STING mediates microglial pyroptosis via interaction with NLRP3 in cerebral ischaemic stroke

Abstract

Background: Ischaemia-evoked neuroinflammation is a critical pathogenic event following ischaemic stroke. Gasdermin D (GSDMD)-associated pyroptosis represents a type of inflammation-associated programmed cell death, which can exacerbate neuroinflammatory responses and brain damage. Stimulator of interferon genes (STING) was recently described as a vital innate immune adaptor protein associated with neuroinflammation. Nevertheless, the regulatory effects of STING on microglial pyroptosis post-stroke have not been well elaborated.

Methods: STING-knockout and wild-type (WT) mice were subjected to middle cerebral artery occlusion (MCAO). STING small interfering RNA (siRNA) was transfected into BV2 cells before oxygen-glucose deprivation/reoxygenation (OGD/R). STING-overexpressing adeno-associated virus (AAV) and NOD-like receptor family pyrin domain containing 3 (NLRP3) siRNA were administered by stereotaxic injection. 2,3,5-Triphenyl tetrazolium chloride (TTC) staining, TdT-mediated dUTP nick end labeling (TUNEL) staining, Fluoro-Jade C (FJC) staining, neurobehavioural tests, immunohistochemistry, cytokine antibody array assay, transmission electron microscopy, immunoblot, Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) were carried out. Co-immunoprecipitation assays were used to investigate the interplay between STING and NLRP3.

Results: STING expression was increased after MCAO and mainly detected on microglia. STING deletion alleviated brain infarction, neuronal damage and neurobehavioural impairment in mice subjected to MCAO. STING knockout suppressed microglial activation and the secretion of inflammatory chemokines, accompanied by mitigation of microglial pyroptosis. Specific upregulation of microglial STING by AAV-F4/80-STING aggravated brain injury and microglial pyroptosis. Mechanistically, co-immunoprecipitation showed that STING bound to NLRP3 in microglia. Supplementation of NLRP3 siRNA reversed AAV-F4/80-STING-induced deterioration of microglial pyroptosis.

Conclusions: The current findings indicate that STING modulates NLRP3-mediated microglial pyroptosis following MCAO. STING may serve as a therapeutic target in neuroinflammation induced by cerebral ischaemic/reperfusion (I/R) injury.

Keywords: cerebral infarction; cerebrovascular disorders; inflammatory response; stroke.

Figure 1

STING expression was upregulated after ischaemic stroke. (A) Immunoblot assessment of p-STING and STING from peri-infarct tissues of mice with experimental MCAO after 1 day or 3 days reperfusion. (B) Protein levels of p-STING and STING in BV2 cells exposed to normal oxygen concentration for 12 and 24 hours after OGD. Data analysis used one-way ANOVA with post hoc Tukey’s test. (C) Representative micrograph demonstrating the peri-infarct area delineated by STING and Iba1 staining. Two random high-power fields in the CTX and STR of a given section were examined. Scale bar: 1 mm. (D) Representative immunohistochemical staining for microglia and STING in ischaemic penumbra tissue samples obtained at day 3 after MCAO. (E) Immunofluorescence of STING in cultured BV2 cells following OGD/R. Iba1 served as a microglial marker. Green, Iba1; red, STING; blue, DAPI. (F) Quantification of co-stained cells after focal ischaemia. (G) MFI of STING was quantitated with Image J. Scale bar: 50 µm; n=6/group. Student’s t-test. Data are mean±SD. *P<0.05, **P<0.01, ***P<0.001 vs WT Sham mice. ^# P<0.05, ^### P<0.001 vs Ctrl microglia. ANOVA, analysis of variance; CTX, cortex; MCAO, middle cerebral artery occlusion; MFI, mean fluorescence intensity; OGD/R, oxygen-glucose deprivation/reoxygenation; STING, stimulator of interferon genes; STR, striatum; WT, wild type.

Figure 2

STING suppression ameliorated brain infarction and neuronal damage following MCAO. (A,B) Immunoblot assessment of STING and quantitation. (C) Representative micrographs depicting TTC staining in various groups poststroke. (D) Quantitative assessment of brain infarct and oedema volume. (E–H) The densities of TUNEL-positive and FJC-positive cells were elevated in WT mice following I/R injury, while STING^-/- reduced these positive signals in the peri-infarct region. Insets showed images with elevated magnification. Scale bar: 100 µm; n=6/group. Data analysis used one-way ANOVA with post hoc Tukey’s test. Data are mean±SD. ***P<0.001 vs Sham mice. ^### P<0.001 vs WT MCAO mice. ANOVA, analysis of variance; FJC, Fluoro-Jade C; MCAO, middle cerebral artery occlusion; STING, stimulator of interferon genes; TTC, 2,3,5-triphenyl tetrazolium chloride; TUNEL, TdT-mediated dUTP nick end labeling; WT, wild type.

