Fig. 5

Disruption of lung microbiota exacerbates inflammatory response and barrier damage following influenza infection. A K-means clustering analysis of the differentially expressed genes (DEGs) in lung tissue was performed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) online database, with a minimum required interaction score of 0.4. The DEGs in lung tissue infected with the influenza virus at 8 dpi, comparing the WT and Abx groups, were primarily categorized into four clusters. B The heatmap illustrates the DEGs in lung tissue infected with the influenza virus at 8 dpi, organized into four clusters comparing the WT and Abx groups. All genes were normalized based on the corresponding genes in the Nor group before conducting differential analysis. All significance levels are determined by the DESeq2 package (Benjamini-Hochberg). C The heatmap depicting the mRNA transcription levels of genes associated with the inflammation (IL6 and TNFα), apoptosis (Bax and Caspase3), and antiviral response (IFNβ1, IFNα, OAS1, Mx1, PKR, ISG15, Myd88, and Mx2) in lung tissues, determined by RT-qPCR. D Correlation analysis between the differentially abundant ASVs at the genus level and the DEGs in the WT and Abx groups through the Mantel test. E Pearson correlations between the differentially relative abundances of A88 (Moraxella) and A15 (Lactobacillus) and the mRNA levels of IFNβ1, Myd88, Caspase 3, and TNFα in the lung tissues of the WT and Abx groups. F The expression levels of viral protein (NP) and several proteins involved in the inflammatory response (TBK1, IRF3 and NF-κB/p65 along with their corresponding phosphorylated forms) in the lung tissues were assessed by Western blot. GAPDH was employed as an internal reference. Grayscale analysis was performed using ImageJ software