Gut microbiota-related lithocholic acid alleviates cgvhd-induced bronchiolitis obliterans syndrome by suppressing CD4+ T cell TCR signaling without impairing the graft-versus-leukemia effect

Abstract Background: Bronchiolitis obliterans syndrome (BOS) is one of the most devastating outcomes of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This remains an area of unmet clinical need for optimal therapy for BOS patients. The gut microbiome influences the pathophysiology of acute GVHD, but its specific role in cGVHD pathogenesis has been unclear. Method: We established a murine cGVHD-BOS model after allo-HSCT. The dynamic variation in the gut microbiome and metabolites during cGVHD-BOS was evaluated. Fecal samples were dynamically collected pre-HSCT and on days 14, 28, 42, and 56 post-HSCT, followed by 16S rRNA and liquid chromatography tandem mass spectrometry (LC-MS/MS) sequencing to analysis gut microbiota and microbiota-derived metabolites. WEHI-3B-based graft-versus-leukemia (GVL) murine models were established to evaluate the impact of exogenous supplementation of lithocholic acid on the GVL effect. Result: We found that the diversity and composition of gut microbiota changed earlier than the occurrence of obvious cGVHD symptoms post-HSCT in cGVHD-BOS mice. On Day 14 and Day 28 after transplantation, before the mice exhibited obvious respiratory symptoms of BOS such as increased airway resistance and decreased alveolar compliance, the α diversity of intestinal microbiota had already significantly decreased with a peak on Day 28 after transplantation. We revealed that high abundances of Proteobacteria, Escherichia-Shigella, Enterococcus, Romboutsia, Erysipelatoclostridium, Corynebacterium were associated with cGVHD-BOS development. Conversely, the abundance of Actinobacteria, Lachnospiraceae, Bifidobacterium, Fecalibacterium, Desulfovibrio, Lachnospiraceae_NK4A136_group, Eubacterium_xylanophilum_group were increased in mice who were remaining cGVHD-free. Furthermore, we found significant alterations in the intestinal microbial metabolites in cGVHD-BOS mice. Induction of cGVHD-BOS resulted in reduced secondary bile acid pools, with the absolute amount of lithocholic acid significantly decreasing in intestinal content as well as in circulation form the early precursor stage after HSCT to evident BOS. Prophylactic administration of lithocholic acid reduces cGVHD-BOS severity in mice. We observed that application of lithocholic acid decreased activation of CD4+ T cells in spleen and lungs. Moreover, lithocholic acid administration did not alter the bacterial composition in the intestine suggesting that its effects are cell-specific and independent of the microbiome. In order to better understand the mechanism by which lithocholic acid inhibits CD4+ T cell activation, we performed gene expression analysis of CD4+T cells cocultured with lithocholic acid or not. The most significantly downregulated pathways upon lithocholic acid treatment included pathways related to TCR signaling pathway, Th1 and Th2 cell differentiation and NF-kappa B signaling pathway. We observed that the transcriptional levels of many genes associated with the KEGG term ‘TCR signaling pathway’ were significantly reduced in cGVHD-BOS mice treated with lithocholic acid. Western blot analysis confirmed that, by activating the takeda G protein-coupled bile acid receptor 5 (TGR5), lithocholic acid reduced the phosphorylation level of PLCγ1, followed by inhibiting Ca2+-nuclear factor of activated T cells (NFAT)2 signaling in CD4+ T cells. Finally, we studied the GVL effect in vivo by injecting WEHI-3B cells. Additional transfer of alloreactive T cells reduced the expansion of leukemic cells in the bone marrow and the spleen. This effect persisted in the mice upon lithocholic acid treatment.Conclusion: Collectively, our findings highlight the importance of dysbiosis of intestinal microbiota and metabolites during the onset and progression of cGVHD-BOS. In particular, we provide a scientific rationale for the systematic use of lithocholic acid in patients undergoing allo-HSCT for prophylaxis and treatment of cGVHD-BOS.