NET stabilization: mechanistic and therapeutic studies in thromboinflammatory disoders

摘要 中性粒细胞（PMN）对炎症刺激的反应是排出中性粒细胞胞外陷阱（NET）、网 带负电荷的无细胞（cf）DNA与带正电荷的组蛋白复合， 而且还损害宿主组织，导致包括脓毒症在内的疾病，在脓毒症中， 急性产生，导致内皮损伤和器官功能障碍，以及镰状细胞病（SCD），其中 NET产生的慢性增加会导致血管炎症和血管疼痛， 闭塞发作（VOE）。不幸的是，阻止NET释放的干预措施增加了细菌 传播，而降解NET的治疗可以释放捕获的微生物和有毒的NET， 降解产物（NDP）导致多系统器官损伤。我认为，净稳定， 其中NET被保留但被修饰以减少NDP释放并增强细菌捕获， 治疗败血症和SCD。血小板因子4（PF 4）是一种带正电荷的趋化因子， 活化的血小板结合并交叉聚集聚阴离子，如肝素和DNA。我发现PF 4 物理上压缩NET，增加其对核酸酶的抗性。PF 4也与带负电荷的 分子的细菌表面上，并显着提高其捕捉NET。KKO，人（h） PF 4：肝素复合物结合单克隆抗体（moAb），稳定PF 4：NET，进一步增加核酸酶 阻力在鼠脓毒症模型中，hPF 4和Fc修饰的去糖基化KKO（DG-KKO）在小鼠脓毒症模型中起作用。 协调一致，以减少NDP释放，增强细菌捕获和改善结果。在这份提案中，我将 比较NET稳定在脓毒症和SCD中的作用，以阐明其作用机制并评估其是否 保护急性和慢性NET释放。具体目标（SA）#1：定义 hPF 4和DG-KKO稳定NET。我将评估hPF 4和DG-KKO如何修饰NET，使用hPF 4变体， 其他阳离子和抗hPF 4单克隆抗体在体外和鼠脓毒症模型中的作用。SA #2：定义以下机制 增强的NET抗菌活性。我将定义PF 4：NET如何结合不同种类的细菌，评估 NET稳定增强细菌杀灭，并测试其他阳离子是否可以复制这些效果。SA #3： 确定在SCD中NET稳定是否具有保护作用。我将定义NET靶向治疗是否 在SA#1中具有保护性，减少输注来自SCD患者的血浆的微流体通道中的细胞损伤， SCD的鼠模型。然后，我将测量PF 4，NDP水平和疾病严重程度之间的关系， SCD患者。我将在职业发展计划的背景下继续这些研究， 教学课程、多学科指导和部门间合作。这项工作将提高我们的 了解NET，并支持两种不同炎症性疾病的潜在新干预。它将 我也促进了我作为一个独立的临床医生，科学家在学术儿科中心的职业生涯的成长 专注于儿童血栓前/炎症前疾病的基础研究和临床护理。

ABSTRACT In response to inflammatory stimuli, neutrophils (PMN) extrude neutrophil extracellular traps (NETs), webs of negatively-charged cell-free (cf) DNA complexed with positively-charged histones, which ensnare pathogens but also damage host tissue, contributing to diseases including sepsis, in which a large burden of NETs is acutely produced causing endothelial damage and organ dysfunction, and sickle cell disease (SCD), in which there is a chronic increase in NET production that contributes to vascular inflammation and painful vaso- occlusive episodes (VOE). Unfortunately, interventions that block NET release increase bacterial dissemination, while treatments that degrade NETs can release entrapped microbes and toxic NET- degradation products (NDPs) that contribute to multisystem organ damage. I posit that NET stabilization, in which NETs are preserved but modified to reduce NDP release and enhance bacterial capture, may be therapeutic in both sepsis and SCD. Platelet factor 4 (PF4) is a positively-charged chemokine released by activated platelets that binds to and cross-aggregates polyanions like heparin and DNA. I have found that PF4 physically compacts NETs, increasing their resistance to nucleases. PF4 also binds to negatively-charged molecules on the bacterial surface and markedly enhances their capture by NETs. KKO, a human (h) PF4:heparin complex-binding monoclonal antibody (moAb), stabilizes PF4:NETs, further increasing nuclease resistance. In murine sepsis models, hPF4 and an Fc-modified, deglycosylated KKO (DG-KKO), work in concert to decrease NDP release, enhance bacterial capture, and improve outcomes. In this proposal, I will compare the effect of NET stabilization in sepsis and SCD, to clarify its mechanism of action and assess if it is protective in both acute and chronic NET release. Specific Aim (SA) #1: Define the mechanism(s) by which hPF4 and DG-KKO stabilize NETs. I will evaluate how hPF4 and DG-KKO modify NETs, using hPF4 variants, other cations, and anti-hPF4 moAbs in vitro and in murine sepsis models. SA#2: Define the mechanism(s) of enhanced NET antimicrobial activity. I will define how PF4:NETs bind different classes of bacteria, assess if NET stabilization enhances bacterial killing, and test whether other cations can replicate these effects. SA#3: Determine whether NET stabilization is protective in SCD. I will define whether NET-targeted therapies protective in SA#1, reduce cellular injury in microfluidic channels infused with plasma from SCD patients and in a murine model of SCD. I will then measure the association between PF4, NDP levels, and disease severity in SCD patients. I will pursue these studies within the context of a career development plan that combines didactic courses, multidisciplinary mentorship, and interdepartmental collaboration. This work will enhance our understanding of NETs and support a potential novel intervention for two distinct inflammatory disorders. It will also facilitate my growth as an independent clinician-scientist with a career at an academic pediatric center focused on the basic research and clinical care of children with prothrombotic/proinflammatory diseases.