Enterococcus faecalis-induced bystander effect causes epigenetic alterations leading to aberrant TGF-β signaling and epithelial-mesenchymal transition

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BACKGROUND: Enterococcus faecalis promotes colorectal cancer (CRC) in murine models through the microbiota-induced bystander effect (MIBE), but its potential to induce epigenetic changes remains unexplored. This study investigates how E. faecalis-infected macrophages induce epigenetic alterations contributing to CRC pathogenesis. METHODS: RAW264.7 macrophages were infected with E. faecalis OG1RF (superoxide-producing), WY84 (superoxide-deficient), or PBS. Conditioned media were collected and used to treat IEC-6 intestinal epithelial cells. Genome-wide H3 acetylation was assessed using chromatin immunoprecipitation sequencing (ChIP-seq). Western blotting and immunofluorescence staining were employed to investigate E. faecalis-induced histone H3 acetylation and associated signaling pathways. Furthermore, colonic organoid cultures and E. faecalis-colonized Il10−/− mouse models were utilized to validate the in vitro findings. RESULTS: Conditioned medium (CM) from E. faecalis-infected macrophages significantly increased histone H3 acetylation at lysines 27 and 9 (H3K27ac/H3K9ac) in IEC-6 intestinal epithelial cells and murine colonic organoids, with similar findings validated in vivo in E. faecalis-colonized Il10−/− mice. ChIP-seq revealed genome-wide alterations in H3K27ac in cells treated with CM from E. faecalis-infected macrophages, leading to activation of CRC-associated signaling pathways. Metabolomic analysis identified 12-hydroxyeicosatetraenoic acid (12-HETE), produced by E. faecalis-infected macrophages, as a mediator of bystander effect promoting H3 acetylation. Furthermore, CM from E. faecalis-infected macrophages and 12-HETE decreased E-cadherin and increased vimentin expression in IEC-6 cells, indicating epithelial-mesenchymal transition—a finding corroborated by reduced E-cadherin levels in intestinal biopsies from E. faecalis-colonized Il10−/− mice. CONCLUSION: Our results indicate that E. faecalis-infected macrophages produce 12-HETE, which promotes epigenetic alterations and induces EMT in intestinal epithelial cells via a bystander effect. These findings strengthen the link between gut microbiota and innate immunity in CRC development, highlighting potential targets for innovative prevention and therapeutic strategies. TRIAL REGISTRATION: Not applicable.

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