Argon mitigates post-stroke neuroinflammation by regulating M1/M2 polarization and inhibiting NF-κB/NLRP3 inflammasome signaling

ABSTRACT

Neuroinflammation plays a vital role in cerebral ischemic stroke (IS). In the acute phase of IS, microglia are activated toward the pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Argon, an inert gas, can reduce neuroinflammation and alleviate ischemia/reperfusion (I/R) injury. However, whether argon regulates M1/M2 polarization to protect against I/R injury as well as the underlying mechanism has not been reported. In this study, we analyzed the activation and polarization of microglia after I/R injury with or without argon administration and explored the effects of argon on NLRP3 inflammasome-mediated inflammation in microglia in vitro and in vivo. The results showed that argon application inhibited the activation of M1 microglia/macrophage in the ischemic penumbra and the expression of proteins related to NLRP3 inflammasome and pyroptosis in microglia. Argon administration also inhibited the expression and processing of IL-1β, a primary pro-inflammatory cytokine. Thus, argon alleviates I/R injury by inhibiting pro-inflammatory reactions via suppressing microglial polarization toward M1 phenotype and inhibiting the NF-κB/NLRP3 inflammasome signaling pathway. More importantly, we showed that argon worked better than the specific NLRP3 inflammasome inhibitor MCC950 in suppressing neuroinflammation and protecting against cerebral I/R injury, suggesting the therapeutic potential of argon in neuroinflammation-related neurodegeneration diseases as a potent gas inhibitor of the NLRP3 inflammasome signaling pathway.

Introduction

Stroke is the second leading cause of death and disability worldwide with >13 million new cases annually, and the incidence in the younger age groups (<50 years) keeps rising (Campbell et al., 2019; Lindsay et al., 2019), which has created a severe social and economic burden. Stroke can be either ischemic (caused by the interruption of the cerebral blood flow by occlusion of cerebral arteries mainly due to thromboembolism) or hemorrhagic (caused by rupture of cerebral vessels). Ischemic stroke is the common type and is usually treated by thrombolysis therapy or mechanical thrombectomy. However, the time window of the therapy is very limited (Campbell et al., 2019), and new treatments are urgently required.

Inflammation is recognized as a critical contributor to the pathophysiology of ischemic stroke (Moskowitz et al., 2010; Ma et al., 2017). The activation of microglia, the resident macrophages in the brain responsible for maintaining homeostasis in the central nervous system (CNS), is the initiation of inflammatory response in the CNS (Ma et al., 2017; Qin et al., 2019). Like macrophages, activated microglia can be polarized to be the pro-inflammatory M1 phenotype or the anti-inflammatory M2 phenotype and produce cytokines and chemokines, which are closely associated with the secondary brain damage or repair, respectively (Qin et al., 2019; Jiang et al., 2020). Therefore, regulating the polarization of macrophage/microglia would be beneficial for repairing brain damage following ischemic stroke (Dong et al., 2021).

Activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome is reported to mediate inflammation after stroke (Alishahi et al., 2019; Feng et al., 2020). NLRP3 inflammasome consists of NLRP3 (sensor), ASC (adaptor), and caspase-1 (effector). Usually, its activation is a two-step process: the first is the ‘priming’ stage, in which the expression of the inflammasome components including NLRP3, caspase-1, and pro-IL-1β is upregulated, and NLRP3 is stabilized through posttranslational modifications. This step is induced through the recognition of various pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by pattern-recognition receptors and the activation of NF-κB. Following the priming stage, when an NLRP3 activator-like stimulation, which occurs during tissue injury, infections, or metabolic imbalances, is recognized, inflammasome assembly and full activation start. Pro-caspase-1 cleaves itself and becomes active caspase-1, resulting in the subsequence processing of pro-IL-1β and pro-IL-18 into active IL-1β and IL-18, two major pro-inflammatory factors. In addition, active caspase-1 cleaves gasdermin D (GSDMD); N-terminus of GSDMD then binds to phosphatidylinositol phosphates and phosphatidylserine in the inner cell membrane, oligomerizes and inserts into the plasma membrane to form 10–14 nm pore, and leads to pyroptosis, a lytic form of cell death (Li et al., 2022) to release DAMPs to trigger further inflammatory action (Swanson et al., 2019; Feng et al., 2020). Pyroptosis also occurs during primary bacterial infection (Li et al., 2022). NLRP3 inflammasome is mainly activated in microglia after cerebral ischemia/reperfusion (I/R) injury onset in mouse transient middle cerebral artery occlusion (tMCAO) model (Gong et al., 2018), and NLRP3 inflammasome inhibitors are potential therapeutics of ischemic stroke (Alishahi et al., 2019; Swanson et al., 2019; Feng et al., 2020).

Argon is an inert gas that shows a favorable neuroprotective effect in a bulk of in vivo and in vitro models (Loetscher et al., 2009; Ulbrich and Goebel, 2016; Li et al., 2018; Liu et al., 2019; Ma et al., 2019). It is more abundant and cheaper than xenon, another noble gas that has been considered the gold standard in gas pharmacology but suffers from its rare availability and high price. Besides, argon lacks anesthesia effects under normobaric pressure and can easily diffuse into deeper brain compartments due to its non-polar characteristic, and no unwanted or even detrimental side effects have been found so far (Ulbrich and Goebel, 2016). Therefore, argon draws more and more attention as a promising new treatment for stroke. It is reported that argon exposure attenuates neuroinflammation and injury whereas enhances M2 microglia/macrophage polarization in rat tMCAO model (Liu et al., 2019, 2022). However, the effect of argon on M1 microglia/macrophage and NLRP3 inflammasome has not been reported, and the mechanism underlying the neuroprotective properties of argon is not fully understood yet.

