Hypoxia Inducible Factor 2a promotes tolerogenic macrophage development during cardiac transplantation through transcriptional regulation of colony stimulating factor 1 receptor

Solid organ transplantation mobilizes myeloid cells, including monocytes and macrophages, which are central protagonists of allograft rejection. However, myeloid cells can also be functionally reprogrammed by perioperative costimulatory blockade to promote a state of transplantation tolerance. Transplantation tolerance holds promise to reduce complications from chronic immunosuppression and promote long-term survival in transplant recipients. We sought to identify novel mediators of transplantation tolerance by performing single cell RNA sequencing of acute rejecting or tolerized cardiac allografts. This led to the unbiased identification of the transcription factor, Hypoxia Inducible Factor (HIF)-2a, in a subset of tolerogenic monocytes. Using flow cytometric analyses and mice with conditional loss or gain of function, we uncovered that myeloid cell expression of HIF-2a was required for costimulatory blockade induced transplantation tolerance. While HIF-2a was dispensable for mobilization of tolerogenic monocytes, which were sourced in part from the spleen, it promoted the expression of colony stimulating factor 1 receptor (CSF1R). CSF1R mediates monocyte differentiation into tolerogenic macrophages and was found to be a direct transcriptional target of HIF-2a in splenic monocytes. Administration of the HIF stabilizer, roxadustat, within micelles to target myeloid cells, increased HIF-2a in splenic monocytes, which was associated with increased CSF1R expression and enhanced cardiac allograft survival. These data support further exploration of HIF-2a activation in myeloid cells as a therapeutic strategy for transplantation tolerance. Overall design: Mice of B6 background were subjected to full MHC-mismatch abdominal heart transplantation with sex matched BALB/c donors. For AR (acute rejecting) group, no additional treatment was given to mice aside from standard analgesics. For Tol (tolerized) group, mice received intravenous infusions of anti-CD40L antibody (CoB, 500 µg per mouse) and donor BALB/c splenocytes (DST, 2 x 10^7 cells) on day 0 prior to transplantation), followed by intraperitoneal injections of anti-CD40L (CoB, 500µg per mouse) on days 7 and 14 post-transplantation. AR allografts were harvested day 8 post transplnatation and day 40 for Tol allografts. Groups were performed in biological replicates (n = 4 mice/group) and pooled together by condition. Each condition was independently enriched utilizing StemCell magnetic enrichment kit for CD45+ cells or Pan-DC and enriched cells were re-pooled at a ratio of 1:1 for processing in the 10x Genomics Chromium System.