Impacto da inativação do gene slpB nos efeitos terapêuticos de Propionibacterium freudenreichii CIRM-BIA 129 em modelo murino de mucosite induzida por 5-Fluorouracil

Abstract

Propionibacterium freudenreichii CIRM-BIA 129 (P. freudenreichii wild-type, WT) is a Gram-positive bacteria used in cheese maturation, which has currently had its probiotic potential investigated. In previous studies, the consumption of this bacterium proved to exert immunomodulatory effects in a murine model of chemical agent-induced colitis. Part of the immunomodulatory effect of this strain depends on the S layer proteins (Slp) to which may be involved in persistence in the intestine, adhesion to the cells and mucus of the host or immunomodulation. Precisely, the inactivation of SlpB protein gene in the P. freudenreichii WT strain resulted in the ΔslpB mutant strain that had its ability to adhere to epithelial cells in vitro reduced, compared to the parental strain. However, the immunomodulatory effect of the SlpB protein of the P. Freudenreichii WT strain is not clear. Thus, it is necessary to elucidate the anti-inflammatory role of this protein in vitro and in vivo. In an in vitro assay, P. freudenreichii WT reduced expression of IL-8 (p<0.0001) and TNF-α (p<0.0001) cytokines in LPS-stimulated HT-29 cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. Subsequently, both strains were investigated in vivo in a 5-FU-induced mucositis mice model. Mucositis is a common side effect of cytotoxic chemotherapy with 5-FU, characterized by mucosal injury, inflammation, diarrhea, and weight loss. The WT strain prevented weight loss, reduced inflammation, and consequently, histopathological scores. Furthermore, it regulated key markers, including Claudin-1 (cld1, p<0.0005) and IL-17a (Il17a, p<0.0001) genes, as well as IL-12 (p<0.0001) and IL-1β (p<0.0429) cytokines levels. Mutant strain displayed opposite regulatory effect on cld1 expression and IL-12 levels. This work emphasizes the importance of SlpB in P. freudenreichii ability to reduce mucositis inflammation. It opens perspectives for the development of probiotic products to decrease the side effects of chemotherapy using GRAS bacteria with immunomodulatory surface protein properties.