Inhibition of double‐strand DNA‐sensing cGAS ameliorates brain injury after ischemic stroke
2020; Springer Nature; Volume: 12; Issue: 4 Linguagem: Inglês
10.15252/emmm.201911002
ISSN1757-4684
AutoresQian Li, Yuze Cao, Chun Dang, Bin Han, Ranran Han, He‐Ping Ma, Junwei Hao, Lihua Wang,
Tópico(s)Inflammasome and immune disorders
ResumoArticle1 April 2020Open Access Inhibition of double-strand DNA-sensing cGAS ameliorates brain injury after ischemic stroke Qian Li Qian Li Department of Neurology, The Second Affiliated Hospital, Harbin Medical University, Harbin, China Search for more papers by this author Yuze Cao Yuze Cao Department of Neurology, The Second Affiliated Hospital, Harbin Medical University, Harbin, China Search for more papers by this author Chun Dang Chun Dang Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China Search for more papers by this author Bin Han Bin Han Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China Search for more papers by this author Ranran Han Ranran Han Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China Search for more papers by this author Heping Ma Heping Ma Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Junwei Hao Corresponding Author Junwei Hao [email protected] Department of Neurology, Xuanwu Hospital, Capital Medical University, Beijing, China Search for more papers by this author Lihua Wang Corresponding Author Lihua Wang [email protected] orcid.org/0000-0003-3946-4032 Department of Neurology, The Second Affiliated Hospital, Harbin Medical University, Harbin, China Search for more papers by this author Qian Li Qian Li Department of Neurology, The Second Affiliated Hospital, Harbin Medical University, Harbin, China Search for more papers by this author Yuze Cao Yuze Cao Department of Neurology, The Second Affiliated Hospital, Harbin Medical University, Harbin, China Search for more papers by this author Chun Dang Chun Dang Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China Search for more papers by this author Bin Han Bin Han Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China Search for more papers by this author Ranran Han Ranran Han Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China Search for more papers by this author Heping Ma Heping Ma Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Junwei Hao Corresponding Author Junwei Hao [email protected] Department of Neurology, Xuanwu Hospital, Capital Medical University, Beijing, China Search for more papers by this author Lihua Wang Corresponding Author Lihua Wang [email protected] orcid.org/0000-0003-3946-4032 Department of Neurology, The Second Affiliated Hospital, Harbin Medical University, Harbin, China Search for more papers by this author Author Information Qian Li1, Yuze Cao1, Chun Dang2, Bin Han2, Ranran Han2, Heping Ma3, Junwei Hao *,4 and Lihua Wang *,1 1Department of Neurology, The Second Affiliated Hospital, Harbin Medical University, Harbin, China 2Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China 3Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA 4Department of Neurology, Xuanwu Hospital, Capital Medical University, Beijing, China *Corresponding author. Tel: +86 1083 198707; E-mail: [email protected] *Corresponding author. Tel: +86 45186 297475; E-mail: [email protected] EMBO Mol Med (2020)12:e11002https://doi.org/10.15252/emmm.201911002 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cytosolic double-stranded DNA (dsDNA) is a danger signal that is tightly monitored and sensed by nucleic acid-sensing pattern recognition receptors. We study the inflammatory cascade on dsDNA recognition and investigate the neuroprotective effect of cyclic GMP-AMP (cGAMP) synthase (cGAS) antagonist A151 and its mechanisms of neuroprotection in a mouse model of experimental stroke. Here, we found that cerebral ischemia promoted the release of dsDNA into the cytosol, where it initiated inflammatory responses by activating the cGAS. A151 effectively reduced the expression of cGAS, absent in melanoma 2 (AIM2) inflammasome, and pyroptosis-related molecules, including caspase-1, gasdermin D, IL-1β, and IL-18. Furthermore, mice