: 762-767
Decrease ininfectious diseases
Decrease ininfectious diseases
Decrease inhost susceptibility
Decrease indisease transmission
Betternutrition
Betterhousing Antibiotics
Hygiene &sanitation
Safer food& water
Immunizations
The hygiene hypothesis:The hygiene hypothesis:an explanation for the rise in allergic and autoimmune diseases?an explanation for the rise in allergic and autoimmune diseases?
Epidemiological evidenceEpidemiological evidence
•• The prevalence of atopy (allergy) is directly related toThe prevalence of atopy (allergy) is directly related tosocioeconomic statussocioeconomic status and and educationeducation
Blackley 1873; Strachan 1989Blackley 1873; Strachan 1989
•• There is lower prevalence of atopy in There is lower prevalence of atopy in developing countriesdeveloping countriesISAAC 1998ISAAC 1998
•• There is a different prevalence of atopy in There is a different prevalence of atopy in WestWest vsvs EastEastGermanyGermany
von Mutius 1994von Mutius 1994
Incidence of diabetes(per 100,000)
24 (77-85)
9.4
7.3 (66-89)
8.05 (54-86)
11.5 (67-90)
19.9
10.9 (85-88)
7.7
19.7
35.8 (87-99)
13.7
Incidence of multiple sclerosis(per 100,000)
5.3
1.9 (56-74)
3.78
1.69 24
0.96 (mortality or incidence)
2.2
5
1.1
2.24
3.94
3.3
1.72
Environmental factorsEnvironmental factors
GenesGenes ImmuneImmunesystemsystem
SmokingSmoking
DietDiet
DrugsDrugs
Geography and social statusGeography and social status
StressStressMicrobes andMicrobes andenteric floraenteric flora
PermeabilityPermeability
AppendectomyAppendectomy
InflammatoryInflammatorybowel diseasebowel disease
Dietary changes and annual incidence rate of IBD in Japan
Crohn’s disease disease
Ulcerative colitis
Th2 Th1CTL
CTLTh1
CTLTh1
Th1 Th2 Th1
Th1Th1 Th2 Th1
Th2 Th1
Th2 Th1
Environmental allergens
Microorganisms
Vaccines
Infections
Standardimmunizations
Th1-promotingconditions CTL Th2 Th1
Th2 Th1
No response
No response
B. Adkins, 2003
Immune responses “imprinted” in early life are maintained into adulthood
Hygiene hypothesis:Hygiene hypothesis:postulated mechanismspostulated mechanisms
““CleanClean”” lifestylelifestyleMatricardi & BoniniClin Exp Allergy 2000““DirtyDirty”” lifestylelifestyle
High allergyLow allergy
Th1Th1 Th2Th2Hay feverHay fever
AsthmaAsthma
Atopic eczemaAtopic eczema
Food allergyFood allergy
Low microbialexposure
Weak immunestimulation
High microbialexposure
?
CrohnCrohn’’ssdiseasedisease
Strong immunestimulation
Yazdanbakhsh M. et al. Science 2002;296:490
Weak regulatory network Strong regulatory network
little Th1 Th2strong
Low IL-10 & TGF-β High IL-10 & TGF-β
Low High
The modified hygiene hypothesis: altered immune regulatory networks
Familial and genetic predisposition to IBD
StrongStronggeneticgenetic
influenceinfluence
WeakWeakgeneticgenetic
influenceinfluence
PsoriasisPsoriasis
MajorMajordepressiondepression
SchizophreniaSchizophrenia
IQIQ
Neurotic/Neurotic/extrovertextrovert
DiabetesDiabetes
AsthmaAsthma