Our previous study demonstrated that timely argon administration during ischemia improves neurological outcomes and decreases infarct volumes in the tMCAO mouse model (He et al., 2022), but the underlying mechanism is not clear. In this study, we tested the hypothesis that argon plays a neuroprotective role through mitigating neuroinflammation by regulating microglia/macrophage M1/M2 polarization and inhitibing NLRP3 inflammasome signaling pathway in microglia in the tMCAO mouse model and primary cultured microglia.

Results

Argon administration suppresses the activation of microglia/macrophage and regulates M1/M2 polarization in the ischemic penumbra during acute phase after stroke

To investigate the effect of argon on neuronal damage and neuroinflammation, we visualized the infarct area with staining for microtubule-associated protein 2 (MAP2), a marker of neuron, and the activated microglia/macrophage in penumbra with staining for Iba1, a marker of microglial cells and subpopulations of the monocyte/macrophage lineage, at 24 h after reperfusion. As shown in Figure 1A, the infarct area identified by the MAP2-negative staining region was significantly increased in striatum and cortex at 24 h after reperfusion, while argon treatment markedly increased the MAP2-positive neurons in both brain areas (Figure 1C). Further analysis revealed that argon reduced the number of Iba1-positive cells compared with that in the I/R group (Figure 1A and D). Moreover, Iba1-positive cells in I/R mice with argon treatment were more ramified and had longer processes compared to the I/R group (Figure 1A and B). Quantification of Iba1 ramification and circularity index in the penumbra of striatum and cortex showed significant changes in both indexes at 24 h post-reperfusion following argon administration (Figure 1E and F). These results reflect that argon administration inhibits the proliferation and activation of microglial cells or monocyte/macrophage in the ischemic penumbra 24 h after I/R, which may be responsible for attenuating the reperfusion injury.

Figure 1.

Argon administration attenuates the infarct size, suppresses the activation of microglia/macrophage, and regulates M1/M2 polarization in the ischemic penumbra during the acute phase after stroke. (A) Representative images of coronal brain sections at bregma 0.5 mm at 24 h after reperfusion. Left panels: MAP2 (neuron marker, red) immunostaining. Scale bar, 2.5 mm. Middle panels: Iba1 (microglia/macrophage marker, green) and DAPI (nuclear, blue) staining of the white box-marked region in the corresponding left panel. Scale bar, 100 μm. Right panels: the zoom-in picture of the white box-marked region in the corresponding middle panel. Scale bar, 10 μm. (B) Illustration of cell ramification and circularity analyses of representative microglia/macrophage of each group as indicated. (C) Quantification of brain infarct size as MAP2-negative area (n = 5–10). (D) Quantification of Iba1-positive microglia/macrophage in the ischemic penumbra of each group as indicated (n = 3). (E and F) Quantitative analysis of ramification (E) and circularity (F) of microglia/macrophage in the indicated groups (46–66 cells per group from 3 mice). (G–J) Relative mRNA levels of the M1 marker genes Fcgr3 (G) and Nos2 (H) and the M2 marker genes Arg1 (I) and Tgfb1 (J) in mouse brain tissues with indicated treatments at 12 h, 24 h, and 72 h post-reperfusion detected by qPCR (n = 3–4). Actb (β-actin)-normalized mRNA levels in the sham group were used to set the baseline value at unity. (K) Protein levels of CD16 (M1 marker) and CD206 (M2 marker) in brain tissues of mice with indicated treatments at 24 h after reperfusion detected by western blotting. β-actin was used as the endogenous control. (L and M) Quantification of CD16 (L) and CD206 (M) protein levels (n = 6). (N) Representative immunostaining images of coronal brain sections at bregma 0.5 mm for CD16 (M1 marker, red), CX3CR1-GFP (microglia/macrophage, green), and DAPI (nuclear, blue) in the ipsilateral striatum at 24 h after reperfusion as indicated. PC represents Pearson's correlation coefficient value to show the signal colocalization status. Scale bar, 100 μm (left panels) and 10 μm (all the zoom-in panels). (O and P) Quantification of CD16-positive cells (O) and CX3CR1-GFP-positive (GFP⁺) cells (P) in the ipsilateral striatum at 24 h after reperfusion (n = 3). (Q and R) Percentage of CD16⁺GFP⁺ cells in GFP⁺ cells (Q) and CD206⁺GFP⁺ in GFP⁺ cells (R) in the indicated groups (n = 3). Data are mean ± SEM. *P < 0.05, **P < 0.01. Ar, argon.

In order to verify the effect of argon on M1/M2 polarization dynamics in the first three days after reperfusion, real-time quantitative polymerase chain reaction (qPCR) of M1 and M2 typical gene markers were performed. As shown in Figure 1G–J, temporal analysis of microglial phenotypes demonstrated that with argon treatment, M1-like (pro-inflammatory type) microglia decreased acutely as early as 12 h after I/R, whereas M2-like (alternative protective type or anti-inflammatory type) response increased from 12 h to Day 3, suggesting that argon shifted the microglia/macrophage polarization from M1-like to M2-like during the acute phase. Furthermore, the protein levels of M1 type (CD16) and M2 type (CD206) microglia/macrophage markers were examined at 24 h after I/R injury. As shown in Figure 1K–M, argon administration significantly reduced the expression level of CD16 after stroke but enhanced that of CD206.

It is known that some of the M1 and M2 signature genes and proteins are expressed not only in microglia/macrophage but also in other nervous system cells or infiltrating immune cells. To further distinguish the effect of argon on the microglial/macrophage-mediated neuroinflammation, CX3CR1^GFP/+ transgenic mice (knock-in of GFP into the CX3CR1 gene locus to label microglia/macrophage under heterozygous condition) were used to determine the effect of argon on microglia/macrophage polarization in the ischemic penumbra. First, the quantity and morphology of CX3CR1-GFP-positive cells in the I/R group were significantly different from that of the sham group; however, when treated with argon, both of them were similar to the sham group (Supplementary Figure S1A). To further identify the phenotype of microglia/macrophage, immunostaining of CD16 or CD206 was conducted. In contrast to the I/R group, the number of CD16⁺, GFP⁺, and CD16⁺GFP⁺ cells with argon administration decreased in the penumbra at 24 h after reperfusion. Cell morphology showed that CD16⁺GFP⁺ cells with argon treatment were more ramified, similar to the sham group (Figure 1N–Q). In contrast, immunofluorescence for CD206 was increased significantly over sham levels at 24 h after reperfusion (Figure 1R; Supplementary Figure S1B–D). The colocalization of CD16 and CD206 with CX3CR1-GFP was shown in Supplementary Figure S2A and B. Taken together, argon administration shifted microglia from M1 toward M2 polarization after I/R injury.

