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PWE-111 Lipopolysaccharide induces small intestinal epithelial cell apoptosis and shedding which is regulated by NFκB signalling

Introduction The gut barrier is formed by a single cell thick, simple columnar epithelium consisting of intestinal epithelial cells (IECs) held together by tight junctions. In the small intestine, IECs are generated in the crypt, migrate up the crypt-villus axis and are shed at the villus tip. When IECs detach from the villus during the process of shedding, discontinuities occur in the epithelium. IEC shedding increases in response to pro-inflammatory cytokines, resulting in larger discontinuities and increased intestinal permeability. Understanding the mechanisms which regulate IEC shedding may allow development of therapeutic strategies which ameliorate its detrimental effects on gut barrier function in intestinal disease states. Aims To investigate whether IEC apoptosis and shedding are increased during induction of endotoxic shock, and the genetic regulation of this process. Methods Lipopolysaccharide (LPS) from Escherichia coli O111:B4, was administered to adult female C57BL/6 mice at 10 mg/kg by intraperitoneal injection (n=6 per group). Animals were euthanased 1–6 h after LPS administration. The duodenum, jejunum and ileum were fixed in formalin. Histopathological examination of H&E stained sections, quantification of villus length, and immunolabelling of IECs for active caspase 3 was performed. Dose response experiments were performed using 0.125 to 20 mg/kg LPS, sacrificing mice (n=4 per group) at 1.5 h. Transgenic mice (NFκB1−/−, NFκB2−/− and cREL−/−) were administered 10 mg/kg or 0.125 mg/kg LPS and euthanased at 1.5 h (n=6). Statistical comparisons were made by t-test with Bonferroni correction. Results In C57BL/6 mice, 10 mg/kg LPS caused a 20–50-fold increase in IEC apoptosis and shedding, with consequent villus atrophy, which was greatest in the duodenum and jejunum at 1.5 h, and in the ileum at 2 h. IEC apoptosis and shedding had subsided by 6 h after LPS administration. LPS at 0.125 mg/kg caused an attenuated response in C57BL/6 mice in terms of villus injury and amounts of apoptosis and shedding at 1.5 h, whereas at this dose NFκB1−/− mice were severely affected, while NFκB2−/− mice were minimally affected. Conclusion These findings suggest that endotoxic shock causes dynamic and hyperacute selective injury to the murine small intestine, prior to obvious pathological alterations in other organ systems. The induced apoptosis and cell shedding occurs within a well defined time period, and varies in intensity and time course in different intestinal segments. The differing sensitivity to LPS in transgenic mice implicates NFκB signalling in the pathogenesis of this type of intestinal injury. Competing interests None declared.