Intestinal NK Cell Plays A Key Role In Mucosal Inflammation And Bacterial Translocation Of Rhesus Monkeys Infected With SIV/SHIV

Intestinal mucosa system plays an important role in preventing harmful microorganisms or virus from invading circulation. However, cellular immunity in mucosa has been influenced seriously during HIV/SIV early infection, which may lead internal environment of intestinal to losing balance and the stable-state being disrupted. Inflammation or complicated infection may appear in intestinal mucosa system. During long-term of SHIV1157ipd3N4infection in mucosa, it’s widely existed that whole of mucosal immunity are seriously disordered and the mucosal barrier that blocks becteria from invading into lamina propria are also largely destructed. In some researches, it has been suggested that high levels of plasma LPS concentration as bacterial metabolities are detected in numbers of patients infected with HIV, which demonstrated HIV infection may lead bacteria through mucosal barrier invading into blood circulation. As the important components of innate immunity, NK cells play significant roles in anti-SIV/HIV infection, which has been published in many papers. However, roles of NK cells in intestinal mucosa system during SIV/SHIV infection are still lack of systematically research. In addition, another NK subset distributed in mucosa tissues is founded in human, which plays mucosal protection roles through IL-22production. For these reasons, immune mechanism of NK cells in inflammation and bacterial translocation during SIV/SHIV infection are explored in this research.Firstly, in order to know about the role of classical NK cells and NKp44+NK cells in intestinal system of rhesus monkeys during SIVmac239early infection, healthy rhesus monkeys of China-origin were selected for this research. To get enough ileum mucosa tissues, operations or euthanasia are handled with these animals, and the corresponding study covers virology, immunology, and cell biology and laboratory animal science. In this study, firstly, we do some explorations on the mechanism of SIVmac239early infection in the intestinal systems of rhesus monkeys. SIV virus can spread into intestinal mucosa system so quickly after inoculation by intravenous injection. In intestinal system, High virus copies can be detected only one day after infection. Serious damage of CD4+T cells in MALTs appears within one week, including depressed CD4+/CD8+ratio, high apoptosis rate. Although CD8+T cells can be greatly activated at this period, increased level of1L-10concentration are detected from ileum tissues, which suggests that inflammation may be occurred at this position. Secondly, we found the mechanism that numbers of NKp44+NK cells are expanded and their IL-22production are enhanced in gut-associated lymph tissues (GALTs) of rhesus monkeys during acute SIVmac239infection. Phenotype and functions of NKp44+NK cells in MALTs are primarily detected though FCM analysis. Phenotype of cells which express NKp44markers also secret IL-22in MALTs of rhesus monkeys is different from that of human. Increased frequencies and IL-22secreting function of NKp44+NK cells occure during early SIVmac239infection. Thirdly, changes of frequencies and functions of different classical NK cell subsets in MALTs of rhesus monkeys during acute SIVmac239infection are explored. Classical NK cells are divided into four subsets in GALTs, according to CD16and CD56. CD56-CD16+NK subset in MALTs is expanded in numbers and the degranulation functions are enhanced during acute phase of SIV infection. The ratio of DN NK subset is reduced but their functions show unchangeable. The ratio of DP and CD56+CD16" NK subsets increase slightly, but the immunoregulation functions are decreased.To study roles of NK cells in bacterial translocation and to explore whether bacterial translocation has correlations with some components of mucosal immunity, further experiments are designed. Animals infected with SHIV1157ipd3N4for one year are grouped into LPShigh and LPSlow groups by plasma LPS level. Then, we design experiments on the correlations between different components of immunity in periphery or mucosa and the LPS levels in infected animals. In this research, Numbers and functions of T cells, B cells, classical NK cells and NKp44+NK cells from periphery and mucosa system are taken into comparison between LPShigh and LPSlow two groups. In this research, experiment methods of multiple colors FCM analysis, ELISA, RNA extraction and qRT-PCR are widely used.These data demonstrate that viral load in plasma and gut tissues has no correlations with levels of LPS concentration in blood. However, ways of SIV infection from rectal mucosa may increase the probability of bacterial translocation. Endotoxemia prompt activated conditions of T cells, which may increase level of SIV/SHIV duplication in intestinal mucosal system and lead more target CD4+T cells lost. Compared with LPSlow group, numbers of NKp44+mucosal NK cells are decreased and it’s function of IL-22production are reduced in animals with high bacterial translocation. Although functions of IFN-γ secretion and degranulation cytotoxicity are enhanced, the results indicate that it cannot have positive effect on bacterial translocation. Functions of classical NK cells are inhibited during SHIV chronic infection. High level of bacterial translocation may lead classical NK cells further activated, however, just low level of cytotoxicity function is detected. These data demonstrated disfunction of NKp44+NK cells and classical NK cells may aggravate extent of bacterial translocation. Thus, normal functions of mucosal NKp44+NK cells plays significant role in protecting mucosal barrier from bacteria invasion.In addition, to improve the intracellular cytokines staining (ICS) assay and serve the detection of SIV-specific cellular immunity in SIV-infected rhesus monkeys, three polyclonal activators are used for PBMC stimulation, and the best positive stimulators and the reasonable stimulating time are tested and determined. Five concentrations of SIVmac239mixed peptides pool are designed to stimulate PBMC from SIV-infected rhesus monkeys, followed by culturing in vitro and detected with flow cytometry at different times, and the optimal concentration of SIV mixed peptides pool and the best stimulating time are ascertained finally. Then, this method is used for detecting of the SIV-specific cellular immunity in the monkeys, combination of PMA+ionomycin can be used as the best positive stimulator. Cytokines secreted from T cells are detected at the highest level by2μg/mL of SIVmac239mixed peptides pool at37℃and in5%CO2culturing for16h in vitro. Optimization of the experimental conditions of ICS has been successfully accomplished, and it may be useful for pre-clinical evaluation of AIDS drugs and vaccine research.In summary, mechanism of mucosa immune system, especially for NK cells as the important part of innate immune system, plays very important roles in early SIV/SHIV infection. Further exploration on the internal significance of NK cells, as well as other immune components, will be benefit for understanding the pathogenesis of AIDS well.