Gingerol ameliorates chronic graft-versus-host disease by regulating treg/Th17 transdifferenciation

Abstract Gingerol reprograms T helper cell differentiationvia P2RY13 suppression to attenuate chronic Graft-versus-host disease Background: Chronic graft-versus-host disease (cGVHD) remains the leading cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT), with limited therapeutic options for steroid-refractory disease. While dysregulated T helper cell differentiation drives fibrosis and organ damage, T cell lineage reprogramming represents an unexplored therapeutic avenue. Our discovery of the lack of serum gingerol in cGVHD murine models prompted investigation into its immunoregulatory functions. Objective: To define the mechanism by which exogenous gingerol modulates T cell lineage differentiation and ameliorates cGVHD. Methods: Two MHC-mismatched cGVHD mouse models (B6→BALB/c and BALB/c→B6) were established. Gingerol (100 mg/ml, 200 μl/day) was administered via oral gavage from day 0 to day 40 post-HSCT. The body weight change, diarrhea, hair loss and survival of recipients were monitored. cGVHD target organ injuries (Liver, Lung, and gastrointestinal (GI) tract) were examined by H&E staining and quantified using histopathological scoring system. Helper T cell (Th) subsets and regulatory T cell (Treg) in the spleen, liver, lung and GI tract were analyzed by flow cytometry. Serum pro-inflammatory cytokines (TNF-α, IFN-γ, IL-17 etc.) were analyzed by ELISA. The transcriptional profile of in vitro activated T cells with or without gingerol treatment was detected by bulk RNA sequencing. Results: Gingerol treatment significantly enhanced overall survival, reduced body weight loss, and lowered clinical cGVHD scores in recipients. Histopathology examination suggested that Gingerol treatment markedly decreased cell infiltration and tissue damage in liver, lung, and GI tract. Serum inflammatory cytokines (IFN-g，TNF-a and IL-17) concentration were significantly reduced after Gingerol treatment. Moreover, gingerol administration decreased TNF-α+, IFN-γ+, and IL-17+ T cells while increased the Foxp3+CD4+ T cells in liver, lung and GI tract. RNA sequencing suggested that gingerol-mediated modulation of the P2Y receptor family, notably significantly suppressed P2RY13. Reduction of P2RY13 protein level was further confirmed by flow cytometry. In addition, gingerol upregulated inhibitory receptors CD244 and PD-1 on T cells. P2RY13 agonist treatment suppressed T cell proliferation, downregulated calcium signaling and glycolysis pathway, ultimately inhibiting Th17 while expanding Tregs in vivo. Conclusion: We identify gingerol as a first-in-class P2RY13 antagonist that ameliorates cGVHD by constraining T cell activation through CD244/PD-1 induction and Th lineage reprograming. This novel immunoregulatory axis offers translational potential for steroid-refractory cGVHD.