Role of macrophages and miRNA in regulating lung macrophage polarization and lung pathogenesis during respiratory virus-induced acute lung injury in normal and diabetic Syrian hamsters.

超过25%的美国退伍军人患有糖尿病，这些退伍军人住院的风险增加 以及严重呼吸道病毒感染后发病率/死亡率的增加，如流感(H1N1)、SARS- 冠状病毒2型(CoVID)和腺病毒(Ad)。感染这些呼吸道病毒会导致急性肺损伤(ALI) 可导致急性呼吸窘迫综合征(ARDS)，死亡率约为40%。几乎没有几个 ALI/ARDS的治疗选择。病毒诱导的ALI/ARDS主要由失控的炎症驱动 回应。肺泡巨噬细胞通过极化/炎症机制诱导和解决ALI/ARDS 州政府。巨噬细胞的可塑性在促炎(M1，促ALI/ARDS)和抗-ALI/ARDS之间变化 炎症(M2，抗ALI/ARDS)表型由其代谢状态驱动。糖尿病是一种新陈代谢 血糖水平高，糖酵解是首选的细胞代谢途径的紊乱。 糖尿病患者的巨噬细胞糖酵解率高，M1表型增加。此外, 糖尿病患者的巨噬细胞从M1变为M2的可塑性较低，因为这一转变 糖酵解。一种可能性是这种糖酵解的转变导致呼吸道病毒的严重后果。 糖尿病患者的感染。 叙利亚仓鼠对流感、SARS-CoV-2和Ad(与其他动物不同)具有天然的容许性 需要病毒适应的啮齿动物)。此外，叙利亚仓鼠自然会因高血糖而患上糖尿病。 脂肪/高糖饮食。Ad14p1是AD14的一种紧急毒株，曾导致严重呼吸道疾病的爆发。 以及世界各地的ALI/ARDS。感染Ad14p1的仓鼠会导致斑片状的支气管肺炎，AS 出现在其他严重的人类病毒性呼吸道感染中。相比之下，AD14的原型菌株几乎没有诱导 肺部发炎。其他研究表明，死于AD14感染的细胞会诱导出类似M2的人类 巨噬细胞反应，而死于Ad14p1感染的细胞不能将M1肺泡巨噬细胞改变为 M2表型。这种垂死的感染细胞活性受Ad基因E1B 20K的表达调节。单元格 感染AD14产生了足够的E1B 20K，使M1巨噬细胞重新极化为M2，而Ad14p1感染则产生了足够的E1B 20K 不能产生足够的E1B 20K，感染细胞不能改变M1巨噬细胞的极化。因此， Ad14p1ALI/ARDS仓鼠模型为研究糖尿病对巨噬细胞的影响提供了一个合适的系统 严重病毒呼吸道感染期间的极化和发病机制。这个项目的长期目标是 了解紧急病毒如何调节巨噬细胞极化以开发新的治疗策略 在糖尿病和非糖尿病退伍军人中，推动巨噬细胞极化到ALI/ARDS解决表型。 为了实现这一目标，将使用多组学方法来鉴定和表型巨噬细胞。 感染Ad14p1的正常和糖尿病仓鼠。使用单细胞RNA测序的转录组学将使用 单个细胞分辨的基因表达谱以鉴定和表型巨噬细胞及其 偏振态。渗入的免疫细胞和其他肺部常驻细胞也将被识别。蛋白质组学将 用于识别推动Ad14p1发病的细胞因子和趋化因子。代谢组学将被用于 了解糖尿病患者在Ad14p1感染期间肺部独特的代谢变化，以及这些变化是如何 改变会影响巨噬细胞的极化。正常人和糖尿病患者感染的病毒学比较研究 具有H1N1流感大流行毒株的仓鼠将被用来确定类似的机制是否 其他严重呼吸道病毒引起的ALI/ARDS的发病机制也与发病机制有关。最后，我们 将测试在原型AD14和Ad14p1感染过程中miRNA表达在调节巨噬细胞中的作用 极化和发病机制，目标是确定免疫调节机制和识别 候选miRNAs可能被用作治疗病毒性ALI/ARDS的药物。

Over 25% of US Veterans have diabetes, and those Veterans are at an increased risk of hospitalization and increased morbidity/mortality following severe respiratory viral infections, such as, influenza (H1N1), SARS- CoV-2 (COVID) and adenovirus (Ad). Infection with these respiratory viruses causes acute lung injury (ALI) that can result in acute respiratory distress syndrome (ARDS), with a mortality rate of ~40%. There are few therapeutic options for ALI/ARDS. Virus induced ALI/ARDS is driven primarily by uncontrolled inflammatory responses. Alveolar macrophages both induce and resolve ALI/ARDS, based on their polarization/inflammatory state. The plasticity of macrophages to vary between pro-inflammatory (M1, pro-ALI/ARDS) and anti- inflammatory (M2, anti-ALI/ARDS) phenotypes is driven by their metabolic states. Diabetes is a metabolic disorder in which levels of blood glucose are high and glycolysis is the preferred cellular metabolic pathway. Macrophages from diabetic patients have a high rate of glycolysis and an increased M1 phenotype. In addition, macrophages from diabetic patients have a lower rate of plasticity to change from M1 to M2 because of this shift to glycolysis. One possibility is that this glycolytic shift contributes to severe outcomes from