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. 2022 Apr 5:13:860977.
doi: 10.3389/fimmu.2022.860977. eCollection 2022.

mtDNA-STING Axis Mediates Microglial Polarization via IRF3/NF-κB Signaling After Ischemic Stroke

Lingqi Kong  1 , Wenyu Li  1 , E Chang  2 , Wuxuan Wang  1 , Nan Shen  1 , Xiang Xu  1 , Xinyue Wang  1 , Yan Zhang  1 , Wen Sun  1 , Wei Hu  1 , Pengfei Xu  1 , Xinfeng Liu  1
Affiliations

mtDNA-STING Axis Mediates Microglial Polarization via IRF3/NF-κB Signaling After Ischemic Stroke

Lingqi Kong et al. Front Immunol. .

Abstract

Neuroinflammation is initiated in response to ischemic stroke, and is usually characterized by microglial activation and polarization. Stimulator of interferon genes (STING) has been shown to play a critical role in anti-tumor immunity and inflammatory diseases. Nevertheless, the effect and underlying mechanisms of STING on microglial polarization after ischemic stroke remain unclarified. In this study, acute ischemic stroke was simulated using a model of middle cerebral artery occlusion (MCAO) at adult male C57BL/6 mice in vivo and the BV2 microglia oxygen-glucose deprivation/reperfusion (OGD/R) model in vitro. The specific STING inhibitor C-176 was administered intraperitoneally at 30min after MCAO. We found that the expression of microglial STING was increased following MCAO and OGD/R. Pharmacologic inhibition of STING with C-176 reduced the ischemia/reperfusion (I/R)-induced brain infarction, edema and neuronal injury. Moreover, blockade of STING improved neurological performance and cognitive function and attenuated neuronal degeneration in the hippocampus after MCAO. Mechanistically, both in vivo and in vitro, we delineated that STING could promote the polarization of microglia towards the M1 phenotype and restrain M2 microglia polarization via downstream pathways, including interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB). In addition, mitochondrial DNA (mtDNA), which is released to microglial cytoplasm induced by I/R injury, could facilitate microglia towards M1 modality through STING signaling pathway. Treatment with C-176 abolished the detrimental effects of mtDNA on stroke outcomes. Taken together, these findings suggest that STING, activated by mtDNA, could polarize microglia to the M1 phenotype following MCAO. Inhibition of STING may serve as a potential therapeutic strategy to mitigate neuroinflammation after ischemic stroke.

