宿主因子血红素加氧酶-1调控流感感染严重程度的作用及机制研究

The severity of respiratory disease caused by influenza virus varies widely among host individuals, which might result in death for the patients with severe symptoms. This process might be linked to the imbalance between antiviral effects and inflammatory response both by the innate immune response. However, the host factors that regulate this process are not clear. Studies have shown that upregulation of type I interferon (IFN-α/β) can cause excessive inflammatory response and increase the severity of influenza. Although our previous studies showed that overexpressing heme oxygenase-1 (HO-1) could induce IFN-α/β production to inhibit viral replication, it didn’t increase the severity of infection, as reflected by the inhibited expression of inflammatory cytokines IL-6 and GM-CSF in cell levels and the reduced lung damage in mice. Combined with the immune-regulatory function articulated by HO-1, we speculate that HO-1 may have a role in balancing its antiviral effects and excessive inflammatory damage, which may be a key host factor affecting prognosis. To confirm this hypothesis, this study intends to clarify the mechanism by which HO-1 regulates the host's antiviral response and excessive inflammatory response by analyzing HO-1 gene polymorphism in clinical influenza patients and performing in vivo and in vitro studies with HO-1 gene-modified mice and macrophage-lung epithelial cell co-culture system. This study will elucidate a new regulatable host factor affecting the severity of influenza infection.

流感感染严重程度存在显著个体差异，重症患者可致死亡。先天免疫应答介导的抗病毒和炎症刺激效应的失衡被认为与之有关，然而调控该过程的宿主因子尚未明确。有研究表明I型干扰素（IFN-α/β）过度激活可致过度炎症反应，增加流感严重程度，然而我们前期研究显示过表达血红素加氧酶-1（HO-1），可通过诱导IFN-α/β上调抑制病毒复制，但未增加感染的严重程度，相反在细胞水平抑制了IL-6、GM-CSF等炎症因子的表达，并减轻了小鼠的肺损伤。上述信息提示，HO-1可能具有调控机体免疫反应，平衡其抗病毒效应与过度性炎症损伤的作用，是影响预后转归的关键宿主因子。结合上述科学假说，本项目拟通过临床流感患者HO-1基因多态性分析，以及HO-1基因修饰小鼠与巨噬-肺上皮细胞共培养体系等体内外研究，进一步明确HO-1调控宿主抗病毒反应、规避过度炎症损伤的作用及机制，全新阐明影响流感感染程度的又一可调控宿主因子。

流感感染严重程度存在显著个体差异，重症患者可致死亡。先天免疫应答介导的抗病毒和炎症刺激效应的失衡被认为与之有关，然而调控该过程的宿主因子尚未明确。前期研究结果提示HO-1可能具有调控机体免疫反应，平衡其抗病毒效应与过度性炎症损伤的作用，是影响预后转归的关键宿主因子。本项目首先明确了HO-1与流感感染严重程度的相关性。动物水平构建了HO-1肺上皮细胞及髓系特异性基因敲除小鼠，敲除HO-1基因后小鼠流感感染程度加重，临床水平初步探索发现HO-1启动子区短GT重复序列对于流感患者具有一定程度的保护作用。其次，我们针对HO-1调控流感感染严重程度的作用机制进行了研究。首先确认了HO-1与IRF-3相互作用的结合位点，包装纯化野生型及三种突变HO-1腺相关病毒，分别为AAV6-HO-1、AAV6-HO-1（H25A）（催化活性缺失）、AAV6-HO-1（K18A）（不能与IRF-3结合）、AAV6-HO-1（H25A/K18A）（双突变），研究其对小鼠流感感染的影响，结果显示AAV6-HO-1能够显著抑制流感感染引起的肺病变及肺组织损伤，且抑制流感病毒蛋白的表达。H25、K18位点在改善肺病变、抗病毒过程中发挥重要作用。另外，AAV6-HO-1能够抑制肺及脾中pDCs细胞及Monocyte细胞的过度产生，增加Macrophage及Tregs细胞数量，激活先天免疫反应但同时维持免疫平衡，其中H25位点在维持免疫平衡过程中发挥重要作用。最后，项目研究了HO-1抵抗IFN-α/β介导的肺上皮细胞凋亡的作用及机制，结果表明HO-1通过下调JAK-STAT通路抑制THP-1细胞中TRAIL的产生，进一步导致与A549细胞表面结合的DR5的蛋白减少，从而抑制IFN-β诱导产生的细胞凋亡。本项目进一步明确HO-1调控宿主抗病毒反应、规避过度炎症损伤的作用及机制，阐明影响流感感染程度的新的可调控宿主因子。