Molecular mechanism of Mincle mediated NET formation:Implications for pneumonic sepsis

 描述(由申请人提供)：中性粒细胞协调对损伤或感染性侮辱的早期免疫反应。中性粒细胞的抗菌功能包括吞噬、脱颗粒和最近描述的中性粒细胞胞外陷阱或Net的形成。网络由脱去变性的染色质纤维组成，上面装饰着抗菌蛋白/颗粒酶，可以捕获和杀死细胞外微生物。肺部感染是败血症的主要原因，在全球范围内构成严重的医疗负担。越来越多的证据表明，中性粒细胞功能异常和根除感染的能力受损是脓毒症高炎症特征的基础之一。尽管越来越多的证据表明Net在清除微生物和调节许多炎症性疾病的免疫反应方面具有优势，但Net形成的病理生理相关性和分子机制尚不清楚。我们实验室最近的研究报道，缺乏哺乳动物C型凝集素受体(CLR)Mincle的Mincle-/-小鼠在肺部感染肺炎克雷伯菌(Kpn)引起的肺炎脓毒症期间表现出肺内净形成功能受损。此外，我们在这里提供的初步数据表明，Mincle/-中性粒细胞在体外对几种激活刺激的反应在净形成方面存在缺陷，尽管它们能够产生与野生型(WT)中性粒细胞相似水平的ROS(据报道是净形成所必需的)。相反，有缺陷的网络形成与Mincle/-中性粒细胞自噬激活受损有关。这些令人兴奋的观察结果表明Mincle参与了自噬激活，并为理解导致网络形成的信号通路提供了机会。在这方面，我们发现，含有适配蛋白、信号转导淋巴细胞激活分子(SLAM)相关蛋白(SAP或SH2D1A)的SH2结构域的siRNA敲除会导致网络形成障碍，并且SAP在激活的中性粒细胞中与Mincle形成复合体。这些新颖的观察结果使我们假设Mincle是中性粒细胞介导的反应的关键组成部分，通过SAP信号调节自噬来驱动网络的形成。为了验证这一假设，我们将承担以下具体目标：首先，我们将Mincle确立为 在体外(在小鼠骨髓中性粒细胞中)和在体内(在KPN感染的肺炎小鼠中)净形成的中心调节器。我们将证明网络的形成依赖于Mincle介导的自噬及其与ROS的关系，以通过siRNA敲除或过表达以及Mincle-/-和WT中性粒细胞的过继转移来响应不同的刺激。其次，我们将通过描述Mincle激活的自噬途径的分子组成及其在网络形成中的作用来阐明Mincle介导的自噬途径，通过检测它们在WT、Mincle-/-和Mincle通过过表达拯救的中性粒细胞中的表达和抑制效果来阐明它们在网络形成中的作用。第三，我们将阐明一个新的Mincle/SAP轴在自噬和网络形成中的作用，以及它在小鼠和人类肺炎败血症中的生物学意义。在这里，我们将建立SAP可以促进自噬，从而形成网络，Mincle提供上游信号，通过SAP信号诱导自噬和网络形成。我们还将确定在KPN诱导的肺炎脓毒症的临床相关模型中以及在脓毒症患者中激活该通路的后果。总之，这一建议提出了一个新的概念，即Mincle通过SAP信号调节自噬来调节网络的形成。这些研究一旦完成，将为微调网络形成提供新的靶点，以获得脓毒症和可能与自噬缺陷和/或放松调节的网络形成相关的其他疾病的治疗益处。

 DESCRIPTION (provided by applicant): Neutrophils orchestrate early immune response to an injury or infectious insult. The antimicrobial functions of neutrophils constitute phagocytosis, degranulation and a recently described formation of Neutrophil Extracellular Traps or NETs. NETs constitute decondenesed chromatin fibrils decorated with antimicrobial proteins/granular enzymes that can trap and kill extracellular microbes. Pulmonary infections are a major cause of sepsis that poses a serious healthcare burden worldwide. Accumulating evidence implicates aberrant neutrophil function and an impaired ability to eradicate the infections as one of the underpinnings for hyperinflammation characteristic of sepsis. Despite accumulating evidence showing advantageous properties of NETs in clearance of microbes and in regulation of immune response in many inflammatory diseases, the pathophysiological relevance and molecular mechanisms underlying NET formation are not well understood. Recent studies from our lab have reported that Mincle-/- mice lacking a mammalian C-type lectin receptor (CLR) Mincle exhibit impaired NET formation in their lungs during pneumonic sepsis caused by pulmonary infection with Klebsiella pneumoniae (KPn). Further, our preliminary data presented here, shows that Mincle-/- neutrophils are defective in NET formation in response to several activation stimuli ex-vivo, despite their ability to produce ROS (reported to be essential for NET formation) at similar levels as wild-type (WT) neutrophils. Instead, the defective NET formation correlated with an impaired activation of autophagy in Mincle-/- neutrophils. These exciting observations implicated Mincle in autophagy activation and presented an opportunity to understand the signaling pathway that leads to NET formation. In this regard, we found that siRNA knockdown of an SH2 domain containing adaptor protein, Signaling lymphocyte-activation molecule (SLAM)-Associated Protein (SAP or SH2D1A) causes impaired NET formation and that SAP forms a complex with Mincle in activated neutrophils. These novel observations led us to hypothesize that Mincle is a critical component of neutrophil-mediated response that drives NET formation by regulating autophagy via signaling through SAP. To test this hypothesis we will undertake the following specific aims: First, we will establish Mincle as a central regulator of NET formation ex-vivo (in murine bone marrow neutrophils) and in-vivo (in KPn infected pneumonic mice). We will show that NET formation is dependent on Mincle-mediated autophagy and its relation with ROS in response to diverse stimuli by siRNA knockdown or overexpression and adoptive transfer of Mincle-/- and WT neutrophils. Second, we will elucidate Mincle mediated autophagy pathway in NET formation by delineating molecular components of autophagy pathway activated by Mincle and their involvement in NET formation by examining their expression and effect of inhibition in WT, Mincle-/- and Mincle rescued neutrophils by overexpression. Third, we will elucidate a novel Mincle/SAP axis in autophagy and NET formation and its biological relevance in murine and human pneumonic sepsis. Here we will establish that SAP can promote autophagy and hence the NET formation and that Mincle provides the upstream signal that induces autophagy and NET formation via SAP signaling. We will also determine the consequence of activation of this pathway in clinically relevant model of KPn induced pneumonic sepsis as well as in sepsis patients. Altogether this proposal puts forth a novel concept that Mincle regulates NET formation by modulating autophagy via signaling through SAP. These studies, when accomplished will provide novel targets for fine tuning the NET formation for therapeutic benefits in sepsis and likely other disease conditions that are associated with a defective autophagy and/or deregulated NET formation.