treated with A151 showed a dampened immune response to stroke, with reduced counts of neutrophils, microglia, and microglial production of IL-6 and TNF-α after MCAO. Moreover, A151 administration significantly reduced infarct volume, attenuated neurodeficits, and diminished cell death. Notably, the protective effect of A151 was blocked in a microglia-specific cGAS knockout mouse. These findings offer unique perspectives on stroke pathogenesis and indicate that inhibition of cGAS could attenuate brain inflammatory burden, representing a potential therapeutic opportunity for stroke. Synopsis Inflammation is involved in the progression of ischemic brain injury. This study focuses on the inflammatory cascade on double-strand DNA (dsDNA) recognition and highlights the possibility of inhibiting dsDNA-sensing cyclic GMP-AMP synthase (cGAS) for treatment of ischemic stroke. The release of dsDNA from necrotic tissue during brain infarction triggers an innate inflammatory cascade. A synthetic oligonucleotide A151 that antagonizes cGAS regulates the microglial immune response and pyroptosis after ischemic stroke. Inhibition of cGAS leads to a decline in neutrophil infiltration into the brain. Suppression of the dsDNA-sensing cGAS pathway reduces ischemic brain injury via mitigating neuroinflammation. The paper explained Problem Ischemic stroke is a highly disabling neurological disease worldwide. Inflammation plays a crucial role in ischemic stroke, which expands brain damage and exists over an extended period until days thus providing more therapeutic opportunities. However, a knowledge gap exists relating to the innate inflammatory cascade triggered by cytoplasmic DNA in the context of ischemic brain. Results In this study, we report that ischemic brain injury triggers cytosolic escape of dsDNA and activates the recently described cGAS (Cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) pathway. cGAS antagonist A151 abolished the cytosolic dsDNA-triggered inflammatory cascade of cGAS gene activation via modulating AIM2 inflammasome- and pyroptosis-associated proteins in mice subjected to transient middle cerebral artery occlusion. A151 effectively regulated microglia activation and decreased the infiltration of peripheral neutrophils into the injured brain following stroke, resulting in reduced infarct volume, and improved the long-term neurological outcome. Impact Collectively, these data highlight that cGAS inhibition is sufficient to govern the emergence of brain inflammation and attenuate innate sterile immune responses that promote ischemic brain injury, and identify potential new therapeutic targets for ischemic stroke. Introduction Ischemic stroke is a devastating neurological disease worldwide, with high global burden (Mukherjee & Patil, 2011). Although the only FDA-approved drug against acute ischemic stroke, recombinant tissue plasminogen activator (rtPA), has shown clinical efficiency, however, due to the narrow therapeutic time window, majority of patients miss the optimal opportunity for vascular recanalization (Wechsler, 2011; Fu et al, 2015). Brain damage induced by initial ischemia is most likely unsalvageable; however, inflammation exists over an extended period until days following ischemia, which may facilitate the secondary neural immunologic deterioration and expand cerebral infarction, thus offering many opportunities for treatment strategies (Iadecola & Anrather, 2011; Fu et al, 2015). After the ictus of brain ischemic attack, cytosolic double-stranded DNA (dsDNA) released by necrotic neuronal cells is a potential damage-associated molecular pattern (DAMP), the underlying mechanisms of brain inflammation on dsDNA recognition by nucleic acid-sensing cyclic GMP-AMP (cGAMP) synthase (cGAS) following stroke have not been explored to date. cGAS has currently been defined as a key cytosolic DNA sensor (Gao et al, 2013; Cai et al, 2014). Upon binding DNA directly, cGAS catalyzes the synthesis of cyclic dinucleotide 2′–3′- cGAMP, a second messenger, which in turn interacts with and activates the stimulator of interferon genes (STING) to induce