Transfusion-driven hyperhemolysis in sickle cell disease

红细胞（RBC）输注仍然是镰状细胞病管理的基础治疗 （SCD）。然而，患者可能会发生迟发性溶血性输血反应（DHTR）， 人群从轻度到危及生命的严重反应的进展不可预测， 并且在溶血危象时患者自身的RBC沿着网织红细胞减少而被破坏， 贫血严重DHTR进展的潜在机制知之甚少， 预防和有效治疗这种不成比例的输血并发症 在SCD患者中。我们最近发现，急性溶血诱导先天性巨噬细胞产生I型干扰素（IFN-I）， 免疫细胞，导致单核细胞衍生的巨噬细胞（MoMΦ）的分化和活化增加， SCD，并加剧抗体（Ab）包被的输注红细胞的破坏。我们的初步数据显示， 单独的抗体致敏红细胞破坏也会导致IFN-I的产生，但如果破坏，IFN-I的水平甚至更高 在溶血条件下发生，有趣的是，这也诱导了镰状红细胞的旁观者溶血， 模拟SCD中的高溶血反应。我们还发现溶血诱导的IFN-I会损害红细胞生成 沿着EPO/EPOR信号传导的抑制。基于这些数据，我们假设Fc受体交联 在SCD的溶血背景下，导致IFN-I水平增加，导致IFN-I信号传导增强， 吞噬细胞和红系细胞，其触发增加的RBC破坏和进一步抑制RBC 生产，分别导致严重的DHTR。在目标1中，我们将集中于识别旁观者的机制 通过检测关键的吞噬激活分子的作用，特别是SCD相关的吃我 信号，包括血小板反应蛋白（TSP-1）及其配体，在高溶血模型中上调。我们 将比较Ab介导的红细胞吞噬作用与Ab非依赖性红细胞吞噬作用在触发 旁观者溶血和询问抑制FcR/SYK磷酸化和血红素的相对贡献 自体镰状红细胞破坏的激活途径。我们还将研究IFN-I作为 通过检查SCD患者样品中IFN-1/STAT 1驱动的单核细胞变化， 比较经历重度和轻度DHTR的患者以及恢复至稳态后的患者。对于目标2，我们将 使用原代红系细胞培养系统确定IFN-1抑制红细胞生成的机制， 靶向删除人成红细胞系和小鼠模型中的关键下游通路。我们还将 测试抑制IFN-I产生/IFN-I信号传导或/和增加EPO/EPOR信号传导的治疗效果 在SCD小鼠中逆转受损的BM红细胞生成以及在体外和培养物中对人红细胞生成的作用 用SCD患者血浆治疗。我们认为，我们提出的研究，以审查进展的基础， DHTR的严重程度可能有助于对风险进行分层，并有助于开发新的靶向治疗，以逆转或预防 高溶血，SCD中挽救生命的治疗的毁灭性并发症。

Red blood cell (RBC) transfusions remain a cornerstone treatment in the management of sickle cell disease (SCD). However, patients may experience delayed hemolytic transfusion reaction (DHTR) which in this patient population has an unpredictable progression from mild to life-threatening severe reactions where both transfused and patient’s own RBCs are destroyed along with reticulocytopenia at the time of hemolytic crisis, exacerbating the anemia. The mechanisms underlying severe DHTR progression are poorly understood, posing challenges for prevention and effective treatments for this transfusion complication which is disproportionately encountered in patients with SCD. We recently found that acute hemolysis induces type I interferon (IFN-I) production in innate immune cells, leading to increased differentiation and activation of monocyte-derived macrophages (MoMΦ) in SCD, and exacerbating destruction of antibody (Ab)-coated transfused RBCs. Our preliminary data showed that Ab-sensitized RBC destruction alone also led to IFN-I production but with even higher levels if destruction occurred under hemolytic conditions, which interestingly also induced bystander hemolysis of sickle RBCs, mimicking hyperhemolysis reaction in SCD. We also found that hemolysis-induced IFN-I impairs erythropoiesis along with inhibition of EPO/EPOR signaling. Based on these data, we hypothesize that Fc receptor crosslinking in a hemolytic backdrop of SCD leads to increase in IFN-I levels, causing heightened IFN-I signaling in phagocytes and erythroid cells which trigger increased RBC destruction and further suppression of RBC production, respectively, leading to severe DHTR. In aim 1, we will focus on identifying mechanisms of bystander hemolysis by examining the role of key phagocytosis activation molecules, specifically SCD associated eat me signals including thrombospondin (TSP-1) and its ligands which are upregulated in hyperhemolytic models. We will compare the role of Ab-mediated erythrophagocytosis versus Ab-independent RBC engulfment in triggering bystander hemolysis and interrogate the relative contribution of inhibiting FcR/SYK phosphorylation and heme activation pathways in autologous sickle RBC destruction. We will also examine the potential of IFN-I as a biomarker of DHTR severity by examining IFN-I/STAT1 driven changes in monocytes in SCD patient samples, comparing patients experiencing severe and mild DHTR and after recovery to steady state. For aim 2, we will define the mechanisms by which IFN-I suppresses erythropoiesis using primary erythroid cell culture system and targeted deletion of key downstream pathways in human erythroblast cell lines and mouse models. We will also test the therapeutic effects of inhibiting IFN-I production/IFN-I signaling or/and increasing EPO/EPOR signaling on reversing impaired BM erythropoiesis in SCD mice and on human erythropoiesis in vitro and in cultures treated with SCD patient plasma. We believe that our proposed studies to examine the basis for progression to DHTR severity may help stratify risk and aid in development of novel targeted therapies to reverse or prevent hyperhemolysis, a devastating complication of an otherwise life-saving treatment in SCD.