NLRP3 inflammasome participates in argon-mediated microglia/macrophage polarization in vivo and in vitro

To evaluate the effect of argon treatment on neuroinflammation, we performed qPCR to test the mRNA levels of certain cytokines, including Tnf (encoding tumor necrosis factor) and Il1b (encoding interleukin 1 beta) (Figure 2A and B). Argon obviously suppressed these inflammation markers induced by I/R damage at 24 h post-reperfusion (all P < 0.01).

Figure 2.

NLRP3 inflammasome participates in argon-mediated microglia/macrophage polarization in vivo. (A and B) Relative mRNA levels of the pro-inflammation factor genes Tnf (A) and Il1b (B) in brain tissue of mice with indicated treatments at 24 h post-reperfusion detected by qPCR (n = 4–6). Actb (β-actin)-normalized mRNA levels in the sham group were used to set the baseline value at unity. (C) Protein levels of pro-IL-1β, cl-IL-1β, NLRP3, and ASC in mouse brain tissues with indicated treatments at 24 h after reperfusion detected by western blotting. β-actin was used as the endogenous control. (D–G) Quantification of pro-IL-1β (D), cl-IL-1β (E), NLRP3 (F), and ASC (G) protein level (n = 5–9). (H) Representative images of coronal brain sections at bregma 0.5 mm for CX3CR1-GFP (microglia/macrophage, green), IL-1β immunostaining (inflammatory factors, red), ASC immunostaining (inflammasome component, violet), and DAPI (nuclear, blue) in the ipsilateral striatum at 24 h after reperfusion as indicated. Scale bar, 100 μm. (I–K) Quantification of fluorescence density of IL-1β immunostaining (I), the number of ASC-positive cells (J), and the percentage of inflammasome-activated microglia represented by ASC⁺/IL-1β⁺/GFP⁺ cells (K) in brain sections as indicated. (L) Representative images of coronal brain sections at bregma 0.5 mm for CX3CR1-GFP (microglia, green), NLRP3 immunostaining (red), and DAPI (nuclear, blue) in the ipsilateral striatum at 12 h, 24 h, and 72 h after reperfusion as indicated. Scale bar, 100 μm. (M and N) Quantification of NLRP3-positive cells (M) and NLRP3⁺/GFP⁺ cells (N) at 12 h, 24 h, and 72 h after reperfusion as indicated (n = 3). PC represents Pearson's correlation coefficient value to show the signal colocalization status. Data are mean ± SEM. *P < 0.05, **P < 0.01. Ar, argon.

Given that the assembly of NLRP3/ASC inflammasome and processing of IL-1β in microglia are the keys to neuroinflammation after ischemic stroke (Ismael et al., 2018), we examined the protein levels of pro-IL-1β, cleaved IL-1β (cl-IL-1β), NLRP3, and ASC. As shown in Figure 2C, argon markedly downregulated the expression levels of these proteins (Figure 2D–G). We further investigated the effect of argon treatment on ASC and IL-1β expression in the microglia/macrophage of ischemic penumbra (Figure 2H). Immunostaining showed that expression levels of IL-1β and ASC increased in CX3CR1^GFP/+ transgenic mice after reperfusion, and argon administration markedly reduced the fluorescent intensity of IL-1β and the quantity of ASC-positive microglia/macrophage in penumbra (Figure 2I–K). The colocalization of IL-1β with CX3CR1-GFP was shown in Supplementary Figure S2C.

Furthermore, the activation of NLRP3 inflammasome in microglia/macrophage is the key trigger for neuroinflammation after I/R. Hence, we adopted the immunostaining of NLRP3 in CX3CR1^GFP/+ transgenic mice to visualize the changes of NLRP3 expression in microglia/macrophage. In contrast to the I/R group, both the number of NLRP3-positive cells and the ratio of NLRP3-positive microglial/macrophage were decreased from 12 h to 72 h post-reperfusion in the I/R group with argon administration (Figure 2L–N). The colocalization of NLRP3 with CX3CR1-GFP was shown in Supplementary Figure S2D–F. These results together suggest that argon suppresses NLRP3 inflammasome in the activated microglia/macrophage, which polarize toward a pro-inflammation state after reperfusion in vivo.

Argon downregulates the production and release of IL-1β via suppressing NF-κB/NLRP3 inflammasome signaling and associated caspase-1/GSDMD cleavage

To mimic the NLRP3-inflammasome-mediated pro-inflammatory in microglia, we applied lipopolysaccharide (LPS), a special agonist of toll-like receptor 4 (TLR4) signaling, combined with nigericin (Nig) (Zhao et al., 2019), a toxin specifically activating NLRP3 inflammasome assembly. The mRNA levels of Fcgr3, Nos2, Arg1, and Tgfb1 were examined to verify the phenotype after stimulation. As shown in Supplementary Figure S3, M1-related gene expression levels were increased after LPS/Nig stimulation, and M2-related genes had no change. Interestingly, argon significantly suppressed M1 polarization and regulated toward M2 phenotype. To further explore whether TLR4/NF-κB, the key pathway of NLRP3 inflammasome priming step, was involved as a major molecule target of argon, we examined NF-κB (p65) nuclear translocation using immunofluorescence staining and found that argon prevented p65 nuclear translocation induced by LPS/Nig stimulation (Figure 3A and B). In line with the suppression of argon in the priming stage, mRNA levels of Nlrp3 and Il1b, two transcriptional targets of NF-κB, were dramatically reduced when treated with argon in the LPS/Nig-treated microglia (Figure 3C and D).

