animal models of ibd - falk aktuell · ibd models • many models have early defects in iec •...
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Moskau 6/2006
Animal models of IBD
Markus F. NeurathI. Medical Clinic
Johannes Gutenberg-University Mainz, Germany
Moskau 6/2006
MurineMurine modelsmodels of IBD 2006of IBD 2006
1. Spontaneous ModelsCotton top tamarin, C3HBir Maus, SAMP/Yit
2. Inducible models/ Hapten reagentsDSS, PG-PS, Oxazolone, acetic acidTNBS, DNBS
3. Adoptive transfer modelsCD45RBhigh model, CD62L+ model, Tgepsilon tg mouse
4. Genetically engineered modelsTransgenic mice: TGFbeta-dom.neg.RII, STAT4, IL-7Knockout mice: IL-2, IL-10, TCR, MDR1, NF-kappaBp50, DARE MausConditional Knockout mice: STAT3 in macrophages
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Mouse models of colitis
Model Pathology Site Pathogenesis/Remarks Costs Spontaneous model SAMP/Yit chronic ileum activation of Th1 T cells towards luminal high antigens in the ileum only Inducible models DSS acute, chronic colon toxic intestinal damage plus activation low mainly mucosal of the mucosal immune system (TH1/TH2)/ addition of azoxymethan induces cancer TNBS acute, chronic colon activation of the immune system (TH1-me- low transmural diated; hapten-specific)/ optimization of TNBS dosage required, only certain strains Adoptive transfer model CD45RB CD62L+ acute, chronic colon, duodenum IL-12 driven TH1 T cells/ induction of colitis high cell transfer into transmural requires 6-12 weeks, appropr. animal facility SCID mice for SCID mice required Genetically eng. model IL-2 knockout mice acute, chronic colon IFN-gamma producing T cells/ colitis 6-15 medium mucosal weeks after birth, variability between mice STAT-4 transgenic acute, chronic colon, ileum TH1 T cells in response to bacterial antigens/ medium transmural requires immunization of mice
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Grossly normal mucosaMethylene blue stained grossly normal mucosa
ReadoutReadout parametersparameters in in murinemurine modelsmodels of IBD of IBD
-weight curves-Histology-Cell isolation: cytokine, FACS, proliferation/apoptosis-endoscopy
Becker et al. Immunity 2004, Gut 2005
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PathogenesisPathogenesis of of mousemouse modelsmodels
1. Bacterial floraGermfree mice fail to develop colitis (Sadlack 1994)Monoassociation with certain strains induces colitis (Rath 1998)Antibiotics suppress colitis activity (Herfarth 1998)T cells in the lamina propria react to their autologouscommensal flora (Duchmann 1996)Adoptive transfer of flora specific T cells induces colitis(Cong 1998)
2. Key cellular playersMacrophages (STAT3 conditional KO)T lymphocytes (adoptive transfer, T cell depletion)- CD4+ - CD8+ -TregB lymphocytes (NFkappaB p50 KO)Epithelial cells
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A key role for IEC in the onsetof ileitis in SAMP/Yit mice
Olson et al. JEM 2006
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Changes in claudin2 and occludin in SAMP/Yit mice
Olson et al. JEM 2006
Occludinileum
Occludincolon
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Epimorphin controls crypt proliferation
• Epimorphin regulates epithelial mesenchymalinteractions
• Epimorphin deficient mice were generated• This protein regulates crypt cell proliferation both in the
small and large intestine• Prolieration is controlled via activation of BMP/TGF-beta
Wang et al. J. Clin. Invest. 2006
WT KO
Moskau 6/2006Wang et al. J. Clin. Invest. 2006
Crypt proliferation protectsfrom experimental colitis
WT KO
WT KO
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Communication between bacteria and the mucosal immune system via DCs
Lumen
Becker et al. JCI 2003, Mac Pherson Science 2003, Konrad Gastro 2006
Hyporesponsive-ness, MucosalCompartmentation,Tolerance
CD11c RNA
CD11c
Dendritic cells
Dendritic cells: Key players in gut inflammation
