In vitro and in vivo immunostimulatory activity of an exopolysaccharide-enriched fraction from Bacillus subtilis

Aims: The aim of this study was to investigate the immunostimulatory effects of an exopolysaccharide-enriched fraction obtained from Bacillus subtilis J92 (B-EPS). Methods and Results: To determine the immunostimulatory activities of B-EPS, we used IFN-γ-primed RAW 264·7 macrophages and CD3/CD28-stimulated splenocytes. Increases in the levels of NO and many cytokines, such as, TNF-α, IL-6, and IL-1β, were observed in IFN-γ-primed RAW 264·7 macrophages by Griess reaction and ELISAs respectively. Using Western blotting and qRT-PCR, we found that B-EPS increased the protein and mRNA expressions of iNOS and the mRNA expressions of TNF-α, IL-6, and IL-1β. A reporter gene assay and EMSA revealed that B-EPS up-regulated the transcriptional activity of NF-κB by increasing its DNA binding and nuclear translocation. Pretreatment with NF-κB inhibitors, that is, BAY11-7082 and PDTC, decreased NO production in IFN-γ-primed RAW 264·7 macrophages by B-EPS. Furthermore, B-EPS increased the proliferation of and cytokine (IL-2 and IFN-γ) production by CD3/CD28-stimulated splenocytes. In a cyclophosphamide-induced immunosuppressed mouse model, B-EPS (5, 15 or 45 mg kg^-1, p.o.) restored thymus and spleen indices. B-EPS also inhibited cyclophosphamide-induced reductions in neutrophil and lymphocyte numbers. Conclusions: B-EPS improves immune function by regulating immunological parameters in IFN-γ-primed macrophages, CD3/CD28-stimulated splenocytes, and in cyclophosphamide-induced immunosuppressed mice. Significance and Impact of the Study: This study suggests that the exopolysaccharides secreted by B. subtilis J92 could be used as immune stimulants.