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，如流感（H1N1）、SARS- CoV-2（COVID）和腺病毒（Ad）。这些呼吸道病毒的感染会导致急性肺损伤（ALI）， 可导致急性呼吸窘迫综合征（ARDS），死亡率约为40%。很少 ALI/ARDS的治疗选择。病毒诱导的ALI/ARDS主要由不受控制的炎症反应驱动。 应答肺泡巨噬细胞基于它们的极化/炎性， 状态巨噬细胞的可塑性在促炎（M1，促ALI/ARDS）和抗炎（M2，促ALI/ARDS）之间变化。 炎症（M2，抗-ALI/ARDS）表型由它们的代谢状态驱动。糖尿病是一种代谢性 血糖水平高且糖酵解是优选的细胞代谢途径的疾病。 来自糖尿病患者的巨噬细胞具有高的糖酵解速率和增加的M1表型。此外，本发明还提供了一种方法， 由于这种转变，糖尿病患者的巨噬细胞从M1变为M2的可塑性较低 糖酵解。一种可能性是这种糖酵解转变导致呼吸道病毒感染的严重后果。 糖尿病患者的感染。 叙利亚仓鼠对流感、SARS-CoV-2和Ad自然是宽容的（与其他仓鼠相反）。 需要病毒适应的啮齿动物）。此外，叙利亚仓鼠可以自然成为糖尿病与高 脂肪/高糖饮食。Ad 14 p1是Ad 14的一种紧急菌株，它导致了严重呼吸道疾病的爆发 和ALI/ARDS。Ad 14 p1的肺感染导致斑片状支气管肺炎， 在其他严重的人类病毒性呼吸道感染中可见。相比之下，Ad 14的原型菌株诱导很少的 肺部炎症其他研究表明，Ad 14感染导致的细胞死亡诱导了M2样人类 巨噬细胞反应，而死于Ad 14 p1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E1 B 20 K的表达调节。细胞 Ad 14感染的巨噬细胞产生足够的E1 B20 K，使M1巨噬细胞向M2巨噬细胞转化，而Ad 14 p1感染的巨噬细胞产生足够的E1 B20 K， 不能产生足够的E1 B 20 K，并且感染的细胞不能改变M1巨噬细胞极化。因此 Ad 14 p1急性肺损伤/急性呼吸窘迫综合征仓鼠模型为研究糖尿病对巨噬细胞的影响提供了合适的系统 严重病毒性呼吸道感染期间的极化和发病机制。该项目的长期目标是 了解新兴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驱动巨噬细胞极化为ALI/ARDS消退表型。 为了实现这一目标，将使用多组学方法来鉴定巨噬细胞， 正常和糖尿病仓鼠感染Ad 14 p1。使用单细胞RNA测序的转录组学将使用 基因表达谱在单个细胞的分辨率，以确定和表型巨噬细胞及其 极化状态还将鉴定浸润的免疫细胞和其他肺驻留细胞。蛋白质组学将 用于鉴定驱动Ad 14 p1发病机制的细胞因子和趋化因子。代谢组学将用于 了解糖尿病患者Ad 14 p1感染期间肺部独特的代谢变化，以及这些变化是如何发生的。 变化影响巨噬细胞极化。正常人和糖尿病患者感染的病毒学比较研究 将使用带有H1N1流感大流行毒株的仓鼠来确定是否有类似的机制， 发病机制与其他严重呼吸道病毒引起的ALI/ARDS发病机制有关。最后我们 将测试在原型Ad 14和Ad 14 p1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，目的是确定免疫调节机制， 这些候选miRNAs可能用作抗病毒性ALI/ARDS的治疗剂。

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.