Improving granulocyte transfusion in neutropenia-related infections

项目摘要 粒细胞输注（又名。中性粒细胞输注或GTX）已被用作治疗方法， 治疗严重贫血患者中危及生命的细菌和真菌感染，包括 造血细胞移植（HCT）受者。GTX的临床结局受到以下因素的不利影响 保存期短和供体中性粒细胞功能受损。中性粒细胞自发性死亡 异质性和多途径介导。利用机理知识和药理学 筛选时，我们确定了一种联合治疗，半胱天冬酶-溶酶体膜透化-氧化剂， 坏死性凋亡抑制加粒细胞集落刺激因子（CLON-G），通过 同时针对多种细胞死亡途径。CLON-G治疗可显著降低中性粒细胞表达， 体内存活，将其半衰期从不到1天增加到超过5天。与新鲜分离的相比 中性粒细胞，储存的CLON-G处理的中性粒细胞显示正常的功能，包括趋化性、ROS 生产、吞噬作用和杀菌作用。这些结果使我们假设CLON-G治疗可以 显著延长粒细胞输注产品的体外保存期，而不损害 GTX接受者的中性粒细胞由于药物的细胞内滞留时间延长，CLON-G 治疗甚至可以改善存活率并增加体内输注的嗜中性粒细胞的半衰期， 从而进一步提高GTX的疗效。在这项研究中，我们将首先在临床上测试这一假设。 粒细胞输注在血小板相关肺炎中的相关小鼠模型。我们将确定 储存的CLON-G处理的中性粒细胞是否可以正常发挥功能并在体内存活更长时间 与新鲜分离的中性粒细胞相比（Aim 1A）。此外，我们还将研究 在嗜中性粒细胞耗尽的NSG小鼠中体内输注人嗜中性粒细胞（Aim 1B）。接下来，我们将检查 用储存的CLON-G处理的中性粒细胞输血是否最终能增强炎症反应， 接受者的反应和杀菌能力与接受者的反应和杀菌能力一样有效，甚至更有效， 输注未经处理的新鲜中性粒细胞（目的2）。最后，鉴定新的CLON-G诱导因子， 在调节中性粒细胞死亡和功能中起关键作用的途径，我们将进行单细胞RNA 测序（scRNA-seq）以揭示CLON-G重编程中性粒细胞的分子特征 (Aim第三章 . 这三个目标中提出的实验将有助于我们更好地了解分子和 导致中性粒细胞死亡的细胞机制，并设计新的长期临床程序 中性粒细胞在输血医学中保存和应用本研究还为 治疗血小板相关性肺炎。通过更好地理解GTX， 粒细胞功能和存活率可能对没有其他选择的患者产生重大影响。这将是一 这是对目前抗生素和G-CSF疗法的重要和必要的补充。

Project Summary Granulocyte transfusion (aka. neutrophil transfusion or GTX) has been utilized as a therapeutic approach for the treatment of life-threatening bacterial and fungal infections in severe neutropenic patients, including hematopoietic cell transplantation (HCT) recipients. Clinical outcomes from GTX are disadvantaged by the short shelf life and compromised function of donor neutrophils. Spontaneous neutrophil death is heterogeneous and mediated by multiple pathways. Leveraging mechanistic knowledge and pharmacological screening, we identified a combined treatment, caspases–lysosomal membrane permeabilization–oxidant– necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which alters neutrophil fate by simultaneously targeting multiple cell death pathways. CLON-G treatment significantly prolongs neutrophil ex vivo survival, increasing their half-life from less than 1 day to greater than 5 days. Compared to freshly isolated neutrophils, the stored CLON-G-treated neutrophils display normal functions, including chemotaxis, ROS production, phagocytosis, and bacteria killing. These results led us to hypothesize that CLON-G treatment can significantly increase the ex vivo shelf life of granulocyte transfusion products without impairing the function of the neutrophils in the GTX recipients. Due to the prolonged intracellular retention of the drugs, CLON-G treatment may even improve the survival and increase the half-life of the transfused neutrophils in vivo and therefore further improve the efficacy of GTX. In this study, we will first test this hypothesis in a clinically relevant murine model of granulocyte transfusion in neutropenia-related pneumonia. We will determine whether the stored CLON-G-treated neutrophils can function normally and survive even longer in vivo compared to freshly isolated neutrophils (Aim 1A). Additionally, we will investigate the survival and function of transfused human neutrophils in vivo in neutrophil-depleted NSG mice (Aim 1B). Next, we will examine whether transfusion with stored CLON-G-treated neutrophils can ultimately enhance the inflammatory response and bactericidal capability of the recipients as effectively as, or even more effectively than, transfusion with untreated fresh neutrophils (Aim 2). Last, to identify novel CLON-G-inducible factors and pathways that play critical role in regulating neutrophil death and function, we will perform single-cell RNA sequencing (scRNA-seq) to reveal the molecular signature of CLON-G reprogrammed neutrophils (Aim 3) . Together, experiments proposed in the three aims will assist us to better understand the molecular and cellular mechanism leading to neutrophil death and to design novel clinical procedures for the long-term storage and application of neutrophils in transfusion medicine. This study also provides a new strategy for treatment of neutropenia-related pneumonia. The ability to utilize GTX through better understanding of granulocyte function and survival could have a major impact on patients with no other options. This will be an important and necessary complementation to the current antibiotic and G-CSF therapies.