K. E. Cunningham1,2, E. A. Novak2, G. Vincent2, J. D. Piganelli3, M. R. Rosengart1, K. P. Mollen2 1University Of Pittsburgh Medical Center,Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh Children’s Hospital,Pediatric Surgery,Pittsburgh, PA, USA 3University Of Pittsburgh Children’s Hospital,Immunogenetics,Pittsburgh, PA, USA
Introduction: The incidence and prevalence of inflammatory bowel disease (IBD) is increasing worldwide. Chronic intestinal inflammation, as seen in IBD, is largely mediated by T-lymphocytes. Ca2+/Calmodulin-dependent kinase IV (CaMKIV) is a multifunctional serine/threonine specific protein kinase predominantly found in lymphocytes and is known to play pivotal roles in immune activation and inflammation by regulating T-lymphocyte differentiation. CaMKIV has been extensively studied in diseases of autoimmunity, but its role in the intestine remains unclear. We have previously shown that activation of CaMKIV contributes to inflammation during murine experimental colitis. However, it is unknown whether CaMKIV activity in immune or non-immune cells primarily contributes to intestinal inflammation. We now hypothesize that CaMKIV upregulation in intestinal T-lymphocytes plays a key role in the pathogenesis of colitis and absence of CaMKIV will confer protection against the development of intestinal inflammation.
Methods: Male CaMKIV KO mice (Camk4tm1Tch/Camk4tm1Tch) and wild-type (WT) counterparts (C57BL/6J) were subjected to dextran sodium sulfate (DSS) colitis for 7 days. Animal weights and Disease Activity Index (DAI) scores were recorded daily. Colonic lamina propria lymphocytes (LPLs) were isolated, stimulated with PMA/Ionomycin, and analyzed by flow cytometry. Intestinal tissue was analyzed for expression of inflammatory cytokines. H&E staining was used to evaluate histological changes. Immunofluorescence was used to observe differences in the quantity and localization of key proteins as well as to identify infiltrating bacteria within the intestinal mucosa.
Results: CaMKIV KO mice subjected to DSS were protected from intestinal inflammation relative to their WT counterparts. Clinical disease severity correlated with CaMKIV activation, as did inflammatory changes demonstrated by H&E staining, PCR, and western blot. Flow cytometry analysis of colon LPLs after DSS treatment verified a population of pathogenic IFNγ+/IL-17+ Th17 T-lymphocytes in WT mice which was absent in CaMKIV KO mice.
Conclusion: We investigate for the first time the role of CaMKIV in the pathogenesis of intestinal inflammation. We demonstrate the presence of a pathogenic Th17 T-lymphocyte population within the colons of WT mice subjected to DSS colitis, but not CaMKIV KO mice, suggesting that CaMKIV activation contributes to the differentiation of IFNγ+/IL-17+ Th17 T-lymphocytes during experimental colitis. Interestingly, this highly inflammatory T-lymphocyte population has been demonstrated to play a key role in the initiation and propagation of inflammation in human IBD. Strategies aimed at suppressing CaMKIV activity may lead to novel immune-based strategies to treat IBD.