Figure 3.

Argon downregulates the production and release of IL-1β via suppressing the NF-κB/NLRP3 inflammasome signaling and associated caspase-1/GSDMD cleavage in vitro. (A) Immunostaining analysis of p65 (NF-κB, red) and DAPI (nuclear, blue) localization in primary microglial cells with indicated treatments. Scale bar, 100 μm and 10 μm (insets). (B) Quantification of nuclear p65 intensity (66–84 cells per group from 9 coverslides). (C and D) Relative mRNA levels of Nlrp3 (C) and Il1b (D) in primary microglial cells with indicated treatments. Actb (β-actin)-normalized mRNA levels in the control group were used to set the baseline value at unity. (E) Immunostaining analysis of inflammasome assembly indicated by ASC (green) speck formation in primary microglial cells with indicated treatments. Nuclei were marked with DAPI (blue). Scale bar, 100 μm and 10 μm (insets). (F) Protein levels of pro-caspase-1, cl-caspase-1, GSDMD, and N-GSDMD in primary microglia with indicated treatments detected by western blotting. β-actin was used as the endogenous control. (G) Quantification of ASC speck-positive cells in each group as indicated (n = 3). (H–K) Quantification of pro-caspase-1 (H), cl-caspase-1 (I), GSDMD (J), and N-GSDMD (K) protein level detected by western blotting (n = 3–9). (L) The released IL-1β level in cell culture detected by ELISA (n = 3). Data are mean ± SEM. **P < 0.01. Ar, argon.

ASC molecules link NLRP3 and pro-caspase-1 and assemble together into large protein dimers, called ASC speck, which becomes a typical biomarker of NLRP3 inflammasome-dependent microglial activation when stimulating primary cultured microglia with LPS/Nig (Zhao et al., 2019). We next explored whether argon influenced ASC speck formation in primary cultured microglia. Immunofluorescence staining results showed that ASC turned from diffused distribution into one speck in each cell upon nigericin stimulation, and argon markedly prevented ASC speck formation (Figure 3E). Quantitative analysis indicated that the number of ASC speck-positive microglia was ∼70% with LPS/Nig stimulation, while in the presence of argon administration, only ∼30% of total cells were observed with ASC speck (Figure 3G).

Previous studies have shown that cleaved caspase-1 (cl-caspase-1) from pro-caspase-1 during NLRP3 inflammasome activation can further cleave GSDMD to produce N-GSDMD fragment, which can bind to the plasma membrane and form membrane pore, leading to a lytic cell death named pyroptosis (Li et al., 2022). As our results showed that argon suppressed nigericin-induced NLRP3 inflammasome activation, we next explored whether argon inhibited the production of NLRP3 downstream effectors, cl-caspase-1 and N-GSDMD, also the indicators of microglial pyrolysis. The expression levels of these proteins were examined in both primary cultured microglia (Figure 3F and H–K) and I/R mouse model (Figure 4). Concomitant with the decreased pro-/cl-caspase-1 production by argon treatment, GSDMD/N-GSDMD were also dramatically downregulated in both in vitro and in vivo experimental models.

Figure 4.

Argon treatment inhibits NF-κB phosphorylation and pyroptosis in the tMACO mouse model. (A) Protein levels of phosphorylated NF-κB (p-p65) and p65 in mouse brain tissues with indicated treatments at 24 h after reperfusion detected by western blotting. β-actin was used as the endogenous control. (B and C) Quantification of p-p65 protein level (B) and p-p65/p65 ratio (C) (n = 3–6). (D) Protein levels of pro-caspase-1, cl-caspase-1, GSDMD, and N-GSDMD in mouse brain tissues with indicated treatments at 24 h after reperfusion detected by western blotting. β-actin was used as the endogenous control. (E–H) Quantification of pro-caspase-1 (E), cl-caspase-1 (F), GSDMD (G), and N-GSDMD (H) protein level (n = 6–9). Data are mean ± SEM. **P < 0.01. Ar, argon.

To visualize the morphology changes of microglia with or without argon treatment and provide directly evidence for pyroptosis, we performed live cell imaging using phase-contrast microscopy. Untreated primary cultured microglia showed the characteristics of resting state with ramification. Upon LPS/Nig stimulation, cells retracted the protrusion and became rounded, with large membrane blebs emerging in ∼85% cells. However, only ∼30% cells showed the morphology evidence of pyroptosis following argon treatment (Supplementary Figure S4).

In hyperactivated cells, N-GSDMD forms pores enough to let IL-1β pass through but not enough to kill the cell (Evavold et al., 2018). We then examined the extracellular IL-1β released into the culture medium using an IL-1β ELISA kit. Results showed that IL-1β levels in cell supernatant increased after LPS/Nig stimulation, while argon treatment decreased the intracellular IL-1β (Figure 3L). These data together indicate that argon protects microglia from LPS/Nig-induced NLRP3 inflammasome-mediated pyroptosis and I/R-induced pyroptosis.

Argon is a promising inhibitor of NLRP3 inflammasome-mediated neuroinflammation to protect against cerebral I/R injury

MCC950 is a potent and specific inhibitor of NLRP3 inflammasome. We then compared the effects of argon and MCC950 on suppressing NLRP3 inflammasome-mediated neuroinflammation in LPS/Nig-stimulated primary cultured microglia (Figure 5A and B) and I/R mice (Figure 5C–K).

Figure 5.

