Figure 7.

Depletion of CD8⁺ cells improved HSV-1 induced intestinal dysmotility. (A) Experimental design: at the 4^th week post HSV-1 OG administration, the mice were intraperitoneally injected with the anti-CD8 monoclonal antibody or IgG2b isotype antibody. The mice were sacrificed 4 weeks later. (B) LMMP preparations were obtained from the ileum of HSV-1-infected mice treated with anti-CD8 monoclonal antibody or IgG2b isotype antibody. LMMP were enzymatically digested, and the resulting cell suspensions were labeled with anti-CD3 and anti-CD8 antibodies and analyzed using flow cytometry. Cells were first selected on a forward scatter and side scatter dot plot and then double positive cells were recorded in 10,000 events. Representative images of 3 independent experiments; n=6 mice per group. (C) Sections of ileum collected from HSV-1 infected mice treated with anti-CD8 monoclonal antibody or IgG2b isotype antibody were subjected to immunohistochemistry for CD3. Representative images of 3 independent experiments. Representative images of 3 independent experiments. n=6 mice per experimental group; 4 independent fields per animal were examined. Scale bar: 40 µm. (D) Immunofluorescence for peripherin on whole mount preparations of distal ileum prepared from HSV-1 infected mice treated with anti-CD8 monoclonal antibody or IgG2b isotype antibody. Representative images of three independent experiments. Representative images of 3 independent experiments. n=6 mice per experimental group; 6 independent fields per animal were examined. Scale bars: 60 µm. (E) Sham infected and 8 wks HSV-1 OG infected mice treated with IgG2b isotype antibody or anti-CD8 antibody were administered via gastric gavage with non-absorbable FITC-labeled dextran and sacrificed 60 minutes later. Gastric emptying was calculated as the percentage of dextran retained in the stomach compared to the total fluorescence in the gastrointestinal tract. Intestinal transit refers to the geometric center that is the center of the distribution of fluorescent dextran in the ileum. Experiments were repeated 3 times; n=6 mice per group. * denotes p < 0.05 vs sham infected mice. (F) Total RNA was extracted from the LMMP of mice at the 8^th wks from the OG HSV-1 inoculum and treated with IgG2b isotype antibody or anti-CD8 antibody. Quantitative PCR was performed to evaluate the expression of HSV-1 genes. The data are reported as the mean ± SEM of gene levels detected in the mice treated with anti-CD8 antibody with respect to those identified in the infected animals dosed with IgG2b isotype antibody. Experiments were repeated 3 times; n=6 mice per group. * denotes p < 0.05 vs 8wks HSV-1 OG inoculum treated with IgG2b isotype antibody.