Hypoxia Inducible Factor 2α 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)-2α, 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-2α was required for costimulatory blockade induced transplantation tolerance. While HIF-2α 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-2α in splenic monocytes. Administration of the HIF stabilizer, roxadustat, within micelles to target myeloid cells, increased HIF-2α in splenic monocytes, which was associated with increased CSF1R expression and enhanced cardiac allograft survival. These data support further exploration of HIF-2α activation in myeloid cells as a therapeutic strategy for transplantation tolerance. 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.

实体器官移植可募集髓系细胞（包括单核细胞与巨噬细胞），此类细胞是同种异体移植排斥反应的核心介导者。然而，围术期共刺激阻断可对髓系细胞进行功能重编程，从而诱导移植耐受状态。移植耐受有望减少慢性免疫抑制带来的并发症，并延长移植受者的长期存活时间。本研究通过对急性排斥或已耐受的心脏同种移植物进行单细胞RNA测序，旨在挖掘移植耐受的新型介导因子。该分析无偏倚地在一群致耐受单核细胞中鉴定出转录因子缺氧诱导因子（Hypoxia Inducible Factor, HIF）-2α。本研究借助流式细胞术分析以及条件性敲除/过表达小鼠模型，证实髓系细胞表达的HIF-2α是共刺激阻断诱导移植耐受所必需的。尽管HIF-2α并非致耐受单核细胞募集（此类细胞部分来源于脾脏）所必需，但它可促进集落刺激因子1受体（colony stimulating factor 1 receptor, CSF1R）的表达。CSF1R可介导单核细胞分化为致耐受巨噬细胞，且在脾脏单核细胞中，CSF1R是HIF-2α的直接转录靶标。将包裹于胶束中以靶向髓系细胞的HIF稳定剂罗沙司他（roxadustat）给予小鼠后，脾脏单核细胞内的HIF-2α水平升高，这与CSF1R表达上调以及心脏同种移植物存活期延长密切相关。上述研究结果支持将髓系细胞中HIF-2α激活作为移植耐受的治疗策略，值得进一步探索。以B6背景小鼠为受体，与性别匹配的BALB/c供体进行完全MHC错配的腹部心脏移植手术。急性排斥（acute rejecting, AR）组小鼠仅给予标准镇痛处理，未施加其他干预措施。耐受（tolerized, Tol）组小鼠于移植前第0天经静脉输注抗CD40L抗体（共刺激阻断剂CoB，每只小鼠500 μg）与供体BALB/c脾脏淋巴细胞（供体脾细胞DST，2×10^7个），并于移植后第7天和第14天经腹腔注射抗CD40L（CoB，每只小鼠500 μg）。急性排斥组同种移植物于移植后第8天取材，耐受组同种移植物于移植后第40天取材。所有组别均设置生物学重复（每组n=4只小鼠），并按实验条件合并样本。各条件下的样本均使用StemCell公司的CD45+细胞或全DC磁珠富集试剂盒进行独立富集，随后将富集后的细胞以1:1的比例重新混合，用于10x Genomics Chromium系统的上机处理。