Targeting NETosis for the Treatment of Tuberculosis

摘要 中性粒细胞是痰液、支气管肺泡灌洗液中最丰富和主要感染的细胞类型 液体和干酪内容物从活动性结核（TB）患者的切除的肺组织。结核病研究 小鼠、非人类灵长类动物和人类已经确定了中性粒细胞丰度和 疾病严重程度增加。尽管越来越多的人认识到中性粒细胞增多与糖尿病的关系， 尽管活动性结核病患者肺内存在大量中性粒细胞， 患者是间接的，或者如果中性粒细胞是对不受控制的感染作出反应的旁观者。特别是， 关于特异性中性粒细胞反应和效应子功能如何影响结核病的细节仍然存在 难以捉摸。为了应对结核分枝杆菌（Mtb）感染，中性粒细胞部署了许多防御措施 包括通过称为NETosis的细胞死亡过程排出中性粒细胞胞外陷阱（NET）。 NETosis通常遵循以下一般步骤：1）组蛋白瓜氨酸酶，2）染色质去凝聚，和3） 释放网状染色质结构，其装饰有抗微生物颗粒蛋白， 结合、捕获和杀死病原体。我们最近发现，NETosis直接促进结核分枝杆菌 复制，其中NETosis的遗传或化学抑制介导体外Mtb感染的更好控制， 在体内，从而验证NETosis作为治疗TB的宿主导向疗法的潜在靶标。我们已经使用 遗传和化学方法来机械地剖析Mtb感染期间NETosis的过程， 它已经确定了一些可以操纵的监管节点，以更好地控制结核分枝杆菌 发病机制我们发现，在中性粒细胞对结核分枝杆菌感染的反应中，蛋白精氨酸脱亚胺酶4（PAD 4） 瓜氨酸使组蛋白去致密化染色质，染色质被包装成囊泡，以NET的形式释放， 促进Mtb复制的方式。我们发现，I型干扰素（IFN），这已经与 与NETosis在许多情况下，但没有一个已知的机制，促进形成 含有染色质的囊泡和NET释放。此外，我们发现了一种新的自噬无关的 ATG5蛋白通过阻断I型IFN依赖的PAD 4活性诱导而抑制NETosis的作用 在Mtb感染期间，在中性粒细胞中缺乏ATG 5表达的情况下NETosis增加导致 结核病易感性多项研究已将I型IFN信号传导水平的增加与结核病病理学联系起来， 老鼠和人类根据我们的数据，NETosis促进Mtb复制和发病机制，NETosis 可能有助于I型IFN信号传导阻碍结核分枝杆菌感染的控制。在本提案中，我们 我将剖析如何NETosis是调节结核分枝杆菌感染（目标1），如何NETosis有助于结核分枝杆菌复制 (Aim 2），以及NETosis如何导致感染失控（目标3）。通过追求我们的目标，我们将 确定HDT靶向的调节机制，目的是在Mtb感染期间阻断NETosis 以确定在Mtb感染期间阻断NETosis的结果。

Abstract Neutrophils are the most abundant and predominantly-infected cell type in the sputum, bronchoalveolar lavage fluid, and caseum contents from resected lung tissue of active tuberculosis (TB) patients. Studies of TB in mice, non-human primates, and humans have identified a correlation between neutrophil abundance and increased disease severity. Although there is a growing appreciation for the association of increased neutrophil abundance with active TB disease, it was still unknown if the presence of neutrophils in the lungs of active TB patients is consequential, or if the neutrophils are bystanders reacting to an uncontrolled infection. In particular, the details on how specific neutrophil responses and effector functions impact TB disease have remained elusive. In response to Mycobacterium tuberculosis (Mtb) infection, neutrophils deploy a number of defenses including the extrusion of neutrophil extracellular traps (NETs) via a process of cell death termed NETosis. NETosis usually follows the general steps of 1) histone citrullination, 2) chromatin decondensation, and 3) release of web-like chromatin structures decorated with antimicrobial granule proteins with the potential to bind, trap, and kill pathogens. We have recently discovered that NETosis directly promotes Mtb replication, where genetic or chemical inhibition of NETosis mediates better control of Mtb infection in vitro and in vivo, thus validating NETosis as a potential target for host-directed therapies to treat TB. We have used genetic and chemical approaches to mechanistically dissect the process of NETosis during Mtb infection, which has identified a number of regulatory nodes that can be manipulated to lead to better control of Mtb pathogenesis. We find that in response to Mtb infection in neutrophils, protein arginine deiminase 4 (PAD4) citrullinates histones to decondense chromatin that gets packaged into vesicles for release as NETs in a manner that promotes Mtb replication. We discovered that type I interferon (IFN), which has been associated with NETosis in numerous contexts but without a known mechanism, promotes the formation of chromatin-containing vesicles and NET release. In addition, we discovered a new autophagy-independent role for the ATG5 protein in suppressing NETosis by blocking type I IFN-dependent induction of PAD4 activity during Mtb infection, where increased NETosis in the absence of ATG5 expression in neutrophils leads to susceptibility to Mtb. Multiple studies have linked increased levels of type I IFN signaling with TB pathology in mice and humans. Based on our data that NETosis promotes Mtb replication and pathogenesis, NETosis could contribute to the ways that type I IFN signaling impedes control of Mtb infection. In this proposal, we will dissect how NETosis is regulated during Mtb infection (Aim 1), how NETosis contributes to Mtb replication (Aim 2), and how NETosis contributes to loss of control of infection (Aim 3). By pursuing our Aims, we will identify regulatory mechanisms to be targeted by HDTs aimed at blocking NETosis during Mtb infection as well as determine what consequences result from blocking NETosis during Mtb infection.