Ion-Current-Based Temporal Proteomic Profiling of Influenza-A-Virus-Infected Mouse Lungs Revealed Underlying Mechanisms of Altered Integrity of the Lung Microvascular Barrier

Investigation of influenza-A-virus
(IAV)-infected lung proteomes
will greatly promote our understanding on the virus-host crosstalk.
Using a detergent-cocktail extraction and digestion procedure and
a reproducible ion-current-based method, we performed the first comprehensive
temporal analysis of mouse IAV infection. Mouse lung tissues at three
time points post-inoculation were compared with controls (n = 4/group), and >1600 proteins were quantified without
missing value in any animal. Significantly changed proteins were identified
at 4 days (n = 144), 7 days (n =
695), and 10 days (n = 396) after infection, with
low false altered protein rates (1.73–8.39%). Functional annotation
revealed several key biological processes involved in the systemic
host responses. Intriguingly, decreased levels of several cell junction
proteins as well as increased levels of tissue metalloproteinase MMP9
were observed, reflecting the IAV-induced structural breakdown of
lung epithelial barrier. Supporting evidence of MMP9 activation came
from immunoassays examining the abundance and phosphorylation states
of all MAPKs and several relevant molecules. Importantly, IAV-induced
MMP gelatinase expression was suggested to be specific to MMP9, and
p38 MAPK may contribute predominantly to MMP9 elevation. These findings
help to resolve the long-lasting debate regarding the signaling pathways
of IAV-induced MMP9 expression and shed light on the molecular mechanisms
underlying pulmonary capillary-alveolar leak syndrome that can occur
during influenza infection.

针对甲型流感病毒（Influenza A Virus, IAV）感染的肺蛋白质组的研究，将极大推动我们对病毒-宿主互作的理解。本研究采用去污剂混合萃取与酶解流程，结合可重现的基于离子电流的分析方法，完成了首次针对小鼠IAV感染的全面时序分析。我们对感染后三个时间点的小鼠肺组织与对照组进行了比较（每组n=4），实现了对超过1600种蛋白质的定量分析，且所有受试动物的定量数据均无缺失。感染后第4天（n=144）、第7天（n=695）及第10天（n=396）均鉴定到显著差异表达蛋白，差异蛋白的假阳性率为1.73%~8.39%。功能注释结果揭示了宿主系统性应答所涉及的多个关键生物学过程。值得注意的是，本研究观察到多种细胞连接蛋白水平下调，以及组织基质金属蛋白酶MMP9水平上调，这反映了IAV诱导的肺上皮屏障结构破坏。针对所有丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinase, MAPK）及相关分子的丰度与磷酸化状态开展的免疫检测实验，为MMP9的激活提供了佐证。重要的是，研究表明IAV诱导的明胶酶型基质金属蛋白酶表达仅特异性对应MMP9，且p38 MAPK可能是驱动MMP9上调的主要通路。本研究结果有助于解决长期以来关于IAV诱导MMP9表达的信号通路争议，并为阐明流感感染期间可能发生的肺毛细血管-肺泡渗漏综合征的分子机制提供了新的见解。