respiratory viral infections in diabetic patients. The Syrian hamster is naturally permissive for influenza, SARS-CoV-2 and Ad (in contrast to other rodents that require viral adaptation). In addition, the Syrian hamster can naturally become diabetic with a high fat/high sugar diet. Ad14p1 is an emergent strain of Ad14 that has caused outbreaks of severe respiratory illness and ALI/ARDS throughout the world. Hamster infection with Ad14p1 results in a patchy bronchopneumonia, as seen in other severe human viral respiratory infections. In contrast, the prototype strain of Ad14 induces little lung inflammation. Other studies have shown that cells dying from Ad14 infection induce an M2-like human macrophage response, while cells dying from Ad14p1 infection fail to change M1 alveolar macrophages to an M2 phenotype. This dying infected cell activity is regulated by the expression of the Ad gene, E1B 20K. Cells infected by Ad14 produced sufficient E1B 20K to repolarize M1 macrophages to M2, while Ad14p1 infection does not produce sufficient E1B 20K, and the infected cells fail to alter M1 macrophage polarization. Therefore, the hamster model of Ad14p1 ALI/ARDS provides an appropriate system to study how diabetes affects macrophage polarization and pathogenesis during severe viral respiratory infections. The long-term goal of this project is to understand how emergent viruses regulate macrophage polarization to develop novel therapeutic strategies to drive macrophage polarization to an ALI/ARDS resolving phenotype in both diabetic and non-diabetic Veterans. To achieve this goal, a multi-omics approach will be used to identify and phenotype macrophages in normal and diabetic hamsters infected with Ad14p1. Transcriptomics using single-cell RNA sequencing will use gene expression profiles at the resolution of individual cells to identify and phenotype macrophages and their polarization states. Infiltrating immune cells and other lung resident cells will also be identified. Proteomics will be used to identify cytokines and chemokines that drive Ad14p1 pathogenesis. Metabolomics will be used to understand the unique metabolic changes in the lungs during Ad14p1 infection in diabetes and how those changes affect macrophage polarization. Comparative virology studies with infection of normal and diabetic hamsters with a pandemic strain of H1N1 influenza will be used to determine whether similar mechanisms of pathogenesis are involved in ALI/ARDS pathogenesis induced by other severe respiratory viruses. Finally, we will test the role of miRNA expression during prototype Ad14 and Ad14p1 infection in regulating macrophage polarization and pathogenesis, with the goals of defining mechanisms of immunomodulation and identifying candidate miRNAs that might be used as therapeutic agents against viral ALI/ARDS.