Genes Mediating Innate Immune Suppression by Hypercapnia in Mammals and Flies

描述(申请人提供)：高碳酸血症，血液和组织中二氧化碳分压升高，通常发生在严重的急性和慢性肺部疾病，如慢性阻塞性肺疾病(COPD)。晚期COPD患者经常发生细菌性肺部感染，高碳酸血症是此类患者死亡的风险因素，就像那些社区获得性肺炎患者一样。我们已经证明，高碳酸血症抑制了人、小鼠和果蝇细胞中宿主防御所需的先天免疫反应基因的转录，并增加了小鼠和果蝇因细菌感染而导致的死亡率。这些发现表明，高碳酸血症不仅是晚期肺部疾病的一个标志，而且通过增加感染的易感性，在不良的临床结果中起着因果作用。我们的数据还强烈地表明，高碳酸血症通过从果蝇到哺乳动物的保守途径(S)抑制了先天性免疫和宿主防御。由于高碳酸血症免疫抑制的分子介体(S)尚不明确，我们在培养的果蝇细胞中进行了全基因组RNAi筛选，鉴定了约140个高碳酸血症抑制抗菌肽基因所需的基因。这些候选CO2介体中最有效的5个编码锌指同源域转录因子Zfh2、组蛋白去乙酰基酶、组蛋白甲基转移酶、染色质相关Ig重复蛋白和RAC相互作用蛋白。这些基因之前都不知道具有免疫调节功能。令人振奋的新数据表明，zfh2突变可以保护成年果蝇免受二氧化碳诱导的宿主防御缺陷。因此，我们推测Zfh2和其他4个候选基因编码的蛋白质是高碳酸血症抑制果蝇天然免疫基因表达和宿主防御的途径(S)的组成部分，这些基因的哺乳动物同源基因介导了小鼠和人类吞噬细胞天然免疫/宿主防御基因的高碳酸化抑制。在Zfh2及其哺乳动物同源基因ZFHX3和ZFHX4的情况下，我们进一步假设，高碳酸血症改变了它们与靶基因启动子或NF-：B转录复合体组件结合的能力，或改变了这些因子的转录活性，从而减少了特定的先天性免疫/宿主防御基因的表达。在拟议的研究中，我们将使用培养的苍蝇、小鼠和人类巨噬细胞在体外验证这些假设，并在体内使用成年果蝇和从铜绿假单胞菌肺炎小鼠获得的肺炎性细胞来验证这些假设。这些研究将首次确定高碳酸血症损害天然免疫和宿主防御的保守途径(S)的成分，并确定其作用机制。这一结果应该为未来旨在预防晚期肺部疾病患者的高碳酸血症免疫抑制的研究奠定基础。

DESCRIPTION (provided by applicant): Hypercapnia, elevation of PCO2 in blood and tissue, commonly occurs in severe acute and chronic lung disorders, such as chronic obstructive pulmonary disease (COPD). Patients with advanced COPD frequently develop bacterial lung infections, and hypercapnia is a risk factor for mortality in such individuals, as in those with community-acquired pneumonia. We have shown that hypercapnia suppresses transcription of innate immune response genes required for host defense in human, mouse and Drosophila cells, and increases mortality due to bacterial infections in mice and Drosophila. These findings suggest that hypercapnia is not simply a marker of advanced lung disease, but plays a causal role in poor clinical outcomes by increasing susceptibility to infection. Our data also strongly suggest that hypercapnia inhibits innate immunity and host defense by pathway(s) conserved from Drosophila to mammals. Because the molecular mediator(s) of hypercapnic immune suppression are undefined, we conducted a genome-wide RNAi screen in cultured Drosophila cells, which identified ~140 genes required for hypercapnic suppression of antimicrobial peptide (AMP) genes. The 5 most potent of these candidate CO2-mediators encode the zinc finger homeodomain transcription factor, Zfh2; a histone deactylase; a histone methyltransferase; a chromatin-associated Ig-repeat protein; and a Rac-interacting protein. None of these genes were previously known to have immunoregulatory function. Exciting new data indicate that mutations in zfh2 protect adult Drosophila against CO2-induced host defense defects. Thus, we hypothesize that Zfh2 and the proteins encoded by the other 4 candidate genes are components of pathway(s) by which hypercapnia suppresses innate immune gene expression and host defense in Drosophila, and that the mammalian orthologs of these genes mediate hypercapnic suppression of innate immune/host defense genes in mouse and human phagocytes. In the case of Zfh2 and its mammalian orthologs, ZFHX3 and ZFHX4, we further hypothesize that hypercapnia alters their abilities to bind target gene promoters or components of the NF-:B transcriptional complex, or modifies the transcriptional activity of these factors, thereby decreasing expression of specific innate immune/host defense genes. In the proposed studies, we will test these hypotheses in vitro using cultured fly, mouse and human macrophages, and in vivo using adult Drosophila and lung inflammatory cells obtained from mice with Pseudomonas aeruginosa pneumonia. These studies will for the first time define components of conserved pathway(s) by which hypercapnia impairs innate immunity and host defense, and determine their mechanisms of action. The results should lay the basis for future studies aimed at preventing hypercapnic immune suppression in patients with advanced lung disease.