The Anti-Inflammatory Properties of Exopolysaccharide from Bacillus Subtilis

Commensal microorganisms can be used therapeutically to ameliorate a variety of diseases. The laboratory of Dr. Katherine L. Knight studies these therapeutic properties in the commensal microorganism known as Bacillus subtilis. Previously, the Knight lab has demonstrated that B. subtilis can protect against colitis caused by Citrobacter rodentium suggesting that B. subtilis possesses anti-inflammatory properties. These properties are specific to the polysaccharide capsule of the microorganism known as exopolysaccharide or EPS. EPS can be produced and purified for study. EPS has been demonstrated to be protective in a variety of diseases such as allergic eosinophilia, sepsis, Graft versus Host disease, and reduce proliferation of tumor cells in vitro. This dissertation focuses on the anti-inflammatory properties of EPS. It focuses on one aspect in vivo and another aspect in vitro. In the context of in vivo, LPS is a inflammatory polysaccharide that signals through TLR4. EPS also signals through TLR4 yet it induces an anti-inflammatory response in vivo. Therefore, the immune response of these molecules are very different and it became of interest to determine if EPS could induce an anti-inflammatory state against LPS known as endotoxin tolerance. Our results demonstrated that EPS could induce a state of endotoxin tolerance and it lasted for 1 week. Moreover, this inhibitory activity was mediated through innate cells,specifically, macrophages found in the small intestine. Endotoxin tolerance by EPS was specific to in vivo, but was not observed in vitro. Furthermore, we observed EPS downregulate cell surface expression of TLR4 in BMDCs in vitro suggesting that this is a potential mechanism by which EPS induces endotoxin tolerance. In addition to looking at the mechanism in the context of inflammation. It became also of interest to determine how EPS generates M2 anti-inflammatory macrophages. It has been previously demonstrated that EPS can generate M2 macrophages in vivo through intraperitoneal injection, but these macrophages cannot be generated in vitro through treating the cells directly with EPS. One potential explanation is that the environment in vitro is not suitable for the generation of M2 macrophages by EPS. We determined that transferred TLR4-KO peritoneal cells could become M2 macrophages upon EPS injection suggesting that the environment of the peritoneal cavity is required for their induction. We also observed the production of an M2 differentiating cytokine IL-4 in the serum suggesting that this is a potential mechanism by which M2 macrophages are being induced. Finally, we also demonstrated that transfer of peritoneal fluid or serum can induce M2 peritoneal macrophages in vitro. Taken collectively, data from this thesis demonstrate a novel mechanism that EPS works in vivo and it gives us mechanistic insights into how EPS generates anti-inflammatory M2 macrophages in vivo. This information regarding this polysaccharide will ideally allow a new therapy and can be utilized to treat diseases in the future.