CardiacCardiacdiseasedisease
CancerCancerMultipleMultiplesclerosissclerosis
Adapted from Chakravarti and Little, Nature 2003
Genetic associations in IBD
Genetic mutations associated with IBDGenetic mutations associated with IBD
CrohnCrohn’’s diseases diseaseChromosome 16 (IBD1): NOD2/CARD15 geneNOD2/CARD15 geneProtein is involved in bacterial recognition andapoptosis
Chromosome 5 (IBD5): OCTN geneOCTN geneProtein is an organic cation transporter
Inflammatory bowel diseaseInflammatory bowel diseaseChromosome 10: GLD5 geneGLD5 geneProtein is involved in epithelial integrity
Courtesy of Dr. M. Silverberg
HLAHLADRB1*1502 associated with UC in Jewish and Japanese populationsDRB1*0103 associated with UC and CD in non-Jewish population
NOD2/CARD15 (IBD1)NOD2/CARD15 (IBD1)No association with CD in Japanese or Chinese populationsDifferent mutation frequencies in Jews
OCTN (IBD5), DLG5OCTN (IBD5), DLG5No association with CD in the Japanese population
NRAMP1, ILNRAMP1, IL--1818Association with CD and UC, respectively, in Japanese patients
Ethnicity and IBD geneticsEthnicity and IBD genetics
LRRTM ?NBDCARD 1 CARD 2
1 121 129 217 270 299 570 744 1020 1040
N terminus C terminus
Apoptosis and Apoptosis and NFNF--κκB activationB activation
Oligomerization Bacterial recognitionBacterial recognition
R702W G908R 1007fs
STRUCTURE AND FUNCTION OF THE NOD2 PROTEIN
Influence of genetics on the response to enteric bacteriaInfluence of genetics on the response to enteric bacteria
EntericEntericfloraflora
NOD2NOD2 TRAF6TRAF6
II--κκBB
NFNF--κκBB
NFNF--κκB/IB/I--κκBB
TLR4TLR4
InflammationInflammation
CD14CD14
PeptidoglycanPeptidoglycan((Muramyl Muramyl dipeptide / MDP)dipeptide / MDP)
LPSLPS(Lipid A)(Lipid A)
No inflammationNo inflammation
VariantNOD2
GeneGene SNPSNP
NF-ΚBIA
NF-ΚB1
MTFHR
MEKK1
MEFV
MEFV
MEFV
MEFV
MDR-1
MDR-1
MCP1
MBL
MBL
MBL
IL-4
IL-4
IL-1RN
IL-1RNIL-1β
NFKBIA 3'UTR (G/A)
-94ins/delATTG
C677T Val(V)222Ala(A)
Asp643Asn (G/A)
P588P (C/T)
D510D (C/T)
Q476Q (G/A)
E474E (A/G)
G2677T/A/C
C3435T exon 26
G>A
Arg52Cys (C/T)
Gly57Glu (G/A)
Gly54Asp (G/A)
34 C>T
590C>T
+2073 C/T intron 2
+2018 T/C exon 2
3954 C/T TaqI
TUCAN TPMTTPMTTPMTTNFRSF1B=TNFR2 TNFRSF1B=TNFR2 TNF-αTNF-αTNF-αTLR9TLR4TLR4STAT6SOD2SLC11A1=NRAMP1PPAR-γPAI1OCTN, 22SLC22A5OCTN, 22SLC22A4NR1I2
Cys10StopA719G
G460A
G238C
C620TG593A
-238G/A
-308G/A
-857C/T
1237 T/CThr399Ile (C/T)
Asp299Gly (A/G) G2964A
Ala16Val T/C
allele 7Pro12Ala C/G
4G/5G(-207G/C)
1672C/T (L503F)
-25385C/TAGTCARD15CARD15CARD15CD14CD16A CSFR1DLG5EPXH1Fas ligandHSP70-2 IBD5 locusIBD5 locusIBD5 locusICAM1ICAM1IKBL IL-10IL-10IL-16IL-18
-6 A/G R702W C2104TG908R G2722C1007insC 3020insC-159T/CV158F (G/T)T2033A113G>A (R30Q) T612C Y113H(-843)C>TPstI 1267 A>G IGR2060a_1IGR2198a_1IGR3096a_1K469E (A/G) R/G241 (G/A)+738T/CG15R G43A(-1082) G>A(-295)T>CTCA/TCC codon35
GeneGene SNPSNP GeneGene SNPSNP
IL-1β -511A/C
IBDchip: a new tool to predict disease outcome?IBDchip: a new tool to predict disease outcome?
