Glutathione S-transferases promote proinflammatory astrocyte-microglia communication during brain inflammation

Abstract

Astrocytes and microglia play critical roles in brain inflammation. Here, we report that glutathione S-transferases (GSTs), particularly GSTM1, promote proinflammatory signaling in astrocytes and contribute to astrocyte-mediated microglia activation during brain inflammation. In vivo, astrocyte-specific knockdown of GSTM1 in the prefrontal cortex attenuated microglia activation in brain inflammation induced by systemic injection of lipopolysaccharides (LPS). Knocking down GSTM1 in astrocytes also attenuated LPS-induced production of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) by microglia when the two cell types were cocultured. In astrocytes, GSTM1 was required for the activation of nuclear factor κB (NF-κB) and the production of proinflammatory mediators, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif chemokine ligand 2 (CCL2), both of which enhance microglia activation. Our study suggests that GSTs play a proinflammatory role in priming astrocytes and enhancing microglia activation in a microglia-astrocyte positive feedback loop during brain inflammation.

Fig. 1.. Enriched expression of GSTM1 in astrocytes in the mouse brain.

(A) A representative Western blot for GSTM1 and GSTT2 in the cerebral cortex, hippocampus, striatum, cerebellum, whole brain, and lung of C57BL/6 WT mice (8 weeks of age, male). β-actin is a loading control. (B) The abundances of GSTM1 and GSTT2 in each brain area relative to that in the lung were quantified by densitometry. Male (8–11 weeks of age), n= 3 mice; Female (8 weeks of age), n = 3 mice. (C) Immunofluorescence showing GSTM1 in the medial prefrontal cortex (mPFC), specifically prelimbic area (PrL), in 8 weeks old C57BL/6J male mice. Cells were co-stained for cell type–specific markers to identify neurons (NeuN), astrocytes (S100β), oligodendrocytes (Olig2), or microglia (Iba1). (D) Quantification of the percentage of each indicated cell type that was positive for GSTM1 staining. Male (8–11 weeks of age), n= 3 mice; Female (8 weeks of age), n = 3 mice. Scale bar, 25 μm. In (B) and (D), error bars represent mean ± SEM. *p<0.05. **p<0.01. n.d., not detected. Significance was determined by two-way ANOVA with Tukey’s post-hoc test.

Fig. 2.. Reduced activation of microglia in astrocyte-specific GSTM1 knockdown mice during brain inflammation induced by systemic injection of LPS.

(A) Experimental design. Mouse Gfap promoter–driven Cre transgenic (mGfap-Cre) mice (3 weeks of age) were stereotactically injected with floxed AAV vector encoding shRNAmir against Gstm1 (AAV-LSL-GFP-Gstm1 shRNAmir) into the medial prefrontal cortex (mPFC) and challenged with intraperitoneal (i.p.) injection of LPS 3–4 weeks later. After 48 hours, the brains were harvested and stained for the presence of virally encoded GFP together with cell-type specific markers (NeuN for neurons and S100β for astrocytes). (B) Slices from the mPFC of LPS-challenged mice injected with AAV encoding the control shRNA or Gstm1 shRNA were stained with the microglia marker Iba1 and their activation status was analyzed by morphological changes in the area of astrocyte-specific GSTM1 knockdown (GFP⁺) by confocal microscopy. (C) To quantify microglial activation, we morphologically classified each Iba1⁺ microglia as ramified, intermediate, amoeboid, or round. These morphologies correspond to surveying (ramified) or activated (intermediate, amoeboid, round) microglia (58). (D) The microglia activation profiles were compared between the mice injected with control shRNA and those injected with Gstm1 shRNA. n = 1,265 microglia from 8 mice for control shRNA; 941 microglia from 8 mice for Gstm1 shRNA). (E) Immunofluorescence showing TNF-α in microglia in the vicinity of astrocytes with GSTM1 knockdown in mice injected with AAV encoding the control shRNA or Gstm1 shRNA. (F) Quantification of the percentages of Iba1⁺ microglia positive for TNF-α in mice in (E). n = 560 microglia from 7 mice for control shRNA; 616 microglia from 8 mice for Gstm1 shRNA. Scale bars, 25 μm (A), 100 μm (B), 10 μm (C), and 25 μm (E). In (D) and (F), each dot represents one animal and the bar represents mean ± SEM. Significance was determined by Mann-Whitney test. *p<0.05, **p<0.01.

