Mechanism Of Cross-talk Between Macrophages And Cd4~+T Cells In The Intestinal Immune System

Object:Intestinal tract is the body’s internal environment direct communication with the outside of the main interface.The intestinal mucosal immune system is the body’s first line of defense against intestinal pathogens infection. Small intestinal lamina propria is a major effect of the intestinal immune system site, which contains a large number of macrophages, CD4+T cells and dendritic cells, etc., in the early resistance plays a key role in the process of intestinal pathogenic bacteria.Among them, the macrophages by producing reactive oxygen intermediates and inducible nitric oxide synthase, play a key in the process of antibacterial effect. CD4+T cells can secrete IFN-gamma that promote macrophage activation. But in the intestinal mucosal immune system against foreign pathogens infection process, about both the existence of mutual regulation and their roles in anti-infection and mechanism, the research is less.A model of Salmonella infection of the intestine by oral infection with the bacteria S. typhimurium strain was established, to observe intestinal lamina propria macrophages and CD4+T cell activation and their roles in the resistance to infection, and discuss the interaction and mechanism, which will provide the intestinal infection and inflammatory diseases such as repair new treatment strategies and ideas.Methods:Firstly, we adopt the method of oral infection with salmonella typhimurium animal model was established. After infection of salmonella typhimurium, we used flow cytometry to detect the mouse small intestine lamina propria lymphocyte percentage of each subgroup, quantity, and the change of activation.Secondly, in order to study the intestinal macrophages and CD4+T cells in the presence of each other after the pathogen invasion, we respectively used antibody to deplet CD4+T cells in mice and used Cl2MDP-liposome to remove macrophages. Then, we detected the change of number and activation of macrophages after removal of CD4+T cells in infected-mice and the change of the number and activation of CD4+T cells after removal of macrophages in infected-mice.Moreover, we used the mice infected with salmonella typhimurium in vitro peritoneal macrophage incubated with magnetic bead separation of CD4+T cell, and add the a-lactose to block galectin-9-Tim-3. Microbial load, activation and cytokines secretion of the macrophages were detected, and activation and cytokines secretion and apoptosis of CD4+T cell were tested by FACS.Finally, we use western blot to test inflammasome NLRP3and NLRC4after infection with salmonella typhimurium in vitro and caspase1protein expression level of peritoneal macrophage, using western blot and ELISA, both FACS and other technology to detect the expression of IL-1β.In addition, we also tested the microbial load of infected macrophages incubated with CD4+T cells in the presence of IL-1β neutralizing antibody. We also tested the microbial load of infected peritoneal macrophage cultured with IL-1β and the impact of galectin-9expression. Our results preliminary explored the important role of galectin-9and Tim-3signaling pathways play in the interaction between macrophages and CD4+T cells and its mechanism.Results:1. Both number and activation of small intestinal lamina propria F4/80+CDllc+macrophage and CD4+T cells increased after infection.We detected the frequency, number and activation of small intestinal macrophages several days after infection by FACS. Interestingly, the frequency and number of small intestinal F4/80+CD11c+macrophages and CD4+T cells increased. These results indicated that these two kinds of cells play an important role in the process of removal of salmonella typhimurium.2. Depletion of CD4+T cells affected the activation of small intestine lamina propria macrophage.We used antibody to remove CD4+T cells in mice, and then compared the changes of the number and activation of small intestine lamina propria macrophages of depleted group with control group after infection.We found that the depleted CD4+T cells infected mice suffered from more microbial load than control infected group, and their intestinal macrophage activation and secretion of cytokines (had the greatest impact on IL-1β) were lower than the control infected group.3. Depletion of macrophages influenced the activation of small intestinal lamina propria CD4+T cell.In addition, we used Cl2MDP liposome to deplet macrophage, and then compared groups the changes of number and activation of the small intestine lamina propria CD4+T cells from depleted group with control group both of that infected with S. typhimurium. We found that the infected mice which were depleted macrophages emerged more microbial load than control infected group, and their intestinal CD4+T cells activation and secretion of cytokines were less than the control infected group. Meanwhile, both the number of CD4+T cells and CD8+T cells increased, but only CD4+T cells appeared enhanced activation.4. The blocking galectin-9-Tim-3pathway by a-lactose affected interaction between macrophages and CD4+T cell interactions.In order to verify the galectin-9and Tim-3signaling pathways involved in the interaction between intestinal macrophages and CD4+T cells, we conducted incubation experiment in vitro. Results show that adding different concentration of alpha lactose to block the combination of the galectin-9and Tim-3, microbial load of the macrophage increased with the rise of a-lactose concentration, but the secretion of IL-1β decreased with it. And activation of CD4+T cells is not affected obviously, but their apoptosis declined as concentration of a-lactose increased.5. The expression level of cytokines and inflammasomes and their downstream signaling molecules of macrophages increased after infection.Determination about various indicators of peritoneal macrophages after infection with salmonella typhimurium (MOI=20:1) in vitro proved that the infected macrophages secrete more IL-β. The expression of inflammasome NLRC4and NLRP3and caspase-1of infected macrophages increased. All of them increased at6hours, reaches their peak at about18hours and present a downward trend after24hours after infection.6. Blocking galectin-9-Tim-3by a-lactose impacted the expression of inflammasomes NLRC4and NLRP3and caspase-1of macrophages.We detecting the expression of inflammasomes and their downstream signaling molecules of macrophages which incubated with CD4+T cell adding different concentration of a-lactose. The results showed that when blocking combination of galectin-9and Tim-3by adding a-lactose, inflammasomes NLRC4and NLRP3, mature caspase-1and the expression of IL-1β showed a trend of decline with the increase of a-lactose concentration. These results suggested that galectin-9-Tim-3can promoted the expression of inflammasomes and downstream molecules in macrophages.7. IL-1β promoted the antibacterial function and the expression of galectin-9of macrophage.We found that with the increase of concentration of IL-1β, microbial load of the infected macrophages which were stimulated with different concentration of IL-β gradually declined. Infected macrophages would suffer from a higher microbial load with the presence of IL-1β neutralizing antibody. And we detected expression of galectin-9on macrophages which culture with10ng/mL IL-1β after the infection by FACS. With the extension of incubation time, expression of galectin-9increased.Conclusions:1. Macrophages in the small intestine, particularly F4/80+CD11c+macrophages, and CD4+T cells are critical for the detection of pathogenic bacteria during S. typhimurium infection. 2. Intestinal macrophages and CD4+T cells interact with each other after infected with S. typhimurium.3. Galectin-9-Tim-3pathway play a important role in cross-talk between intestinal macrophages and CD4+T cells.4. IL-1β induced by galectin-9-Tim-3pathway led to control of bacterial infection or IL-1β stimulated galectin-9expression.5. Galectin-9-Tim-3interaction triggered IL-1β secretion in an inflammasome-dependent manner.