Courtesy of Dr. M. Sans
Fold
cha
nge
-20
0
20
40 Ulcerative colitisUlcerative colitis
Crohn's diseaseCrohn's disease
-20
0
20
40
Fold
cha
nge
II VIIIVIIIVIIVIIVIVIVVIVIVIIIIIIIIIIS. Chakravarti, 1999S. Chakravarti, 1999
GENE EXPRESSION PROFILES IN INFLAMMATORY BOWEL DISEASEGENE EXPRESSION PROFILES IN INFLAMMATORY BOWEL DISEASE
UPPER BOWELUPPER BOWEL(Stomach and Duodenum)(101-103 CFU/ml)Lactobacilli, Streptococci, Yeasts
SMALL BOWELSMALL BOWEL(Jejunum and ileum)(104-108 CFU/ml)Lactobacilli, Streptococci,Enterobacteriaceae,Bacteroides, Fusobacteria,Bifidobacteria
LARGE BOWELLARGE BOWEL (Colon)(1010-1012 CFU/ml)Bacteroides, Clostridia,Pseudomonas, Bifidobacteria,Streptococci, Lactobacilli,Veillonella, Protozoa, Yeasts,Fusobacteria, Proteus, Enterobacteriaceae,Staphylococci
Courtesy of C. De Simone
Different types and concentration of bacteria inDifferent types and concentration of bacteria inthe human gastrointestinal tractthe human gastrointestinal tract
Mucosal Mucosal immuneimmune
responseresponseEntericEnteric
floraflora
The enteric flora contains 2x10The enteric flora contains 2x101414
bacteriabacteria
Composed by over 2 billionsComposed by over 2 billionscells per gram of stoolcells per gram of stool
Constituted of over 400 speciesConstituted of over 400 species
About 80% of stool dry weightAbout 80% of stool dry weightis made up by bacteriais made up by bacteria
““PhysiologicalPhysiological”” intestinal inflammationintestinal inflammation
Ogawa H et al. Am J Ogawa H et al. Am J Physiol Physiol 2000; 279:G4922000; 279:G492
Effect of bacterial reconstitution on colon histologyEffect of bacterial reconstitution on colon histology
Germ freeGerm free
Specific pathogen freeSpecific pathogen free
3 day reconstituted3 day reconstituted
14 day reconstituted14 day reconstituted
Sampling of intestinal bacteria by mucosal dendritic cells
Rescigno M, et al. Nature Immunol 2:361, 2001
Mowat A.McI., Nature Rev 3:331, 2003
Do dendritic cell abnormalities underlieDo dendritic cell abnormalities underlieIBD pathogenesis?IBD pathogenesis?
TOLLTOLL--LIKE RECEPTORS AND THEIR LIGANDSLIKE RECEPTORS AND THEIR LIGANDS
TLRTLR--11TLRTLR--22
Han J & Ulevitch RJ. Nat Immunol 2005;12:1198
LRRLRR
TIRTIR
TLRTLRTLRTLR--22TLRTLR--66
TLRTLR--22TLRTLR--??
TLRTLR--33TLRTLR--??
TLRTLR--44TLRTLR--44
TLRTLR--55TLRTLR--??
TLRTLR--77TLRTLR--88
TLRTLR--99TLRTLR--99
LipoproteinsLipoproteinsLipoproteinsLipoproteins
zymosanzymosan GIPLsGIPLs Poly(I:C)Poly(I:C)LPSLPS
F proteinF protein FlagellinFlagellin ssRNAssRNA CpGCpG
MyD88MyD88 MalMal TRIFTRIF TRAMTRAM
Defensins: immune and non-immune functions
Selsted ME & Oulette AJ, Nat Immunol 2005;5:551LTA
LPS (TLR4) MDP (NOD2)
Bacteria CpG (TLR9)
T-cell, monocyte,mast cell chemotaxis
Dendritic cellrecruitment
and maturation
Neovascularization,wound closure
Mast celldegranulation
Epithelial cellproliferation
Defensins and otherantimicrobial peptides
NOD2 expression in terminal ileum crypt cells
NOD2
NOD2
NOD2
NOD2
NOD2
NOD2
Lysozyme
Lysozyme
Control
Ogura Y et al., Gut 2003;52:1591
Reduced Paneth cell α-defensins and antimicrobial activityin ileal Crohn’s disease
Wehkamp Wehkamp J et al. PNAS 2005; 102:18129J et al. PNAS 2005; 102:18129
The study of the flora is as challenging, but potentially asThe study of the flora is as challenging, but potentially asrewarding as the study of geneticsrewarding as the study of genetics
The flora plays a key role in most animal models of IBD, asThe flora plays a key role in most animal models of IBD, aswell as in humanswell as in humans
The flora of IBD patients differs from that of control subjectThe flora of IBD patients differs from that of control subjects,s,is is unstableunstable and shows and shows reduced diversityreduced diversity
Some Some E. coliE. coli and and B. B. vulgatusvulgatus may play a special detrimentalmay play a special detrimentalrole in IBDrole in IBD
Characteristics of the enteric flora in IBDCharacteristics of the enteric flora in IBD
Adapted from:Adapted from: Marteau Marteau P. et al., AlimentP. et al., Aliment Pharmacol Ther Pharmacol Ther 2004; 20 (2004; 20 (SupplSuppl.):18.):18--2323
Is an IBD infectious agent like Waldo in a crowd?
