Fig 2: TET2-STAT5 complex facilitates TRPM4 expression in irradiated microglia by enhancing the demethylation of Trpm4 promoter. (A) Global 5-mC and 5-hmC levels in irradiated BV2 cells. (B) Prediction of binding site for STAT5 to the promoter of TRPM4 by JASPAR database. (C) ChIP-qPCR to verify the binding of pSTAT5 to the TRPM4 promoter in irradiated BV2 cells. IgG antibodies were used as the negative control. (D) The association of TET2 and pSTAT5 detected by Co-IP, in irradiated BV2 cells. Samples are immunoprecipitated with isotype IgG as a control. (E-F) ChIP-qPCR to verify the binding of TET2 (E) or 5-hmC (F) to the TRPM4 promoter in irradiated BV2 cells. IgG antibodies were used as the negative control. Rad, radiation. Data are represented as mean ± SD. ***P < 0.001 versus control. ###P < 0.001 versus siControl. n = 3 per group

Fig 3: TET2 participates in the regulation of TRPM4 expression and its association with SUR1 within microglia following radiation. (A-C) qRT-PCR (A) and western blotting (B-C) results of TRPM4 within the sorted microglia from post-RIBI mouse brain after the pharmacological inhibition of TET2. (D) Confocal analysis of TRPM4, SUR1, and Iba-1 staining within hippocampus of post-RIBI mice. (E-G) qRT-PCR (E) and western blotting (F-G) results of TRPM4 within the irradiated BV2 cells after Tet2 gene knockdown. (H) Confocal analysis of TRPM4 and SUR1 staining within the irradiated BV2 cells after Tet2 gene knockdown. Scale bar = 50 μm. Data are represented as mean ± SD. ***P < 0.001 versus sham or control. ##P < 0.01, ###P < 0.001 versus vehicle or siControl. n = 3 or 5 per group

Fig 4: 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 (100 μM) 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. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 5: Schematic representation of the regulatory effects of SUR1-TRPM4 on microglial NLRP3-mediated neuroinflammation after whole-brain radiation. This study unveils the pivotal role and neuroprotective potential of SUR1-TRPM4 in driving microglial NLRP3-mediated persistent neuroinflammation following whole-brain radiation. The specific mechanism by which SUR1-TRPM4 regulates NLRP3 inflammasome activation in microglia has been reported in our previous research [21]. Here, we have further demonstrated that irradiation causes mitochondrial damage to microglia, leading to violent release of ROS which enhance the transcription of SUR1, TRPM4, and NLRP3 inflammasome-related molecules. Moreover, inducibly expressed TET2 by ROS forms a complex with pSTAT5, which subsequently enters the nucleus to mediate demethylation in gene promoters, thereby enhancing TRPM4 expression in irradiated microglia. To sum up, our findings decipher that SUR1-TRPM4 crucially mediates the persistent activation of microglial NLRP3 inflammasome under the action of ROS after whole-brain radiation, offering novel therapeutic strategies for delayed RIBI as well as other NLRP3-related neurological disorders involving excessive ROS production