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Intestinal and MLN dendriticcells regulate colitis activity
Annacker et al. JEM 2006
CD8alpha+, CD11b+ CD11c+ CD103+
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Basic concepts in IBD animal models
IL-5, IL-13
TGF-βcirculation
Dendritic cells
TTH1
CD4
CD4TH2 TH3
Antigens
Pathogenic Protective
TNF, IFN-γ
CD4
TH1
TH1 and Th2 cytokines Tr1 and Th3 cytokines
Tr1
Treg
IL-10
contact
FoxP3
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Immunopathogenesis of colitis:Decision making by APCs/dendritic cells
TT
T
T helper 1
TT
T
IL-4/5, IL-13
Low IL-12High EBI3
BacterialAntigens
DendriticCells
T
High IL-12Low EBI3
TT
T
Nieuwenhuis et al. PNAS 2003
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Anti-IL-12 p40 antibodies induce T cell apoptosis
IL-12R
β1
STAT-3STAT-4
NaiveT cells IL-23R
β1
STAT-3STAT-4STAT-5
p40p35
p40p19
MemoryT cells
β2
Anti p40 antibodies suppress established Th1 colitis via induction of T cell apoptosis(JEM 1995, Gastro 1999)
P19 transgenic mice exhibit multiorgan inflammation including GI tract(JI 2001 166 7563)
P19 but not p35 knockout mice fail to develop EAE (Nature 2003 421 744)
T cell activation/survival
Th1 T celldifferentiation
IL-12 IL-23
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Healthyp19
Colitisp19
IL-23 is expressed in lamina propria DCs of mice with transfer colitis
ColitisCD11c
ColitisCD11cp19
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Generation of IL-23 p19 knockin mice
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wei
ght i
n %
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0 1 2 3 7 8 9 10
IL-23-p19 KOWT
wei
ght i
n %
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85
90
95
100
105
0 1 2 3 4 5 6 7 8
IL-23-p19 HETWT
IL-23-p19 KO
DSS
IL-23 KO mice are highly susceptible to inducible colitis models
TNBS
daysdays
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2
4
6
8
10
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Wild-type IL-23 KO
endo
scop
ic s
core
Wild-type Wild-type
IL-23 KO IL-23 KO
IL-23 KO mice are highly susceptible to TNBS induced colitis
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0
1
2
3
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5
wt KOhi
stol
ogic
sco
re
Wild-type
IL-23 KO
IL-23 KO mice are highly susceptible to TNBS induced colitis
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IL-6
0100200300
400500600
IL-12
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050
100150200250300350400450
0 h 1 h 2 h 4 h 8 h 16 h
WTIL-23p19 KO
0100200300400500600700800
WTIL-23p19 KO
0 h 1 h 2 h 4 h 8 h 16 h
pg/ m
lpg
/ ml
IL-12 p35
IL-12 p40
WTp19 KO
WTp19 KO
Crossregulation of IL-12 p35 and p40 expression by IL-23 p19
US LPS CpG
US LPS CpG
LPS
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PBS
anti IL-12 p40
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wei
ght i
n %
anti-IL-12p40control
0 1 2 3 4 5 6 7 8 11 12 13 14 15 16
0 5 10 15 200,00
0,25
0,50
0,75
1,00
PBS
anti-p40
Days
Surv
ival
Anti-IL-12 therapy rescues p19knockin mice from lethal colitis
P19 knockin mice/ TNBS
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A A rolerole forfor bothboth ILIL--12/IL12/IL--23 in 23 in CrohnCrohn´́ss diseasedisease
TT
T
Crohn´sdisease
T helper 1
Bacterialantigens
LPDC
p35/p40(IL-12)
Memory responsesPerpetuation
p19/p40(IL-23)
T cell survival
T helper IL-17
T TT
IEC
perpetuates
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IL-23 p19 controls colitisactivity in IL-10 KO mice
Yen et al. J. Clin. Invest. 2006
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IL-23 induces a T cell subsetproducing IL-6 and IL-17
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Blocking IL-6 and IL-17in experimental colitis
Yen et al. J. Clin. Invest. 2006
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IL-4
IL-12
TGF-βnaive
TH1
TH2
iTreg
THIL17+
-
IL-6IL-1β
IL-23stabilization
A A role for role for ILIL--12/IL12/IL--23 in 23 in colitis colitis ??
