Stabilization of HIF-1α By the Prolyl Hydroxylase Inhibitor Roxadustat Reduces Gastrointestinal Graft-Versus-Host Disease

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for high-risk hematologic malignancies. The benefits of allo-HSCT are limited by the onset of graft-versus-host disease (GVHD) associated with morbidity and mortality. Acute GVHD (aGVHD) primarily affects epithelial cells of the gastrointestinal tract, skin, and liver by involving alloreactive T-cells and pro-inflammatory cytokines in the effector phase. The barrier-protective role of hypoxia-inducible factor (HIF) regulated by hydroxylase inhibitors was significantly implicated in hypoxic inflammatory diseases, including inflammatory bowel disease. In this study, roxadustat, a prolyl hydroxylase inhibitor was tested in the murine model of aGVHD which may have beneficial effects in decreasing hypoxia-mediated early gastrointestinal injury in aGVHD. Methods: Using a murine HSCT model, recipient BALB/c mice received a single dose of total body irradiation (750cGy), followed by intravenous infusion of 5 million bone marrow and 4 million splenocytes of either syngeneic (BALB/c) or allogeneic (C57BL/6) donors and were subsequently treated with roxadustat (25mg/kg body weight) or vehicle control once daily until day +7 and observed until day +42. Mice were monitored for clinical signs of GVHD, survival and analyzed on day +7 and day +42. Results: Roxadustat treatment significantly improves survival by day +42 (p = 0.01) with improved clinical GVHD symptoms from day 7 to 42 (day 7: p = 0.0009). Significantly reduced colon (p = 0.03) and small bowel pathology (p = 0.01) on day +7 in the Roxadustat was associated with significant reduction of serum inflammatory cytokines IFN- γ (p = 0.04) and TNF-α (p = 0.03). Roxadustat treatment with allogeneic recipients significantly decreased T-cell infiltration in the gut and prevented TNF-α induced intestinal epithelial cell apoptosis through repression of Fas-associated death domain protein (FADD), caspase 3, caspase 8 in an HIF-1α dependent manner. Moreover, roxadustat treatment significantly improved the integrity of tight junction proteins including ZO-1 and occludin in allogeneic recipients. Additionally, in vitro experiments demonstrated that roxadustat treatment suppressed TNF-α production by peritoneal macrophages and showed a significant reduction in antigen-presenting cells dependent T cell proliferation in mixed lymphocyte reaction. Conclusion: These results suggest that the administration of prolyl hydroxylase inhibitor roxadustat attenuates early gut injury and shows clinical benefit in a mouse model of allogeneic hematopoietic stem cell transplantation, which could represent an alternative preventive and possible treatment strategy for acute GVHD.