Presentation on theme: "TL1A Expression in Human IBD and Animal Models"— Presentation transcript:
1TL1A Expression in Human IBD and Animal Models Bala ManickamPfizer ACVP-STP fellow, Anatomic pathology residentUniversity of Georgia, Athens GA
2TL1A & DR3 - BiologyTL1A (aka TNFSF15) is a recently identified member of TNF superfamily.Expressed by monocytes, macrophages, dendritic cells, synovial fibroblasts, chondrocytes and endothelial cellsIncreased expression when stimulated by cytokines, immune complexes and micro-organisms.Its Cell surface receptor DR3 is (aka TNFSF25, WSL-1, TRAMP ands LARD) is mainly expressed by T cellsTL1A-DR3 interaction amplifies the DR3 expression and mediates the inflammatory effects.DR3 is a type I cytokine receptor with an intracytoplasmic death domainDcR3 is the decoy receptor that does not have an intracytplasmic domain and hence inhibits the downstream signalingNat Rev Rheumatol Feb;6(2):67-8Biochem Pharmacol Apr 1;81(7):838-47
3Inflammatory Bowel Disease Human IBDIBD represents an important chronic disease affecting the GI tract of man and domesticated animal species.The 2 IBD entities in humans are Crohn’s disease (CD) and Ulcerative colitis (UC).Immune mediated - responds to immunomodulatory drugsThe pathogenesis of IBD involves:Failure of immune regulation.Genetic susceptibility.Environmental triggers (microbial flora).Disruption of the mucosal barrier.Mouse models of IBDDSS (Dextran sulphate) colitis: Oral administration of this sulphated polysaccharide to mice induces a self limiting colitisTNBS (Trinitrobenzene sufonic acid) colitis: Colonic inflammation is induced by intrarectal administration of TNBS dissolved in 50% ethanolCanine IBDSmall intestine: Lymphocytic-plasmacytic enteritis (LPE), eosinophilic enteritis and eosinophilic gastro-enteritis (EGE)Large intestine; Lymphocytic-plasmacytic colitis, eosinophilic colitis, histiocytic ulcerative colitis (HUC) (mainly PAS-positive macrophages), and regional granulomatous colitis (mainly PAS-negative macrophages)
4TL1A & DR3 expression in human IBD Healthy Person CD UCTL1ATL1A: CD- lymphocytes and macrophages; UC- Plasma cellsDR3: Increased numbers of immunoreactive cells were detected in the lamina propria lymphocytes from involved areas of IBD patientsDR3J Immunol 2003;171;
5TL1A & DR3 : Evidence of Efficacy Gut bacteriaControl anti TL1ATNBScolitisDSScolitisFig 1: Attenuation of TNBS colitis by anti-TL1A mAb.Fig 2: DSS model colitis; both prophylactic and therapeuticFig 3: TL1A transgenic mice spontaneously develop colitis; DR3 KO attenuates colitisProposed mechanismControl DR3 KOGASTROENTEROLOGY 2008;135:552–567Mucosal Immunology (2011) 4, ;
6Rationale for current study Up-regulation of TL1A and DR3 in human CD and UC and in rodent IBD modelsTL1A and DR3 deficiency attenuates murine IBDIBD 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.Studying IBD in spontaneous and induced models may provide insights into how we model the disease in laboratory mice
7HypothesisBy characterizing and comparing expression profile of TL1A and other critical immunological markers in murine IBD to people, we may further validate (and perhaps even uncover) therapeutic targets for human IBD.
8ObjectivesCompare and characterize the staining of TL1A & DR3 in intestinal tissues obtained from DSS and TNBS mouse colitis models, human IBD, canine IBD and normal colons from cynomolgus monkeys.Compare the staining characteristics of mast cells in intestinal tissues obtained from DSS and TNBS mouse colitis models, human IBD and canine IBD.Compare the expression of T&B cells in different models of IBD.
