The Impact Of Alveolar Epithelial Cells On Toll-Like Receptor-Mediated Inflammatory Responses Of Macrophages Against Mycobacterium Tuberculosis Infection

Pulmonary tuberculosis is a chronic fatal zoonotic infectious disease caused by an infection of Mycobacterium (Mtb).As the etiologic agent for Tuberculosis disease is known to induce strong immune responses and successfully survive within its host macrophages. The same as pulmonary epithelial cells are also targets of primary infection and are routes for the spread of pulmonary pathogens. But an organism to resist exogenous pathogens depends on the interaction between various cells. So, it’s necessary to explore the immunoregulatory role of alveolar epithelial cells to macrophages in response to Mtb infections in an air-liquid interface (ALI) model mimicking the vivo environment.An ALI of alveolar epithelial cells A549 and U937 mononuclear cell-derived macrophages co-culture model was employed in our study. We trying to discover the immune regulation of the epithelial cells to macrophage in the process of co-culture model infected by H37Rv through detecting macrophages TLR signal and its downstream inflammation factors expression between mycobacterium tuberculosis H37Rv infection respectively or co-infection. Some results as follows:(1) The study showed a trend of increase in Toll signaling molecules TLR2/4/6/8, MyD88 and TRAF6, NF-κB as well as their down-stream inflammatory cytokines IL-1(3/2/6/8/10/12β in U937 cells when U937 macrophages were infected with H37Rv. But both of A549 cells and U937 cells were infected with H37Rv, the inflammatory responses of U937 macrophages were alleviated in comparison with U937 were infected alone in the ALI co-culture model, as determined by the expression of above signaling molecules and cytokines. This result revealed a molecular mechanism potentially involved in this ALI co-culture model, A549 epithelial cells may be plays a special role in the regulation of U937 macrophage in immune response, make A549 cells can alleviative TLR signal mediated inflammatory response to U937 macrophage in the process of infected by H37Rv.(2) Compared with no inhibitors, LY294002 can reduce holistic inflammatory response intensity in U937 macrophages in the process of co-culture model infected by H37Rv. But in the presence of LY294002, both of A549 cells and U937 cells were infected with H37Rv, the inflammatory responses of U937 macrophages were aggravate in comparison with U937 were infected alone in the ALI co-culture model, as determined by the expression of above signaling molecules and cytokines. Make A549 cells loss of the roles of alleviative TLR signal mediated inflammatory response to U937 macrophage in the process of infected by H37Rv. Meanwhile, we find out LY294002 can reduce pGSK3β expression to improve the activity of GSK3β. The existence of LY294002 make A549 lost the function that relieve TLR signal mediated inflammatory response in U937 cells.(3) In the presence of LiCl, the result from gene and protein levels test show:A trend of increase in Toll signaling molecules and their down-stream inflammatory cytokines in U937 cells when U937 macrophage were infected alone with H37Rv. But both of A549 cells and U937 cells were infected with H37Rv, the inflammatory responses of U937 macrophages is lower than the group that U937 infected alone in the ALI co-culture model. In the presence of LiCl, the down-regulation of TLR signal mediated inflammatory response in U937 macrophage is exist simultaneously with the down-regulation of GSK3β. From the above suggesting GSK3β played an important balancing role between epithelial cells A549 and U937 macrophage inflammatory response interaction in this co-culture infected model.(4) When mTOR inhibited by rapa, the expression of TLR4, TLR6, IL-6 and IL-12β in U937 significantly upregulated in co-cultured co-infected group. In experimenting we find out that in the presence of rapa in this co-culture model, the expression of GSK3β in co-infected group is significantly higher than U937 infected alone, but whether mTOR through GSK3P raised to stimulate the expression of pro-inflammatory factor still need more verification.In conclusion, we find out that alveolar epithelial cells can alleviate TLR signal mediated inflammatory response in the process of mononuclear macrophage infected by H37Rv to some extent, and this effect may be realized via PI3K pathway in macrophages by reducing the activity of GSK3β.