Epigenetic reprogramming of dendritic cells by DNMT1 inhibition attenuates Th2 skewing in allergic airway inflammation

Allergic asthma is driven by Th2-polarized immune responses, yet the epigenetic mechanisms underlying dendritic cell (DC)-mediated Th2 skewing remain unclear. Using a house dust mite (DME)-induced murine asthma model, we combined pharmacological DNMT1 inhibition (5-Aza-2′-deoxycytidine, 5-azadC), DC-specific Dnmt1 ablation (Dnmt1fl/fl Itgax-Cre mice), chromatin immunoprecipitation (ChIP), RT-qPCR, and ubiquitination assays to dissect methylation-dependent regulation of IL-12b and TIM4 in DCs. Sensitization increased DNMT1 occupancy at the Il12b promoter, inducing hypermethylation (2.5-fold vs. naïve, p < 0.001) and suppressing IL-12b expression (60% reduction, p < 0.001). DNMT1 inhibition with 5-azadC or genetic Dnmt1 ablation restored IL-12b levels (p < 0.01), reduced BALF Th2 cytokines (40–60%), eosinophils (62%), and mast cell mediators (p < 0.01), and attenuated airway inflammation. IL-12b promoted TIM4 degradation via Trim28-mediated K48-linked ubiquitination (p < 0.01), while Il12b deficiency sustained TIM4 expression and Th2 polarization. DNMT1 enrichment at the Il12b promoter correlated with TIM4 upregulation (r = 0.87, p < 0.01), forming a self-reinforcing loop disrupted by Timd4 knockdown. DNMT1 in DCs orchestrates Th2 polarization via Il12b silencing and TIM4 stabilization, positioning DNMT1 inhibitors and TIM4-targeted therapies as novel strategies to rebalance Th1/Th2 responses in asthma.