Postbiotic Enterococcus faecalis EF-2001 induces IL-10 production through macrophage scavenger receptor 1-mediated phagocytosis

Postbiotics have attracted attention owing to their safety and immunomodulatory effects. The heat-inactivated lactic acid bacterium Enterococcus faecalis EF-2001 (EF-2001) is one such postbiotic. Extensive studies on EF-2001 have demonstrated its safety and versatile biological activities, particularly immunomodulatory effects such as anti-inflammatory responses. However, the molecular mechanisms underlying these regulatory processes remain unclear. Here, we investigated phagocytosis and cytokine production of EF-2001 using murine macrophage-like cells, RAW264 cells. After stimulation with EF-2001, phagocytosis was evaluated using flow cytometry and microscopy. Secreted cytokines were evaluated using ELISA. RNA-seq and gene set enrichment analyses were performed to identify candidate receptors. To validate the candidate receptors, the receptor genes were downregulated by siRNA knockdown, followed by Quantitative PCR and immunoblotting to determine knockdown efficacy. The effects of gene silencing on cell function were characterized by flow cytometry and ELISA. A neutralizing antibody was used to confirm knockdown experiments. The intracellular signaling pathways were examined using specific inhibitors of key kinases. EF-2001 was phagocytosed by RAW264 cells with cytokine production, including IL-10, TNF-α, and IL-6, in a dose-dependent manner. Bioinformatic analysis identified Msr1 , Cd36 and Colec12 as candidate receptor genes for phagocytosis by EF-2001. Phagocytosis was significantly reduced by suppressing macrophage scavenger receptor 1 (MSR1) expression. The neutralizing antibody against MSR1 showed similar suppression of phagocytosis. Thus, MSR1 was identified as the primary receptor for EF-2001 recognition and phagocytosis. MSR1 suppression also affects cytokine production, especially that of IL-10. In terms of intracellular signaling pathways, Syk and PI3K inhibition substantially reduced EF-2001 phagocytosis. The inhibition of Syk, PI3K, and MAPKs hampered IL-10 production induced by EF-2001 stimulation. EF-2001 is recognized and phagocytosed primarily via MSR1 in macrophages, accompanied by the induction of IL-10. Syk and PI3K signaling are involved in phagocytosis and IL-10 release, whereas their downstream MAPK pathways predominantly contribute to IL-10 production. Our data provide insights into the immunomodulatory mechanisms through which biotic components interact with macrophages.