Abstract Rationale Aging is an important risk factor for severe disease or death in patients with viral pneumonia, including influenza (IAV). T cell-mediated protection from IAV decreases with age, however, the precise molecular bases of such dysfunction are unknown. Metformin is an anti-diabetic drug that modulates cellular metabolism. However, the effects of metformin on age-related dysfunction in T cell function are not well understood. In this study, we studied the effects of aging on viral clearance and immune responses to influenza pneumonia and the effects of metformin on viral clearance in aged mice. Methods Aged mice (20 month) and young mice (2 month) were infected with influenza A virus H1N1; mRNA and protein expression in whole lungs were analyzed by western blotting, unbiased RNA-seq and single cell (sc) RNA-seq. To investigate functional CD8+ T cells responses, IAV-specific CD8+ T cells were assayed with IAV tetramer, along with their proliferative capacity and transcriptional alterations (with RNA-seq). Aged mice received daily intraperitoneal injections of metformin (65 mg/kg, i.p., every other day), beginning on day 3 post-infection. Lung tissues were collected on day 9 post-infection and analyzed by scRNA-seq in addition to functional T cell responses. Results Aged mice showed persistently increased viral mRNA and protein expression as well as enhanced inflammation in the lung. Both scRNA-seq and flow cytometric analyses showed a decrease in CD4+ and IAV-specific CD8 T cells T cells. This was supported by RNAseq analysis of IAV-specific CD8+ T cells that showed involvement of the p53 pathway and reduced oxidative phosphorylation pathway in aging mice. Metformin treatment post-infection markedly reduced pulmonary viral loads, mechanistically by alleviating proliferative defects linked to elevated T cell senescence and DNA damage biomarkers. Single-cell transcriptomics revealed metformin-induced enrichment of E2F targets, type I interferon responses and oxidative phosphorylation pathways in CD4+ and CD8+ T cells. Conclusions We demonstrate aged mice exhibit impaired clearance of IAV compared to young mice. The failure to efficiently clear the virus in aged mice correlates with sustained lung inflammation. Using scRNA sequencing, determination of IAV-specific T cell responses, and computational approaches, we report an aging-associated proliferative/metabolic defect in CD4+ T cells and a senescent-like CD8+ T cell phenotype that impairs antigen-specific responses to IAV infection. Metformin treatment enhances viral clearance by restoring the proliferative capacity of CD4+ T cells and modulating DNA damage responses in CD8+ T cells. This abstract is funded by: NIH grants, R01 HL173154, R01 HL139617, R01 HL151702, and the U.S. Department of Veterans Affairs Merit Award I01BX003056
Matsunaga et al. (Fri,) studied this question.