Hypercapnia and Suppression of Anti-viral Host Defense

 描述(申请人提供)：高碳酸血症血液和组织中二氧化碳分压的升高，通常发生在晚期COPD和急性呼吸衰竭中。慢性阻塞性肺疾病患者经常发生细菌和病毒性肺部感染，包括流感，高碳酸血症是此类患者死亡的风险因素。我们已经证明，高碳酸血症抑制了多个由NF-κB调节的、宿主防御所需的先天免疫基因的转录，并抑制了巨噬细胞的吞噬和自噬介导的细菌杀伤(M？S)。此外，我们发现，高碳酸血症会增加小鼠和果蝇因细菌感染而导致的死亡率。这些发现有力地表明，高碳酸血症不仅是晚期肺部疾病的一个标志，而且它通过抑制免疫功能和增加感染的易感性，在不良的临床结果中起到因果作用。高碳酸血症在果蝇和哺乳动物系统中的作用相似，表明高二氧化碳通过保守途径抑制先天性免疫基因的表达(S)。因此，我们在果蝇中进行了全基因组RNAi筛选，并确定锌指同源框转录因子ZFH2是CO2‘S免疫抑制作用的候选介体。值得注意的是，缺乏zfh2的突变果蝇可以防止二氧化碳导致的细菌感染死亡率的增加。接下来，我们发现哺乳动物ZFHX3基因在M？S中表达；ZFHX3以二氧化碳敏感的方式与多个高碳酸血症调节的M？基因结合；ZFHX3的siRNA敲除阻断了高碳酸血症免疫基因的抑制。在目前应用的初步实验中，我们观察到高碳酸血症抑制了内毒素和甲型流感病毒诱导的人和小鼠体内I型干扰素途径抗病毒基因的表达。值得注意的是，ZFHX3结合了干扰素调节因子和NF-κB基因，这种结合是二氧化碳敏感的，提示ZFHX3通过阻断IRF和NF-κB激活的干扰素-α和干扰素-β的转录来介导高碳酸血症对抗病毒基因表达的影响。我们还发现，高碳酸血症抑制了IAV诱导的自噬，这是另一个抗病毒途径，这是在高碳酸血症诱导负性自噬调节因子Bcl2和Bclxl表达增加以及Akt激活之后进行的。最后，至关重要的是，暴露在高二氧化碳环境中会增加S和其他细胞中新城疫病毒的复制，增加小鼠新城疫病毒感染的死亡率。这些研究将验证以下假设：高碳酸血症通过ZFHX3介导的CO2依赖的转录变化抑制干扰素途径抗病毒基因的表达；高碳酸血症通过增加Bcl2和Bclxl的表达以及Akt的激活来抑制IAV诱导的自噬；以及髓系ZFHX3缺乏或阻断高碳酸血症对自噬的抑制将减少IAV相关的肺损伤和死亡率。这项研究将确定二氧化碳水平升高抑制抗病毒宿主防御的新机制，这是一种以前未被认识到的高碳酸血症的不良后果，并将为未来旨在预防晚期肺部疾病患者高碳酸血症诱导的免疫抑制的研究奠定基础。

 DESCRIPTION (provided by applicant): Hypercapnia the elevation of PCO2 in blood and tissue, commonly occurs in advanced COPD and in acute respiratory failure. Patients with COPD frequently develop bacterial and viral lung infections, including influenza, and hypercapnia is a risk factor for mortality in such individuals. We have shown that hypercapnia suppresses transcription of multiple NF-κB-regulated innate immune genes required for host defense and inhibits phagocytosis and autophagy-mediated bacterial killing by macrophages (MØs). Moreover, we found that hypercapnia increases mortality due to bacterial infections in both mice and Drosophila. These findings strongly suggest that hypercapnia is not simply a marker of advanced lung disease, but that it plays a causal role in poor clinical outcomes by suppressing immune function and increasing susceptibility to infection. The similarity of hypercapnia's effects in Drosophila and mammalian systems suggested that elevated CO2 inhibits innate immune gene expression by conserved pathway(s). Thus, we conducted a genome-wide RNAi screen in Drosophila and identified the zinc finger homeobox transcription factor, zfh2, as a candidate mediator of CO2's immunosuppressive effects. Notably, mutant Drosophila deficient in zfh2 were protected against the CO2-induced increase in mortality from bacterial infection. Next, we found that ZFHX3, a mammalian zfh2 ortholog, is expressed in MØs; that ZFHX3 binds in a CO2-sensitive manner to multiple hypercapnia-regulated MØ genes; and that siRNA knockdown of ZFHX3 blocked hypercapnic immune gene suppression. In preliminary experiments for the current application, we observed that hypercapnia inhibited LPS- and influenza A virus (IAV)-induced expression of type I interferon (IFN) pathway antiviral genes in human and mouse MØs. Notably, ZFHX3 bound IFN regulatory factors (IRFs) and NF-κB genes, and this binding was CO2-sensitive, suggesting that ZFHX3 mediates hypercapnia's effect on antiviral gene expression by blocking IRF- and NF-κB- activated transcription of IFN-α and IFN-β. We also found that hypercapnia suppressed IAV-induced autophagy, another antiviral pathway, and that this followed hypercapnia-induced increases in expression of the negative autophagy regulators, Bcl-2 and Bcl-xL, and activation of Akt. Finally, of critical importance, exposure to elevated CO2 enhanced IAV replication in MØs and other cells and increased the mortality of IAV infection in mice. The proposed studies will test the hypotheses that hypercapnia inhibits expression of IFN pathway antiviral genes via CO2-dependent changes in transcription mediated by ZFHX3; that hypercapnia inhibits IAV-induced autophagy via increased expression of Bcl-2 and Bcl-xL and activation of Akt; and that myeloid ZFHX3 deficiency or blocking hypercapnic inhibition of autophagy will decrease IAV-associated lung injury and mortality in mice. This investigation will define novel mechanisms by which elevated levels of CO2 suppress antiviral host defense, a previously-unrecognized adverse consequence of hypercapnia, and will lay the basis for future studies aimed at preventing hypercapnia-induced immunosuppression in patients with advanced lung disease.