OIDs activates cGAS - STING -IRF3 pathway in BV2 cells
Because ischemia in MI activates the cGAS-STING pathway[10, 11], we hypothesized that OGD-induced substances can activate the cGAS-STING in microglia. Increasing evidence indicates that cGAS-STING pathway plays a control role in defense and responds to various microbial invasions and diseases[12-20]. cGAS is a cytosolic DNA sensor that can activate innate immune responses by synthesis of the second messenger cGAMP, which then activates the adaptor STING[21]. STING triggers downstream activation of interferon regulatory factor 3 (IRF3)[22] which regulates expression of type 1 interferon (IFN) and IFN-stimulated genes (ISG). It has been reported that microglia mediate neuroinflammation via this pathway.
Although cGAS-STING pathway plays an important role in various diseases, it relies on the dual stimuli of local hypoglycemic and hypoxic environments but its mechanism in post-IS is still unclear. We first established an OGD cell model to simulate the ischemic environment in vivo. Along with this, BV2 cells were cultured with supernatant produced by OGD to study the changes of cGAS-STING pathway. RT-PCR was used to check the mRNA expression level of cGAS, STING and p-IRF3. We found that OGD-induced DAMPs (OIDs) strongly increased the transient expression of cGAS, STING and p-IRF3 peaking at 24h after culture, thereafter the expression levels of these genes declined in varying degrees(fig. 1B). Interestingly, the morphological changes of BV2 microglia and mRNA expression of cGAS-STING-pIRF3 showed an amoebiform pattern (fig. 1A).
Using western blot assay, we estimated the expression changes of cGAS, STING and p-IRF3 proteins in BV2 cells. Compared with CM and Ctrl groups, the expressions level of STING, cCAS, p-IRF3 in OGD group was significantly higher than that in other three groups, on the other hand, and the expression of IRF3 did not change significantly (fig. 1C-D). Of note, the supernatants derived from the cell culture medium treated with glucose-free hypoxia and glucose-free hypoxia could not activate the expression of STING, cGAS and p-IRF3.
To further investigates whether mtDNA can directly activate cGAS-STING pathway, and then up-regulate the expression of related genes. To authenticate the role of mtDNA in the cGAS-STING signaling pathway, we utilized dideoxycytidine (ddC), a deoxyribonucleoside analog that specifically inhibits mtDNA replication and decreases mtDNA nucleoid size[23-25].Treatment of OGD with ddC resulted in a significant decrease in the expression of STING, cGAS and p-IRF3, and ddC significantly inhibited the activation of the cGAS-STING pathway (fig. 1C-D). Our results indicate that mtDNA contributes significantly to the activation of the cGAS-STING-pIRF3 signaling pathway, which may also indicate that mtDNA is an important DAMP for microglia in response to inflammatory responses.
OIDs triggers transformation of M1 and M2-like BV2 cells
To further analyze the effect of OIDs on microglia polarity, we used flow cytometry to separate different phenotypes of microglia. First, CD16/32 was stained, and then CD206 and ARG1 were co-stained. The results showed that the fluorescence signal of CD16/32 was significantly enhanced when stimulated by OIDs (fig. 2A). No significant changes were observed in the Ctrl and CM groups, neither distinct changes were observed in OGD-mtDNA treated group(OGD)(fig. 2A). CD16/32 is thought to be highly expressed in M1-like microglia, this indicated that the proportion of M1 cells in BV2 cells increased after the stimulates of OIDs.
Arg1-positive cells decreased significantly after stimulation with OIDs, and remained at a low level in the OGD (fig. 2B). The results indicated that Arg1 gene expression was decreased after OIDs stimulation, CD206 expression was not significantly affected by OIDs, it is well documented that CD206 is expressed more in M2 microglia, likewise Arg1 expression is more pronounced in M2 microglia than in M1. Overall, the signal of Arg1-CD206 co-stained cells decreased after OIDs treatment, moreover, the ratio of M1/M2 was markly increased. On the other hand, in OGD+ddC, RFS of M1/M2 was significantly decreased (fig. 2C). These results indicated that OID triggered and promoted phenotype transformation from M2 to M1 phenotype, ddC treatment reversed the transformation from M2 to M1 and confirming that mtDNA is involved in the process.
We next analyzed the expression of marker genes of M1 and M2 cells by ELISA. These factors include TNFa, iNOS, TGF-β and IL-10. TNFa and iNOS are proinflammatory mediators, while TGF-β and IL-10 are antiinflammatory cytokines and are regarded as markers related to tissue repair. The results showed that iNOS and TNFa expression was up-regulated in BV2 cells of OGD group, but there were no significant changes in TGF-β and IL-10(fig. 3). The expression of IL-10, iNOS, TGF-β and TNFa in OGD mtDNA group did not change significantly(fig. 3). It can be seen that TNFa and iNOS expression increased significantly with the stimulation of OIDs. This indicates that microglia-mediated inflammation is enhanced in cerebral ischemia.
cGAS-siRNA inhibited the cGAS-STING pathway and further promoted the polarization BV2 cells
cGAS gene is a key gene in the cGAS-STING pathway, but it can be used as a potential therapeutic target in neuroinflammation is still uncertain. We used siRNA technology to inhibit the expression of cGAS in BV2 cells, following which the expression changes of downstream cascade genes were analyzed. The results showed that the expression of STING and p-IRF3 genes was down-regulated by the decrease of cGAS expression(fig. 4A).
