Unfolded protein response in Influenza virus infection and inflammation

项目总结 感染后对流感病毒(IAV)蛋白质合成和加工的需求，包括二硫化物的形成 肺上皮细胞内质网(ER)中IAV蛋白的半胱氨酸结合可诱导ER应激。 内质网应激反过来诱导基于内质网的未折叠蛋白反应(UPR)。病毒激活并维持UPR 然而，它们的复制，慢性UPR会导致促炎反应和诱导细胞凋亡。它不是 明确特定的UPR组件是否被IAV捕获以进行传播，以及它们是否也有助于大规模 IAV诱导的促炎反应和对肺的损伤增加。 我们的新的初步结果表明，IAV感染激活了潜在的转录因子ATF6α。ATF6α 已知与UPR相关基因的特定DNA元件结合并上调其转录。 IAV诱导的细胞因子/趋化因子启动子的计算分析表明存在保守的 细胞因子和趋化因子启动子区的ATF6α结合元件。接下来我们观察到， 血管内皮生长因子6α的缺失/抑制可减少炎症、细胞因子/趋化因子、主动脉内静脉负荷和呼吸道 小鼠高反应性(AHR)。此外，我们发现IAV感染会特别增加一种PDI- PDIA3在HBE细胞和感染IAV的小鼠体内。肺上皮细胞特异性消融PDIA3的研究 减少IAV蛋白中的二硫桥、细胞因子/趋化因子、IAV负荷和肺内AHR反应。 这些结果提示ATF6、α和PDIA3在IAV感染和免疫病理过程中起着重要作用。我们的 最重要的假说是IAV诱导的ATF6α和PDIA3的激活支持显性上皮亲 炎症反应、IAV蛋白和促炎细胞因子中的二硫键增加IAV 繁殖和肺部免疫病理学。我们设计了两个具体的目标来检验我们的假设。 在特定的目标#1中，我们将确定IAV诱导的UPR激活的转录因子ATF6α的功能作用 在调节细胞因子/趋化因子的表达中，随后免疫病理的诱导和发展 对肺部健康的长期影响。具体目标#2试图剖析IAV诱导的关键需求 PDIA3在二硫键介导的IAV蛋白(HA&NA)、促炎细胞因子/趋化因子加工中的作用 随后的免疫病理诱导。在这两个目标中，我们将使用转基因小鼠模型、细胞培养和 敏感的氧化还原试验和生化试验。最重要的是，我们将评估特定抑制剂的疗效。 ATF6α(Ceapin-A7)和PDIA3(PACMA31)在缓解IAV诱导的UPR中的作用，减少随后的PRO 炎症反应，并最终导致IAV诱导的免疫病理改变。这些研究 将阐明IAV诱导的肺上皮细胞UPR在IAV增殖和显性PRO中的重要性。 炎症反应，并提供了对IAV感染和急需的新的治疗方法的洞察 严重的呼吸窘迫超过了支持性护理。

PROJECT SUMMARY After infection the demand for influenza (IAV) protein synthesis and processing, including formation of disulfide bonds in cysteines of IAV-proteins in the endoplasmic reticulum (ER) of lung epithelial cells induces ER stress. ER stress in turn induces an ER based unfolded protein response (UPR). Viruses activate and sustain UPR for their replication, however, chronic UPR results in pro-inflammatory response and induction of apoptosis. It is not clear whether specific UPR components are seized by IAV for propagation and they also contribute to massive increases in IAV-induced pro-inflammatory responses and injury to the lung. Our novel preliminary results suggest that IAV infection activates the latent transcription factor, ATF6α. ATF6α is known to bind to specific DNA elements of UPR related genes and upregulates their transcription. Computational analysis of IAV-induced cytokine/chemokine-promoters showed the presence of conserved ATF6α binding elements in the promoter regions of cytokines and chemokines. We next observed that deletion/inhibition of ATF6α decreases inflammation, cytokines/chemokines, IAV burden and airway hyperresponsiveness (AHR) in mice. Furthermore, we found that IAV infection increases specifically a PDI- PDIA3 in HBE cells and in mice infected with IAV. Lung epithelial specific ablation of PDIA3 significantly decreased disulfide bridges in IAV-proteins, cytokines/chemokines, IAV burden, and AHR responses in the lung. These results suggested a prominent role for ATF6α and PDIA3 during IAV infection and immunopathology. Our overarching hypothesis is that the IAV-induced activation of ATF6α and PDIA3 supports overt epithelial pro- inflammatory responses, disulfide bonds in IAV proteins and in pro-inflammatory cytokines to increase IAV propagation and lung immunopathology. We have designed two specific aims to test our hypothesis. In Specific Aim #1 we will determine the functional role of IAV-induced UPR-activated transcription factor ATF6α in regulating expression of cytokines/chemokines, subsequent induction of immunopathology and development of long lasting effects on lung health. The specific Aim #2 seeks to dissect the critical requirement of IAV-induced PDIA3 in disulfide mediated processing of IAV-proteins (HA & NA), pro-inflammatory cytokines/chemokines subsequent induction of immunopathology. In both aims we will use transgenic mouse models, cell culture and sensitive redox assays and biochemical assays. Most importantly we will assess the efficacy of specific inhibitors of ATF6α (Ceapin-A7) and PDIA3 (PACMA31) in easing IAV-induced UPR, decreasing subsequent pro- inflammatory responses, and ultimately resulting in resolution of IAV-induced immunopathology. These studies will shed light on the importance of the IAV-induced lung epithelial UPR in IAV propagation and overt pro- inflammatory responses and offers insight into new and highly needed treatment modalities for IAV infection and severe respiratory distress beyond supportive care.