Objectives Loss of intestinal barrier function plays an important role in the pathogenesis of inflammatory bowel disease (IBD). Shedding of intestinal epithelial cells is a potential cause of barrier loss during inflammation. The objectives of the study were (1) to determine whether cell shedding and barrier loss in humans can be detected by confocal endomicroscopy and (2) whether these parameters predict relapse of IBD. Methods Confocal endomicroscopy was performed in IBD and control patients using intravenous fluorescein to determine the relationship between cell shedding and local barrier dysfunction. A grading system based on appearances at confocal endomicroscopy in humans was devised and used to predict relapse in a prospective pilot study of 47 patients with ulcerative colitis and 11 patients with Crohn's disease. Results Confocal endomicroscopy in humans detected shedding epithelial cells and local barrier defects as plumes of fluorescein effluxing through the epithelium. Mouse experiments demonstrated inward flow through some leakage-associated shedding events, which was increased when luminal osmolarity was decreased. In IBD patients in clinical remission, increased cell shedding with fluorescein leakage was associated with subsequent relapse within 12 months after endomicroscopic examination (p<0.001). The sensitivity, specificity and accuracy for the grading system to predict a flare were 62.5% (95% CI 40.8% to 80.4%), 91.2% (95% CI 75.2 to 97.7) and 79% (95% CI 57.7 to 95.5), respectively. Conclusions Cell shedding and barrier loss detected by confocal endomicroscopy predicts relapse of IBD and has potential as a diagnostic tool for the management of the disease.

The intestinal epithelium is a critical component of the gut barrier. Composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, this delicate structure prevents the transfer of harmful microorganisms, antigens, and toxins from the gut lumen into the circulation. The equilibrium between the rate of apoptosis and shedding of senescent epithelial cells at the villus tip, and the generation of new cells in the crypt, is key to maintaining tissue homeostasis. However, in both localized and systemic inflammation, this balance may be disturbed as a result of pathological IEC shedding. Shedding of IECs from the epithelial monolayer may cause transient gaps or microerosions in the epithelial barrier, resulting in increased intestinal permeability. Although pathological IEC shedding has been observed in mouse models of inflammation and human intestinal conditions such as inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This process may also be an important contributor to systemic and intestinal inflammatory diseases and gut barrier dysfunction in domestic animal species. This review aims to summarize current knowledge about intestinal epithelial cell shedding, its significance in gut barrier dysfunction and host-microbial interactions, and where research in this field is directed.

Background & Aims Tumor necrosis factor (TNF) increases intestinal epithelial cell shedding and apoptosis, potentially challenging the barrier between the gastrointestinal lumen and internal tissues. We investigated the mechanism of tight junction remodeling and barrier maintenance as well as the roles of cytoskeletal regulatory molecules during TNF-induced shedding. Methods We studied wild-type and transgenic mice that express the fluorescent-tagged proteins enhanced green fluorescent protein–occludin or monomeric red fluorescent protein 1–ZO-1. After injection of high doses of TNF (7.5 μg intraperitoneally), laparotomies were performed and segments of small intestine were opened to visualize the mucosa by video confocal microscopy. Pharmacologic inhibitors and knockout mice were used to determine the roles of caspase activation, actomyosin, and microtubule remodeling and membrane trafficking in epithelial shedding. Results Changes detected included redistribution of the tight junction proteins ZO-1 and occludin to lateral membranes of shedding cells. These proteins ultimately formed a funnel around the shedding cell that defined the site of barrier preservation. Claudins, E-cadherin, F-actin, myosin II, Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK) were also recruited to lateral membranes. Caspase activity, myosin motor activity, and microtubules were required to initiate shedding, whereas completion of the process required microfilament remodeling and ROCK, MLCK, and dynamin II activities. Conclusions Maintenance of the epithelial barrier during TNF-induced cell shedding is a complex process that involves integration of microtubules, microfilaments, and membrane traffic to remove apoptotic cells. This process is accompanied by redistribution of apical junctional complex proteins to form intercellular barriers between lateral membranes and maintain mucosal function.

SUMMARY 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 into the blood. 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 can be disturbed as a result of increased IEC shedding. Such pathological IEC shedding can 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, because 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 in mice deficient in TNF-receptor 1 (Tnfr1^−/−), Tnfr2 (Tnfr2^−/−), nuclear factor kappa B1 (Nfκb1^−/−) or Nfĸb2 (Nfĸb2^−/−). Apoptosis and cell shedding was quantified using immunohistochemistry for active caspase-3, and gut-to-circulation permeability was assessed by measuring plasma fluorescence following fluorescein-isothiocyanate–dextran gavage. LPS, at doses ≥0.125 mg/kg body weight, induced rapid villus IEC apoptosis, with peak cell shedding occurring at 1.5 hours after treatment. 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 5 hours. TNFR1 was essential for LPS-induced IEC apoptosis and shedding, and the fate of the IECs 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.

Gastric colonization with Helicobacter pylori induces diverse human pathological conditions, including superficial gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma and its precursors. The treatment of these conditions often relies on the eradication of H. pylori, an intervention that is increasingly difficult to achieve and that does not prevent disease progression in some contexts. There is, therefore, a pressing need to develop new experimental models of H. pylori-associated gastric pathology to support novel drug development in this field. Here, we review the current status of in vivo and ex vivo models of gastric H. pylori colonization, and of Helicobacter-induced gastric pathology, focusing on models of gastric pathology induced by H. pylori, Helicobacter felis and Helicobacter suis in rodents and large animals. We also discuss the more recent development of gastric organoid cultures from murine and human gastric tissue, as well as from human pluripotent stem cells, and the outcomes of H. pylori infection in these systems.

Purpose: Cytokines such as interleukin (IL)-6 and granulocyte colony-stimulating factor (G-CSF) are important metastasis promoters. This study has investigated the functional significance of the increased circulation of galectin-3, a common feature in patients with cancer and in particular those with metastasis, on cytokine secretion from the blood vascular endothelium in cancer. Experimental Design: The effects of galectin-3 on secretion of cytokines from human microvascular lung endothelial cells were assessed in vitro by cytokine array and in vivo in mice. The consequences of galectin-3–induced cytokine secretion on endothelial cell behaviors were determined, and the relationship between the levels of circulating galectin-3 and cytokines in patients with colorectal cancer with and without metastasis was investigated. Results: Galectin-3 at pathologic concentrations found in patients with cancer induces secretion of IL-6, G-CSF, sICAM-1, and granulocyte macrophage colony-stimulating factor from blood vascular endothelial cells in vitro and in mice. These cytokines autocrinely/paracrinely interact with the vascular endothelium to increase the expressions of endothelial cell surface adhesion molecules integrinα_vβ₁, E-selectin, ICAM-1, and VCAM-1, resulting in increased cancer cell–endothelial adhesion and increased endothelial cell migration and tubule formation. In patients with metastatic colon cancer, higher serum galectin-3 levels correlated significantly with increased serum G-CSF, IL-6, and sICAM1 concentrations. Conclusion: The increased circulation of galectin-3 in patients with cancer induces secretion of several metastasis-promoting cytokines from the blood vascular endothelium that enhances endothelial cell activities in metastasis. Targeting the actions of circulating galectin-3 in patients with cancer therefore represents a promising therapeutic strategy to reduce metastasis and improve survival. Clin Cancer Res; 19(7); 1693–704. ©2013 AACR.