After that, flow cytometry was performed in the si-cGAS group and the Ctrl group. As described above, CD16/32 staining and CD206-Arg1 co-staining were performed. The results showed that CD16/32 signal was not altered significantly after si-cGAS treatment(fig. 4B). This may suggest that the cGAS-STING pathway is not directly related to the expression of CD16/32. In the CD206-Arg1 double-staining system, we found that the signals of Arg1 and CD206 were significantly enhanced after si-cGAS(fig. 4C), suggesting that after inhibiting the cGAS-STING-IRF3 signaling pathway, the frenquency of M2 cells was increased but that of M1 cells was decreased. Additionally, the ratio of M1/M2 was less than 1 after cGAS-siRNA treatment, indicating that the incidence of M2 BV2 cells was increased(fig. 4E).
The expression of IL-10 and TGF-β then followed which showed that it was significantly increased (p < 0.01, P < 0.05, Student's t test), however, the expression of TNFα was significantly decreased (p <0.05, Student's t test), and iNOS expression was not significantly decreased(fig. 4D). This suggests that following inhibit cGAS expression, the expression of inflammation-related genes decreased, while that IL-10 and TGF-β in activated microglia increased and indeed shifted for the repair type. This suggest that cGAS is potential target in treatment of IS. In other words, inhibition of cGAS expression may attenuate microglia-mediated inflammation and prevent neuronal damage.
Establishment of mouse ischemic stroke model
In vitro,we have confirmed our hypothesis that the cGAS-STING signaling pathway in activated BV2 microglia affects the expression of downstream inflammatory factors. It promotes the MI phenotype polarization, which may exacerbate inflammation. Moreover, we demonstrated that inhibiting cGAS expression can effectively reduce the expression level of inflammation-related genes. We then constructed a mouse MCAO model to further demonstrate the role of cGAS-STING pathway in vivo. At 3, 5 and 7 days after MCAO, brain section were prepared and processed for immunefluorescence. Images were captured and shown (fig. 5). In the control group, there was only modulate cGAS, STING and p-IRF3 immunefluorescence in activated microglia double labeled with iba1. In MCAO group at 3, 5 and 7 days, cGAS, STING and p-IRF3 immunefluorescence in iba1 labeled microglia was noticeably augmented(fig. 5).
Longa neurological score(Table1) showed that scores of neurological MCAO model at 1 day and 3 day were 2.78 ±0.67 and 1.89 ± 0.6, respectively. This shows that the MCAO model we constructed is reliable for further assessment.
Table 1 Neurological scores of MCAO mice. Longa scores were used to evaluate the neurological function. The scoring criteria were as follows: 0 points: no neurological deficits; 1 point: left forepaw limb can not fully extend; 2 points: circle to the left; 3 points: tilt to the left when walking; 4 points: unable to walk on its own, conscious loss; 5 points: death. Score > 1 may be used as a criterion for the successful construction of MCAO model.
|
1d
|
3d
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Sham
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0.00 ± 0.00
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0.00 ± 0.00
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Ctrl-siRNA
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2.78 ± 0.67
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1.89 ± 0.60
|
cGAS-siRNA
|
2.44 ± 0.53
|
1.56 ± 0.53
|
si-cGAS inhibited MIDs-induced apoptosis
OIDs can cause atrophy of microglia cells in vitro, and the number of BV2 cells is reduced. To verify the effects of MCAO-induced DAMPs (MIDs) on mice neurons, we performed frozen sections derived form normal mice and MCAO mice as well as injected ctrl-siRNA and cGAS-siRNA lentivirus respcetively,were processed for Tunel labeling. TUNEL labeled cells were absent in normal mice brain tissue,but in ctrl-MCAO at 3 days, a large number of TUNEL positive cells were observed.(fig. 6A)
In mice transfected with cGAS-siRNA lentivirus, the signal of apoptosis was significantly weaker in comparison with the ctrl-MCAO mice, indicating that si-cGAS inhibited MIDs-induced apoptosis (fig. 6A). In Nissl stained section in the ctrl-MCAO, many neurons appears atrophic,also the number of neurons was greatly reduced. In cGAS-siRNA lentivirus-transfected mice brain tissue (fig. 6B). the neurons appears normal in external morphology. In summary, the ctrl-MCAO group showed decreased in neural population,however, after inhibiting the expression of cGAS, the number of neurons was increased significantly (fig. 6C).
si-cGAS reduced MIDs-activated gene expression
To further explore the molecular mechanism of MIDs-induced apoptosis, we performed immunofluorescence experiments on three groups of brain tissue sections mentioned above. cGAS, STING and p-IRF3 gene immunofluorescence was located separated in area containing many iba1 positive cells. cGAS, STING, and p-IRF3 signals were absent in the control group, but intense cGAS, STING, and p-IRF3 immunofluorescence signals were detected in area rich in iba1 positive cells in the ctrl-MCAO group (fig. 7A). Such an expression pattern immunofluorescence induced by MIDs was significantly inhibited by cGAS-siRNA, .WB and ELISA assays confirmed the expression of cGAS, STING, and p-IRF3 as detected by immunofluorescence(fig. 7B,C).
si-cGAS suppressed the expression of pro-inflammatory factors in MCAO models
Recent studies have provided mechanistic insights into how DNA damage activated a cytosolic DNA sensing pathway(cGAS-STING) and thus result in inducing type I IFNs and other immune-regulatory cytokines[26-30]. To further analyze the effects of the cGAS-STING pathway in vivo, we collected mouse microglia at different stages of treatment. Our results indicate that the expression levels of pro-inflammatory mediators such as iNOS, TNFα, etc were effectively reduced by cGAS-siRNA(fig. 8C-D), whereas Arg1 levels were elevated under cGAS-siRNA stimulation (fig. 8B). Flow cytometry sorting showed that the CD16/32-labeled cell signal was attenuated, whereas that of the Arg1/CD206 co-labeled signal was enhanced. This indicates a decrease in the M1/M2 ratio and a shift of microglia to the repair phenotype (fig. 8E).