Role of miR-181a-5p in Adenovirus 14p1 induced Acute Lung Injury

项目总结 已经有一种新的腺病毒株(Ad14p1)在全球范围内、地区性地爆发。 引起急性肺损伤，类似于其他Ad血清型感染暴发时观察到的 (例如，广告3、4和7)。然而，以前还不可能进行比较病毒学和发病机制的研究。 研究，对比一个原型，亲本Ad株，如AD14，与一个单一的，全球分布的基因 变体，如Ad14p1。 这些Ad14p1感染严重程度增加的原因尚不清楚。我们已经证明了 受Ad14感染诱导的细胞病变效应(CPE)的细胞诱导免疫抑制活性 通过抑制IL-1b、IL-6、IL-8和TNF-α激活的人肺泡巨噬细胞。相比之下，Ad14p1 CPE身体由于病毒E1B 20K表达减少而增加了这些相同细胞因子的产生 Ad14p1具CPE身体。与这些体外观察相一致的是，感染叙利亚仓鼠的原型 AD14导致轻微的炎症和肺损伤，而Ad14p1感染导致明显的急性肺损伤 损伤和ARDS样病理。 已有研究表明，凋亡细胞改变了microRNA(MiRNA)的表达，从而抑制了原核糖核酸的表达。 这些来自凋亡细胞的miRNA可以传递给巨噬细胞。我们 推测类似的机制可能发生在感染AD14的细胞中，而不是感染了 Ad14p1变异体。为了测试这一点，我们用AD14或Ad14p1感染人类细胞，并在 各自的CPE身体。数据显示，AD14 CPE身体富含四种miRNA(AD14 MiRNA)， 所有这些都被证明可以抑制依赖于核因子-kB的促炎细胞因子的转录。 至Ad14p1 CPE身体。我们的假设是在AD14 CPE中这些特异性miRNA的表达增加 身体及其向应答巨噬细胞的转移是AD14 CPE身体的机制 抑制促炎反应。 我们在这里提出的研究将检验这些假设。我们将测试AD14 miRNA在调制中的作用 利用miRNA模拟巨噬细胞的炎症反应及E1B 20K是否调节AD14 miRNA 在感染过程中表达。我们将测试表达最丰富的AD14miRNA，miR-181a-5p， 作为AD14 CPE免疫抑制的介导物，通过使用3‘UTR报告结构和抑制miR-181a- 5便士，带miRZips。最后，我们将进行翻译研究，以确定miR-181a-5p在 叙利亚仓鼠Ad14p1感染的免疫发病机制。这个项目完成后，我们预计将 对病毒诱导的细胞miRNAs在巨噬细胞调节中的作用有了更深入的了解 急性肺损伤时对Ad14p1的反应。我们研究的积极影响将是 确定miRNAs作为ARDS治疗干预措施的潜力。

PROJECT SUMMARY There have been globally distributed, regional outbreaks of a novel strain of adenovirus (Ad) 14, Ad14p1, that induces acute lung injury similar to that observed during outbreaks of infections with other Ad serotypes (e.g., Ad 3, 4 and 7). It has not, however, previously been possible to do comparative virology and pathogenesis studies, contrasting a prototype, parental Ad strain, such as Ad14, with a single, globally distributed genetic variant, such as Ad14p1. The reasons for the increased severity of these Ad14p1 infections are poorly defined. We have shown that cells undergoing Ad-induced cytopathic effect (CPE) from infection with Ad14 elicit an immunorepressive activity of activated human alveolar macrophages by repressing IL-1b, IL-6, IL-8 and TNF-alpha. In contrast, Ad14p1 CPE corpses enhance production of these same cytokines, due to decreased expression of viral E1B 20K in Ad14p1 CPE corpses. Consistent with these in vitro observations, infection of Syrian hamsters with prototype Ad14 resulted in minimal inflammation and lung injury, whereas Ad14p1 infection induced a marked acute lung injury and ARDS-like pathology. It has been shown that apoptotic cells have altered expression of microRNA (miRNA) that can repress pro- inflammatory responses and that these miRNA from apoptotic cells can be delivered to macrophages. We postulated that a similar mechanism could be occurring with Ad14 infected cells but not cells infected with the Ad14p1 variant. To test this, we infected human cells with Ad14 or Ad14p1 and did small RNA-seq on the respective CPE corpses. The data showed that Ad14 CPE corpses are enriched in four miRNA (Ad14 miRNA), all of which have been shown to repress NF-kB dependent transcription of pro-inflammatory cytokines, compared to Ad14p1 CPE corpses. Our hypothesis is that increased expression of these specific miRNA in Ad14 CPE corpses and their transfer to responder macrophages are the mechanisms through which Ad14 CPE corpses repress pro-inflammatory responses. The studies we propose here will test those hypotheses. We will test the role of Ad14 miRNA in modulating inflammatory responses of macrophages using miRNA mimics and whether E1B 20K modulates Ad14 miRNA expression during infection. We will test the role of the most abundantly expressed Ad14 miRNA, miR-181a-5p, as the mediator of Ad14 CPE immunorepression by using 3’ UTR reporter constructs and inhibiting miR-181a- 5p with miRZips. Finally, we will do translational studies to determine the role miR-181a-5p plays in the immunopathogenesis of Ad14p1 infection in the Syrian hamster. Upon completion of this project, we expect to have an increased understanding of the role of virally induced cellular miRNAs in modulation of macrophage responses during acute lung injury in response to Ad14p1. The positive impact of our studies will be the determination of the potential of miRNAs as therapeutic interventions for ARDS.