ANIMAL MODELS OF IBDANIMAL MODELS OF IBDSpontaneousSpontaneous InducedInduced
Administration of Administration of Gene targeting:Gene targeting: Cell transfer intoCell transfer intoexogenous agents knock out/transgenic immunodeficienexogenous agents knock out/transgenic immunodeficient animals t animals
•• ILIL--2 2 --//--•• ILIL--2R2Rαα chain chain --//--•• ILIL--10 10 --//--•• TGFTGFββ --//--
•• TCRTCRαα --//--
•• Keratin 8 Keratin 8 --//--•• GGααii22 --//--
•• HLA B27 tgHLA B27 tg•• ILIL--7 tg7 tg•• mdr1a mdr1a --//--•• Stat 4 tg Stat 4 tg •• CRF2CRF2--4 4 --//--•• MfMf--PMN Stat 3 PMN Stat 3 --//--•• WASP WASP --//--•• TNFTNFΔΔAREARE
•• EE--cadherin tgcadherin tg
•• CD4+ CD45RBCD4+ CD45RB hihi into into scidscid or or Rag Rag --//-- micemice
•• Bone marrow intoBone marrow intoTgTgεε26 mice26 mice
•• Cotton top tamarinCotton top tamarin
•• C3HC3H--HeJBir HeJBir mousemouse
•• SAMP1/SAMP1/Yit Yit mouse mouse
EnemaEnema•• TNBS TNBS •• Oxazolone Oxazolone •• Acetic acidAcetic acid•• Immune complexImmune complex
OralOral•• IndomethacinIndomethacin•• CarageenanCarageenan•• DSSDSS
SubcutaneousSubcutaneous•• Cyclosporin ACyclosporin A
IntracolonicIntracolonic•• PGPG--PSPS
Blumberg R et al. Blumberg R et al. Curr Curr Op Op Immunol Immunol 1999: 11:6481999: 11:648
Courtesy of Dr. R.B. Sartor
Conventional environment
Germfree environment
HLA-B27 transgenic ratmodel of colitis
Completely different and independent defects can result in IBD
The genetic background influences themanifestations and severity of IBD
Most models fail to develop IBD in agermfree environment
Components of the normal flora arenecessary to develop IBD
Lessons from animal models of IBD
Low mucosal bacterial concentration (300 cfu/μl)
Swidsinski A et al. Gastroenterology 2002;122:44High mucosal bacterial concentration (72000 cfu/μl)
Density of enteric bacteriain human mucosal biofilm
Control
Crohn’s disease
Mucosal bacteria concentration is related to severity of gut inflammation
0
20
40
60
80
% p
atie
nts
100
Asymptomaticcontrols
Self-limitedcolitis
Indeterminatecolitis
Ulcerativecolitis
Crohn’sdisease
0 - < 1000 cfu/μl 1000 - < 10000 cfu/μl 10000 - < 50000 cfu/μl > 50000 cfu/μl
Swidsinski A et al. Gastroenterology 2002; 122:44
DD’’ Haens Haens G et al., Gastroenterology 114:262, 1998G et al., Gastroenterology 114:262, 1998
Recurrence of ileal CrohnRecurrence of ileal Crohn’’s diseases diseasebefore and after infusion of intestinal contentsbefore and after infusion of intestinal contents
Courtesy of Dr. L.Courtesy of Dr. L. DielemanDieleman
Loss of tolerance to autologous enteric flora in IBDLoss of tolerance to autologous enteric flora in IBD
Proliferative response (Proliferative response (CPMCPM))MediumMedium PHAPHA AnaerobicAnaerobic AerobicAerobic
CD activeCD activePMBCPMBC 317317 1677416774 15641564 16511651LPMC LPMC -- 268268 1702917029 18521852 16711671LPMC +LPMC + 276276 2344923449 1755317553 1546715467
UC activeUC activePMBCPMBC 700700 2505425054 995995 11011101LPMC LPMC -- 479479 2604826048 579579 589589LPMC +LPMC + 276276 2810628106 1635616356 1533715337
