Project description:Neutrophil accumulation in crypt abscesses is a pathological hallmark of ulcerative colitis. Based on recent evidence that mucosal metabolic changes influence disease outcomes, we hypothesized that transmigrating neutrophils influence the transcriptional profile of intestinal epithelia. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by neutrophil-epithelial crosstalk. Real-time O2 sensing indicated that transmigrating neutrophils rapidly deplete microenvironmental O2 sufficient enough to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). Utilizing HIF reporter mice in a TNBS colitis model, we investigated the relative contribution of neutrophils and the respiratory burst to M-bM-^@M-^\inflammatory hypoxiaM-bM-^@M-^] in vivo. Gp91phox-null mice, which mirror human chronic granulomatous disease, developed accentuated colitis compared to control with exaggerated neutrophil infiltration and diminished inflammatory hypoxia. In conclusion, transcriptional imprinting of host tissue by infiltrating neutrophils modulates the host response to inflammation. Likewise, the respiratory burst contributes fundamentally to localized O2 depletion, resultant microenvironmental hypoxia and effective inflammatory resolution. Two models were employed, direct and indirect. The M-bM-^@M-^\DirectM-bM-^@M-^] migration model entailed establishing a chemotactic gradient (using fMLP) across monolayers of T84 intestinal epithelial cells grown on the underside of permeable supports (3um pore). Neutrophils (PMN) were induced to migrate in the physiologically relevant the physiologically relevant basolateral-to-apical direction. Following migration, T84s were rested in complete media and 2hrs later harvested for RNA isolation. In the Indirect model, PMN were applied to T84s as with the direct model. After migration, conditioned supernatants were collected, cells pelleted and supernatants filtered through 0.2um pore. Conditioned supernatants were transferred to naive T84 monolayers for 2hrs, followed by RNA harvest. Each model was exposed to neutrophils (PMN) or not. All monolayers contained chemotactic peptide fMLP on the apical side. Total of 12 samples, 4 conditions in triplicate: Direct migration without neutrophils (T84 +fMLP -PMN), Direct migration with neutrophils (T84 +fMLP +PMN), Indirect migration without neutrophils (T84 +fMLP -PMN), Indirect migration with neutrophils (T84 +fMLP +PMN)

Project description:This project was done in collaboration with Dr. Richard Cummings to examine sialylation of CD44v6 and its role in clearance of neutrophils from inflamed intestinal epithelium. Specifically, we have employed a functional approach using membrane preparations from interferon gamma-stimulated intestinal epithelial cells to generate a monoclonal antibody, designated GM35, which blocks neutrophil transepithelial migration through the promotion of neutrophil adhesion at the apical surface of the intestinal epithelium. Protein biochemistry, sequencing, confocal microscopic analysis, and immunoprecipitation studies all identify the protein ligand for this antibody as CD44v6. However, selective inhibition of O- or N-linked glycosylation reveal that the antibody is specific for an O-linked glycotope and glycoarray analysis of the GM35 antibody by Core H of the Consortium for Functional Glycomics reveal that this antibody binds with high affinity and specificity to a carbohydrate epitope consistent in structure with sLeA. Inhibition of O-linked glycosylation attenuated both GM35 binding and its functional effects as did specific cleavage of sialic acid residues from the cell surface, using neuraminidase, although the functional effects of cleavage were smaller and harder to assess. CD44v6 has previously been studied as a marker of inflammation in Inflammatory Bowel Disease, and GM35 staining is clearly upregulated by interferon gamma in T84 and HT29 intestinal epithelial cells. The specific enzymes governing the sialylation of CD44v6 in intestinal epithelium have yet to be determined. Analysis of a panel of intestinal epithelial cells including T84, HT29, and Caco-2 cells revealed that cell-type specific regulation of both splicing and glycosylation is essential for the regulation of the expression of the GM35 epitope. And, forcible expression of CD44 in Caco-2 cells, which are negative for CD44v6 at baseline, results only in upregulation of previously expressed isoforms of CD44, but not in GM35 expression. Based on western blot and immunofluorescent staining, GM35 expression is greatest in interferon gamma-treated T84 cells. Thus, we analyzed RNA samples for the expression of glycosylation specific genes in interferon gamma-stimulated T84 cells relative to that of both non-stimulated T84 cells and GM35-negative Caco-2 cells in order to identify glycosylation-specific targets contributing to the interferon gamma-dependent upregulation of sialylated CD44v6 in intestinal epithelial cells.

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Homo sapiens

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