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.

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.