Intermittent ketogenic diet promotes Treg differentiation and alleviates Crohn’s disease via the β-hydroxybutyrate driven AHCY–DNMT1–Foxp3 axis

BACKGROUND: Crohn's disease (CD) is characterized by epithelial barrier dysfunction and mucosal immune dysregulation. Dietary interventions have gained increasing attention as important therapeutic strategies. We evaluated whether an intermittent ketogenic diet (IKD), which elevates β-hydroxybutyrate (BHB), ameliorates CD via lysine β-hydroxybutyrylation (Kbhb) and elucidated the underlying mechanisms. METHODS: IL-10 knockout mice were allocated to three groups: continuous ketogenic diet (KD), IKD, or a normal diet. Disease activity index (DAI), histopathological changes, cytokine levels, and epithelial barrier integrity were evaluated. Regulatory T cells (Tregs) and Foxp3 expression were assessed by flow cytometry and quantitative real-time PCR (qRT-PCR). To elucidate the underlying mechanisms, we conducted metabolomic and transcriptomic analyses, pyrosequencing, and additional experiments. RESULTS: Both KD and IKD alleviated colonic inflammation, but IKD better prevented epithelial senescence. IKD increased colonic Treg infiltration and Foxp3 expression. Metabolomics revealed elevated BHB in IKD colons. BHB reduced inflammation and promoted Treg differentiation in a dose-dependent manner. Mechanistically, BHB induced Kbhb of S-adenosylhomocysteine hydrolase (AHCY), inhibiting its activity, leading to S-adenosylhomocysteine (SAH) accumulation, downregulating DNA methyltransferase 1 (DNMT1), and promoting Foxp3-TSDR demethylation to enhance Foxp3 transcription and Treg differentiation. IKD also enriched Akkermansia muciniphila, supporting gut and systemic BHB levels. CONCLUSIONS: In an IL-10 knockout model, IKD improves intestinal barrier function and reshapes the gut microbiota. It is associated with BHB-induced Kbhb-mediated inhibition of AHCY, leading to SAH accumulation and DNMT1 downregulation, thereby promoting Treg differentiation. These findings suggest protective effects of IKD in the IL-10 knockout colitis model.