CD inactiveCD inactivePMBCPMBC 270270 1153911539 274274 302302LPMCLPMC 574574 1497914979 689689 800800
UC inactiveUC inactivePMBCPMBC 417417 1605416054 700700 894894LPMCLPMC 482482 1713217132 779779 808808
ControlsControlsPMBCPMBC 10481048 3030530305 474474 396396LPMCLPMC 942942 2448124481 751751 710710
Duchmann Duchmann R et al., R et al., Clin Clin ExpExp Immunol Immunol 102:448, 1995102:448, 1995
““PhysiologicalPhysiological””inflammationinflammation
ToleranceTolerance
Normal mucosaNormal mucosa
Persistent pathologicalPersistent pathologicalinflammationinflammation
Loss of toleranceLoss of tolerance
IBD mucosa
Result of hostResult of host--enteric flora interactionsenteric flora interactions
Beneficial interactionBeneficial interaction Detrimental interactionDetrimental interaction
““PhysiologicalPhysiological””inflammationinflammation
PathologicalPathologicalinflammation (IBD)inflammation (IBD)
BacterialBacterialmodulationmodulation
AntibioticsAntibiotics ProbioticsProbiotics
Evidence implicating the gut flora in theEvidence implicating the gut flora in thepathogenesis of IBDpathogenesis of IBD
Occurrence of IBD lesions in gut segments with the Occurrence of IBD lesions in gut segments with the highest concentrations of bacteriahighest concentrations of bacteria
Increased numbers of bacteria in the mucosa of IBDIncreased numbers of bacteria in the mucosa of IBDpatientspatients
Beneficial effect of fecal stream diversion in preventingBeneficial effect of fecal stream diversion in preventingCD and recurrence upon restoration of fecal flowCD and recurrence upon restoration of fecal flow
Attenuation of IBD by antibiotics and probioticsAttenuation of IBD by antibiotics and probiotics
Immunological reactivity against bacterial antigens inImmunological reactivity against bacterial antigens inIBD patients IBD patients
Patterns of intestinal immune responsesPatterns of intestinal immune responses
A. Levine, 2002
Acute inflammation Acute inflammation (Host defense)(Host defense)
Pathologic/uncontrolled inflammation (IBD)
Physiologic/”controlled”inflammation
Return to Return to tolerancetolerance
Loss of Loss of tolerancetolerance
Food Flora
Increased antibody production in IBD mucosa
Serum or mucosal antibodies against autoantigens:
Intestinal epithelial cell antigens• Epithelial cell-associated components (ECAC)
• 40 KD/human tropomyosin fraction 5 (hTM5)
Neutrophil antigens• pANCA
Other antigens• Lymphocytic, pancreatic, cardiolipin
Humoral immunity in IBD
J Clin Invest 1985;76:311-318
IgG eluted from colons of UC but IgG eluted from colons of UC but not CD patientsnot CD patients
recognizes a 40 Kd protein recognizes a 40 Kd protein
Expression of 40 Kd/human Expression of 40 Kd/human tropomyosin 5 (hTM5) in tropomyosin 5 (hTM5) in
colon and extracolon and extra--intestinal organsintestinal organs
Dig Dis Sci 1999;44:1-13
Skin
Colon
Ciliary processChondrocytes
Bile ducts
40 KD
Gut 1993;34:650-7
Co-localization of 40 KD, IgG1, and C3b in UC colonic mucosa
Serum antibodies against bacterial antigens:
Saccharomyces cerevisiae (Baker’s yeast) (ASCA)
