Isoallolithocholic acid attenuates acute liver allograft rejection by promoting Treg differentiation through the NR4A1/FOXP3 signaling axis

Background Acute allograft rejection driven by disordered CD4 + T cell alloimmunity severely limits long-term graft survival following orthotopic liver transplantation (OLT). Conventional broad-spectrum immunosuppressants cause severe systemic side effects, while therapeutic approaches relying on metabolites to induce transplant tolerance remain insufficiently explored. Isoallolithocholic acid (isoallo-LCA), a secondary bile acid produced by gut microbiota, maintains Treg homeostasis, yet its biological function and molecular mechanisms in regulating hepatic alloimmune rejection have not been clarified. Objective To systematically explore the therapeutic efficacy of isoallo-LCA against acute liver allograft rejection and uncover its underlying molecular regulatory mechanism. Methods An MHC-mismatched murine orthotopic liver transplantation acute rejection model was established. Recipient survival, liver pathological injury, BANFF rejection scores and serum hepatic injury indicators (ALT, AST, TBIL) were detected to evaluate the protective effect of isoallo-LCA. Flow cytometry was applied to analyze intrahepatic Th1 and Treg proportions. Transcriptomic sequencing combined with in vivo and in vitro western blot assays was used to screen and verify key signaling molecules, and an NR4A1 inhibitor was administered to conduct rescue validation experiments. Results Isoallo-LCA intervention significantly prolonged recipient survival, alleviated intrahepatic inflammatory damage, lowered BANFF rejection grades and decreased serum ALT, AST and TBIL levels. Isoallo-LCA inhibited the differentiation of pro-inflammatory T-bet + Th1 cells and promoted the expansion of FOXP3 + Tregs, restoring the balance of intrahepatic CD4 + T cell subsets. NR4A1 was identified as the core downstream effector of isoallo-LCA. Blockade of NR4A1 pharmacologically nearly eliminated the protective phenotypes mediated by isoallo-LCA, confirming the essential role of the NR4A1/FOXP3 signaling pathway. Conclusion Isoallo-LCA relieves acute liver allograft rejection by activating the NR4A1/FOXP3 signaling axis to remodel intrahepatic Th1/Treg immune balance. These findings provide preclinical evidence supporting gut microbial bile acid metabolites as potential low-toxic adjuvant candidates for inducing transplant immune tolerance, although clinical translation requires further validation in human studies.