Effects of ATF2/TSC1 on epilepsy by modulating the microphages polarization of microglia

Abstract

Epilepsy (EP) is a chronic nervous system disease characterized by recurrent attacks, and its causes are complicated. Inflammatory reaction mediated by microglia is an important factor in the progression of EP. Activating transcription factor 2 (ATF2) can be used as a transcription factor to regulate the microglia-mediated inflammatory response, but its role in EP is unclear. In this study, kainic acid (KA) was used to induce the EP cell and mouse model. Real-time polymerase chain reaction was used to detect ATF2, TNF-α, IL-6, TGF-β, and IL-10 mRNA expression. ATF2, INOS, ARG1, and TSC1 protein levels was examined by western blot. The fluorescence intensity of ATF2, IBA1, CD80, and CD206 was examined by immunofluorescence staining. The cell ratios of CD80, IL-1β, CD206, and CD63 were detected by flow cytometry. Dual-luciferase reporter and chromatin immunoprecipitation assays were conducted to verify the interaction between ATF2 and TSC1. Hematoxylin & eosin and Nissl staining were used to observe the structure of hippocampus and Nissl bodies. The results indicated that KA induced M1 polarization of HMC3 cells and increased the levels of TNF-α and IL-6 mRNA by activating KA receptors, and inhibiting KA receptors attenuated the M1 polarization of KA-induced HMC3 cells. ATF2 expression was increased in KA-induced HMC3 cells and hippocampal tissues of mouse, while TSC1 expression was repressed. ATF2 knockdown diminished the M1 polarization of KA-induced HMC3 cells, enhanced the M2 polarization, and relieved neuroinflammation in EP mouse. TSC1 overexpression inhibited M1 polarization in KA-induced HMC3 cells. Dual luciferase and chromatin immunoprecipitation results revealed that ATF2 bound to the promoter of TSC1 and negatively regulated the transcription of TSC1. In conclusion, inhibition of ATF2 and promotion of TSC1 transcription may be a new pathophysiological mechanism for the treatment of EP neuroinflammation.

Keywords: Activating transcription factor 2; Epilepsy; Microglia; Microphages polarization; Tuberous sclerosis complex 1.

Fig. 1

KA-induced M1 polarization of microglia HMC3. Flow cytometry was used to detect M1 macrophage markers CD80 (A-B) and IL-1β (C-D), as well as M2 macrophage markers CD206 (E-F) and CD163 (G-H). RT-qPCR was performed to measure the mRNA levels of inflammatory cytokines TNF-α (I), IL-6 (J), TGF-β (K), and IL-10 (L). Western blot analysis was conducted to assess the protein expression of KA receptors GLUK5 and GLUK2 (M-O). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 2

Inhibition of KA receptor attenuates the M1 polarization effect of KA on HMC3 cells. Flow cytometry was used to detect M1 macrophage markers CD80 (A-B) and IL-1β (C-D), as well as M2 macrophage markers CD206 (E-F) and CD163 (G-H). RT-qPCR was employed to assess the mRNA levels of inflammatory cytokines TNF-α (I), IL-6 (J), TGF-β (K), and IL-10 (L). Western blot analysis was performed to determine the protein expression of KA receptors GLUK5 and GLUK2 (M-O). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 3

ATF2 is overexpressed in KA-induced HHMC3 cells. (A) ATF2 mRNA expression in KA-induced HMC3 cell. (B-C) ATF2 protein expression in KA-induced HMC3 cell. (D). The fluorescence intensity of ATF2 and IBA1. Analysis of the relative fluorescence intensity of ATF2 (E) and IBA1 (F). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 4

ATF2 inhibition represses the M1 polarization of HMC3 cells. (A-B) ATF2 protein level in HMC3 cells transfected with sh-ATF2. (C-D) ATF2 protein expression in KA-induced HMC3 cells transfected with sh-ATF2. Flow cytometry was performed to access cell ratio of M1 microphage CD80 (E-F), IL-1β (G-H) and M2 microphage marker CD206 (I-J), CD163 (K-L). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 5

ATF2 inhibition represses the M1 polarization of HMC3 cells. The mRNA expression of TNF-α (I), IL-6 (B), TGF-β (C), and IL-10 (D) in HMC3 cells transfected with sh-ATF2. (E) The gel images of INOS and ARG1. (F) Relative expression of INOS protein. (G) Relative expression of ARG1 protein. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 6

ATF2 regulates TSC1 transcription. (A) The binding sites of ATF2 obtained from JASPAR database. (B) The promoter regions (P1: promoter region 1, P2: promoter region 2, P3: promoter region 3) of TSC1. (C) The relative enrichment of ATF2 in TSC1 promoter regions. Dual luciferase reporter (D) and western blot (E) assay revealed that ATF2 inhibited the transcription of TSC1. (F) The luciferase activity of wild-type (WT) and mutant (MUT) TSC1 in HMC3 cells. (G) Relative expression of TSC1 in KA-induced HMC3 cells. (H-I) Transfection with sh-ATF2 enhanced TSC1 protein. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 7

Overexpressed TSCI represses M1 polarization of KA-induced HMC3 cells. (A-B) TSC1 protein level in KA-stimulated HMC3 cells transfected with pcDNA-TSC1. (C-D) The relative expression of IBA1 protein. Flow cytometry detected the cell ratios of CD80 (E-F), IL-1β (G and J), CD206 (H and K), and CD163 (I and L). The relative expression of TNF-α (M), IL-6 (N), TGF-β (O), and IL-10 (P) mRNA. (Q) The gel images of INOS and ARG1. (R) Relative expression of INOS protein. (S) Relative expression of ARG1 protein. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Fig. 8

ATF2 regulates microglia polarization in KA-induced mouse by medicating TSC1 transcription. (A) Racine scale scores were used to evaluate the seizure severity of mice. (B) HE staining of mouse hippocampal tissues. (C-D) Nissl staining of mouse hippocampal tissues. (E) The gel images of ATF2 and TSC1. The relative expression of ATF2 (F) and TSC1 (G). (H-I) The fluorescence staining of IBA1 and CD80. (J-K) The fluorescence staining of IBA1 and CD206. The relative expression of TNF-α (L), IL-6 (M), TGF-β (N), and IL-10 (O). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.