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

Mincle 介导的 NET 形成的分子机制：对肺炎脓毒症的影响

基本信息

批准号：
9929103
负责人：
Jyotika Sharma
金额：
$ 15.99万
依托单位：
UNIVERSITY OF NORTH DAKOTA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-12-01 至 2020-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9929103
关键词：
Adaptor Signaling Protein Adoptive Transfer Antimicrobial Resistance Autophagocytosis Biological Bone Marrow C Type Lectin Receptors Cells Cessation of life Characteristics Chromatin Fibril Communicable Diseases Complex DNA Data Development Disease Enzymes Exhibits Goals Gram-Negative Bacteria Healthcare Human Immune Immune response Immunologic Receptors Impairment In Vitro Infection Infection Control Inflammatory Klebsiella pneumonia bacterium Knock-out Link Lung Lung infections Mediating Microbe Modeling Molecular Mus Outcome Pathway interactions Peptide Hydrolases Phagocytosis Play Pneumonia Production Property Proteins Reactive Oxygen Species Regulation Reporting Research Role SH2D1A gene Sepsis Side Signal Pathway Signal Transduction Signaling Molecule Small Interfering RNA Stimulus Testing Therapeutic Tissues antimicrobial base care burden clinically relevant combat extracellular first responder immunoregulation in vivo knock-down microbial mortality mouse model neutrophil novel overexpression programs public health relevance receptor-mediated signaling response response to injury septic patients src Homology Region 2 Domain

项目摘要

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.