2009 Annual Report 1a.Objectives (from AD-416) 1) To identify the mechanisms of local type 1 and type 2 cytokine-dependent alterations in gut function in the colon and in unaffected areas in the small intestine.. 2) Determine the impact of specific nutritional deficiencies alone (Vitamin E and Selenium) on baseline levels of cytokines and cytokine receptor expression and on gut function.. 3)Determine the impact of specific nutritional deficiencies (Vitamin E and Selenium) on immune-induced alterations in gut function. 1b.Approach (from AD-416) Experiments will compare responses in Wild Type mice or mice deficient in the signaling factors Stat4 or Stat6 after treatment with agents known to elevate Th1 (inflammation using trinitrobenzene sulfonic acid) or Th2 cytokines (enteric helminth infection). Specific nutritional deficiencies that impact these responses and affect gut function will be examined; treatment groups will include. 1)vehicle,. 2)TNBS,. 3)infection,. 4)VE and Se deficient diet). Molecular (real time RT-PCR) will be used to determine diet-induced alterations in cytokine profiles and histological evaluation will be performed to identify modifications in infiltrating cells as a result of the diet deficiencies. Laser capture microscopy (LCM) will be used to localize cytokine receptors to specific cell types including epithelial mucosa, smooth muscle and nerves. Preliminary data indicate that diets deficient in both VE and Se impair the ability of the intestine to clear enteric nematodes and affect intestinal function. Function is defined as changes in epithelial cell secretion and absorption in vitro (Epithelial cell transport in Ussing chambers) and smooth muscle contraction in vitro (Isometric smooth muscle contraction). Evaluation of histological changes and assessment of cytokine and cytokine receptor expression will be determined routinely using LCM and tissue cytokines using RT-PCR. 3.Progress Report The intestinal tract is in a continuous state of flux affected by routine and novel materials entering the gut, but requires appropriate absorption of nutrients and exclusion of harmful agents to maintain host health. This project tested the hypothesis that specific agents that trigger immune activation in the intestine can influence both immune and non-immune cells and induce physiological changes that regulate nutrient absorption in the face of inflammation. Models of natural parasitic infection of the intestinal tract were used to induce activation of the Th2-based arm of the immune system and to study the adaptation to infection and the control of the intensity of inflammation. Parasite infection also mimics a patterned response that is physiologically and immunologically similar to that activated by food allergens. Exposure of the intestine to tri-nitrobenzene sulfulfuric acid (TNBS) induced a patterned response similar to that resulting from bacterial and viral infections that activates the Th1-based arm of the immune response. These interactions were studied to evaluate the role of host cytokines and cytokine receptors (compounds involved in immune regulation) that also engage non-immune pathways in the intestine and influence nutrient absorption and smooth muscle contractility. The results from the project demonstrated that the interplay of different immune activators that induced regulatory T cell and macrophage populations in the intestine produced cytokines that activated a generalized inflammatory response that could interfere with normal intestinal function if not appropriately regulated. The studies included the specific role of dietary selenium and vitamin E and their contribution to optimal immune and intestinal function. 4.Accomplishments 1. The presence of parasites in the intestine act to signal molecules that regulate host intestinal function. Parasites infect over one quarter of the world’s population, generally in impoverished areas, but they also stimulate host immune responses comparable to those induced by food allergens that have importance in the U.S. Our studies demonstrated that parasite infection caused a key receptor for the cytokine IL-13, termed IL-13Ralpha2, to be present in increased amounts on intestinal smooth muscle cells. This receptor binds IL-13 tightly and prevents its action in the intestine. If the receptor is genetically removed in specially treated mice, then there is enhanced contraction of intestinal smooth muscle and enhanced resistance to parasitic infection by the epithelial cells lining the intestine compared to the responses in infected normal mice. These data showed that IL-13Ralpha2 plays an important protective role in parasite infection by limiting the availability of IL-13, thereby regulating both the immune and biological effects of IL-13. In a separate study, parasite infection induced a T cell-signaling molecule called TGF-beta affected the levels of IFN-gamma, a pro-inflammatory molecule that contributes to chronic intestinal colitis. Examination of these mechanisms systematically has resulted in the development of a strategy to control responses to parasitic infection and similar changes induced by food allergens. Review Publications Guo, L., Urban Jr, J., Jankovic, D., Zhu, Z., Paul, W. 2008. Calcium-dependent p38 phosphorylation is important for memory CD4 T cell effector cytokine transcription and mRNA stabilization. Journal of Immunology. 18(6):3984-3993. Patel, N., Kreider, T., Urban Jr, J.F., Gause, W. 2009. Characterization of effector mechanisms at the host: parasite interface during the immune response to tissue-dwelling intestinal nematode parasites. International Journal for Parasitology. 39(1):13-21. Morimoto, M., Zhao, A., Sun, R., Stiltz, J., Madden, K.B., Mentink-Kane, M., Ramalingam, T., Wynn, T.A., Urban Jr, J.F., Shea-Donohue, T. 2009. IL-13 receptor alpha-2 regulates the immune and functional response to Nippostrongylus brasiliensis infection. Journal of Immunology. 183(3):1934-1999. Ince, M.N., Elliott, D.E., Setiawan, T., Metwali, A.A., Blum, A., Chen, H.L., Urban Jr, J.F., Flavell, R.A., Weinstock, J.V. 2009. Role of T cell TGF beta signaling in intestinal cytokine responses and helminthic immune modulation. European Journal of Immunology. 39:(7):1870-8. Bazzone, L., Smith, P., Rutitzky, L., Shainheit, M., Urban Jr, J.F., Setiawan, T., Blum, A., Weinstock, J., Stadecker, M. 2008. Co-Infection with intestinal helminths markedly reduces proinflammatory cytokines and disease severity in natural and induced high-pathology Schistosomiasis. Infection and Immunity. 76(11):5164-72.