Argon is a promising inhibitor of NLRP3 inflammasome-mediated neuroinflammation to protect against cerebral I/R injury. (A) The flowchart of in vitro experiments using primary cultured mouse microglia (PMG). Red represents LPS stimulating duration, green indicates nigericin stimulating duration, blue represents argon treatment time, and grey shows MCC950 treatment time. The short arrow represents the sample collection timepoint. (B) Levels of released IL-1β in cell supernatant with indicated treatments detected by ELISA (n = 4–9). (C) The flowchart of animal experiments. Red represents ischemia duration, blue represents argon treatment time, and grey shows MCC950 treatment time. The short arrow represents the sample collection timepoint. (D) TTC staining of consecutive brain sections showing the infarct region of the indicated group. Scale bar, 10 mm. (E) Quantitative analysis of infarction volume of the whole mouse brain with indicated treatments (n = 6–7). (F–H) Neurological deficits of mice with indicated treatments evaluated by Bederson score (F), general deficits (G), and focal deficits (H) (n = 6–7). (I) Protein levels of pro-IL-1β and cl-IL-1β in mouse brain tissues with indicated treatments at 24 h after reperfusion detected by western blotting. β-actin was used as the endogenous control. (J and K) Quantification of pro-IL-1β (J) and cl-IL-1β (K) protein level (n = 5–6). Data are mean ± SEM. *P < 0.05, **P < 0.01. Ar, argon.

As the consequence of forming pyroptotic pore by N-GSDMD, the release of IL-1β in cell supernatant was increased by LPS/Nig stimulation. We found that both argon and MCC950 addition prevented the release of IL-1β, with a stronger inhibition observed in the argon addition group. Moreover, argon or MCC950 without LPS/Nig (as negative control) did not affect the IL-1β level in cell supernatant. These data indicate that argon suppresses LPS/Nig-induced microglia/macrophage IL-1β release more prominently than MCC950 (Figure 5B).

This was further verified in vivo. 2,3,5-triphenyltetrazolium chloride (TTC) staining showed smaller infarct areas in the argon-treated group than in the MCC950-treated group (Figure 5D and E). Argon treatment displayed better outcomes in neurological deficit assessments, including Bederson score, general deficits, and focal deficits, compared with MCC950 treatment (Figure 5F–H). Western blot analysis also showed that argon inhibited the protein levels of pro-IL-1β and cl-IL-1β more prominently than MCC950 (Figure 5I–K). Collectively, these results indicate that argon is a promising inhibitor of NLRP3 inflammasome-mediated neuroinflammation and has a higher neuroprotective efficacy than MCC950.

Discussion

Although the neuroprotective effect of argon has been recognized for decades in a variety of models (Hollig et al., 2014; Gardner and Menon, 2018; Li et al., 2018), the underlying mechanism has not been fully understood. In this study, we provide evidence that argon inhalation during ischemia mitigates microglia/macrophage activation, inhibits M1 whereas enhances M2 polarization in the ischemic penumbra of the striatum in the murine tMCAO model, attenuates the activation of NLRP3 inflammasome, and reduces NLRP3 inflammasome-mediated pyroptosis in microglia in vitro and in vivo. Moreover, argon works better than the widely used NLRP3 inhibitor MCC950 in suppressing neuroinflammation and protecting neurons after ischemic stroke. The anti-inflammation effect of argon is probably through restraining the phosphorylation and nuclear translocation of NF-κB.

Microglia as the microenvironment monitors and initiators of immune response in CNS have dual functions in various brain injuries including ischemic stroke. After activated, microglia are either polarized to the pro-inflammatory M1 phenotype or the anti-inflammatory M2 phenotype and play critical roles in secondary brain damage or repair, respectively (Qin et al., 2019; Jiang et al., 2020). Several studies have shown that argon inhibits the activation of microglia in an array of animal models, including the tMCAO model (Liu et al., 2019), traumatic brain injury (Moro et al., 2021), subarachnoid hemorrhage (Kremer et al., 2020), and retinal I/R injury model (Goebel et al., 2021). Besides activation, post-stroke argon administration enhances M2 microglia/macrophage polarization in the tMCAO model (Liu et al., 2019) and mouse traumatic brain injury model (Moro et al., 2021). However, the effect of argon on M1 microglia has not been reported. In our study, we observed that the number of M2 microglia increased whereas the number of M1 microglia decreased at 12 h, 24 h, and 72 h post-reperfusion, by qPCR detection of microglial polarization marker gene expression. Therefore, we consider argon as a dual modulator of microglial polarization in this context.

Argon probably regulates microglial polarization in two ways. First, NF-κB is one of the primary transcription factors that activate and transform microglia into the M1 phenotype (Jiang et al., 2020). Our study showed that argon deters NF-κB from phosphorylation and translocation to nuclear. Thus, argon is likely to inhibit M1 polarization via directly blocking NF-κB activation. Second, NLRP3 inflammasome-mediated pyroptosis, as well as the maturation and release of IL-1β and IL-18, provides a pro-inflammatory microenvironment to enhance the M1 polarization (Alishahi et al., 2019; Jiang et al., 2020). Our data showed that argon treatment reduces the level of cl-IL-1β and GSDMD and dramatically limits IL-1β release from microglia, removing the signals that further stimulate microglia to polarize to M1 phenotype.

MD2, which mediates the interaction between LPS and the TLR4–MD2 complex that recognizes PAMP or DAMP signals and activates inflammatory responses like NLRP3 inflammasome signaling pathway (Park et al., 2009), is a potential target of argon. On the one hand, given that argon inhibits the phosphorylation and translocation of NF-κB, the molecular target of argon is probably located upstream of the NF-κB/NLRP3 signaling pathway, where MD2 locates (Park et al., 2009). On the other hand, MD2 has hydrophobic pockets that act in the interaction between LPS and the TLR4–MD2 complex (Park et al., 2009). Argon, as a noble gas, shares structure similarities with xenon, a known anesthetic (Ezzeddine, 2011; Nair, 2019), and may act by van der Walls forces in the hydrophobic pockets within MD2, like other noble gases causing anesthesia (Hameroff, 1998), to block the interaction between LPS and the TLR4–MD2 complex and abolish the downstream signaling transduction from the very beginning. Moreover, the hydrophobic pocket also exists in ASC (Pal et al., 2019) and caspase-1 (Wang et al., 2020), making them potential targets too. Whether it is the case needs further research.

