IL-12 family members in experimental colitis. Markus F. Neurath I. Medical Clinic Johannes Gutenberg-University Mainz, Germany

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1 IL-12 family members in experimental colitis Markus F. Neurath I. Medical Clinic Johannes Gutenberg-University Mainz, Germany

2 IBD - Pathogenesis Genetic Predisposition Bacterial Antigens Activation of the Mucosal Immune System Th1 some Th2 NK T FDG-PET Crohn s disease Ulcerative colitis Neurath et al. Nature Med. 2002

3 Immunopathogenesis of IBD: Decision making by APCs/dendritic cells Bacterial Antigens High IL-12 Low EBI3 T T T T helper 1 Crohn s disease T T T Dendritic Cells Low IL-12 High EBI3 T IL-5, IL-13 Ulcerative colitis

4 Anti-IL-12 p40 antibodies induce T cell apoptosis IL-12 Naive T cells p40 p35 IL-12R IL-23 Memory T cells p40 p19 IL-23R Anti p40 antibodies suppress established Th1 colitis via induction of T cell apoptosis (JEM 1995, Gastro 1999) β1 β2 STAT-3 STAT-4 Th1 T cell differentiation β1 STAT-3 STAT-4 STAT-5 T cell activation/ survival P19 transgenic mice exhibit multiorgan inflammation including GI tract (JI ) P19 but not p35 knockout mice fail to develop EAE (Nature )

5 A role for IL-12/IL 12/IL-2323 in Crohn s s disease? - IL-12 TH1 T-bet naive TGF-β IL-4 TH2 GATA3 itreg FoxP3 + IL-6 IL-1β TH IL17 RORgt IL-23 stabilization

6 IL-23 is expressed in lamina propria DCs of mice with transfer colitis Healthy p19 Colitis CD11c Colitis p19 Colitis CD11c p19

7 Generation of IL-23 p19 knockin mice

8 IL-23 KO mice are highly susceptible to inducible colitis models TNBS 105 DSS weight in % WT IL-23-p19 KO days weight in % WT IL-23-p19 HET IL-23-p19 KO days

9 IL-23 KO mice are highly susceptible to TNBS induced colitis Wild-type Wild-type endoscopic score Wild-type IL-23 KO IL-23 KO IL-23 KO

10 IL-23 KO mice are highly susceptible to TNBS induced colitis Wild-type 5 IL-23 KO histologic score wt KO

11 Crossregulation of IL-12 p35 and p40 expression by IL-23 p19 pg/ ml pg/ ml IL-6 WT p19 KO US LPS CpG IL-12 WT p19 KO US LPS CpG IL-12 p35 WT IL-23p19 KO 0 h 1 h 2 h 4 h 8 h 16 h LPS IL-12 p40 WT IL-23p19 KO 0 h 1 h 2 h 4 h 8 h 16 h

12 Anti-IL-12 therapy rescues p19 knockin mice from lethal colitis 105 P19 knockin mice/ TNBS 100 weight in % control 70 anti-il-12p PBS Survival 1,00 0,75 0,50 0,25 anti-p40 PBS 0, Days anti IL-12 p40 Becker et al. Cutting Edge JI 2006

13 A role for both IL-12/IL 12/IL-2323 in Crohn s s disease p35/p40 (IL-12) T helper 1 TT T Memory responses Perpetuation Bacterial antigens p19/p40 (IL-23) perpetuates LPDC T helper IL-17 T T T Crohn s disease T cell survival IEC

14 Anti-IL-12 p40 antibody therapy in Crohn s disease 79 patients with active Crohn s disease (CDAI ) Randomisation: Placebo or 7 weeks 1 or 3mg/kg ABT874 s.c.: Induction of T cell apoptosis % ** ** ** AB = Antibody KO = control ** = p< ** 20 0 KO AB KO AB KO AB KO AB 7 Wks 12 Wks 7 Wks 12 Wks response remission Mannon et al. New Engl. J. Med. 2004

