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Graft versus host disease (GVHD) is a severe, often lethal, complication of hematopoietic stem cell transplantation, and although prophylactic regimens are given as standard pre-transplantation therapy, up to 60% of these patients develop aGVHD, and require additional immunosuppressive intervention. Previously we showed that treatment of mice with a purified probiotic molecule, exopolysaccharide (EPS) from Bacillus subtilis, significantly increased survival of GVHD mice compared to control PBS-treated mice (70% vs 10%, respectively), with EPS-treated mice surviving more than 100 days after aGVHD induction. To test if EPS can also ameliorate GVHD in humans, we used humanized NSG-HLA-A2 mice and induced GVHD by i.v. injection of HLA-A2neg human PBMCs. After deriving dendritic cells (DC) from CD34+ cord blood cells, we pretreated them with EPS or PBS, and injected them together with PMBCs into the NSG-HLA-A2 mice. We found that EPS-treated DC significantly prolonged the survival of these mice, compared to PBS-treated DC. We also found that EPS-treated DC downregulated expression of activation markers (CD80, CD86), upregulated expression of inhibitory molecules (PD-L1, PD-L2), and inhibited activation of alloreactive T cells in MLR. We conclude that EPS induces tolerogenic human DC, which ameliorate GVHD in humanized NSG-HLA-A2 mice, likely by inhibiting alloreactive T cell response. Supported by NIH R41 AI155281

Acute graft versus host disease (aGVHD) is a severe and often lethal complication of allogeneic bone marrow transplantation (BMT). We found that mice injected with exopolysaccharide (EPS) secreted by a commensal bacterium, Bacillus subtilis, significantly improved survival, reduced weight loss and decreased production of systemic pro-inflammatory cytokines in GVHD mice. We utilized a novel biosensor mouse model to assess the activation of donor T cells in live mice during aGVHD and found that EPS inhibited the activation of alloreactive donor T cells. EPS also inhibited alloreactive T cell activation in mixed lymphocyte reactions (MLR) in vitro and this inhibition required DCs. Treating DCs with EPS led to upregulation of the inhibitory molecule PD-L1 and secretion of the anti-inflammatory cytokine IL-10, characteristic of inhibitory DCs. We determined that EPS induces inhibitory DCs through TLR4, signaling through both MyD88 and TRIF signaling pathways. The inhibition of alloreactive T cells by these inhibitory DCs is mediated by indoleamine 2,3-dioxygenase (IDO). We hypothesized that inhibition of alloreactive donor T cell activation in GVHD mice by EPS is also mediated by DCs and that EPS induces inhibitory DCs in vivo as well as in vitro. The data demonstrate the potential of EPS or EPS-treated DCs as novel reagents for preventing GVHD.

We previously showed that exopolysaccharide (EPS) produced by a commensal bacterium, Bacillus subtilis, induces alternatively-activated, anti-inflammatory M2 macrophages that protect mice from T cell-dependent inflammatory disease, induced by the enteric pathogen Citrobacter rodentium. Here, we show that EPS induces clinical improvement in another T cell-mediated disease, graft versus host disease (GVHD). GVHD is a common complication of allogenic bone marrow transplantation and is caused by the activation of alloreactive donor T cells. To monitor the inflammatory response of allogeneic bone marrow cells in recipient mice, we used donor cells from transgenic mice that express a luciferase-based biosensor that is cleaved by caspase-1, a protein activated during inflammation. We found that donor inflammatory responses and T cell activation was decreased in EPS-treated mice. Our previous data indicate that EPS does not have a direct effect on T cells. To identify the cell type affected by EPS, we tested the activity of EPS on bone marrow-derived dendritic cells (BMDCs) and found that these cells have upregulated expression of the inhibitory molecule, PD-L1, and the cells inhibited alloreactive T cell activation and proliferation in mixed lymphocyte reactions (MLR). We also found that in vivo injection of EPS resulted in the upregulation of PD-L1 expression on splenic dendritic cells. We conclude that EPS ameliorates clinical manifestations of GVHD by decreasing the activation of donor T cells, and we suggest that EPS affects T cells by inducing inhibitory dendritic cells.

Exopolysaccharide (EPS) secreted by a commensal bacterium, Bacillus subtilis, induces alternatively-activated, anti-inflammatory M2 macrophages that protect mice from T cell-dependent inflammatory disease induced by the enteric pathogen Citrobacter rodentium. We tested the activity of EPS on dendritic cells (DCs), and found that in vitro, EPS not only induced the expression of inhibitory molecules, PDL1 and PDL2, but also that these DCs inhibited alloreactive T cell activation and proliferation in mixed lymphocyte reactions (MLR). We also found that in vivo injection of EPS resulted in the generation of DCs expressing PD-L1 and PD-L2. To test if the EPS-induced DCs are inhibitory in vivo, we used a model that mimics Systemic Lupus Erythematosus (SLE) and tested if the level of autoantibody was decreased. We injected Bm12 cells into EPS-treated C57BL/6 mice to induce SLE-like symptoms, and found that the titer of autoantibodies was significantly decreased in the EPS-injected mice compared to control mice. We conclude that EPS can generate inhibitory DCs both in vitro and in vivo, and we suggest that these DCs have the potential to protect from SLE and other autoimmune diseases.