I2 (Pseudomonas fluorescens-associated
sequence)
Outer membrane porin C of E. coli (OmpC)
Bacterial flagellins (CBir-1)
Humoral immunity in IBD
Increased frequency and titers ofanti-flagellin antibodies in Crohn’s disease
Targan SR et al. Gastroenterology 2005; 128:2020
Enhanced cell-mediated immunity to bacterial antigens in Crohn’s disease
Pirzer U et al. Lancet 1991; 338: 1238
ControlControln = 31n = 31
CrohnCrohn’’s s small bowel small bowel remissionremission
n = 17n = 17
CrohnCrohn’’s s small small bowel bowel
relapserelapsen = 14n = 14
CrohnCrohn’’s s large bowel large bowel remissionremission
n = 25n = 25
CrohnCrohn’’s s large bowel large bowel
relapserelapsen = 26n = 26
Ulcerative Ulcerative colitis colitis
remissionremissionn = 23n = 23
Ulcerative Ulcerative colitis colitis
relapserelapsen = 15n = 15
Distal Distal colitis colitis
relapserelapsen = 6n = 6
>10>10
2020
3030
4040
5050100100150150
TNF
TNF
∝∝(p
g/m
l)(p
g/m
l)
Elevated serum TNFElevated serum TNF--αα concentrations in children concentrations in children with active IBDwith active IBD
Murch SH et al. Gut 1991, 32:913
Elevated stool TNFElevated stool TNF--αα concentrations in children concentrations in children with active IBDwith active IBD
ControlControl DiarrheaDiarrhea ActiveActiveCrohnCrohn’’ss
Inactive Inactive CrohnCrohn’’ss
ActiveActiveUCUC
Inactive Inactive UCUC
1010
100100
10001000
1000010000
TNF
TNF --
αα(p
g/g
stoo
l)(p
g/g
stoo
l)
Braegger CP et al. Lancet 1992, 339:89
F. Pallone, 2002
POLARIZATION OF IMMUNE RESPONSES IN IBDPOLARIZATION OF IMMUNE RESPONSES IN IBD
Th1Th1 (IL(IL--12, IL12, IL--18, IFN18, IFN--γγ))
Th2Th2 (IL(IL--4, IL4, IL--5, IL5, IL--13)13)
Th1Th1 (IL(IL--2, IFN2, IFN--γγ))
““Th2Th2””(IL(IL--4, IL4, IL--5, IL5, IL--13)13)
UlcerativeUlcerativecolitis (UC)colitis (UC)
CrohnCrohn’’ssdisease (CD)disease (CD)
F. Pallone, 2002
POLARIZATION OF IMMUNE RESPONSES IN IBDPOLARIZATION OF IMMUNE RESPONSES IN IBD
Th1Th1 (IL(IL--12, IL12, IL--18, IFN18, IFN--γγ))
Th2Th2 (IL(IL--4, IL4, IL--5, IL5, IL--13)13)
Th1Th1 (IL(IL--2, IFN2, IFN--γγ))
““Th2Th2””(IL(IL--5, IL5, IL--13)13)
UlcerativeUlcerativecolitis (UC)colitis (UC)
CrohnCrohn’’ssdisease (CD)disease (CD)
Fuss IJ, et al. J Clin Invest 2004;113:1490
Distinct mucosal cytokine profiles in control, CD and UC patients
IL-1
3
IFN
-γIL
-4IL-5
0
50
100
150N
umbe
r of T
- ce l
lsX 1
04
0 1 2 3 4 5Weeks in culture with IL-2
Ulcerative colitis
Chronic nonspecific colitis
Indeterminate colitisCrohn’s disease
Normal control
Differential proliferation of biopsyDifferential proliferation of biopsy--derived Tderived T--cells incells invarious forms of intestinal inflammationvarious forms of intestinal inflammation
Differential proliferation and apoptosis ofnormal, Crohn’s disease and ulcerative colitis mucosal T-cells
ControlCDUC
0
50
100
150
200
250
300**
* *
**
*
*
*
*
Unstimulated CD2 CD3
*
*
*
*
*
0
20
40
60
* *
*
% a
popt
osis
CPM
X103
Sturm A, et al. Gut 2004;53:1624
DiDi Sabatino Sabatino A, et al. Gut 2004; 53:70A, et al. Gut 2004; 53:70