Our study proved that argon inhibits NLPR3 inflammasome-mediated inflammation in microglia; however, this can be only part of the story. First, the comparison between argon and MCC950, one of the most potent and selective inhibitors that block NLRP3 inflammasome activation (Coll et al., 2015, 2019; Zahid et al., 2019), supports this hypothesis. Argon worked better in suppressing the release of IL-1β in vitro and in reducing brain infarct volume and improving neurological outcomes in the tMCAO model. Since NLRP3 is only part of the TLR4–MD2 and NF-κB downstream signaling pathways (Lu et al., 2008; Lin et al., 2021), it makes sense that argon works better than MCC950. Also, the mRNA level of TNF-α, a cytokine not involved in the NLRP3 inflammasome pathway, was downregulated in argon-treated tMCAO mice, which is in line with the hypothesis. Second, although NLRP3 inflammasome plays a predominant role in the inflammation after ischemic stroke (Yang et al., 2014; Ismael et al., 2018; Ma et al., 2018; Alishahi et al., 2019; Feng et al., 2020; Franke et al., 2021), inflammasome is a big family including different kinds of sensors other than NLRP3, like NLRP1, NLRP6, NLRP7, and NLRP12 in NLR family, CARD domain containing 4 (NLRC4), and AIM2 (absent in melanoma 2), which are all involved in the processing of caspase-1, IL-1β, IL-18, and GSDMD (Latz et al., 2013; Malik and Kanneganti, 2017). Studies showed that besides NLRP3, the expression of other sensors like AIM2, NLRC4, and NLRP1 were elevated after stroke (Fann et al., 2013; Lammerding et al., 2016; Franke et al., 2021; Lu et al., 2021). Some studies even showed that the inflammatory response after stroke was independent of NLRP3, as NLRP3 knockout or inhibition had no effect on the extent of injury caused by stroke in mice (Denes et al., 2015; Lemarchand et al., 2019). Instead, AIM2 and NLRC4 inflammasome contribute to the inflammation and injury (Denes et al., 2015). The conflict between these results may be attributed to the different ischemic time to cause different inflammatory response. Nevertheless, NLRP3 seems not to be the only path for inflammasome activation post-stroke. The executors of inflammasome may vary depending on the disease context, and argon may affect other inflammasomes as well. This idea needs to be tested in the future. Third, despite microglia are the primary source of NLRP3 inflammasome-mediated inflammation, NLRP3 also expresses and functions in other cell types like endothelial cells and neurons post-ischemic stroke. Yang et al. (2014) found that NLRP3 is mainly expressed in microglia and endothelial cells, by reverse transcription–PCR in primary cultured cells in vitro and confocal immunofluorescent analysis in vivo. NLRP3 expression is elevated in endothelial cells under oxygen and glucose deprivation in vitro (Cao et al., 2016; Bellut et al., 2021; Wang et al., 2021) and is responsible for the endothelial cell death (Bellut et al., 2021) and blood–brain barrier disruption in vivo in murine stroke model (Bellut et al., 2021) and in the stroke of diabetic rats (Ward et al., 2019). In a hemorrhagic transformation model induced by delayed recombinant tissue plasminogen activator (rt-PA) treatment in thromboembolic stroke rat, NLRP3 was elevated in neurons, microglia, and endothelial cells (Guo et al., 2018). NLRP3 expression in neurons was also detected in some other studies (Sun et al., 2020; Franke et al., 2021). In our confocal immunofluorescent analysis of the brain section of tMCAO mice, the expression of NLRP3 in neurons, endothelial cells, as well as microglia were also observed (data not shown), consistent with these reports. Whether argon inhibits the activation of NLRP3 inflammasome in neurons and endothelial cells needs further assessment and has already been in our study plan.

Besides the obvious anti-inflammation effect of argon, it has some incomparable advantages: (i) argon can penetrate the blood–brain barrier and diffuse to the deep region of tissue, therefore suitable for brain injury treatment; (ii) argon is easy to administrate and modified in resuscitation; (iii) argon has no sedating effect under normal pressure and appeared to be safe in preliminary human trials (Gardner and Menon, 2018); (iv) it is relatively cheap and easy to transport. Altogether, argon is an attractive option for treating NLRP3 inflammasome-mediated post-stroke brain damage. Given that NLRP3 inflammasome is widely involved in aseptic inflammatory diseases (Wang and Hauenstein, 2020), autoimmune disorders (Li et al., 2020), cancers, and metabolic disorders like type 2 diabetes and obesity (Sharma and Kanneganti, 2021), argon, as a gas inhibitor of NLRP3 inflammasome signaling pathway, may have a much broader application prospect in the clinic.

In conclusion, our study proves that argon is a potent gas inhibitor of NLRP3 inflammasome to block microglial activation and polarization toward M1 phenotype, attenuates inflammation, and mitigates brain injuries post-ischemic stroke. Hence, argon is a promising treatment for ischemic stroke damage.

