Mechanistic insights into the systemic inflammation and organ failure in sepsis

描述（由申请人提供）：脓毒症是一种严重的疾病，是危重病人死亡的主要原因。它的特点是全身性炎症反应综合征（SIRS）及其相关的多器官功能衰竭（MOF）。脓毒症相关SIRS/MOF的发病机制仍然知之甚少，治疗/诊断选择仍然有限。SIRS/MOF发展的剩余问题之一是局灶性感染如何被夸大为全身性炎症。根据我们发表的和初步的研究，我们认为外泌体穿梭mirna在将炎症信号运输到远处的靶细胞和器官中起着至关重要的作用。miR-15a和miR-16紧密聚集在同一染色体位置，因此携带相似的细胞功能。我们认为miR-15a/16介导巨噬细胞过度激活，并通过巨噬细胞释放的外泌体（细胞外囊泡）将炎症信号转运到远处的靶标。miR-15a/16的生成和被封装到外泌体中需要PTRF， PTRF是一种穿梭于质膜和细胞核之间的新型脂筏蛋白。我们发表的研究一致表明，PTRF的缺失保护小鼠免受CLP后SIRS/MOF/死亡和死亡巨噬细胞衍生的NO/ROS。在本提案中，我们假设外泌体（EV）穿梭miR-15a/16在脓毒症诱导的SIRS及其相关的MOF中起关键作用。我们的研究可能为败血症后SIRS/MOF的治疗和诊断提供新的靶点，这与临床实践高度相关。巨噬细胞是免疫系统的第一道防线，在炎症信号的传递和放大中起着至关重要的作用。因此，我们选用巨噬细胞作为细胞模型。盲肠结扎穿刺（CLP）作为体内脓毒症模型。我们将检验我们的假设，具体目的如下：目的一：确定体外巨噬细胞中miR-15a/16的产生、分泌和调控。目的二：体外检测miR-15a/16在巨噬细胞中的细胞功能。目的III：在体内确定外泌体（EV）穿梭体miR-15a/16在脓毒症中的功能作用。

DESCRIPTION (provided by applicant): Sepsis is a serious entity and the leading cause of death in the critically ill patients. It is characterized by a systemic inflammatory response syndrome (SIRS) and its associated multi-organ failure (MOF). The pathogenesis of sepsis associated SIRS/MOF is still poorly understood and therapeutic/diagnostic options remain limited. One of the remaining questions for the development of SIRS/MOF is how the focal infections are exaggerated to systemic inflammation. Based on our published and preliminary studies, we believe that exosomal-shuttle miRNAs play crucial roles in transporting inflammatory signals to the distant target cells and organs. miR-15a and miR-16 are clustered closely at the same chromosome position, thus, carrying similar cellular functions. We believe that miR-15a/16 mediate macrophage over-activation and transport the inflammatory signals to distant targets, via the macrophage released exosomes (extracellular vesicles). The generation and encapsulation of miR-15a/16 into the exosomes require PTRF, a novel lipid raft protein shuttling between plasma membrane and nuclei. Consistently, our published studies have demonstrated that deletion of PTRF protected mice from SIRS/MOF/death after CLP and deceased macrophage derived NO/ROS. In this proposal, we hypothesize that exosomal (EV)-shuttle miR-15a/16 plays crucial roles in sepsis induced SIRS and its associated MOF. Our studies potentially provide novel therapeutic and diagnostic targets for SIRS/MOF after sepsis, which is highly relevant to clinical practice. Macrophages, the first arm of defense in the immune system, play crucial roles in the transmission and amplification of inflammatory signals. Therefore, we use macrophages as the cellular models. Cecal ligation and puncture (CLP) is used as an in vivo sepsis model. We will test our hypotheses in the following specific aims: Aim I: To determine the generation, secretion and regulation of miR-15a/16 in macrophages in vitro. Aim II: To determine the cellular functions of miR-15a/16 in macrophages in vitro. Aim III: To determine the functional roles of exosomal (EV)- shuttle miR-15a/16 in sepsis in vivo.