Irf9 signalling in vagal sensory ganglia shapes neuroimmune responses and disease severity during influenza A virus infection

Influenza A virus (IAV) remains a major cause of global morbidity and mortality. Although the role of pulmonary immune cells has been extensively studied, there is increasing evidence that airway-innervating vagal sensory neurons play an important role during respiratory viral infection. These neurons have been shown to influence pulmonary inflammation, and disease outcomes. We previously showed that vagal sensory neurons undergo transcriptional changes following IAV infection, including upregulation of interferon regulatory factor 9 ( Irf9 ). IRF9 is a key mediator in type I and III interferon signalling, yet its role in vagal sensory neurons remains undefined. Here, we investigate the effects of targeted Irf9 knockdown within vagal sensory ganglia on the clinical and pathological presentation of IAV disease. Using bilateral intraganglionic microinjections of AAV-delivered short hairpin RNA, we selectively knocked down Irf9 within the vagal sensory ganglia. Transduction efficiency was confirmed by the reporter protein GFP, and Irf9 gene expression was significantly reduced in IAV-infected knockdown animals compared with IAV-infected scramble controls. Knockdown of vagal Irf9 was associated with exacerbated clinical disease severity, reflected by greater weight loss, higher clinical severity scores, and altered respiratory parameters during infection. Despite these clinical effects, Irf9 knockdown did not alter pulmonary viral load or cytokine responses in lung or serum, indicating that worsened disease was not driven by peripheral anti-viral immune responses. However, Irf9 knockdown was accompanied by increased number of inflammatory cells within the vagal sensory ganglia and changes in the local ganglia proteomic profile. These findings raise the possibility that Irf9 may contribute to regulating neuronal signalling pathways and help shape physiological determinants of disease severity.