Effects Of Intraepithelial Lymphocyte On Intestinal Epithelium Inmurine Models Of Acute Intestinal Injury

Background:The intestinal mucosa constitutes an important organ in which defense against harmful organisms develops. At the time when body is injured by trauma, burns, shock, ischemia/reperfusion (I/R) and radiation induced injury, the intestinal barrier dysfunction occurs and enterogenous infection ensues. Increased inflammation activity might aggravate the primary disease. Although there have been many research studies of various aspects of intestinal barrier function (IBF) over the last ten years, the immune mechanism by which IBF are regulated remains unclear. The objective here was to focus on the underlying mechanism of the exaggerated immune response following intestinal injury, to clarify some key points (especially those perceived as points of controversy), and present new strategies.Specialized intestinal intraepithelial lymphocyte (IELs), located between the epithelium basolateral surfaces, are considerable as the first line of defense as well as in the maintenance of intestinal barrier homeostasis. In both the small and large intestines, IELs are primarily heterogeneous T cells. Most IELs, especially in the small intestine, express the homodimeric form of CD8a (CD8aa), which is barely detectable in the systemic immune compartments. Moreover, IELs from the small intestines of mice and humans contain more ??+T-cell receptor (TCR) T cells than do peripheral T cells. In mice, the proportion of TCR ??+T cells is approximately 60%, whereas in humans, it is approximately 10-14%. Although a major task of IELs is to mediate barrier homeostasis, the increased activation of IELs and the close proximity of these cells to the intestinal epithelium might contribute to immunopathological responses and initiate and/or exacerbate inflammation.Results from our laboratory have revealed that I/R induced the epithelial apoptosis and the disruption of TJ proteins (Claudin-1 and Occludin). More interestingly, we found that IL-7, which is produced by intestinal epithelial cells and influences IELs development and function, increased in the mild ischemia small intestinal tissues of patients with intestinal obstruction and in a murine model of acute intestinal I/R.Strategically located at the epithelial basolateral surface, IELs are intimately associated with epithelial cells and maintain the epithelial barrier integrity. Intestinal I/R-induced acute injury not only damages the epithelium but also affects the IBF. Therefore, we hypothesized that I/R-induced mucosal damage would affect IEL phenotype and function.Immunohistochemistry, Western blot, flow cytometry and other techniques were used to study the changes of the IELs subtype variation and function under conditions of I/R and radiation induced injury. We also wanted to examine the activation status of IEL subsets and its role in mucosalimmune-regulation, including TJs, TER. This proposal would give insightinto the mechanism of IELs in maintaining intestinal barrier homeostasis and mayprovide novel strategy to prevent the loss of intestinal barrier function.Motheds:AnintestinalI/Rmodel and a radiation induced injury model in mice were used to study the changes of intestinal morphologyand permeability. Western blot analysis and immunohistochemistry were used to detect the expression of TJs.IEL phenotype, activation, and apoptosis under the condition of I/R were examined by using flow cytometry.Co-culture model was used to detect if the IELs-derived cytokine(s) was involved in the regulation of intestinal barrier function, including TJs expression and structure.Mice were treated with anti-TNF-?, the changes of intestinal morphologyand permeability were detected with TER assay and microscopy analysis.Results:Severe mucosal damage, including villous blunting, epithelial denudation, and lamina propriadisintegration, were observed in the I/R group.After intestinal I/R, the continuityof occludin expression was disrupted, and this protein wasdiffusely presented along the apical membrane and withinthe subapical region, and the lateral space was enlarged, and the IELs detached fromthe epithelium.Intestinal I/R also significantlyincreased the CD8ap+, CD4+, and TCR??+IEL subpopulations. Furthermore, intestina I/R enhanced activation andpromoted apoptosis in IELs. The RT-PCR results showed that TNF-? and IL-10 mRNA expression in IELssignificantly increased after I/R (P<0.05).Mouse CT26 cell line was co-cultivated with IELs'supernatant.When co-cultured with I/R treated IELs'supernatant, CT26 expressed low levels of Claudin-1 and Occludin. Moreover, the TJs structure was disrupted and permeability was decreased.Intestinal I/R was linked to production of TNF-a, which was also detected a higher level in IELs'supernatant. Administration of a blocking anti-TNF-a could significantly reduce intestinal damage.The histological features occurring in the radiation induced injury model were characterized by the shortening of the villi, loss of villousepithelium, multiple erosions, inflammatory cell infiltration. Radiation caused an significantly decrease of IEC derived IL-7 expression. Furthermore,radiation casued a loss of IELwhen compared tothe control group.Conclusion:I/R resulted in intestinal epithelium destruction andIEL detachment. In addition, the phenotype and function ofIELs were altered. IELs-derived TNF-a disrupted the formation of TJs and decreased the permeability. Blocking anti-TNF-a could significantly attenuate these intestinal damages.Our results demonstrated thatirradiation significantly damaged the intestinalmucosa, increased the epithelial apoptosis andcaused the IELs loss, which affects the immune function in gut.