the production of type I interferons via transcription factors interferon regulatory factor 3 (Chen et al, 2016; Xia et al, 2016) and of pro-inflammatory cytokines (e.g., IL-6 and TNF-a) via transcription factors nuclear factor κB (NF-κB) (Ablasser et al, 2013; Cai et al, 2014). Recently, it was shown that cytosolic DNA detected by the cGAS-STING axis could induce a cell death program and that was associated with inflammasome activation, thus targeting cGAS signaling is likely to be a logical approach of controlling inflammatory response triggered by cytoplasm dsDNA and ameliorating the related pathology (Gaidt et al, 2017). In addition, a series of studies have demonstrated that a protein complex called inflammasome absent in melanoma 2 (AIM2) plays a central role in various sterile self-DNA triggered inflammatory conditions, such as radiation-induced gastrointestinal syndrome and hematopoietic failure (Hu et al, 2016). AIM2 recognizes cytosolic dsDNA and directly binds it through its C-terminal HIN-200 domain, and then changes its conformation and triggers the "prion-like" polymerization of the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) into a filamentous helical structure, offering a multitude of activation sites for the inflammasome effector caspase-1. Consequently, caspase-1 is activated to drive the proteolytic cleavage and maturation of precursor cytokines, including pro-interleukin-1β (IL-1β) and pro-interleukin-18 (pro-IL-18), leading to the extracellular release of pro-inflammatory cytokines (Hornung et al, 2009). In addition to the post-translational assembly of inflammasome that is directly triggered by dsDNA, type I interferons stimulated by the cGAS-STING pathway have been shown to be capable of enhancing the AIM2 inflammasome response by promoting the expression of inflammasome platforms and substrates (Martinon et al, 2009; Lugrin & Martinon, 2018). Moreover, caspase-1 has been demonstrated to trigger a form of programmed cell death called pyroptosis (Broz & Dixit, 2016), which is inherently high inflammatory (Bergsbaken et al, 2009). Gasdermin D (GSDMD) has recently emerged as a major substrate of caspase-1 responsible for pyroptosis; of note, this inflammatory caspase-mediated program death differs from caspase-mediated apoptosis in that it causes localized cellular swelling and disrupts membrane integrity, thereby leading to the extravasation of cellular contents and inflammatory mediators into the extracellular milieu (Bergsbaken et al, 2009; Broz & Dixit, 2016; Aglietti & Dueber, 2017; Shi et al, 2017). We are optimistic that manipulating the innate DNA-sensing signaling may be an optimistic therapeutic intervention to improve the stroke outcomes. The synthetic oligodeoxynucleotide, A151, comprised of the immunosuppressive motif TTAGGG, has recently been shown to be capable to abrogate activation of cytosolic nucleic acid-sensing cGAS and AIM2 inflammasome by binding to these molecules in a manner that is competitive with immune-stimulatory DNA (Kaminski et al, 2013; Steinhagen et al, 2018). However, whether pharmacologically antagonizing dsDNA-sensing cGAS via A151 could govern pyroptosis and the overall neuroinflammation in the context of brain ischemia still remains poorly defined. Results Upregulation of double-strand DNA (dsDNA) and dsDNA sensors triggered by brain ischemia dsDNA is a potent DAMP in sterile inflammation following stroke. To detect the expression of this specific DAMP molecule in the ischemic brain, we examined dsDNA levels using immunofluorescent staining, although dsDNA staining of the sham brain was limited and distributed predominantly as ring-like nuclear contours; after the induction of MCAO, the intensities of dsDNA increased with a nucleoplasmic relocation in the penumbra; disintegration of the nucleus was also detected at time points of 6 and 24 h, as well as 3 days after MCAO (Fig 1A). Moreover, following stroke, the majority of dsDNA stainings appeared to be nuclear, with close spatial co-localization with DAPI and 53BP1, while cytoplasmic dsDNA could also be detected in the ischemic penumbra (Fig 