Fig. 3.. Impaired production of microglial TNF-α by GSTM1 silencing in co-cultured astrocytes.

(A) Amplification of inflammatory responses between astrocytes and microglia through soluble mediators. Previous studies suggest that microglia produce pro-inflammatory cytokines such as TNF-α and IL-1β, which in turn stimulate astrocytes to produce pro-inflammatory mediators such as GM-CSF and CCL2, which amplify inflammatory responses in microglia. (B) Experimental design. Primary mouse glial cultures were prepared from 6–8 P2–5 pups (mixed male and female) and infected with lentivirus encoding shRNA. Astrocytes were enriched and then co-cultured with BV2 microglia overnight before LPS stimulation for 6 hours. Cells and culture supernatants were harvested for qRT-PCR analysis and ELISA, respectively. The graph shows TNF-α production in response to LPS stimulation in astrocyte and microglia monocultures and in co-culture as measured by ELISA. Representative data from two biologically independent cell culture experiments were shown. (C) Quantification of TNF-α production in co-cultures of BV2 microglia with astrocytes expressing the control shRNA or Gstm1 shRNA. Quantification was performed by ELISA. (D) Quantification of Tnf and Il1b expression in co-cultures of BV2 microglia with control or GSTM1 knockdown astrocytes. Quantification was performed by qRT-PCR of cell extracts. (E) Quantification of Tnf, Csf2, and Ccl2 expression in co-cultures of BV2 microglia and WT astrocytes in the presence of TNF-α and IL-1β signaling-blocking antibodies. Quantification was performed by qRT-PCR of cell extracts. Each bar represents mean ± SD of triplicate measurements. For (C–E), representative data from three biologically independent cell culture experiments were shown. In (C) and (D), significance was determined by two-way ANOVA with Sidak’s post-hoc test for control vs. Gstm1 shRNA. In (E), significance was determined by one-way ANOVA with Sidak’s post-hoc test for no-antibodies vs. anti-TNF-α/IL-1β. *p<0.05. **p<0.01. n.d., not detected. h, hours.

Fig. 4.. Altered induction of inflammatory mediators in cultured GSTM1-knockdown astrocytes.

(A) Quantification of GM-CSF and CCL2 in supernatants from control and GSTM1 knockdown primary mouse cortical astrocytes in response to TNF-α and IL-1β stimulation. Quantification was performed by ELISA. (B) Expression of Ccl2, Csf1, Csf2, Tgfb1, Nos2, and Il33 mRNAs in control and GSTM1 knockdown primary mouse cortical astrocytes stimulated with TNF-α or IL-1β. Transcripts were quantified by qRT-PCR. Each bar represents mean ± SD of triplicate measurements. Representative data from three biologically independent cell culture experiments are shown. Significance was determined by two-way ANOVA with Sidak’s post-hoc test for control shRNA vs. Gstm1 shRNA. ** p<0.01.

Fig. 5.. GSTM1 knockdown impairs NF-κB activation in primary astrocytes.

(A) Western blot for the p65 subunit of NF-κB (p65), JNK, ERK, and IkBα and the phosphorylated forms of these proteins (p-p65, p-JNK, p-ERK, and p-IkBα) in primary mouse cortical astrocytes expressing a control or Gstm1 shRNA. β-actin is a loading control. (B) Quantification of p65, JNK, ERK, and IkBα phosphorylation relative to the total abundance of each protein in primary mouse cortical astrocytes expressing a control or Gstm1 shRNA. (C) Western blot and quantification of p65 phosphorylation in astrocytes treated with the GSH depletor DEM during TNF-α stimulation. (D) Schematic model of the role of GSTM1 in astrocytes and pro-inflammatory astrocyte-microglia interactions. Our findings support the role of GSTM1 in activating NF-κB and inducing the expression of Ccl2 and Csf2 in astrocytes. In the absence of GSTM1, microglia activation is attenuated by insufficient amounts of astrocyte-derived GM-CSF and CCL2. This also results in a decrease in TNF-α production by microglia due to reduced positive feedback mediated by GM-CSF and CCL2. For (A) and (C), representative blot data from three independent experiments were shown. For quantification in (B) and (C), bar graphs represent mean ± SD of three biologically independent cell cultures. In (B), significance was determined by two-way ANOVA with Sidak’s post-hoc test for control shRNA vs. Gstm1 shRNA. In (C), significance was determined by two-way ANOVA with Tukey’s post-hoc test. ** p<0.01.