Journal: Journal of Advanced Research

Article Title: Gut microbial GABAergic signaling improves stress-associated innate immunity to respiratory viral infection

doi: 10.1016/j.jare.2023.06.008

Figure Lengend Snippet: GABA acts through the αKG/Tet2/ PPARγ axis to mediate metabolic-epigenetic regulation of AMs. (A) Schematic diagram of the TCA cycle, with the key enzymes regulated upon stress labeled. Red arrow, down-regulated, and Green arrow, up-regulated in stressed AMs versus non-stressed AMs post IAV infection. (B) Serum levels of αKG and succinate analyzed by GC-MS, with the ratio of αKG to succinate provided. (C-F) Stressed and Non-stressed (NS) mice were infected or uninfected with IAV for 3 days. CD45 + CD11b - CD64 + CD11c + SiglecF + AMs were sorted for subsequent analysis. (C) Immunoblotting of Tet2; (D) Dot-blot analysis of 5hmc or 5mc level. MB, methylene blue staining (as loading control). (E) ChIP test of 5hmc or 5mc enrichment at the pparγ locus; (F) Immunoblotting of PPARγ; (G) Immunoblotting of Tet2 and PPARγ in MH-S cells treated with GABA at the indicated doses; (H) Immunoblotting of Tet2 or PPARγ in GABA-treated macrophages with or without bicuculline (Bic; 40 μM). (I-K) MH-S cells were transfected with Tet2 siRNA (siTet2) or siRNA (siNC), and then treated with GABA (100μM) or 0.01% DMSO prior to IAV infection. (I) PPARγ luciferase activity; (J) Immunoblotting of Tet2 and PPARγ; (K) Heatmapping of AM-associated genes. Shown are representative images. The bar graph is the densitometric quantification of western blot bands. Data from three individual experiments are shown as means ± SD. * P < 0.05, ** P < 0.01, with one-way ANOVA (three or more groups) followed by Bonferroni post hoc test.

Article Snippet: Solution chromatin was immunoprecipitated with anti-5hmc (#39069, 2 μg), anti-5mc (#ab10805, 2 μg), or normal rabbit IgG (CST, #2729, 2 μg), respectively.

Techniques: Labeling, Infection, Gas Chromatography-Mass Spectrometry, Western Blot, Dot Blot, Staining, Transfection, Luciferase, Activity Assay