TL 1 A Expression in Human IBD and

TL 1 A Expression in Human IBD and Animal Models Bala Manickam Pfizer ACVP-STP fellow, Anatomic pathology resident University of Georgia, Athens GA

TL 1 A & DR 3 - Biology Nat Rev Rheumatol. 2010 Feb; 6(2): 67 -8 Biochem Pharmacol. 2011 Apr 1; 81(7): 838 -47 2

Inflammatory Bowel Disease Human IBD Mouse models of IBD Ø IBD represents an important chronic disease affecting the GI tract of man and domesticated animal species. Ø DSS (Dextran sulphate) colitis: Oral administration of this sulphated polysaccharide to mice induces a self limiting colitis Ø The 2 IBD entities in humans are Crohn’s disease (CD) and Ulcerative colitis (UC). Ø TNBS (Trinitrobenzene sufonic acid) colitis: Colonic inflammation is induced by intrarectal administration of TNBS dissolved in 50% ethanol Ø Immune mediated - responds to immunomodulatory drugs Ø The pathogenesis of IBD involves: 1. Failure of immune regulation. 2. Genetic susceptibility. 3. Environmental triggers (microbial flora). 4. Disruption of the mucosal barrier. Canine IBD Ø Small intestine: Lymphocytic-plasmacytic enteritis (LPE), eosinophilic enteritis and eosinophilic gastro -enteritis (EGE) Ø Large intestine; Lymphocytic-plasmacytic colitis, eosinophilic colitis, histiocytic ulcerative colitis (HUC) (mainly PAS-positive macrophages), and regional granulomatous colitis (mainly PASnegative macrophages) 3

TL 1 A & DR 3 expression in human IBD Healthy Person CD UC TL 1 A DR 3 J Immunol 2003; 171; 4868 -4874 4

TL 1 A & DR 3 : Evidence of Efficacy Gut bacteria Control Gut bacteria anti TL 1 A TNBS colitis DSS colitis Control GASTROENTEROLOGY 2008; 135: 552– 567 DR 3 KO Mucosal Immunology (2011) 4, 172 -185; 5

Rationale for current study Ø Up-regulation of TL 1 A and DR 3 in human CD and UC and in rodent IBD models Ø TL 1 A and DR 3 deficiency attenuates murine IBD Ø IBD occurs spontaneously in dogs. Because of this, and because it shares a similar immunopathology, it may provide valuable mechanistic insight into the disease in humans. Ø Studying IBD in spontaneous and induced models may provide insights into how we model the disease in laboratory mice 6

Hypothesis Ø By characterizing and comparing expression profile of TL 1 A and other critical immunological markers in murine IBD to people, we may further validate (and perhaps even uncover) therapeutic targets for human IBD. 7

Objectives ØCompare and characterize the staining of TL 1 A & DR 3 in intestinal tissues obtained from DSS and TNBS mouse colitis models, human IBD, canine IBD and normal colons from cynomolgus monkeys. ØCompare the staining characteristics of mast cells in intestinal tissues obtained from DSS and TNBS mouse colitis models, human IBD and canine IBD. ØCompare the expression of T&B cells in different models of IBD. 8

Experimental design Groups Number of subjects Number of slides/subject Total number of slides DSS (Mouse) 5 10 50 TNBS (Mouse) 5 10 50 Normal colon (Mouse) 5 10 50 UC (Human) 6 6 36 CD (Human) 6 6 36 Normal colon (Primate) 5 5 25 IBD (Dog) 5 10 50 Normal colon (Dog) 5 10 50 9

Reagents • Anti-human TL 1 A m. Ab (made by Pfizer, optimized for IHC) – Homology to mouse, rat: 92% – Homology to dog (predicted): 86% • Anti-human DR 3 m. Ab (made by Novus Biol) – The immunogenic peptide shares – 44% homology with canine DR 3 (XP_546752 ) – 88% homology with primate DR 3 (XM_003274297 • CD 3, CD 45 RB 220, CD 20, Toluidine blue Pfizer Internal Use 10

Results: TL 1 A expression Normal CD UC Naïve DSS TNBS Human Mice 11

Results: TL 1 A expression Cynomolgus Human Mice Vasculature (+) Germinal center (-) DR 3 CD Lamina propria 12

Mast cells in IBD – Dog, Mice Naive IBD Naive DSS H&E T-blue TNBS 13

B-lymphocytes-Human IBD Normal CD UC Germinal center Lamina propria 14

B-lymphocytes-Mice IBD Naive DSS TNBS Germinal center Lamina propria 15

T-lymphocytes-Human IBD Normal CD UC Germinal center Lamina propria 16

T-lymphocytes-Mice IBD Naive DSS TNBS Germinal center Lamina propria 17

B & T lymphocytes- Cynomolgus B-cells T-cells Germinal center Lamina propria 18

Conclusion TL 1 A: Ø The expression of TL 1 A was similar and consistent across the species Ø TL 1 A was up regulated in IBD when compared to the Naive/healthy patients Ø This data not only validates the existing murine IBD models but also indicates that TL 1 A could be a druggable target in IBD Ø We also uncovered that Cynomolgus shares similar staining characteristics with human and murine IBD tissues and thus can probably be a good model to study human IBD B&T-lymphocytes: Ø Increased numbers of both B & T cells in the lamina propria in cases of IBD, suggests a potential role for immune activation in IBD Mast cells: Ø In our study, We could not detect any difference in the expression of mast cells in both naïve and or IBD patients in both murine and Dog cases. 19

Acknowledgement Ø Ø Ø Ø Ø Dr. Timothy La. Branche (Industry Mentor) Dr. Elizabeth Howerth (Academic Mentor) Dr. Zaher Radi (Pfizer) Dr. Shawn O’ Neil (Pfizer) Jameel Syed (Pfizer) Zachery Stewart (Pfizer) University of Georgia ACVP-STP Coalition Studies were funded and supported by DRSD, Pfizer 20

Thank you! Pfizer Internal Use 21