The purpose of this proposal is to define the cellular and molecular mechanisms by which the NF-kappaB subunits p50 and p65 inhibit inflammation within the lower bowel. The inhibitory functions of p50 may be especially relevant to the control of inflammation within the colon, as our laboratory has shown that mice lacking p50 (p50-/-) are sensitive to colitis induced by Helicobacter hepaticus, and this sensitivity is significantly exacerbated in mice that both lack p50 and are heterozygous for p65 (p50-/-p65+/-). These mice are sensitized to the development of colitis by a defect intrinsic to the innate immune system. This defect may reflect an inability to control H. hepaticus-induced inflammatory gene expression within antigen presenting cells (APCs), as H. hepaticus infection induces higher levels of the critical inflammatory cytokines IL-12p40 and IP-10 in p50-/- and p50-/-p65+/- macrophages than in WT macrophages. The goals of this proposal are: 1) To determine the mechanism by which p50 and p65 within cells of the innate immune system contribute to inhibiting the inflammatory response to H. hepaticus. Using a novel mouse strain created in our laboratory (p50-/-p65+/-RAG-2-/-), we will evaluate the possibility that p50/p65 activity is required within the innate immune system to facilitate the inhibitory function of regulatory T cells. 2) To determine the mechanisms by which p50 and p65 inhibit H. hepaticus induced inflammatory gene expression. We will use molecular techniques to compare the function of endogenous IL-12p40 and IP-10 promoters in WT, p50-/-, and p50-/-p65+/- macrophages. 3) To determine whether p50 and p65 prevent an overaggressive immune response that injures the host, or alternatively, whether p50 and p65 prevent an immune deficiency that leads to increased bacterial burden. We will compare bacterial burden in RAG-2-/- and p50-/-p65+/-RAG-2-/- mice, and determine whether introduction of functional innate immune cells into p50-/-p65+/-RAG-2-/- mice can prevent H. hepaticus-induced inflammation. Taken together, we believe that these studies will lead to insights regarding the molecular pathogenesis of inflammatory bowel disease that could lead to novel therapeutic strategies.
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