Unfolded protein response in Influenza virus infection and inflammation

项目摘要 感染后需要进行流感（IAV）蛋白质的合成和加工，包括形成二硫键 肺上皮细胞内质网（ER）中IAV蛋白的半胱氨酸中的键诱导ER应激。 ER应激反过来诱导基于ER的未折叠蛋白反应（UPR）。病毒激活并维持普遍定期审议， 然而，它们的复制，慢性UPR导致促炎反应和细胞凋亡的诱导。不 明确是否特定的普遍定期审议成分被IAV抓住传播，它们也有助于大规模的 增加IAV诱导的促炎反应和肺损伤。 我们的新的初步结果表明，IAV感染激活了潜伏转录因子ATF 6 α。ATF6α 已知其与UPR相关基因的特异性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显示存在保守的 细胞因子和趋化因子启动子区的ATF 6 α结合元件。我们接着观察到， ATF 6 α的缺失/抑制可降低炎症、细胞因子/趋化因子、IAV负荷和气道 高反应性（AHR）。此外，我们发现，IAV感染增加，特别是一个PDI- PDIA 3在HBE细胞和感染IAV的小鼠中的表达。肺上皮特异性消融PDIA 3显著 IAV蛋白质、细胞因子/趋化因子、IAV负荷和肺中AHR反应中的二硫键减少。 提示ATF 6 α和PDIA 3在IAV感染和免疫病理过程中起重要作用。我们 总体假设是IAV诱导的ATF 6 α和PDIA 3的激活支持明显的上皮促增殖， 炎症反应，IAV蛋白和促炎细胞因子中的二硫键增加IAV 繁殖和肺免疫病理学。我们设计了两个具体目标来验证我们的假设。 在具体目标#1中，我们将确定IAV诱导的UPR激活的转录因子ATF 6 α的功能作用。 调节细胞因子/趋化因子的表达，随后诱导免疫病理学和发育 对肺部健康的长期影响。具体目标#2旨在剖析IAV诱导的 PDIA 3在IAV蛋白质（HA & NA）、促炎细胞因子/趋化因子的二硫化物介导的加工中 随后诱导免疫病理学。在这两个目标中，我们将使用转基因小鼠模型，细胞培养和 灵敏的氧化还原测定和生物化学测定。最重要的是，我们将评估特定抑制剂的疗效 ATF 6 α（Ceapin-A7）和PDIA 3（PACMA 31）在减轻IAV诱导的UPR，降低随后的pro-RNA表达方面的作用。 炎症反应，并最终导致IAV诱导的免疫病理学的解决。这些研究 将阐明IAV诱导的肺上皮UPR在IAV传播中的重要性， 炎症反应，并提供了深入了解新的和高度需要的治疗方式，为IAV感染， 严重呼吸窘迫，无法支持治疗。

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.