The gut barrier, composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, prevents the entrance of harmful microorganisms, antigens and toxins from the gut lumen. Small intestinal homeostasis is normally maintained by the rate of shedding of senescent enterocytes from the villus tip exactly matching the rate of generation of new cells in the crypt. However in various localized and systemic inflammatory conditions, intestinal homeostasis may be disturbed as a result of increased IEC shedding. Such pathological IEC shedding may cause transient gaps to develop in the epithelial barrier and result in increased intestinal permeability. Although pathological IEC shedding has been implicated in the pathogenesis of conditions such as inflammatory bowel disease, our understanding of the underlying mechanisms remains limited. We have therefore developed a murine model to study this phenomenon, as IEC shedding in this species is morphologically analogous to humans. IEC shedding was induced by systemic lipopolysaccharide (LPS) administration in wild-type C57BL/6 mice, and mice deficient in TNF-receptor 1 (Tnfr1-/-), Tnfr2-/-, Nuclear Factor kappa B1 (Nfκb1-/-) or Nfκb2-/-. Apoptosis/cell shedding was quantified using immunohistochemistry for active Caspase-3 and gut lumen to systemic circulation permeability was assessed by measuring plasma fluorescence following fluorescein isothiocyanate-dextran gavage. LPS, at doses ≥0.125mg/kg induced rapid villus IEC apoptosis with peak cell shedding occurring at 1.5h. This coincided with significant villus shortening, fluid exudation into the gut lumen and diarrhea. A significant increase in gut to circulation permeability was observed at 5h. TNFR1 was essential for LPS-induced IEC apoptosis and shedding and the fate of the IEC was also dependent on NFκB, with signaling via NFκB1 favoring cell survival, and via NFκB2 favoring apoptosis. This model will enable investigation of the importance and regulation of pathological IEC apoptosis and cell shedding in various diseases.
- Received June 13, 2013.
- Accepted August 15, 2013.
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