Increased numbers of apoptotic cells in the lamina propriaIncreased numbers of apoptotic cells in the lamina propriaof infliximabof infliximab--treated Crohntreated Crohn’’s disease patientss disease patients
BeforetreatmentBeforeBefore
treatmenttreatmentAfter 10 week
treatmentAfter 10 weekAfter 10 week
treatmenttreatment
Crohn’s disease patientsCrohnCrohn’’s disease patientss disease patients Control patientsControl patientsControl patients
Enteric floraEnteric flora
T cell activationT cell activation
ProliferationProliferation
ApoptosisApoptosis
Defective apoptosisDefective apoptosis
CrohnCrohn’’s diseases disease
ProliferationProliferation ApoptosisApoptosis
T cell activationT cell activation
Increased apoptosisIncreased apoptosis
Controlled inflammationControlled inflammation
T cell activationT cell activation
ProliferationProliferation ApoptosisApoptosis
Normal apoptosisNormal apoptosis
Physiological inflammationPhysiological inflammation
PathogensPathogensDietary antigensDietary antigens
Physiological Physiological intestinal intestinal
inflammationinflammation
Controlled IFNControlled IFN--γγ and TNFand TNF--ααproductionproduction
TGFTGF--ββpp--SMAD3SMAD3
SMAD7SMAD7
Healthy subjectsHealthy subjects
Excessive IFNExcessive IFN--γγ and TNFand TNF--ααproductionproduction
TGFTGF--ββpp--SMAD3SMAD3SMAD7SMAD7
Pathological Pathological intestinal intestinal
inflammationinflammation
CD patientsCD patients
Environmental factors (Enteric Environmental factors (Enteric flora, food, smoking, etc.)flora, food, smoking, etc.)
Intestinal immune Intestinal immune responseresponse
Genetic factors (NOD2 and other Genetic factors (NOD2 and other mutations)mutations)
Impaired suppressor activity of TGFImpaired suppressor activity of TGF--ββ in IBDin IBD
Marks DJB et al., Lancet 2006;367:668Marks DJB et al., Lancet 2006;367:668
Impaired neutrophil accumulation and ILImpaired neutrophil accumulation and IL--8 production8 productionin acutely injured Crohnin acutely injured Crohn’’s disease mucosas disease mucosa
Marks DJB et al., Lancet 2006;367:668Marks DJB et al., Lancet 2006;367:668
Impaired erythema, swelling and blood flowImpaired erythema, swelling and blood flowupon upon E. coliE. coli injection in Crohninjection in Crohn’’s diseases disease
ControlControl
CrohnCrohn’’s diseases disease
ControlControl
Leukocyte-endothelial interactions: molecular locks and keys
E n d o t h e l i a l c e l l
E n d o t h e l i a l c E n d o t h e l i a l c e l l e l l
T h e l o c k:T h e l o c k:ce l l a d h e s i o n m o l e c u l ece l l a d h e s i o n m o l e c u l e
T h e k e y:T h e k e y:c e l lc e l l--b o u n d / s e c r e t e d l i g a b o u n d / s e c r e t e d l i g a
n dn d
CD
UC
CD
NL
Increased angiogenesis in IBD mucosa
Danese S et al. Gastroenterology 2006 (in press)Danese S et al. Gastroenterology 2006 (in press)
Adh
eren
t MO
LT4
cells
/ m
m2
0
500
1000
1500
2000
2500
Unst. IL-1β LPS TNF-α IL-4 IFN-γ0
500
1000
1500
2000
2500
Unst. IL-1β LPS TNF-α IL-4 IFN-γ
CONTROL IBD
Increased leukocyte adhesiveness of IBD microvasculatureIncreased leukocyte adhesiveness of IBD microvasculature
Stress!!!Stress!!!
