[Exploring the impact of macrophage depletion on influenza viral pneumonia and its mechanism of action through the cGAS-STING pathway].

Objective This study investigated the impact of macrophage depletion on influenza A virus (IAV)-induced pneumonia and its underlying mechanism. Methods A mouse model of IAV-induced pneumonia with macrophage depletion was created using CL2MBP followed by IAV inoculation. Experiments were conducted on days 1, 3, and 5 post-infection. Hematoxylin and eosin (HE) staining was used to assess tissue pathology, and the bicinchoninic acid (BCA) assay was employed to measure protein levels in bronchoalveolar lavage fluid (BALF). ELISA was used to quantify inflammatory factors and cGAMP in lung tissues, and flow cytometry was performed to detect macrophages in BALF. qPCR was used to assess mitochondrial DNA (mtDNA) expression levels. cGAS and STING protein levels in lungs were evaluated by immunohistochemistry. Single-cell sequencing was used to detect the expression levels of IFI16 gene in humans, while immunohistochemical staining was employed to assess IFI204 protein expression in mouse lung tissues. In vitro, RAW264.7 macrophages were treated with STING agonist MSA-2 or inhibitor H-151. Real-time quantitative PCR (qPCR) was used to detect the expression levels of STING, viral nucleoprotein (NP), interleukin 1β (IL-1β), and IFI204 in cells from each group. ResultsFlow cytometry confirmed effective macrophage depletion. BALF protein and inflammatory cytokines (TNF-α, IFN-γ) were significantly higher in macrophage-depleted mice. Histopathological damage worsened, with the most severe effects in macrophage-depleted mice. Macrophage depletion reduced mtDNA, cGAMP, and cGAS/STING protein expression in lungs. Elevated IFI16 expression was observed in COVID-19 or IAV-infected patients. Following macrophage depletion, IFI204 expression in mouse lung tissues was significantly reduced. In vitro, MSA-2 increased IFI204 mRNA, suppressed IL-1β, and inhibited IAV NP replication. H-151 reversed these effects and promoted viral NP gene expression. ConclusionMacrophage depletion impairs the cGAS-STING pathway activation in response to IAV infection, disrupting the innate immune response and worsening viral lung injury.