Figure 3

STING deletion improved long-term sensorimotor and cognitive deficits after cerebral ischaemia. (A) mNSS was assessed in male mice before and up to 21 days after injury. (B,C) Sensorimotor impairment was tested by the adhesive removal (B) and corner (C) tests 1–21 days after MCAO. n=10/group. Data analysis used two-way ANOVA with post hoc Tukey’s test. Cognitive function was examined by the NOR test on day 21 post-MCAO. (D) Representative tracks. Blue rectangle: novel object. Green circle: familiar object. (E) Recognition index. n=10/group. (F,G) Representative FJC-stained images and quantitation of FJC⁺ cells in CTX and hippocampal regions (CA1, CA3, and DG) 21 days after MCAO. Scale bar: 200 µm; n=6/group. Data analysis used one-way ANOVA with post hoc Tukey’s test. Data are mean±SD. *P<0.05, ***P<0.001 vs Sham animals. ^# P<0.05, ^## P<0.01, ^### P<0.001 vs WT MCAO group. ANOVA, analysis of variance; CTX, cortex; FJC, Fluoro-Jade C; MCAO, middle cerebral artery occlusion; mNSS, modified neurological severity score; NOR, novel object recognition; STING, stimulator of interferon genes; WT, wild type.

Figure 4

STING promoted post-stroke microglial and inflammatory activation in the peri-infarct area. (A) Altered morphology of microglia in the peri-infarct CTX and STR of WT and STING^-/- mice 3 days after MCAO. (B) Quantitation of various parameters (numbers of Iba1-positive cells per 0.1 mm², circularity, numbers of branches and average branch lengths) for microglia in each animal. (C,D) Immunostaining and quantification of CD68 in WT and STING^-/- mice. Scale bar: 50 µm; n=6/group. Data analysis used one-way ANOVA with post hoc Tukey’s test. (E) Representative blots of the cytokine antibody array in experimental groups. (F) Quantitative analysis showed significantly decreased amounts of inflammatory cytokines in the ischaemic brain of STING^-/- mice vs WT control mice 3 days after MCAO, including C5a, G-CSF, CXCL1, CCL2, CCL12, CCL3, IL-16 and CXCL2. n=4/group. Student’s t-test. Data are mean±SD. **P<0.01, ***P<0.001 vs Sham animals. ^# P<0.05, ^## P<0.01, ^### P<0.001 vs WT MCAO animals. ANOVA, analysis of variance; CTX, cortex; MCAO, middle cerebral artery occlusion; STING, stimulator of interferon genes; STR, striatum; WT, wild type.

Figure 5

STING knockout alleviated microglial pyroptosis against ischaemic insult. (A,B) Double immunostaining of GSDMD and Iba1 in the CTX and STR of peri-infarct regions and quantitative analysis 3 days following ischaemic insult. (C) Representative transmission electron micrographs of microglial cells in the peri-infarct area. Rightmost panel showed the inset of the left panel at higher magnification. The orange arrows showed pyroptosis pores on the plasma membrane. (D) Immunoblots showing GSDMD and GSDMD-N protein amounts in WT and STING^-/- mice with experimental stroke. (E) ELISA analysis of IL-1β and IL-18 levels in brain tissue specimens. (F,G) Immunofluorescence staining of GSDMD and Iba1 in the si-NC and si-STING groups and quantitation of signals; insets depict images at higher magnification. (H,I) Western blot analysis of GSDMD and GSDMD-N in BV2 microglia 24 hours post-reoxygenation. (J) Extracellular releases of IL-1β and IL-18 in OGD/R-induced BV2 microglia. Scale bar: 50 µm; n=6/group. Data analysis used one-way ANOVA with post hoc Tukey’s test. Data are mean±SD. ***P<0.001 vs Sham animals. ^### P<0.001 vs WT MCAO animals. ^&& P<0.01, ^&&& P<0.001 vs si-NC or si-STING-transfected Ctrl microglia. ^$ P<0.05, ^$$$ P<0.001 vs si-NC transfected OGD/R microglia. ANOVA, analysis of variance; CTX, cortex; GSDMD, gasdermin D; GSDMD-N, N-terminal domain of GSDMD; MCAO, middle cerebral artery occlusion; NC, negative control; OGD/R, oxygen-glucose deprivation/reoxygenation; STING, stimulator of interferon genes; STR, striatum; WT, wild type.

