Several viruses induce intestinal epithelial cell death during enteric infection. T3D-RV

Several viruses induce intestinal epithelial cell death during enteric infection. T3D-RV reassortant viruses, we recognized the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral contamination to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric contamination of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral contamination. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease. IMPORTANCE Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Contamination with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this computer virus in the development of celiac disease, an autoimmune intestinal disorder brought on by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal contamination. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease. = 7 to 10 mice per computer virus strain). (A) Titers of T1L and T3D-RV in different regions of the intestine and secondary lymphoid organs were determined at the times shown by plaque assay. The small intestine was sectioned into thirds, approximating the duodenum, jejunum, and ileum. Viral titers are expressed as PFU per tissue. The 24-hpi titer values were previously published in reference 3; data are used with permission of the publisher. (B) One day after inoculation, intestines LEE011 novel inhibtior were resected, and the distal half was flushed, Swiss rolled, and processed for histology. Sections were stained with a polyclonal antiserum specific for reovirus. Representative sections of jejunum are shown (scale bar, 100 m). Error bars show SEMs. *, 0.05; LEE011 novel inhibtior **, 0.01; ****, 0.0001; one-way LEE011 novel inhibtior ANOVA and Sidak’s multiple-comparison test. To determine cell types in the intestine targeted by T1L and T3D-RV, mice were inoculated perorally and euthanized at 1 day postinoculation (dpi). Intestines were dissected, Swiss rolled, Rabbit polyclonal to ZNF286A and processed for immunohistochemistry. In intestines from both T1L- and T3D-RV-infected mice, cells displaying morphological characteristics of mature IECs stained positive for reovirus antigen (Fig. 1B). Consistent with previous LEE011 novel inhibtior observations (19), the incidence of reovirus-positive cells was low. Thus, both T1L and T3D-RV infect mature enterocytes in intestines of adult mice. T3D-RV contamination induces caspase-3 activation and villus shedding in the gut. To determine whether T1L and T3D-RV induce cell death and cause tissue damage = 5 to 18 mice per group). (C) Cleaved-caspase-3 staining in the lumen was quantified by outlining the luminal region using the Digital Histology Shared Resource tool (= 3 mice per computer virus). The percent luminal staining was decided as follows: (area in the lumen positive for cleaved-caspase-3 staining/area in the whole tissue positive for cleaved-caspase-3 staining) 100. (B) Error bars indicate SEMs. (C) Error bars indicate SDs. *, 0.05; ***, 0.001. values were determined by one-way ANOVA and Tukey’s multiple-comparison test (B) and Mann-Whitney test (C). Since T1L and T3D differ in the capacity to induce apoptosis, we hypothesized that T3D-RV induces more apoptosis in the gut, which could stimulate sloughing of infected enterocytes to mediate the quick viral clearance observed in Fig. 1A. To determine whether T1L and T3D-RV differ in the capacity to trigger apoptosis, epithelial cells positive for cleaved caspase-3 were enumerated and normalized to the total quantity of villi examined. T3D-RV-infected mice experienced significantly more epithelial cells positive for cleaved caspase-3 per villus than did those infected with T1L (Fig. 2B). To test whether T1L and T3D-RV differ in the shedding of apoptotic enterocytes into the intestinal lumen, the luminal region was layed out using Ariol Review software, the area positive for cleaved caspase-3 was demarcated, and the percentage of positive staining in the lumen was quantified relative to the positive staining.