School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, University of Newcastle, Callaghan, NSW, Australia.
Priority Research Centre for Health Lungs, Hunter Medical Research Institute (HMRI), University of Newcastle, New Lambton Heights, NSW, Australia.
Front Immunol. 2022 Feb 25;13:805558. doi: 10.3389/fimmu.2022.805558. eCollection 2022.
Virus-induced asthma exacerbation is a health burden worldwide and lacks effective treatment. To better understand the disease pathogenesis and find novel therapeutic targets, we established a mouse model of steroid (dexamethasone (DEX)) resistant asthma exacerbation using ovalbumin (OVA) and influenza virus (FLU) infection. Using liquid chromatography-tandem mass spectrometry (LC-MC/MS), we performed a shotgun proteomics assay coupled with label-free quantification to define all dysregulated proteins in the lung proteome of asthmatic mice. Compared to control, 71, 89, and 30 proteins were found significantly upregulated by at least two-fold (p-value ≤ 0.05) in OVA-, OVA/FLU-, and OVA/FLU/DEX-treated mice, respectively. We then applied a Z-score transformed hierarchical clustering analysis and Ingenuity Pathway Analysis (IPA) to highlight the key inflammation pathways underlying the disease. Within all these upregulated proteins, 64 proteins were uniquely highly expressed in OVA/FLU mice compared to OVA mice; and 11 proteins were DEX-refractory. IPA assay revealed two of the most enriched pathways associated with these over-expressed protein clusters were those associated with MHC class I (MHC-I) antigen-presentation and interferon (IFN) signaling. Within these pathways, signal-transducer-and-activator-of-transcription-1 (STAT1) protein was identified as the most significantly changed protein contributing to the pathogenesis of exacerbation and the underlying steroid resistance based on the label-free quantification; and this was further confirmed by both Parallel Reaction Monitoring (PRM) proteomics assay and western blots. Further, the pharmacological drug Fludarabine decreased STAT1 expression, restored the responsiveness of OVA/FLU mice to DEX and markedly suppressed disease severity. Taken together, this study describes the proteomic profile underpinning molecular mechanisms of FLU-induced asthma exacerbation and identifies STAT1 as a potential therapeutic target, more importantly, we provided a novel therapeutic strategy that may be clinically translated into practice.
病毒诱导的哮喘恶化是全球范围内的健康负担，目前缺乏有效的治疗方法。为了更好地了解疾病的发病机制并找到新的治疗靶点，我们使用卵清蛋白（OVA）和流感病毒（FLU）感染建立了一种类固醇（地塞米松（DEX））抵抗性哮喘恶化的小鼠模型。使用液相色谱-串联质谱（LC-MC/MS），我们进行了一种 shotgun 蛋白质组学测定，结合无标记定量法来定义哮喘小鼠肺部蛋白质组中所有失调的蛋白质。与对照组相比，在 OVA-、OVA/FLU-和 OVA/FLU/DEX 处理的小鼠中，分别有 71、89 和 30 种蛋白质的表达至少上调了两倍（p 值≤0.05）。然后，我们应用 Z 分数变换的层次聚类分析和 IPA 分析来突出疾病潜在的关键炎症途径。在所有这些上调的蛋白质中，有 64 种蛋白质在 OVA/FLU 小鼠中与 OVA 小鼠相比表达显著上调；并且有 11 种蛋白质对 DEX 有抗性。IPA 分析显示，与这些过表达蛋白簇最相关的两个最富集途径是与 MHC Ⅰ类（MHC-I）抗原呈递和干扰素（IFN）信号相关的途径。在这些途径中，基于无标记定量法，转录激活因子 1（STAT1）蛋白被鉴定为导致恶化发病机制和潜在类固醇抵抗的最显著变化蛋白，并且这一点通过平行反应监测（PRM）蛋白质组学测定和 Western blot 进一步得到了证实。此外，药物氟达拉滨降低了 STAT1 的表达，恢复了 OVA/FLU 小鼠对 DEX 的反应性，并显著抑制了疾病的严重程度。总之，本研究描述了 FLU 诱导的哮喘恶化的蛋白质组学特征，并确定了 STAT1 作为一个潜在的治疗靶点，更重要的是，我们提供了一种新的治疗策略，可能在临床上转化为实践。
