Title : Vaccine adjuvant potential of exopolysaccharides from pediococcus pentosaceus EIR/HM-1 as a novel immunostimulatory agent
Abstract:
Recent studies have increasingly emphasized the immunomodulatory potential of Exopolysaccharides (EPS) derived from probiotic bacteria, identifying them as promising natural, biocompatible, and effective candidates for immune modulation. In the present study, we investigated the in vitro immunostimulatory effects of EPS isolated from Pediococcus pentosaceus EIR/HM-1, a commensal bacterium derived from the human milk microbiota, on RAW 264.7 murine macrophage cells. EPS was extracted from the culture supernatant of P. pentosaceus EIR/HM-1 through ethanol precipitation, followed by dialysis and lyophilization. The total carbohydrate content of the EPS was quantified using the phenol-sulfuric acid method. To assess biocompatibility, cytotoxicity of EPS was evaluated via the MTT assay in RAW 264.7 cells. Following a 24-hour stimulation with EPS, gene expression levels were measured using quantitative real-time PCR, and cytokine production was quantified through enzyme-linked immunosorbent assay. Additionally, Nitric Oxide (NO) levels were determined using a colorimetric assay, while the expression of surface markers CD80, CD86, and MHC-II was analyzed by flow cytometry. Our results revealed that the total carbohydrate content of the EPS was 12,034.04 ± 1.9 mg/L. EPS concentrations ranging from 1 to 100 µg/mL showed no cytotoxic effects on macrophage cells. Transcriptomic analysis demonstrated that EPS significantly upregulated the expression of several immune-related genes, including TNF-α, IL-1β, IL-10, IL-6, iNOS, NOD1, NOD2, ARG1, CD206, COX-2, CCL2, CCL3, CCL5, and CXCL10, when compared to untreated controls. Similarly, ELISA results indicated that EPS treatment (10–100 µg/mL) significantly enhanced the secretion of pro-inflammatory cytokines IL-6 and TNF-α, eliciting an immune response comparable to that triggered by Lipopolysaccharide (LPS). Furthermore, NO production was markedly elevated at 100 µg/mL of EPS. Flow cytometric analysis showed that MHC-II expression in EPS-treated cells was comparable to that in the LPS group, while CD80 and CD86 levels were significantly upregulated relative to untreated controls. Collectively, these findings suggest that EPS derived from P. pentosaceus EIR/HM-1 exhibits potent immunostimulatory properties and holds significant promise as a novel vaccine adjuvant capable of enhancing immunogenicity. Nevertheless, further in vivo studies are required to elucidate the underlying mechanisms and to assess its broader potential in immunotherapy and vaccine development.
