C107-14 Exploring How Mitochondrial Damage and Dysfunction Influences Tlr9 and Cgas-sting Pathways in Obesity and Influenza

Abstract Rationale Obesity increases the risk of acute respiratory distress syndrome (ARDS) following viral infections, but the underlying mechanisms remain unclear. Mitochondrial DNA (mtDNA) plays a dual role in immune regulation—activating beneficial antiviral defenses via Type I Interferons (IFNs) and promoting detrimental systemic inflammation through (toll-like receptor 9) TLR9 signaling. Obesity-related mitochondrial dysfunction is associated with increased mtDNA release. However, mtDNA-mediated Type I Interferon production via cGAS/STING is dependent on a healthy pool of mitochondria while TLR9-mediated inflammation is not. Therefore, we hypothesized that obesity exacerbates viral ARDS by increasing mtDNA-driven TLR9 inflammation and impairing antiviral Type I Interferon responses. Methods Mice were fed either a high-fat diet or standard chow for 16 weeks, followed by intranasal infection with 50 µL of PR8 influenza virus. MtDNA concentrations in plasma and BALF were quantified by ddPCR. Cytokines and Type I Interferons were measured using MSD multiplex assays and ELISA, respectively. Lung pathology was assessed via histology and electron microscopy. Mitochondrial function was analyzed using OROBOROS and Seahorse assays, and STING and TBK1 expression were evaluated by Western blot. Results Following influenza infection, obese mice exhibited persistent weight loss and neutrophilia compared to controls. Obese mice displayed increased mtDNA release in plasma and BALF at before versus 5 days after infection (7.045 v 113.8 p = 0.004, 30.48 v 164.2 p 0.001, respectively, Figure 1A). Then, obese mice showed increased TLR9 expression at day 5 versus day 7 after infection (1.664 v 47.48, p = 0.02, Figure 1B). Furthermore, obese mice had increased serum mtDNA, a trend towards increased BALF IL-6, and reduced BALF Type I Interferon compared to lean mice (5.2 v 7.9 p = 0.03, 287.2 v 363.3 p = 0.3, 1.959 v 1.828 p = 0.05 respectively, Figure 1C-E). Electron microscopy revealed fragmented and damaged mitochondria in alveolar macrophages of obese lungs, although no differences in STING or TBK1 expression were detected. Conclusions Obesity is associated with baseline mitochondrial dysfunction and elevated mtDNA release in the lungs. After influenza infection, obese mice show enhanced mtDNA-TLR9 signaling and reduced antiviral Type I Interferon responses, independent of STING and TBK1. Ongoing mechanistic studies are investigating the role of mitochondrial integrity in modulating antiviral immunity, potentially at the downstream IRF3 level. Ultimately, we aim to define mitochondrial function and mtDNA-mediated inflammation as modifiable targets to improve outcomes of influenza-induced ARDS in obesity. This abstract is funded by: T32 HL166127 (to AJK and ANP), R01DK136756 (to MLH), American Thoracic Society Research Foundation Unrestricted Critical Care Grant (to MLH), BX006238 (to MLH), K24 HL155884 (to LCA), BX006447 (to LCA)