1B). Since astrocytes and microglia are the main immune cells that quickly respond following ischemia and participate in the neuroinflammation, next, we performed double immunofluorescent analysis of dsDNA with cell type-specific markers for microglia (Iba1) and astrocytes (GFAP). Cytosolic dsDNA immunofluorescent signals could be detected in microglial Iba-1 and astrocytic GFAP (Fig 1B). Figure 1. Accumulation of dsDNA and upregulation of cGAS after brain ischemia. A. Representative single immunofluorescent staining for dsDNA at 6, 24 h, and 3 days of reperfusion after MCAO in the penumbra as well as in the sham brain, arrowheads indicate disintegration of the nucleus. Scale bars, 5 μm. B. Representative images of double immunofluorescent staining for 53BP-1 and dsDNA (upper panel), Iba-1 and dsDNA (middle panel), and for GFAP and dsDNA (lower panel), arrowheads indicate cytoplasmic dsDNA. Scale bars, 5 μm. C. Western blot analysis of cGAS and STING using lysates prepared from the indicated tissues of mouse brain following MCAO. D. Quantitative analysis for Western blot analysis. n = 6 mice per group. **P < 0.01, two-tailed unpaired Student's t-test. E. Immunohistochemical staining for cGAS at 3 days of reperfusion after tMCAO in peri-ischemic area as well as in the corresponding regions of sham control brains. Scale bars, 50, 20 μm in insets. F. Quantitative analysis of cGAS immunohistochemical staining. n = 6 mice per group. **P < 0.01, two-tailed unpaired Student's t-test. G. Representative double immunofluorescent stainings for cGAS (red) and 53BP1 (green). n = 6 mice per group. Scale bars, 50 μm. Data information: Data are presented as mean ± SEM. P-values are reported in Appendix Table S2. Download figure Download PowerPoint cGAS is a key cytosolic dsDNA sensor. In parallel with the heightened deposition of dsDNA during ischemia, we identified abundant expression of cGAS and STING in the ipsilateral hemisphere compared with the contralateral hemisphere on day 3 following stroke (Fig 1C and D). Immunohistochemical analysis also showed an elevation of cGAS in ischemic mouse brains, and the upregulated cGAS primarily located in the cytoplasm of cells (Fig 1E and F). Furthermore, cGAS was mainly expressed in 53BP1-positive cells, indicating that cGAS was induced where DNA double-strand breaks caused by ischemia (Fig 1G). Taken together, these data suggest that increased release of self-derived dsDNA within the cytosol may trigger the activation of cGAS-STING pathway during brain ischemia. Inhibition of cGAS signaling, AIM2 inflammasome, and pyroptosis by A151 after MCAO Given the striking changes in cGAS expression of MCAO mice, modulation of cGAS may be a logical experimental approach to confer protection from the effects of MCAO. The effect of A151 on cGAS signaling was examined in brain tissues of MCAO mice. Transcript levels in brain tissues were evaluated on day 3 after MCAO. qRT–PCR revealed that cGAS and STING were highly induced in the brain of vehicle-treated MCAO animals, whereas A151 treatment abrogated the induction of both cGAS and STING (Fig 2A). Western blot analysis further confirmed that the upregulation of cGAS, STING, along with the expression of downstream NF-κB, was significantly attenuated by the administration of A151 (Fig 2B and C). Figure 2. A151 inhibits MCAO-induced expression of cGAS, and AIM2 inflammasome- and pyroptosis-associated moleculesMice received daily intraperitoneal (IP) injections of A151 (300 μg) or an equal volume of vehicle for three consecutive days after MCAO induction. A. The mRNA expression levels of the cGAS and STING after MCAO were determined using qRT–PCR. n = 6 mice per group. **P < 0.01, one-way ANOVA followed by Tukey post hoc test. B. The expression levels of cGAS, STING, and NF-κB proteins in the brains of indicated groups were measured using Western blot analysis. C. Bar graph shows the relative protein expression levels of cGAS, STING, and NF-κB in the brains of indicated groups. n = 6 mice per group. *P < 0.05, **P < 0.01, one-way ANOVA followed by Tukey post hoc test. D. The mRNA expression levels of