Figure 6

Specific STING upregulation in microglia aggravated infarct volume and microglial pyroptosis after ischaemic stroke. (A) Representative TTC staining indicating ischaemic infarct volume in WT or STING^-/- MCAO mice injected with AAV-NC or AAV-STING. (B,C) Quantification of infarct and oedema volume. (D) Quantitation of mNSS scores. Mice in the AAV-STING group had starkly higher scores. (E) Immunoblotting analysis of GSDMD and GSDMD-N in WT or STING^-/- MCAO mice receiving AAV, with quantification. (F) Representative images and quantification showing the numbers of GSDMD-positive microglia at the CTX and STR of the peri-lesional tissue. Scale bar: 50 µm. (G) IL-1β and IL-18 production in the ipsilateral CTX were determined with specific ELISA assay kits. n=6/group. Data analysis used one-way ANOVA with post hoc Tukey’s test. Data are mean±SD. *P<0.05, **P<0.01, ***P<0.001 vs AAV-NC infected MCAO animals. AAV, adeno-associated virus; ANOVA, analysis of variance; CTX, cortex; GSDMD, gasdermin D; GSDMD-N, N-terminal domain of GSDMD; MCAO, middle cerebral artery occlusion; mNSS, modified neurological severity score; NC, negative control; STING, stimulator of interferon genes; STR, striatum; WT, wild type.

Figure 7

NLRP3 was involved in STING-induced microglial pyroptosis on I/R injury. (A) Immunofluorescent staining of Iba1 (green), NLRP3 (red) and STING (blue) in the ischaemic penumbra of MCAO mice. White arrows indicate a co-localization of STING and NLRP3. (B) Lysates from brain tissues underwent immunoprecipitation with anti-STING antibody. Immunoprecipitates were assessed by immunoblot using antibodies targeting STING and NLRP3. (C) Representative micrographs depicting co-localization of NLRP3 (red) and STING (blue) examined immunohistochemically in microglia (Iba1, green) following OGD/R. (D) Microglia lysates from the control and OGD/R groups underwent immunoprecipitation with anti-STING antibody, followed by Western blot for STING and NLRP3 detection. (E,F) Double immunostaining of NLRP3 and Iba1 in the peri-infarct area and quantitation 72 hours following reperfusion. Insets depict images at higher magnification. (G) NLRP3, IL-1β and IL-18 mRNA levels in WT and STING^-/- mice after MCAO or non-injury control procedures. (H) Representative Western blot images and quantitation for NLRP3, ASC, pro-caspase-1, cleaved caspase-1, IL-1β, IL-18, p65 and p-p65 in vivo. (I) Immunoblots showing the protein expression amounts of NLRP3, ASC, pro-caspase-1, cleaved caspase-1, IL-1β, IL-18, p65 and p-p65 in BV2 microglia transfected with si-NC or si-STING. Scale bar: 50 µm. In B, D and G, n=4/group. In A, C, E, F, H and I, n=6/group. Data analysis used one-way ANOVA with post hoc Tukey’s test. Data are mean±SD. *P<0.05, ***P<0.001 vs Sham mice. ^# P<0.05, ^### P<0.001 vs WT MCAO mice. ANOVA, analysis of variance; I/R, ischaemic/reperfusion; MCAO, middle cerebral artery occlusion; NC, negative control; OGD/R, oxygen-glucose deprivation/reoxygenation; STING, stimulator of interferon genes; WT, wild type.

Figure 8

NLRP3 silencing abolished the detrimental effects of STING after MCAO attack. (A) Immunoblot and (B) quantification of STING, NLRP3, ASC, cleaved caspase-1, GSDMD, GSDMD-N, IL-1β, and IL-18 in WT MCAO mice infected with AAV-STING and si-NC or si-NLRP3. (C) Confocal micrographs of MCAO brain samples after double immunofluorescent staining for GSDMD and Iba1. (D) Quantified results for GSDMD expression were shown. (E–G) Representative micrographs depicting TTC staining and quantitation of infarct volume and brain oedema following MCAO. (H) mNSS of AAV-STING and si-NC or si-NLRP3 transfected WT MCAO mice. Scale bar: 50 µm; n=6/group. Student’s t-test. Data are mean±SD. **P<0.01, ***P<0.001 vs AAV-STING+si-NC WT MCAO mice. AAV, adeno-associated virus; GSDMD, gasdermin D; GSDMD-N, N-terminal domain of GSDMD; MCAO, middle cerebral artery occlusion; mNSS, modified neurological severity score; NC, negative control; ns, no significant difference; STING, stimulator of interferon genes; TTC, 2,3,5-triphenyl tetrazolium chloride; WT, wild type.