NeuroNeuro--endocrineendocrineresponseresponse
MucusMucus
MucosalMucosalimmune systemimmune system
activationactivationBowel inflammationBowel inflammation
Enteric floraEnteric floraDietary antigensDietary antigens
PermeabilityPermeability
Collins SM, Am J Collins SM, Am J Physiol Physiol 280:G318, 2001280:G318, 2001
Potential contribution of stress to IBD pathogenesisPotential contribution of stress to IBD pathogenesis
Evolution of IBD therapy:Evolution of IBD therapy:the result of understanding IBD pathogenesisthe result of understanding IBD pathogenesis
19401940’’ss SulfasalazineSulfasalazine19501950’’ss Cortisone, ACTHCortisone, ACTH19601960’’ss Azathioprine Azathioprine 19701970’’ss 55--ASAASA19801980’’ss 66--MP, metronidazole, MP, metronidazole,
elemental dietselemental diets19901990’’s (early)s (early) Cyclosporine, budenoside, Cyclosporine, budenoside,
methotrexate, antibioticsmethotrexate, antibiotics19901990’’s (late)s (late) Biologicals, probiotics,Biologicals, probiotics,
leukapheresisleukapheresis20002000’’ss Combination therapiesCombination therapies
Chronic gut inflammation: Chronic gut inflammation: imbalance of immune mediatorsimbalance of immune mediators
ProPro--inflammatoryinflammatory
AntiAnti--inflammatoryinflammatory
TNF-αTNF-αIL-1βIL-1β IL-8IL-8 IL-12IL-12
IFN-γIFN-γ TGF-βTGF-βIL-10IL-10
IL-1raIL-1raIL-4/13IL-4/13
TNF-αTNF-α IL-1βIL-1β IL-8IL-8 IL-12IL-12 IFN-γIFN-γ TGF-βTGF-β IL-10IL-10IL-1raIL-1raIL-4/13IL-4/13
Recombinant cytokineRecombinant cytokine--basedbasedtherapy for IBDtherapy for IBD
ProPro--inflammatoryinflammatory AntiAnti--inflammatoryinflammatory
ProPro--inflammatoryinflammatory AntiAnti--inflammatoryinflammatory
AntiAnti--cytokine antibodycytokine antibody--basedbasedtherapy for IBDtherapy for IBD
TNF-αTNF-α IL-1βIL-1β IL-8IL-8 IL-12IL-12 IFN-γIFN-γ TGF-βTGF-β IL-10IL-10IL-1raIL-1raIL-4/13IL-4/13
Dis
ease
act
ivity
inde
xD
isea
se a
ctiv
ity in
dex
0
1
2
3
4
5
6
1.0
ILIL-- 1
2 p4
0 12
p40
(ng/
ml)
(ng/
ml)
1.5
2.0
2.5
0.5
0
IFN
IFN -
- γγ(n
g/m
l)(n
g/m
l)
10
15
20
0
5
Switch in cytokine profiles (Th1Switch in cytokine profiles (Th1 Th2) in ILTh2) in IL--1010--//-- micemiceduring the clinical course of colitisduring the clinical course of colitis
0
100
200
300
ILIL-- 4
(pg
/ml)
4 (p
g/m
l)
PrePre EarlyEarly LateLate0
100
200
300
ILIL-- 1
3 (p
g/m
l)13
(pg
/ml)
PrePre EarlyEarly LateLate
PrePre EarlyEarly LateLate PrePre EarlyEarly LateLate PrePre EarlyEarly LateLate
Prolonged response to infliximab therapy in earlyProlonged response to infliximab therapy in earlybut not late pediatric Crohnbut not late pediatric Crohn’’s diseases disease
0
25
50
75
100Fr
actio
nal r
emis
sion
Weeks following infliximab infusion20100 30
Kugathasan S. et al. 1999
40
EarlyEarly(n=6)(n=6)
LateLate(n=6)(n=6)
CrohnCrohn’’s disease and ulcerative colitis share epidemiological ands disease and ulcerative colitis share epidemiological andclinical features, but represent distinct entities with uniqclinical features, but represent distinct entities with uniqueuemechanisms of inflammation in each conditionmechanisms of inflammation in each condition
Environmental changesEnvironmental changes, , genetic predispositiongenetic predisposition, the , the entericentericcommensal floracommensal flora, and the , and the mucosal immune responsemucosal immune response are the keyare the keycomponents of IBD pathogenesiscomponents of IBD pathogenesis
Loss of immune toleranceLoss of immune tolerance against the autologous enteric floraagainst the autologous enteric floraappears to be a central event in IBD pathogenesis, and appears to be a central event in IBD pathogenesis, and
modulationmodulationof the floraof the flora and/or the hostand/or the host’’s s immune responseimmune response against it seemagainst it seemessential to control gut inflammationessential to control gut inflammation
Current biological therapies are a direct result of an improveCurrent biological therapies are a direct result of an improveddunderstanding of IBD pathogenesis, and further progress understanding of IBD pathogenesis, and further progress willwillcontinuecontinue to generate to generate better formsbetter forms of therapyof therapy
Summary and ConclusionsSummary and Conclusions