9Experimental design Groups Number of subjects Number of slides/subject Total number of slidesDSS (Mouse)51050TNBS (Mouse)Normal colon (Mouse)UC (Human)636CD (Human)Normal colon (Human)Normal colon (Primate)25IBD (Dog)Normal colon (Dog)
10Reagents Anti-human TL1A mAb (made by Pfizer, optimized for IHC) Homology to mouse, rat: 92%Homology to dog (predicted): 86%Anti-human DR3 mAb (made by Novus Biol)The immunogenic peptide shares44% homology with canine DR3 (XP_ )88% homology with primate DR3 (XM_CD3, CD45RB220, CD20, Toluidine bluePfizer Internal Use
11Results: TL1A expression Normal CD UCHumanNaïve DSS TNBSScattered cells within the lamina propria of the normal/naïve are positive for TL1ATL1A expression is increased in IBD conditions in both human cases as well as in murine models.A moderate to variably strong staining of the cytoplasm and the membrane of the Non T, non B, Non neutrophilic cell type was consistently present within the inflamed areas in mice and humansMice
12Results: TL1A expression Cynomolgus Human MiceVasculature(+)Germinalcenter(-)TL1A expression in the vasculature (endothelial cells) were consistent in cynomolgus, humans and mice tissues and served as an internal controlGALT did not stain positive for TL1A in both mice and human IBD tissuesDR3: The lymphocytes infiltrating the lamina propria and the submucosa in crohn’s disease were strongly positive for DR3. The staining was localized to the nucleus, cytoplasm and the cell membraneCDLamina propriaDR3
13Mast cells in IBD – Dog, Mice Naive IBDH&ET-blueDog: Only rare mast cells are present within the control and the IBD dogsMice: Only rare mast cells are present in Naïve, DSS and TNBS colitis; There is no difference in the expression of Mast cells between the groupsNaive DSS TNBS
14B-lymphocytes-Human IBD Normal CD UCGerminal centerLamina propriaGALT hyperplasia is present in patients with CD and UCIncreased numbers of B-lymphocytes are present within the lamina propria of the CD and UC patients when compared to normal individual
15B-lymphocytes-Mice IBD Naive DSS TNBSGerminal centerLamina propriaGALT hyperplasia is present in DSS and TNBS colitisIncreased numbers of B-lymphocytes are present within the lamina propria of the CD and UC patients when compared to normal individualBasal level expression of B cells within the normal colon was similar in mice and humansB & T cells within the GALT and the lamina propria were markedly increased in IBD patients when compared to healthy controls that was consistent with mice models
16T-lymphocytes-Human IBD Normal CD UCGerminal centerLamina propriaGALT hyperplasia is present in patients with CD and UCIncreased numbers of T-lymphocytes are present within the lamina propria of the CD and UC patients when compared to normal individual
17T-lymphocytes-Mice IBD Naive DSS TNBSGerminal centerLamina propriaIncreased numbers of B-lymphocytes are present within the lamina propria of the CD and UC patients when compared to normal individualBasal level expression of T cells within the normal human colon was increased when compared to the mice models
18B & T lymphocytes- Cynomolgus B-cells T-cellsGerminal centerThere are few scattered B-lymphocytes within the lamina propria of naïve Cynomolgus colonGerminal center stains positive for B-lymphocytesThere are few scattered T-lymphocytes within the lamina propria of naïve Cynomolgus colonLamina propria
19Conclusion TL1A: B&T-lymphocytes: Mast cells: The expression of TL1A was similar and consistent across the speciesTL1A was up regulated in IBD when compared to the Naive/healthy patientsThis data not only validates the existing murine IBD models but also indicates that TL1A could be a druggable target in IBDWe 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 IBDB&T-lymphocytes:Increased numbers of both B & T cells in the lamina propria in cases of IBD, suggests a potential role for immune activation in IBDMast cells: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.
20Acknowledgement Dr. Timothy LaBranche (Industry Mentor) Dr. Elizabeth Howerth (Academic Mentor)Dr. Zaher Radi (Pfizer)Dr. Shawn O’ Neil (Pfizer)Jameel Syed (Pfizer)Zachery Stewart (Pfizer)University of GeorgiaACVP-STP CoalitionStudies were funded and supported by DRSD, Pfizer