Improving granulocyte transfusion in neutropenia-related infections

项目概要 粒细胞输注（又名中性粒细胞输注或 GTX）已被用作以下疾病的治疗方法： 治疗严重中性粒细胞减少症患者危及生命的细菌和真菌感染，包括 造血细胞移植（HCT）受者。 GTX 的临床结果受到以下因素的不利影响： 供体中性粒细胞的保质期短且功能受损。中性粒细胞自发死亡是 异质性并由多种途径介导。利用机械知识和药理学 筛选后，我们确定了一种联合治疗方法，半胱天冬酶-溶酶体膜透化-氧化剂- 坏死性凋亡抑制加上粒细胞集落刺激因子（CLON-G），通过改变中性粒细胞的命运 同时针对多种细胞死亡途径。 CLON-G 治疗显着延长中性粒细胞寿命 体内存活，将其半衰期从不到 1 天增加到大于 5 天。与刚隔离的相比 中性粒细胞，储存的 CLON-G 处理的中性粒细胞显示正常功能，包括趋化性、ROS 产生、吞噬和杀灭细菌。这些结果使我们推测 CLON-G 治疗可以 显着延长粒细胞输血产品的离体保质期，且不损害其功能 GTX 受体中的中性粒细胞。由于药物在细胞内滞留时间较长，CLON-G 治疗甚至可以提高输注中性粒细胞的存活率并延长体内的半衰期 从而进一步提高GTX的功效。在这项研究中，我们将首先在临床上检验这一假设 中性粒细胞减少症相关肺炎中粒细胞输注的相关小鼠模型。我们将确定 储存的CLON-G处理过的中性粒细胞在体内是否能正常发挥功能并存活更长时间 与新鲜分离的中性粒细胞相比（目标 1A）。此外，我们将调查其生存和功能 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，我们将检查 输注储存的 CLON-G 处理的中性粒细胞是否最终会增强炎症 接受者的反应和杀菌能力与以下效果一样有效，甚至更有效： 输注未经处理的新鲜中性粒细胞（目标 2）。最后，确定新的 CLON-G 诱导因子和 在调节中性粒细胞死亡和功能中发挥关键作用的途径，我们将进行单细胞 RNA 测序 (scRNA-seq) 揭示 CLON-G 重编程中性粒细胞的分子特征 （目标 3） 。 总之，这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并设计新的长期临床程序 中性粒细胞的储存及在输血医学中的应用。这项研究还提供了新的策略 治疗中性粒细胞减少症相关肺炎。通过更好地理解来利用 GTX 的能力 粒细胞功能和生存可能对没有其他选择的患者产生重大影响。这将是一个 对当前抗生素和 G-CSF 疗法的重要且必要的补充。

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.