Heme-Nrf2 signaling shifts myeloid differentiation. Heme-Nrf2 signaling shifts myeloid differentiation

Heme is an erythrocyte-derived toxin that drives disease progression in hemolytic anemias. During hemolysis, specialized bone marrow-derived macrophages with a high heme-metabolism capacity orchestrate disease adaptation by removing damaged erythrocytes and heme-protein complexes from the blood and supporting iron recycling for erythropoiesis. Here, we performed single-cell RNA sequencing with RNA velocity analysis of GM-CSF-supplemented mouse bone marrow cultures to assess myeloid differentiation under heme stress. We found that heme-activated NRF2 signaling shifted the differentiation trajectories of cells towards antioxidant, iron-recycling macrophages at the expense of dendritic cells, as these cells were selectively deficient in heme-exposed bone marrow cultures. Heme eliminated the capacity of GM-CSF-supplemented bone marrow cultures to activate antigen-specific T cells. The generation of functionally competent dendritic cells was restored by NRF2 loss. The heme-induced phenotype was reproduced in hemolytic mice with sickle cell disease and spherocytosis and associated with reduced dendritic cell functions in the spleen. Our data provide a novel mechanistic underpinning how hemolytic stress may provoke hyposplenism-related secondary immunodeficiency, which is a critical determinant of mortality in patients with genetic hemolytic anemias. Overall design: Two-factorial multiplexed scRNA-seq experiment using GM-CSF BM cultures from Nrf2-/- mice and wild-type littermates that were treated with heme or with the selective NRF2 activator ML-334

血红素（heme）是一种红细胞来源的毒素，可促进溶血性贫血（hemolytic anemia）的疾病进展。在溶血过程中，具备高血红素代谢能力的特化骨髓来源巨噬细胞（bone marrow-derived macrophages），可通过清除血液中的受损红细胞与血红素-蛋白质复合物，并支持红细胞生成过程中的铁循环，从而调控疾病适应过程。
本研究对经粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor, GM-CSF）处理的小鼠骨髓体外培养物开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）结合RNA速率（RNA velocity）分析，以评估血红素应激下的髓系分化过程。研究发现，血红素激活的核因子红细胞2相关因子2（nuclear factor erythroid 2-related factor 2, NRF2）信号通路可将细胞分化轨迹转向抗氧化、铁循环型巨噬细胞，并以树突状细胞（dendritic cells）的生成为代价——在血红素暴露的骨髓体外培养物中，树突状细胞呈现选择性缺失。
血红素会削弱经GM-CSF处理的骨髓体外培养物激活抗原特异性T细胞（antigen-specific T cells）的能力。敲除NRF2可恢复功能完备树突状细胞的生成能力。
血红素诱导的表型可在镰状细胞病（sickle cell disease）与球形红细胞增多症（spherocytosis）的溶血性小鼠模型中重现，且与脾脏内树突状细胞功能受损相关。本研究数据为溶血性应激如何诱发脾功能减退（hyposplenism）相关继发性免疫缺陷（secondary immunodeficiency）提供了全新的机制依据，而该免疫缺陷是遗传性溶血性贫血（genetic hemolytic anemias）患者死亡的关键决定因素。
实验设计：采用双因素多重scRNA-seq实验方案，将来自Nrf2敲除（Nrf2-/-）小鼠及其野生型同窝仔鼠的GM-CSF处理骨髓体外培养物，分别用血红素或选择性NRF2激活剂ML-334进行处理。