HVEM调控肺泡巨噬细胞极化影响脓毒症急性肺损伤转归的作用和机制

In the early stage of sepsis-induced acute lung injury (ALI), excessive M1 polarization of alveolar macrophages is a key link in the uncontrolled inflammatory response, while its polarization regulation mechanism is unknown. The applicant reported that herpes simplex virus invading medium (HVEM) and its ligand BTLA signaling can aggravate ALI,. Preliminary experiments showed that HVEM expression in alveolar macrophages was significantly up-regulated the in ALI mice. After HVEM-siRNA intervention, M1 cytokine levels in alveolar macrophages and lung STAT1 phosphorylation levels decreased simultaneously, and the inflammation in mice lung was alleviated. We hypothesized that HVEM may promote alveolar macrophage towards M1 polarization through the STAT1 pathway and affect ALI outcome. This study intends to use a sepsis-induced ALI model and extracted primary macrophages, and intervene HVEM by siRNA and its ligand recombinant protein, to determine the role of HVEM in promoting alveolar macrophage towards M1 polarization during ALI. Pathway molecular agonist or antagonist would be used to explore the downstream signaling pathway of HVEM. Moreover, the effect of HVEM intervention on macrophage polarization on ALI prognosis was demonstrated. The aim of this study is to elucidate the polarization mechanism of alveolar macrophages in sepsis-induced ALI, as well as to explore new targets for ALI treatment.

脓毒症急性肺损伤(ALI)早期，肺泡巨噬细胞(AM)过度M1极化是炎症反应失控的重要环节，其极化机制不明。申请者文章报道，疱疹病毒侵入介质(HVEM)及其配体BTLA信号均可加重ALI炎症反应。预实验发现，HVEM在ALI小鼠AM中表达显著上调，HVEM-siRNA干预后小鼠AM内M1型细胞因子与肺组织STAT1磷酸化水平同时降低、小鼠肺部炎症减轻。综上提出假说：ALI中HVEM可能通过STAT1途径促进AM向M1极化、影响ALI转归。本研究拟利用脓毒症ALI小鼠模型和原代巨噬细胞，通过siRNA干扰HVEM表达和配体重组蛋白激活HVEM，确定HVEM在ALI中促进AM向M1极化的作用;通过信号通路分子阻断剂和激动剂，探索HVEM下游信号通路；并在体验证干预HVEM调控AM极化对ALI转归的影响。旨在阐明脓毒症ALI中肺泡巨噬细胞极化的机制、为脓毒症ALI的治疗探索新的靶点。

急性肺损伤(ALI)早期，肺泡巨噬细胞过度M1极化是炎症反应失控的重要环节，其极化机制不明。本研究明确了疱疹病毒侵入介质-B和T淋巴细胞衰减因子（HVEM-BTLA）在ALI中调控肺泡巨噬细胞极化的作用，发现在ALI中HVEM表达上调BTLA/p-SHP2通路促进肺泡巨噬细胞向M2极化，验证了在体干预HVEM表达调控肺泡巨噬细胞极化对ALI转归的影响。另外首次发现肺内细胞群表达Sca-1+CD45-CD31-的肺内源性间充质干细胞（LRMSCs）对体外LPS环境中以及LPS诱导的ALI中巨噬细胞和淋巴细胞具有免疫调控作用，该免疫调控作用亦通过HVEM-BTLA通路来实现。数据表明：ALI时肺组织HVEM表达升高，肺巨噬细胞及淋巴细胞BTLA表达升高。LRMSCs对LPS诱导的M1型肺泡巨噬细胞具有免疫调控作用，与之共培养可下调肺泡巨噬细胞炎症相关通路，巨噬细胞炎症因子基因TNF、IL-6、IL-1b下调，趋化因子MCP-1及MIP-2基因下调，IL-10基因上调，提示其向M2型巨噬细胞极化。LPS刺激的炎症环境及LPS诱导的ALI大鼠模型中，肺泡巨噬细胞和CD4淋巴细胞均受LRMSCs免疫调控。建立ALI模型后，气道内输入LRMSCs，可改变肺内细胞HVEM及BTLA表达，从而改变肺内炎症状态及肺损伤程度。过表达HVEM的LRMSCs在体内体外具有更强的免疫调控作用。ALI时肺泡巨噬细胞及CD4淋巴细胞表面BTLA表达升高。LRMSCs通过上调巨噬细胞和CD4淋巴细胞表面BTLA表达及其下游分子SHP-2磷酸化来实现免疫调控作用。HVEM和BTLA表达广泛，本研究表明HVEM/BTLA在不同细胞中的表达可能起到不同的炎症调控结果，其信号转导在ALI炎症反应发生发展和调控中起到重要的作用。本研究为ALI的发生机制和防治提供了新的证据和靶点, 为新药物的研发奠定了一定的理论基础。