The mechanisms whereby chemically defined diets induce remission in Crohn's disease are not understood. Progress will depend on the examination of dietary factors in in vivo and in vitro model system where nutrients can be accurately administered and their effects measured by well- defined immunological markers. We will examine how dietary constituents influence the mucosal immune system by examining the hypothesis the dietary constituents (specifically dietary fat or antigenic load) alter the immune system of the gut by influencing the expression of genes in the epithelium. We will study the genes coding for cytokines (particularly IL-6 and IL-8-related """"""""chemokines"""""""", IL-8 and macrophage inflammatory protein-2 [MIP-21) and the surface expression of class II major histocompatibility complexes (MHC) on the cell surfaces. We will pursue the following specific aims:
Specific aim#1 Determine the influence of dietary factors on the expression IL-6, MIP-2 and class II MHC by enterocytes in vivo. We will use the differences between commercially available Mead-Johnson products to study 1) the role of fat and 2) the role of antigenic protein on the mucosal immune system. We will examine their effect on the epithelial expression of cytokines and class II MHC expression in an inflammatory bowel disease model (reovirus infected mouse) and during weaning. Our previous studies have shown that an elemental diet will alter the expression of class II MHC genes in the epithelium. We will study IL-6, MIP-2 and class II MHC and its associated invariant chain. The messenger RNAs for these molecules will be detected by RNA transfer analysis (northern blots) and by quantitative RT- PCR of RNA isolated from small intestinal epithelial cells; and by in situ hybridization of frozen intestinal sections. Proteins for class II MHC and invariant chain will be examined by Western blots. Cytokines will be studies by the generation of IL-6 and MIP-2 in freshly isolated cells using ELTSA assays.
Specific aim #2 : Determine the importance of nutritional factors on the expression of IL- 6, IL-8 and class IIMHC in cultured human intestinal epithelial cell lines. We will complement the in vivo studies by studying nutrients directly on the expression of cytokines and class II MHC related molecules in vitro. We will examine 1) short chain fatty acids and 2) protein and nucleotides in human intestinal epithelial cells. We will induce IL-6 and IL-8 expression by (permissive) cytokines (IL-1 and IFN-gamma) at concentrations at which expression is evident, but not maximal. We will study the effect of sodium butyrate, nucleotides and protein antigens the expression of these genes with the techniques described in specific aim #1.
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