the AIM2 inflammasome components (AIM2, ASC, and caspase-1), effector of pyroptosis GSDMD, IL-1β, and IL-18 after MCAO were determined using qRT–PCR. n = 6 mice per group. *P < 0.05, **P < 0.01, one-way ANOVA followed by Tukey post hoc test. E. The expression of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups was measured using Western blot analysis. F. Bar graph shows the relative protein expression levels of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups. n = 6 mice per group. *P < 0.05, **P < 0.01, one-way ANOVA followed by Tukey post hoc test. Data information: Data are presented as mean ± SEM. P-values are reported in Appendix Table S2. Download figure Download PowerPoint AIM2 is another dominant player of sterile inflammation in response to cytoplasmic dsDNA. It is worth noting that in addition to directly triggering by dsDNA, AIM2 can also be activated and amplified by an initiate signal type I interferón that is stimulated by cGAS signaling (Martinon et al, 2009; Lugrin & Martinon, 2018). Moreover, another important target of inflammasome activation is to drive pyroptosis ("fiery death"), a type of caspase-1-dependent inflammatory programmed cell death (Lamkanfi & Dixit, 2014). However, it is currently unclear whether pyroptosis occurs within brain cells following stroke and whether A151 treatment could influence AIM2 activation and pyroptosis in ischemic brain. To clarify these issues, transcript levels in brain tissues were measured on day 3 after MCAO. qRT–PCR revealed that AIM2, ASC, caspase-1, GSDMD, IL-1β, and IL-18 were highly induced in the brains of vehicle-treated MCAO animals, whereas A151 treatment inhibited the induction of these genes (Fig 2D). We also observed that administration of A151 dampened the expression of AIM2 inflammasome-related molecules (AIM2/caspase-1/ASC) at early time point of 24 h after MCAO and that A151 reduced levels of IL-1β, and several downstream inflammatory cytokines/chemokines (IL-6, MCP-1) at early time points of 6 and 24 h (Appendix Fig S1), suggesting the anti-inflammatory effect of A151 treatment initiated after the onset of cerebral ischemia. Additionally, pronounced increase of protein levels of AIM2 inflammasome components (AIM2/ASC/caspase-1), IL-1β, and pyroptosis effector GSDMD was also increased in ischemic brain compared with sham-operated group, whereas markedly repressed in MCAO mice treated with A151 (Fig 2E and F). These results suggest that AIM2 inflammasome is activated and ischemic mouse brains undergo pyroptosis after stroke, leading to the processing of pro-inflammatory factors such as IL-1β and cell death, whereas A151 could effectively suppress AIM2 activation and pyroptosis. A151 prevents microglial pyroptosis after MCAO Pyroptosis, an inflammatory programmed cell death, was recently shown to be mediated by GSDMD, which could subsequently amplify the inflammation through the concomitant release of neurotoxic and inflammatory mediators. We then studied expression of related molecules using immunohistochemical staining. Immunohistochemistry analysis indicated that caspase-1, IL-1β, and GSDMD expression were significantly induced in MCAO+vehicle mice (Fig 3A and B). However, the levels of these molecules were reduced with A151 treatment (Fig 3A and B). Notably, cells displayed GSDMD immunoreactivity at the plasma membrane (Fig 3A, Inset), which is consistent with pyroptosis. Figure 3. A151 suppresses microglial pyroptosis after focal ischemic stroke A. Representative images of immunohistochemical staining of IL-1β, caspase-1, and GSDMD in the brains of indicated group. Scale bars, 50 μm. B. Quantitative analysis for immunohistochemical staining. n = 6 mice per group. **P < 0.01, one-way ANOVA followed by Tukey post hoc test. C. Representative images of double immunofluorescent staining for GFAP and GSDMD, for Iba-1 and GSDMD, and for NeuN and GSDMD in post-ischemic brains after tMCAO. Scale bars, 20 μm. D. Representative images of double immunofluorescent staining for Iba-1 and GSDMD, for Iba-1 and caspase-1, and for Iba-1 and IL-1β of sham+vehicle, MCAO+vehicle, and MCAO+A151 