Abstract
BACKGROUND: Early outcomes of influenza-induced lung injury depend on the rapid activation of the macrophage-type I interferon (IFN) axis, a process that requires tightly regulated glutathione-reactive oxygen species (GSH-ROS) buffering. OBJECTIVE: The aim of this study was to determine whether oral Lactobacillus pentosus B240 amplifies the macrophage-IFN response by pre-modulating the GSH-ROS pathway, thereby enhancing innate immunity against the H1N1 virus. METHODS: The public dataset GSE43764 (48 Agilent one-color microarrays) was re-analyzed using ComBat batch correction, limma differential analysis, gene set variation analysis (GSVA) functional scoring, weighted gene co-expression network analysis (WGCNA) co-expression, CIBERSORTx deconvolution, and mixed-effects modeling; all statistics were Benjamini-Hochberg adjusted. RESULTS: In uninfected mice, pulmonary Gclc was significantly upregulated in the B240 group (p (adj) < 0.05) and overall GSH-ROS and Nrf2 GSVA scores were higher than controls; after the viral challenge, GSH-ROS, Nrf2, macrophage activation, and type I IFN GSVA scores showed significant interactions from 1 to 6 days (q < 0.05). At 1 dpi, B240+CA04 versus Saline+CA04 had 418 upregulated and 289 downregulated genes (p (adj) < 0.05, |log(2)FC| ≥ 0.58), dominated by macrophage markers and ISGs. Gclc and Nqo1 were elevated at 1-3 days (p < 0.05), and Adgre1 and Rsad2 were elevated at 1-6 days (p < 0.01). The WGCNA red module (312 genes) correlated most strongly with GSH-ROS GSVA and macrophage abundance (q < 0.05); GSH-ROS core genes (Gclc and Nqo1) and macrophage-IFN genes (Adgre1, Mx1, and Rsad2) were co-expressed (Pearson r > 0.2, FDR < 0.05). Enrichment covered viral response, type I IFN, RIG-I-like, Toll-like, NOD-like, and JAK-STAT antiviral pathways, plus GSH metabolism, oxidative stress, and macrophage activation (all q < 0.05). CIBERSORTx showed macrophage abundance and the M1/M2 index remained higher at 1, 3, and 6 dpi with B240, with three-way interaction estimates of 0.28 and 0.59 (q = 0.002). Single-sample analysis revealed stronger GSH-ROS versus macrophage-ISG correlation in B240 (r = 0.81, q = 0.001) than control (r = 0.53, q = 0.008); Fisher z p (adj) = 0.015. Integrative network nodes had Spearman ρ 0.58-0.72 (q < 0.05), confirming oxidative-IFN coupling. CONCLUSIONS: B240 elevates baseline GSH-ROS thresholds and reshapes a red co-expression module, synchronously amplifying macrophage infiltration and type I IFN feedback, thereby strengthening early innate responses to H1N1 infection and suggesting nutritional-immunological prophylaxis for high-risk respiratory viral exposure.