Unraveling the regulatory role of intercellular communication in intestinal immune cells mediated by H₂ in sepsis recovery through single-cell RNA sequencing

BACKGROUND: Sepsis is a life-threatening condition with dysregulated immune responses, where the intestinal immune system plays a pivotal role. While hydrogen gas (H₂) has shown therapeutic potential, its precise impact on intestinal immune cell communication remains elusive. METHODS: We employed a murine model of sepsis induced by cecal ligation and puncture (CLP). Mice were divided into control, sepsis, and H₂-treated sepsis groups. A comprehensive approach combining single-cell RNA sequencing (scRNA-seq) of intestinal tissues with histological, biochemical, and functional analyses was used to dissect the immune landscape and H₂'s effects. RESULTS: H₂ treatment significantly ameliorated intestinal injury, reduced epithelial barrier leakage (evidenced by decreased serum DAO and FITC-dextran), and suppressed systemic inflammation. scRNA-seq revealed that H₂ restored sepsis-induced perturbations in both B and T cell subsets. It normalized the aberrant expansion of non-functional B cells and rectified T cell differentiation trajectories, promoting a functional balance. Mechanistically, H₂ modulated key genes (e.g., Stat3, Ccl5, Foxo1) and critical pathways including oxidative phosphorylation, B cell receptor signaling, and Foxo signaling. Furthermore, cell-cell communication analysis demonstrated that H₂ robustly enhanced the weakened interactions between T cells, B cells, and epithelial cells, particularly via the CCL signaling network. CONCLUSION: Our study elucidates that H₂ therapy alleviates sepsis by systemically reprogramming the intestinal immune micro environment at the single-cell level. This is achieved by normalizing immune cell subsets, rewiring intercellular communication, and ultimately rebalancing the inflammatory response, positioning H₂ as a promising immunomodulatory agent for sepsis.