groups. Scale bars, 50 μm. E. Bar graph shows the relative immunofluorescence intensity of GSDMD, caspase-1, and IL-1β in the brains of indicated groups. n = 6 mice per group. **P < 0.01, one-way ANOVA followed by Tukey post hoc test. DAPI staining: nuclei. Data information: Data are presented as mean ± SEM. P-values are reported in Appendix Table S2. Download figure Download PowerPoint Furthermore, cell type-specific analysis on GSDMD expression profiles in the ischemic penumbra showed that GSDMD immunofluorescent signals were apparently colocalized with microglial Iba1, a few merged signals were observed in the NeuN presenting neurons, while GFAP-labeled astrocytes did not co-express GSDMD in the brain of MCAO mice (Fig 3C). These data indicate that microglia are the predominant cell subset in the central nervous system (CNS) that undergo pyroptosis in MCAO. In addition, as shown in Appendix Fig S2, we further evaluated the localization of GSDMD (red) compared to microglia marker Iba-1 (green) in the ischemic brain and found that the Iba-1 expression diminished with increasing GSDMD expression. Healthy microglia that are GSDMD− exhibited the highest Iba-1 expression. Additionally, immunofluorescent staining of GSDMD, caspase-1, and IL-1β with Iba-1 generated another line of evidence for enhanced microglial pyroptosis following stroke, whereas A151 treatment significantly suppressed such an increase (Fig 3D and E). These results suggest that A151 can prevent microglial pyroptosis in the context of ischemic conditions. A151 attenuates poly (dA:dT)-induced cGAS activation and caspase-1-induced pyroptosis in vitro To further investigate dsDNA-sensing cGAS activation and pyroptosis in microglia with a more dsDNA-relevant stimulus, comparable analyses were performed in vitro by transfection of primary microglia with poly (dA:dT), a synthetic analogue of dsDNA, in combination with A151. Consistent with the in vivo results, the expression levels of cGAS axis and pyroptosis-associated proteins (caspase-1, IL-1β, and GSDMD) were elevated in both lysates (Fig 4A and B) and supernatants (Fig 4C and D) of LPS-primed primary microglia stimulated with poly (dA:dT) compared with the blank control group, whereas the elevation of these proteins was suppressed by A151. MTS assay and LDH analysis were performed to clarify the microglial loss in vitro studies. Primary microglia were treated with A151 or PBS. MTS values were normalized to PBS-treated controls and showed no difference between the two groups (Appendix Fig S3A). Lactate dehydrogenase (LDH) activity was evaluated in cell culture supernatants, poly(dA:dT) exposure increased LDH release from microglia, while A151 treatment reduced its release (Appendix Fig S3B). Immunofluorescence analysis in BV2 cells further revealed the upregulation of cGAS, IL-1β, caspase-1, and GSDMD and nuclear translocation of NF-κB induced by LPS/poly(dA:dT) stimulation (Fig 4E and F), while this induction was attenuated by A151. These results suggest that pharmacological inhibition of dsDNA-sensing cGAS and AIM2 could ameliorate dsDNA-induced pyroptosis. Figure 4. A151 alleviates cGAS activity and expression of pyroptosis-associated proteins in vitro A. Expression of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 (FL), and GSDMD in primary microglia lysates was detected using Western blot. B. Quantitative analysis for Western blot analysis of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1, and GSDMD. n = 3 per group. P-values are reported in Appendix Table S3, one-way ANOVA followed by Tukey post hoc test. *P < 0.05, **P < 0.01 compared with the untreated group, #P < 0.05, ##P < 0.01 compared with the LPS+poly(dA:dT) group. C. The protein levels of caspase-1 p20 and IL-1β (p17) in the supernatant of cultured primary microglia were determined by Western blot. D. Quantitative analysis for Western blot analysis of caspase-1 and IL-1β. n = 3 per group. P-values are reported in Appendix Table S3, one-way ANOVA followed by Tukey post hoc test. **P < 0.01 compared with the untreated group, #P < 0.05 compared with the