15 IL-12 related cytokines

16 EBI3 is rapidly induced by TLR ligation in dendritic cells Wirtz et al. J. Immunol. 2005

17 Reconstitution of EBI3 promoter activity in 293 cells Wirtz et al. J. Immunol. 2005

18 IL-12 related cytokines in T helper cell differentiation and activation IL-27 + T-bet TH1 Cellular immunology Autoimmunity naive IL-27 IL-12 - IL-4 TGF-β IL-6 TGF-β GATA-3 FoxP3 RORγt IL-23 TH2 itreg TH IL17 Humoral immunity Atopy, asthma, allergy Immunoregulatory function Inflammation Autoimmunity

19 CLP model of polymicrobial sepsis CLP : cecal ligation and puncture

20 IL-27 protein is upregulated in serum and peritoneal lavage fluid post CLP Wirtz et al. J. Exp. Med. 2006

21 IL-27 EBI3 deficiency protects mice from CLP induced lethality

22 EBI3 deficiency protects mice from sepsis induced by gram - bacteria

23 Increased leucocyte migration into the peritoneal cavity in EBI3 deficient mice

24 Decreased numbers of bacteria in EBI3 -/- mice 24 h post CLP better local control of infection?

25 Increased oxidative burst in EBI3 -/-

26 IL-27 decreases TLR mediated ROI production in vitro

27 Increased lethality after administration of ril-27

28 sil-27ra Fusion protein

29 Treatment with sil-27ra protects mice from CLP dependent lethality

30 Conclusion IL-27 is upregulated early in murine septic peritonitis IL-27 EBI3 deficiency or treatment with a neutralizing IL-27 R fusion protein protects mice from septic peritonitis IL-27 acts as a downregulator of innate immune cell function Blockade of IL-27 emerges as a potentially novel therapy for sepsis

31 IL-27/EBI3 in IBD EBI3 UC > CD (Christ et al. Gastroenterology, 1998) CU and subset of CD (Omata et al., Inflamm. Bowel. Dis 2001) active MC (Schmidt et al., Inflamm. Bowel. Dis 2005) IL-27 p28 MC > CU (Schmidt et al., Inflamm. Bowel. Dis 2005) Mouse studies : EBI3 -/- mice do not develop Oxazolone colitis (Nieuwenhuis et al., PNAS 2002)

32 Experimental procedures EBI3 -/- (C57BL/6) 1,5 3 % DSS for 7 days in drinking water daily weight monitoring Evaluation of colitis severity Colonoscopy Colon length Histological scoring Isolation of LPMC or full thickness organ culture for cytokine multiplexing or Realtime-PCR Immunofluorescence

33 Increased expression of EBI3/p28 in DSS colitis Intestinal epithelium LPMC LPMC from DSS colitis

34 Increased acute DSS colitis in EBI3-/- mice

35 Increased acute DSS colitis in EBI3 -/- mice EBI3 +/+ EBI3 -/- **

36 High numbers of infiltrating myeloid cells in EBI3 -/- mice EBI3 +/+ MPO CD11c EBI3 -/-

37 Increased proinflammatory cytokine levels in EBI3 -/- mice full thickness organ culture LPMC

38 Conclusion EBI3 and p28 are early upregulated in murine DSS colitis EBI3 deficiency is associated with severe acute DSS colitis EBI3 is potentially a citical down-regulator of intestinal immune responses

39 Ralf Kiesslich, Martin Holtmann Jonas Mudter, Raja Atreya Kai Hildner, Thanka Nadar. Con Schneider, Jürgen Siebler Clemens Neufert Clinic Thank you Lab Cooperations Jan Schmidt Richard Blumberg Stefan Rose-John Radovan Dvorsky Hans A. Lehr Jeff Molkentin Manolis Pasparakis Gerhard Fritz Xose Bustelo Imke Tiede, Christoph Becker Daniela Poppe, Stefan Wirtz Benno Weigmann, Alexej Nikolaev Brigitte Bartsch, Massimo Fantini Ute Teichgräber