IL-23activation
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Anti-IL-12 p40 antibody therapy in Crohn´s disease
Mannon et al. New Engl. J. Med. 2004
• 79 patients with active Crohn´s disease (CDAI 220-450)• Randomisation: Placebo or 7 weeks 1 or 3mg/kg ABT874 s.c.:
Induction of T cell apoptosis
remissionresponse
0
20
40
60
%
7 Wks 12 Wks 7 Wks 12 WksKO AB AB AB ABKO KO KO
AB = AntibodyKO = control** = p< 0.001
**
**
****
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Induction of T cell apoptosisvia mTNF
Van den Brande et al. Gastroenterology 2003
Matsuoka et al.Gastroenterology 2005
SerSerSer
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Blockade of the IL-6/ sIL6R system induces apoptosis in colitis
Atreya Nat. Med. 2000
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Blocking the IL-6 signaling pathwayin Crohn´s disease
Ito et al. Gastroenterol. 2004
before MRA after MRA
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T cell apoptosis
Azathioprine/ Rac1
Clinical therapy of IBD and T cell apoptosis
Induction of T cell apoptosis(cell death)
rapid
delayed
Mitoma et al. 2005, Ten Hove et al. 2002, Tiede et al. 2003
Anti-TNF/IL-6R antibodies
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IBD no 6-MP IBD 6-MP (n=13) IBD 6-MP (n=5) (n=7) responder non responder
Tiede et al. J. Clinical Invest. 2003
6-MP responsiveness correlates with the induction of apoptosis
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A molecular mechanism of action of azathioprine
CD28
Rac1/2
IκΒ
NF-κΒ
Bcl-xL
antiapoptotic signalInduction of apoptosis
Rac-GTP(active)
ERM
T cell-APC conjugationInhibition of T cell-APC conjugates
-thioGTP
STAT3
azathioprinevav
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Rac1-GDP Rac1-GTP
Vav
Vitality, T cell-APC conjugation
6-Thio-GTP
Apoptosis, Inhibition of T cell-APC conjugation
Rac1-Thio-GDP Rac1-Thio-GTP
CD28
Azathioprine blocks vavactivity on Rac1
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Steric modelling of ThioGTP
Cyan = switch 1 region of Rac1 (residues27-35) Magenta = switch 2 region of Rac1 (residues 59-71)Yellow = contact area of vav1 with Rac1
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Risk factors for cancer: duration and extent of disease, flares, PSC
Intraepi-thelial Neo-plasia
Cancer
Flat neoplasia
Ulcers
Ulcerative colitis and colon cancer
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Chronic intestinal inflammationfavours cancer development
Berg et al. JCI 1998Spencer Gastroenterology 2002
IL-10 knockout Colitis
Early TH1 (IFN-gamma) ColitisLate TH2 Colitis with colon cancer
CD4+
CD8+
Early phase
Late phase
Colon cancer
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D0 D80
DSS DSS DSS
D20 D40 D60
Tumorigenesis in the AOM/DSS model
AOM
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Control
antibodyAnti-IL-6R Ab
IL-6 blockade suppressestumorigenesis in the DSS/AOM model
Becker et al. Immunity 2004
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WT UntreatedWT AOM/DSS treated
Shift from mIL-6R expression towards sIL-6R during tumorigenesis
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Blockade of the sIL-6R suppresses tumorigenesis
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How does colitis drive tumor growth ?
TGFβ
sIL-6R
Growth
IL-6
IL-6/sIL-6R DC
T
TACE
EGFR Ligands
mIL-6R
Bacteria
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Summary
• There are numerous experimental IBD models• There is no ideal model: the choice of the model depends on the
questions of the investigators• Pathogenesis: The intestinal flora drives gut inflammation in most
IBD models• Many models have early defects in IEC• Additional key players are DCs and T cells• The data have important implications for designing novel
therapeutic approaches for IBD: in particular, targeting of cytokine signal transduction and T cell apoptosis emerge as important approaches for therapy of IBD
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Ralf Kiesslich, Martin HoltmannJonas Mudter, Raja AtreyaKai Hildner, Thanka Nadar.Con Schneider, Jürgen SieblerClemens Neufert
Jan Schmidt Richard BlumbergStefan Rose-JohnRadovan DvorskyHans A. LehrJeff MolkentinManolis PasparakisGerhard FritzXose Bustelo
CooperationsClinic
Lab
Imke Tiede, Christoph BeckerDaniela Poppe, Stefan WirtzBenno Weigmann, Alexej NikolaevBrigitte Bartsch, Massimo FantiniUte Teichgräber
Thank you