LPS+poly(dA:dT) group. E, F. Immunofluorescence was conducted to detect the expression levels of cGAS, NF-κB (arrows indicate NF-κB translocation), and pyroptosis-associated molecules (GSDMD, caspase-1, and IL-1β) in BV2 cells. n = 3 in each group. Scale bars, 50 μm. Data information: Data are presented as mean ± SEM. P-values are reported in Appendix Table S2. Download figure Download PowerPoint A151 suppresses neutrophil infiltration and production of microglia pro-inflammatory factors in MCAO Next, we sought to determine the impact of A151 on brain's post-ischemic inflammation is mediated by modulating the activation of local resident cells (microglia) or by recruitment of peripheral immune cells, or both. We examined such cellular components in the brains of MCAO mice (Fig 5A). We found that A151 significantly reduced neutrophils (CD45highCD11b+Ly6G+) infiltration in the brain on day 3 after MCAO compared with those in vehicle-treated controls (Fig 5B). In addition, A151 decreased neutrophil-attracting chemoattractants CCL2 and CXCL10 (Appendix Fig S4A), which may also have a direct effect on neutrophils since several cells expressing cGAS were also positive for ly6G of neutrophils marker (Appendix Fig S4B). Interestingly, the spleens of A151-treated mice exhibited increased numbers of neutrophils but no increase of any other leukocyte subtype (Fig 5C). Figure 5. A151 attenuates neutrophil infiltration and production of pro-inflammatory factors in microglia after tMCAO A. Gating strategy of brain-infiltrating immune cells including CD4+ T cells (CD3+ CD4+), CD8+ T cells (CD3+CD8+), B cells (CD3−CD19+), NK cells (CD3−NK1.1+), monocyte/macrophages (CD11b+CD45highF4/80+), neutrophils (CD11b+CD45highLy6G+), and microglia (CD11b+CD45int) and their expression of IL (interleukin)-6, TNF-α (tumor necrosis factor-α), TGF-β (transforming growth factor-β), and IL-4. FMO, fluorescence minus one. B. Quantitative analysis of the counts of brain-infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. **P < 0.01, two-tailed unpaired Student's t-test. C. Bar graph shows counts of leukocyte subtype in the spleens of MCAO mice receiving indicated treatment. n = 6 mice per group. **P < 0.01, two-tailed unpaired Student's t-test. D. Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. *P < 0.05, two-tailed unpaired Student's t-test. E. Bar graph shows percentages of microglia expressing IL-6, TNF-α, TGF-β, and IL-10 in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. *P < 0.05, **P < 0.01, two-tailed unpaired Student's t-test. F. Representative images of immunofluorescent staining for ly6G in peri-infarct areas of MCAO mice receiving indicated treatment. Scale bar, 50 μm. G. Bar graph shows the number of ly6G+ neutrophils. n = 6 mice per group. **P < 0.01, two-tailed unpaired Student's t-test. H. Representative images of immunofluorescent staining for Iba-1 in peri-infarct areas of MCAO mice receiving indicated treatment. Scale bar, 50 μm. I. Bar graph shows the number of Iba-1+ microglia. n = 6 mice per group. **P < 0.01, two-tailed unpaired Student's t-test. Data information: Data are presented as mean ± SEM. P-values are reported in Appendix Table S2. Download figure Download PowerPoint In addition, microglia (CD45int CD11b+) numbers decreased as did microglial expression of tumor necrosis factor-α (TNF-α) and factor interleukin-6 (IL-6). In contrast, the microglial expression of transforming growth factor-β (TGF-β) was increased in MCAO mice receiving A151 (Fig 5D and E). An increase in IL-4 expression was also noticed, while that was not statistically significant (Fig 5E). Moreover, macrophages also co-expressed cGAS in the penumbra of MCAO animals (Appendix Fig S5A), A151 also affected the production of IL-4 and TNF-α from blood-derived macrophages (Appendix Fig S5B and C), suggesting that A151 effectively inhibited the activation of microglia/macrophage in the brain after MCAO. Double immunofluorescence staining for microglial proliferation and apoptosis as revealed by Iba-1/Ki-67 and Iba-1/TUNEL was performed in the penumbra as wel
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