Keywords: STING; ischemia/reperfusion (I/R) injury; microglia; neuroinflammation; polarization.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Temporal expression and cellular localization of STING following ischemic stroke. (A) Western blot and quantitative analysis of cGAS, p-STING and STING at 6 h, 1d, 3d, and 7d after MCAO. n = 6. (B) Western blotting showing cGAS, p-STING and STING expression at 12 h, 24 h, 48h after reoxygenation. n = 4. (C, D) STING/Iba1 double immunostaining in Sham and MCAO mice 1d after reperfusion and in Control and OGD/R BV2 microglia 24h after reoxygenation. STING signal was increased after I/R injury both in vivo and in vitro. n = 4. Data are expressed as mean ± SD. ** P < 0.01, *** P < 0.001 vs Sham group. § P < 0.05, §§§ P < 0.001 vs Ctrl group. Scale bar = 20 μm.
Figure 2
Figure 2
C-176 treatment rescued infarct volume, brain edema, and neuronal injury. (A) Representative images for TTC staining in the indicated groups.(B, C) Quantitative analysis of infarct volume and brain edema in treated mice 1d post-modeling. (D, E) Representative microphotographs and quantitative analysis of NeuN/TUNEL co-staining. The dotted lines designate infarct borderlines. (F, G) FJC staining showed that the density of FJC-positive cells was increased in the peri-infarct area, while C-176 treatment reversed this trend. Insets show a higher magnification view. n = 6. Data are expressed as mean ± SD. *** P < 0.001 vs Sham group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs MCAO+vehicle group. n. s., no significant difference. Scale bar = 50 μm.
Figure 3
Figure 3
Inhibition of STING improved neurological performance and cognitive function after MCAO. (A, B) The mNSS and Corner test were assessed at day 1, 3, 7, 14, and 21 after MCAO. Mice were tested before MCAO surgery. MCAO 1-7 d: n = 15; MCAO 14-21 d: n = 10. Long-term cognitive functions were assessed by the Morris water maze. (C, D) The escape latency and swim path length were recorded at days 22-28 after MCAO. n = 12. (E) Representative swimming trajectories of the three groups in probe trials. (F, G) The crossovers of the platform location and the percentage of time spent in the probe quadrant. n = 12. (H, I) FJC staining was performed to assess the neuronal degeneration in hippocampus at day 28 after MCAO. n = 5. Data are expressed as mean ± SD. *** P < 0.001 vs Sham group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs MCAO+vehicle group. Scale bar = 100 μm.
Figure 4
Figure 4
The STING inhibitor C-176 promoted M2 polarization after MCAO. (A) Immunoblotting analysis of cGAS, p-STING, STING, p-p65, p65, p-IRF3 and IRF3 in the peri-infract tissue of Sham, MCAO+vehicle and MCAO+C-176 mice 1d post-MCAO. n = 4. (B) Immunoblotting images and quantitative analysis of cGAS, p-STING, STING, p-p65, p65, p-IRF3 and IRF3 in BV2 microglia of Ctrl, OGD/R+vehicle and OGD/R+C-176 group 24h after reoxygenation. n = 4. (C, D) Western blot and quantification analysis for iNOS and Arginase-1 in treated mice 3d post-MCAO and BV2 microglia 24h post-reoxygenation. n = 4. (E, F) mRNA expression of pro-inflammatory genes (TNF-α, iNOS, IL-1β and IL-6) and anti-inflammatory genes (IL-10 and Arg-1) were measured by real-time PCR 3d after MCAO and 24h after OGD/R. n = 4. (G) Representative confocal images of two microglia phenotypes were obtained from the peri-infarct cortex. (H) Quantification of CD16/32 positive M1 microglia and CD206 positive M2 microglia in the treated mice. n = 6. (I, J) Representative immunostained images and statistical analysis of M1 state (CD16/32+/Iba1+) and M2 state (CD206+/Iba1+) BV2 microglia. n = 4. Data are expressed as mean ± SD. ** P < 0.01, *** P < 0.001 vs Sham group; ## P < 0.01, ### P < 0.001 vs MCAO+vehicle group. §§ P < 0.01, §§§ P < 0.001 vs Ctrl group; † P < 0.05, †† P < 0.01, ††† P < 0.001 vs OGD/R+vehicle group. n. s., no significant difference. Scale bar = 20 μm.
Figure 5
Figure 5
STING knockdown ameliorated brain infarction and edema, promoted neurological recovery and rescued the expression of anti-inflammatory genes. (A) si-STING (red) co-stained with Iba1 (green) 1d post-MCAO in mice receiving Cy5-conjugated si-STING. (B, C) Immunoblots and quantitative analysis of STING-related proteins, including cGAS, p-STING, STING, p-p65, p65, p-IRF3 and IRF3 of Sham, MCAO+vehicle, MCAO+si-NC and MCAO+si-STING mice 1d after MCAO. n = 4. (D-F) Representative images of TTC staining and quantitative analysis of infarct volume and brain edema 1d post-MCAO. n = 6. (G) mNSS score of the indicated groups at day 1 and day 3 after MCAO. n = 8. (H, I) Relative mRNA expression of M1 microglia-specific transcripts (TNFα, iNOS, IL-1β and IL-6) and M2 microglia-specific transcripts (IL-10 and Arg-1). n = 4. Data are expressed as mean ± SD. ** P < 0.01, *** P < 0.001 vs Sham group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs MCAO+si-NC group. Scale bar = 20 μm.
Figure 6
Figure 6
Blockade of STING suppressed microglial mtDNA leakage after MCAO. (A, B) Representative confocal, 3D-reconstructed images of dsDNA+/HSP60+/Iba1+ microglia and quantification of microglial mtDNA 1d post-MCAO. Images were reconstructed from confocal images using Imaris software. n = 5. (C) Relative mRNA expression of mtDNA-specific genes (UUR, COXI, ND1 and COX3) 1d after MCAO. n = 4. (D, E) BV2 microglia cells were stained with anti-dsDNA and anti-HSP60 to detect mtDNA at 24h after reoxygenation. Representative dsDNA/HSP60 double immunostaining images and quantification of mtDNA. n = 4. Data are expressed as means ± SD. *** P < 0.001 vs Sham group; ## P < 0.01, ### P < 0.001 vs MCAO+vehicle. §§§ P < 0.001 vs Ctrl group; ††† P < 0.001 vs OGD/R+vehicle group. Scale bar = 10 μm.
Figure 7
Figure 7
mtDNA induced microglial M1 polarization by activating STING signaling pathway. (A, B) Western blot and quantification analysis of cGAS, p-STING, STING, p-p65, p65, p-IRF3 and IRF3 in Sham-, MCAO+vehicle-, MCAO+mtDNA-, and MCAO+mtDNA+C-176-treated mice 1d after reperfusion. n = 4. (C-E) Representative TTC staining images and quantification of infarction volume and brain edema 1d post-MCAO. n = 6. (F) mNSS score was assessed at day 1, 3, 7, 14 and 21 after MCAO. MCAO 1-7 d: n = 10; MCAO 14-21 d: n = 8. (G, H) TUNEL (red) and NeuN (green) double immunostaining with quantitative analysis. Dotted line designates the infarct borderline. n = 6. (I–L) Representative confocal images and quantification of M1 modality microglia (CD16/32+/Iba1+) and M2 modality microglia (CD206+/Iba1+) in the indicated groups. n = 6. Scale bar = 20 μm. Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 vs Sham group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs MCAO+vehicle group; $$ P < 0.01, $$$ P < 0.001 vs MCAO+mtDNA group. n. s., no significant difference.
Figure 8
Figure 8
Proposed mechanism of STING-mediated microglial polarization following ischemic stroke. I/R injury induces the leakage of mtDNA to cytoplasm and the activation of STING in microglia. Blockade of STING shifts the microglia toward M2 modality through IFR3 and NF-κB, thereby rescuing neuroinflammation and stroke outcomes. mtDNA can promote microglial polarization to the M1 phenotype via activating STING signaling pathway.
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