A Study of Immune-Regulation of Microbiota Supernatant on Macrophage Function

The diverse human microbial ecosystem colonizes many different body sites. The gastrointestinal (GI) tract, especially, is a major region that that harbors a complex microbial community composed of trillions of commensal bacteria. Interactions between the commensal bacteria and the host immune system begin at birth and continue through the human lifespan. In the gut, commensal bacteria help the host to ensure the maintenance of intestinal homeostasis as well as the mucosal barrier structure through host stimulation of mucus and antimicrobial molecules. The secretion of these host factors functions to inhibit pathogenic invasion and maintain the population of commensal bacteria. The microbial communities also secrete their own metabolites to regulate host metabolism as well as host immunity. Together, this complex crosstalk allows a series of host-microbe interactions and host responses that are necessary to manipulate the host immunity, maintenanmucosal homeostasis and regulate the composition of the microbiota. In the gastrointestinal tract, the major immune cells that actively participate in the host defense mechanism are resident intestinal macrophages. These cells are primarily responsible for the first-line host defense that protects the host from an enormous number of potentially pathogenic bacteria and/or antigenic stimuli that are present in the intestinal lumen. Normally, during steady state, these cells are very anergic and tolerant towards the commensal bacteria. Yet, they still retain their phagocytic and bactericidal activity. Although the mechanisms surrounding the anergy of the intestinal macrophages are not well understood, the abundance of commensal bacteria present in the gut lumen may suggest that the possible function of commensal bacteria is to influence the activity of gut macrophages. Little is known about the commensal bacteria’s ability to exert their function on the host intestinal macrophages through the production of commensal metabolites. It is well understood that commensal bacteria can secrete metabolites to regulate host metabolism as well as influence host immunity. We believe that the commensal bacteria might regulate intestinal macrophages in the same manner. In this study, we sought to understand the involvement of the secreted commensal bacterial factors/metabolites in response to the anergic function of intestinal macrophages. We hypothesized that the secreted bacteria factors/metabolites present in the bacterial supernatant can suppress macrophage activation upon the induction of TLR or NLRP3 agonists on macrophages. To test our hypothesis, we will use four different strains of commensal bacteria that are normally present in the gastrointestinal tract. Those are Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus rhamnosus, and Streptococcus oralis. We will determine whether the secreted bacterial factors present in the commensal culture supernatants can actually suppress the activation of macrophages in the presence of LPS or MSU crystal stimuli in vitro. Furthermore, we will also test whether the bacterial factors present in the bacterial supernatant can suppress the recruitment of neutrophil in the peritoneal cavity in the presence of LPS stimulation in vivo. If the secreted commensal factors/metabolites can suppress the activation of macrophage or the recruitment of netrophils upon the induction of TLR or NLRP3 agonists on macrophages, we will then seek to determine whether the secreted commensal factors can suppress macrophages activation in the transwell system, a system that mimics the gastrointestinal tract consisting of both epithelial cells and macrophages. By taking advantage of the transwell system, we will determine whether the secreted bacterial factors in the commensal supernatants can actually suppress macrophage activation during the induction of TLR or NLRP3 agonists on macrophages in the presence of epithelial layers. Together, these studies provide insights into how commensal bacteria can regulate macrophage responses in the gastrointestinal system. We discovered that the secreted commensal factors, which have molecular weights of less than 10Kda, can suppress macrophage activation as well as neutrophil recruiment in both in vivo and in vitro as well as in the transwell system upon MSU crystal or LPS crystal stimuli. In contrast, the secreted factors present in the commensal supernatants which have molecular weight greater than 10KDa act as a stimulatory mediator and promote the activation of macrophages in TLR-2 dependent manner