Materials and methods

Animals

ICR mice used in this study were purchased from the Experimental Animal Center of Nantong University (Institutional License: SYXK(SU)-2012-0030). CX3CR1^GFP/GFP transgenic mice were purchased from Jackson Laboratories, and the CX3CR1^GFP/+ heterozygous mice used in the study were bred from C57BL/6 and CX3CR1^GFP/GFP mice. Mice were maintained under a 12-h light/12-h dark cycle in the individually ventilated cages, with a constant temperature (25°C ± 0.5°C) and relative humidity (55% ± 5%), and were allowed to access to food and water freely throughout the study. Animal experiments in this study were approved by the Animal Ethics Committee at Nantong University (Approval No: S20190920-303), and all the experiments were conducted according to the relevant regulatory standards. The research protocol and animal care procedures of this study were inspected and approved by the Laboratory Animal Center and Lab Animal Ethical Committee of Nantong University.

tMCAO animal model

Male ICR mice and CX3CR1^GFP/+ mice weighed 24–30 g were used for the tMCAO model construction. Mice were anesthetized with 5% isoflurane (R510-22-8, RWD) using a face mask and continuously inhaled with a pipeline maintenance respirator (BD21 4LZ, Medical supplies & Services) during the surgery. tMCAO was induced as previously described (Xu et al., 2021). A 6-0 monofilament with a silicon-coated tip (6023PK5Re, Doccol Corporation) was introduced to the right external carotid artery and advanced along the internal carotid artery to occlude the MCA. The same surgical procedure was performed in sham-operated animals except for occlusion of the artery. Occlusion was confirmed by a laser Doppler (Moor Instruments) via measurement of blood flow, and animals showing <80% signal drop compared with baseline or absence of ischemia in the striatum were excluded pre-hoc. The filament was removed 3 h after MCAO in order to allow reperfusion. Body temperature was maintained at 37°C ± 0.5°C by the heating mat (1116, Biomart) during the surgery. Neurological behavior assessments were carried out 24 h post-reperfusion by researchers double-blinded to the experimental groups. At the designed time point after reperfusion, the mice were euthanized via intraperitoneal injection of a compound anesthetic, 2.5% Avertin (0.15 ml/10 g, 2,2,2-tribromoethanol, Cat#T48402, Sigma; 2-methyl-2-butanol, Cat#152463, Sigma), and sacrificed, and samples were collected for the following experiments.

Primary microglia culture and cell stimulation

Primary microglial cells were cultured using a standard protocol as previously described (Wayman et al., 2012). Briefly, cultures were prepared from postnatal Day 0–2 mouse pups. Brains were immediately removed and placed in ice-cold Hank's balanced saline solution (HBSS). Cortical hemispheres were dissected, and meninges and blood vessels were removed. All cortical tissues were cut into pieces and transferred to pre-warmed HBSS with 0.25% trypsin for 12 min at a 37°C water bath, then neutralized by Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), and suspended up and down for 20 times. Cells were collected by centrifugation and resuspended with DMEM with FBS. Cell suspension was then seeded onto T75 flasks. Cells were maintained with DMEM/F12/10% FBS medium at 37°C in the culture incubator of 5% CO₂ and allowed to mature in vitro for 7 days, and then cultured with DMEM/F12/10% FBS/GMCSF (5 ng/ml) for another 7 days. Then, microglia were harvested by gently vortexing the flask for 30 min at the speed of 180 rpm in the incubated shaker (MaxQ^TM 4000, Thermo Fisher Scientific). The supernatant containing detached microglial cells were collected by centrifuging at 1000 g for 10 min at room temperature. All culture medium and reagents were purchased from Gibco (Thermo Fisher Scientific).

To specifically activate NLRP3 inflammasome signaling pathway in the primary cultured microglia, 5 × 10⁵–1 × 10⁶ microglia for qPCR and western blotting and 1 × 10⁴–1 × 10⁵ microglia for immunostaining were seeded with culture media without GMCSF. Twenty-four hours later, cells were treated with 100 ng/ml LPS (Cat#00-4976-93, Invitrogen) for 3 h and then stimulated with nigericin (Cat#M7029, AbMole) for another 1 h (Supplementary Figure S5).

Argon administration and experimental procedures

The device used for animal argon administration is illustrated in Supplementary Figure S5A, with a 3.5-L volume. The system was flushed with the argon–oxygen mixture (79% argon + 21% oxygen) four times the system volume before mouse exposure. The experiment flowchart is shown in Supplementary Figure S5B. Briefly, 1 h after tMCAO, mice were randomly assigned into different groups. Then, the mice in argon-treated groups were exposed to the argon–oxygen mixture for 3 h. After being exposed to argon for 2 h, the mice were taken out of the chamber for a very short time to remove the filament that blocked the blood flow. Air was used as the control.

The chamber used for cell argon administration is illustrated in Supplementary Figure S5C, with the temperature kept at 37°C using the water circulation equipment. The argon–oxygen–carbon dioxide mixture (74% argon + 21% O₂ + 5% CO₂) was used to treat cells, and the chamber was washed with the gas mixture for 5 min before sealed. The experiment flowchart is shown in Supplementary Figure S5D. Briefly, after LPS treatment, cells were immediately deposited in the chamber and exposed to the gas mixture for 3 h. Then, cells were moved out, added with nigericin, and kept in the cell chamber for another 1 h before sample collection.

Evaluation of neurological behavior

The neurologic assessments were conducted double-blinded. The neurological behavior was scored at 24 h after reperfusion according to the Clark's scoring system (Clark et al., 1997), using Bederson neurological score (0–3), general neurological scale (0–28), and focal neurological scale (0–28).

Measurement of cerebral infarct volume

After neurological evaluation, the mice were euthanized and brains were collected for measurement of infarct volume. The brain was removed rapidly, placed at −20°C for 20 min, and cut into five 2-mm-thick coronal slices. Then, the sections were immersed in 1% TTC in phosphate-buffered saline (PBS) at 37°C for 20 min followed by overnight immersion in 4% paraformaldehyde (PFA). The infarct area of each slice was demarcated and analyzed using Image J. Infarct volume was calculated by integration of the infarct areas of all brain slices. The infarct area was determined by subtracting the area of non-infarcted tissue in the ipsilateral hemisphere from that of the intact contralateral hemisphere to correct for brain swelling (Swanson et al., 1990).

