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 严重中性粒细胞减少患者危及生命的细菌和真菌感染的治疗，包括 造血细胞移植(HCT)受者。GTX的临床结果因 供者中性粒细胞保质期短，功能受损。自发性中性粒细胞死亡 异质性，并由多条途径介导。利用机械学知识和药理学 经过筛选，我们确定了一种联合处理方法，即半胱氨酸酶-溶酶体膜通透性-氧化剂- 坏死性下垂抑制加粒细胞集落刺激因子(Clon-G)，通过 同时靶向多条细胞死亡途径。Clon-G治疗显著延长中性粒细胞ex 体内存活，半衰期从不到1天增加到5天以上。与刚刚隔离的 中性粒细胞，储存的克隆G处理的中性粒细胞表现出正常的功能，包括趋化，ROS 生产、吞噬和杀菌。这些结果使我们假设克隆-G治疗可以 显著延长粒细胞输注制品的体外保质期而不损害其功能 GTX受者的中性粒细胞。由于药物在细胞内的滞留时间延长，Clon-G 治疗甚至可能提高体内输注的中性粒细胞的存活率和半衰期 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检验这一假设。 中性粒细胞减少相关肺炎小鼠输注粒细胞模型的建立。我们将决定 经克隆G处理后储存的中性粒细胞能否在体内正常发挥功能并存活更长时间 与新分离的中性粒细胞相比(目标1A)。此外，我们还将研究细胞的存活和功能 在中性粒细胞耗尽的NSG小鼠体内输注人中性粒细胞(目标1B)。接下来，我们将考察 输注储存的克隆G处理的中性粒细胞是否最终能增强炎症 接受者的反应和杀菌能力与，甚至比， 输注未经处理的新鲜中性粒细胞(目标2)。最后，确定新的克隆性G诱导因子和 在调节中性粒细胞死亡和功能中起关键作用的通路，我们将进行单细胞RNA 测序(scRNA-seq)揭示克隆性G重编程中性粒细胞的分子特征 (目标3) 。 总之，在三个目标中提出的实验将帮助我们更好地理解分子和 导致中性粒细胞死亡的细胞机制和设计新的长期临床程序 中性粒细胞在输血医学中的保存与应用。这项研究还提供了一种新的战略 中性粒细胞减少相关性肺炎的治疗。通过更好地了解以下内容利用GTX的能力 粒细胞功能和存活率可能会对没有其他选择的患者产生重大影响。这将是一个 对目前的抗生素和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.