Control of gvhd by probiotics with a single strain of muribaculaceae bacterium through secretory proteins

Abstract Background Hematopoietic cell transplantation (HCT) is an effective approach to cure hematologic malignancies, but graft-versus-host disease (GVHD) and malignant relapse are the major causes of mortality and mobility of the patients. Conditioning regimens and GVHD damage the gut integrity and reduce the diversity of gut microbiota in patients after allo-HCT. Probiotics were reported to increase gut microbiota content and diversity resulting in ameliorated GVHD. In the previous study, we identified B. fragilis altered gut microbiota and alleviated GVHD, providing a proof of concept that a single strain of commensal bacteria can be a safe and effective means to protect gut integrity and ameliorate GVHD. In clinic, Muribaculaceae abundance was lower in patients with GVHD than those without after allo-HCT. Here we investigated the effects of Muribaculaceae on GVHD development and tumor relapse using murine HCT models. Methods Multiple murine allogeneic bone marrow transplantation (allo-BMT) models with luciferase-transduced leukemia were used, including MHC-mismatched (B6 to BALB/c and FVB to B6) and haplo-identical (B6 to BDF1). GVHD severity was monitored by survival, weight loss and clinical scores, and leukemia relapse was monitored by bioluminescent imaging (BLI). Muribaculanceae bacterium (M. bacterium) was confirmed by long-range whole-genome sequencing. M. bacterium was cultured in hypoxic chamber and administered via oral gavage into recipients. Host microbiota was measured by 16S rRNA sequencing. Results We administered live M. bacterium orally into BMT recipients and observed that the recipients treated with M. bacterium had better survival and lower GVHD clinical scores without leukemia relapse reflected by BLI signal. The results were reproduced in multiple allo-BMT models, indicating that live M. bacterium alleviates GVHD severity while preserving the graft-vs.-leukemia (GVL) effect. In contrast, heat-killed M. bacterium was no longer effective in preventing GVHD, suggesting that bacterialcolonization is required for M. bacterium to mediate the beneficial effect. Through 16S rRNA sequencing, we found that M. bacterium increased microbiota diversity in recipient gut. While the abundance of clostridium and lactobacillus was increased, Lactococcus and Candidatus arthromitus abundance was decreased in recipients, suggesting that M. bacterium shifted recipient gut microbiota towards healthier composition. When using antibiotics-treated or germ-free mice as recipients, administration of M. bacterium also attenuated GVHD although less effectively as compared to the recipients with normal gut microbiota. Taken together, we reason that host microbiota contributes to M. bacterium-mediated GVHD protection. To investigate how M. bacterium dampens GVHD, we measured the effect of M. bacterium on recipient gut permeability, M. bacterium reduced gut permeability and improved intestinal barrier function after allo-BMT. To explore the molecular mechanisms by which M. bacterium protects intestinal integrity, we investigated a variety of molecules in recipient small intestines, including cytokines, inflammation markers, SCFA receptors, and tight junction proteins. M. bacterium increased IL-22, Reg3γ, GPR109A, Claudin 1 and Jam-C. Furthermore, M. bacterium promoted the proliferation of intestinal epithelial cells, inhibited cytokine production of donor T cells in the recipient. We next asked whether M. bacterium impacts T-cell allogeneic responses through its soluble factors. Evidently, the supernatant of M. bacterium significantly suppressed the proliferation and cytokine production of T cells, including IFNγ, TNFα and GM-CSF, upon allogeneic stimulation, but it did not affect T-cell survival. Further, proteinase K-treated supernatant lost the suppressive activity, indicting that proteins produced by M. bacterium play an essential role. Conclusion In summary, orally administration of a single strain M. bacterium mitigates GVHD in mice while preserving the GVL effect. M. bacterium promotes intestinal epithelial cell proliferation, improves recipient intestinal integrity and reduces donor T-cell response. Host microbiota contributes to M. bacterium-mediated GVHD protection. Soluble proteins secreted by M. bacterium play a substantial role in GVHD protection. The study reveals M. bacterium as a new probiotic that attenuates GVHD while preserving GVL activity at least partially through soluble proteins.