Dependency of TLR4 signaling for the anti-inflammatory effect of exopolysaccharide (EPS) of Bacillus subtilis (MPF4P.731)

Abstract

Abstract Although probiotics offer a promising source of therapeutic compounds, few studies have identified the bacterial molecules or host signaling pathways required for the immunomodulatory effects. We previously demonstrated that exopolysaccharide (EPS) from probiotic Bacillus subtilis is the bacterial molecule that prevents disease induced by the murine pathogen Citrobacter rodentium, a pathogen that shares many similarities to the human pathogen enteropathogenic E. coli. To begin to elucidate how EPS functions, we determined which cells are required for protection and found that mice depleted of macrophages using clodronate loaded liposomes are not protected from the pathogen. Further, transfer of macrophage-rich peritoneal cells from wt, but not TLR4 KO, EPS-treated mice conferred protection from pathogen-induced colitis; these data suggest a role for TLR4 and macrophages. Using flow cytometry and microscopy, we find that EPS binds F4/80+CD11b+ macrophages and co-localizes with TLR4. We also find that EPS binds both wt and TLR4 KO macrophages, suggesting that EPS binds to a component of the TLR4 signaling complex instead of TLR4 directly. We are immunoprecipitating the EPS receptor, predicted to be a co-receptor of the TLR4 signaling complex, and determining if EPS is an agonist or antagonist of TLR4 signaling. Our studies should lead to a new class of bacteria-derived immunomodulatory molecules.