NET stabilization: mechanistic and therapeutic studies in thromboinflammatory disoders

摘要 作为对炎症刺激的反应，中性粒细胞(PMN)排出中性粒细胞胞外陷阱(Net)，网络 带负电荷的无细胞(Cf)dna与带正电荷的组蛋白复合，从而诱捕病原体。 但也会损害宿主组织，导致包括败血症在内的疾病，在这些疾病中，NETS的大量负担是 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 有一种慢性的净产量增加，导致血管炎症和血管疼痛。 闭塞性发作(VOE)。不幸的是，阻止净释放的干预措施增加了细菌 传播，而降解蚊帐的治疗方法可以释放被困的微生物和有毒的蚊帐- 导致多系统器官损伤的降解产物(NDP)。我假设净稳定化，在 哪些网络被保留但被修改以减少NDP的释放并增强细菌捕获，可能是 对脓毒症和SCD均有治疗作用。血小板第4因子(PF4)是一种正电的趋化因子，由 激活的血小板，结合并交叉聚集多阴离子，如肝素和DNA。我发现PF4 物理上压缩网络，增加它们对核酸酶的抵抗力。PF4还与带负电荷的 细菌表面的分子，显著增强了它们被Net捕获的能力。Kko，人类(H) PF4：肝素复合体结合型单抗(MOAB)，稳定PF4：Net，进一步增加核酸酶 抵抗。在小鼠脓毒症模型中，HPF4和Fc修饰的脱糖KKO(DG-KKO)在 协调减少NDP的释放，增强细菌捕获，并改善结果。在这份提案中，我将 比较净稳定在脓毒症和SCD中的作用，以阐明其作用机制并评估其是否 对急性和慢性净释放均有保护作用。具体目标(SA)#1：定义机制(S) HPF4和DG-KKO稳定网。我将评估HPF4和DG-KKO如何使用HPF4变体修改网络， 其他阳离子，以及体外和小鼠脓毒症模型中的抗HPF4单抗。SA#2：定义机制(S) 增强了净抗菌活性。我将定义PF4：Net如何结合不同类别的细菌，评估是否 净值稳定增强了对细菌的杀灭，并测试其他阳离子是否可以复制这些效果。SA#3： 确定网络稳定在SCD中是否具有保护性。我将定义网络靶向治疗是否 在SA#1中进行保护，减少输入SCD患者和患者血浆的微流体通道中的细胞损伤 一种SCD小鼠模型。然后，我将测量PF4、NDP水平与疾病严重程度之间的关联 SCD患者。我将在职业发展计划的背景下进行这些研究， 授课课程、多学科指导和部门间协作。这项工作将增强我们的 了解NETS，并支持对两种不同的炎症性疾病进行潜在的新干预。会的 也有助于我成长为一名独立的临床医生兼科学家，在一家儿科学术中心工作 专注于血栓前/炎症性疾病儿童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.