Glutathione-ROS Pathway Activation by Probiotic B240 Augments Macrophage Response to Influenza

Zhen Ye^*Ying Zhang

• Tianjin University Chest Hospital, Tianjin, China

Background: Early outcomes of influenza‑induced lung injury depend on rapid activation of the macrophage–type I interferon axis, a process that requires tightly regulated glutathione‑reactive oxygen species (GSH‑ROS) buffering. Objective: To determine whether oral Lactobacillus pentosus B240 amplifies the macrophage– interferon response by pre‑modulating the GSH‑ROS pathway, thereby enhancing innate immunity against H1N1 virus. Methods: Public dataset GSE43764 (48 Agilent one‑color microarrays) was re‑analysed using ComBat batch correction, limma differential analysis, GSVA functional scoring, WGCNA co‑expression, CIBERSORTx deconvolution and mixed‑effects modelling; all statistics were Benjamini‑Hochberg adjusted. Results: In uninfected mice, pulmonary Gclc was significantly up‑regulated in the B240 group (adj.P < 0.05) and overall GSH‑ROS and Nrf2 GSVA scores were higher than controls; after viral challenge, GSH‑ROS, Nrf2, macrophage activation and I‑IFN GSVA scores showed significant interactions from 1–6 d (q < 0.05). At 1 dpi B240+CA04 versus Saline+CA04 had 418 up‑regulated and 289 down‑regulated genes (adj.P < 0.05, |log₂FC| ≥ 0.58), dominated by macrophage markers and ISGs. Gclc and Nqo1 were elevated at 1–3 d (P < 0.05); Adgre1 and Rsad2 at 1–6 d (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, Nqo1) and macrophage‑IFN genes (Adgre1, Mx1, 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 glutathione metabolism, oxidative stress and macrophage activation (all q < 0.05). CIBERSORTx showed macrophage abundance and M1/M2 index remained higher at 1, 3, 6 dpi with B240, three‑way interaction estimates 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‑interferon coupling. Conclusions: B240 elevates baseline GSH‑ROS thresholds and reshapes a red co‑expression module, synchronously amplifying macrophage infiltration and type I interferon feedback, thereby strengthening early innate responses to H1N1 infection and suggesting nutritional‑immunological prophylaxis for high‑risk respiratory viral exposure.