IL-12 family members in experimental colitis Markus F. Neurath I. Medical Clinic Johannes Gutenberg-University Mainz, Germany
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
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
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 2001 166 7563) P19 but not p35 knockout mice fail to develop EAE (Nature 2003 421 744)
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
IL-23 is expressed in lamina propria DCs of mice with transfer colitis Healthy p19 Colitis CD11c Colitis p19 Colitis CD11c p19
Generation of IL-23 p19 knockin mice 1 2 3 4 5 6
IL-23 KO mice are highly susceptible to inducible colitis models TNBS 105 DSS weight in % 100 95 90 85 80 75 WT IL-23-p19 KO 0 1 2 3 7 8 9 10 days weight in % 100 95 90 85 80 WT IL-23-p19 HET IL-23-p19 KO 0 1 2 3 4 5 6 7 8 days
IL-23 KO mice are highly susceptible to TNBS induced colitis 14 12 10 Wild-type Wild-type endoscopic score 8 6 4 2 0 Wild-type IL-23 KO IL-23 KO IL-23 KO
IL-23 KO mice are highly susceptible to TNBS induced colitis Wild-type 5 IL-23 KO histologic score 4 3 2 1 0 wt KO
Crossregulation of IL-12 p35 and p40 expression by IL-23 p19 pg/ ml pg/ ml 600 500 400 300 200 100 0 0 250 200 150 100 50 IL-6 WT p19 KO US LPS CpG IL-12 WT p19 KO US LPS CpG 450 400 350 300 250 200 150 100 50 0 800 700 600 500 400 300 200 100 0 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
Anti-IL-12 therapy rescues p19 knockin mice from lethal colitis 105 P19 knockin mice/ TNBS 100 weight in % 95 90 85 80 75 control 70 anti-il-12p40 65 0 1 2 3 4 5 6 7 8 11 12 13 14 15 16 PBS Survival 1,00 0,75 0,50 0,25 anti-p40 PBS 0,00 0 5 10 15 20 Days anti IL-12 p40 Becker et al. Cutting Edge JI 2006
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
Anti-IL-12 p40 antibody therapy in Crohn s disease 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 % 60 40 ** ** ** AB = Antibody KO = control ** = p< 0.001 ** 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
IL-12 related cytokines
EBI3 is rapidly induced by TLR ligation in dendritic cells Wirtz et al. J. Immunol. 2005
Reconstitution of EBI3 promoter activity in 293 cells Wirtz et al. J. Immunol. 2005
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
CLP model of polymicrobial sepsis CLP : cecal ligation and puncture
IL-27 protein is upregulated in serum and peritoneal lavage fluid post CLP Wirtz et al. J. Exp. Med. 2006
IL-27 EBI3 deficiency protects mice from CLP induced lethality
EBI3 deficiency protects mice from sepsis induced by gram - bacteria
Increased leucocyte migration into the peritoneal cavity in EBI3 deficient mice
Decreased numbers of bacteria in EBI3 -/- mice 24 h post CLP better local control of infection?
Increased oxidative burst in EBI3 -/-
IL-27 decreases TLR mediated ROI production in vitro
Increased lethality after administration of ril-27
sil-27ra Fusion protein
Treatment with sil-27ra protects mice from CLP dependent lethality
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
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)
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
Increased expression of EBI3/p28 in DSS colitis Intestinal epithelium LPMC LPMC from DSS colitis
Increased acute DSS colitis in EBI3-/- mice
Increased acute DSS colitis in EBI3 -/- mice EBI3 +/+ EBI3 -/- **
High numbers of infiltrating myeloid cells in EBI3 -/- mice EBI3 +/+ MPO CD11c EBI3 -/-
Increased proinflammatory cytokine levels in EBI3 -/- mice full thickness organ culture LPMC
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
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