Immunofluorescence staining analysis

Immunofluorescence staining was performed on the 25-μm-thick brain sections as previously described (Johann et al., 2015). Briefly, brain sections were permeabilized, blocked, and incubated overnight with the following primary antibodies at 4°C: mouse anti-NLRP3 (1:200, Cat#AG-20B-0014-C100, AdipoGen Life Sciences), rabbit anti-ASC (1:800, Cat#67824s, Cell Signaling Technology), goat anti-Iba1 (1:500, Cat#Ab5076, Abcam), anti-MAP2 (1:800, Cat#56438s, Cell Signaling Technology), IL-1β (1:500, Cat#sc-52012, Santa Cruz), and CD16 (1:1000, Cat#ab246222, Abcam). Sections were then washed by PBS and incubated for 2 h with fluorochrome-conjugated secondary antibodies. DAPI (1:5000, Cat#C1006, Beyotime Biotechnology) was used to stain the nucleus. Images were acquired by Leica TSC SP8 confocal microscope (Leica).

Circularity and ramification analyses were carried out in microglial cells with Iba1 antibody (Figure 1A) to quantify the microglial activation, using Fiji software (Version 2.1.0/1.53c, NIH). To avoid bias, cell selection and image processing were executed in a double-blinded way. Cells were selected and cropped according to two criteria: (i) randomly selecting all intact cell soma and apparent branches in the imaging field; (ii) no overlapping with neighboring cells. Ramification analysis is performed with concentric circles appearing at a radius of five pixels from the soma (place the soma center in the center of image) and running sholl analysis in Fiji plugin to calculate the number of intersections between every cell branch and concentric circles. The total number of intersections is recorded as ramification. The higher the degree of activation, the fewer the number of intersections between the concentric circles. Cell circularity analysis represents the cell shape with the function of 4πA_cell/P_cell² (A, cell area; P, cell perimeter), with 0 representing an imperfect shape (ramified) and a score closer to 1 representing a perfect circle (amoeboid) (Leyh et al., 2021).

For colocalization analysis, colocalization value (Pearson's correlation coefficient) was calculated from images using Image-Pro Plus software (version 7.0, Windows). The closer the Pearson's coefficient is to 1, the more the two signals colocalize together (Bolte and Cordelières, 2006).

The average number of immunostaining-positive cells and the relative fluorescence intensity were counted using Fiji software.

Western blot analysis

Western blotting was conducted as previously described (Wu et al., 2021). Briefly, equal amounts of total protein samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then blotted onto a nitrocellulose membrane. The membrane was blocked using 5% nonfat milk for 2 h at room temperature and then incubated with the following primary antibodies overnight at 4°C: mouse anti-β-actin (1:1000, Cat#4970s, Cell Signaling Technology), rabbit anti-NLRP3 (1:1000, Cat#15101s, Cell Signaling Technology), rabbit anti-ASC (1:1000, Cat#67824s, Cell Signaling Technology), mouse anti-IL-1β (1:1000, Cat#AF5103, Affinity Biosciences), rabbit anti-caspase-1 (1:1000, Cat#ab179515, Abcam), CD16 (1:1000, Cat#ab246222, Abcam), CD206 (1:1000, Cat#24595T, Cell Signaling Technology), GSDMD (1:1000, Cat#ab219800, Abcam), p65 (1:10000, Cat#8242, Cell Signaling Technology), and p-p65 (1:10000, Cat#3033S, Cell Signaling Technology). Thereafter, the membrane was incubated with the appropriate secondary antibodies for 2 h at 26°C. Blot bands were detected using an ECL detection system with a Tanon 5200 Multi imaging system. The bands were quantified by densitometry analysis using the Fiji software.

Cell imaging and analysis

Cellular immunofluorescence staining was performed as previously reported (Hwang et al., 2019). Cells seeded on slides were fixed with 4% PFA for 20 min at room temperature, washed with PBS, and blocked in 10% bovine serum albumin (Cat#4240GR100, BioFroxx) for 2 h in before being probed with p65 (1:500, Cat#8242, Cell Signaling Technology) or ASC (1:500, Cat#67824s, Cell Signaling Technology) antibodies. Thereafter, cells were incubated with donkey-anti-rabbit 488 (1:500, 711-545-152, Jackson) at room temperature and protected from light for 1 h. Images were acquired by Leica TSC SP8 confocal microscope. For p65 nuclear translocation analysis, Image J was used to frame nuclei and then the nuclear p65 signal fluorescence intensity was calculated. For ASC speck analysis, 5–6 slides were analyzed in each group, and ∼6–10 fields were analyzed for each slide. The percentage of ASC speck-positive cells as well as total cells per field was calculated using the Fiji software (Nagar et al., 2021).

For pyroptosis-like morphology analysis, images were acquired by Leica DMIL microscope (Leica) with the phase-contrast mode. Images were statistically analyzed using the Fiji software. Three wells (20 fields per well) of each group were detected. Percentage of pyroptosis-like blebs in total cells per field was calculated (Herr et al., 2020).

Real-time qPCR analysis

Total RNAs were extracted from cell or tissue samples with TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA (1 μg) was converted to cDNA. HiScript II RT SuperMix for qPCR was used for reverse transcription (+gDNA wiper) (Cat#R323-01, Vazyme Biotech Co., Ltd). qPCR was performed using Power SYBR Green PCR Master Mix (Vazyme Biotech Co., Ltd) with a 7900HT Fast Real-Time PCR System (Applied Biosystems). Data were normalized to the expression of the housekeeping gene Hmbs. Expression levels of genes of interest were calculated as follows: relative mRNA expression = Ct of the gene of interest/average Ct of housekeeping gene, where Ct is the threshold cycle value. Primer sequences are listed in Supplementary Table S1.

Cytokine measurement via ELISA

To measure the released IL-1β in cell culture, the supernatants were harvested from different treatment groups. The concentrations of IL-1β in the culture medium were measured using ELISA kits (Cat70-EK201B/3-96, Multisciences) according to the manufacturer's instructions. Briefly, samples and biotinylated antibody working solution were separately added to the microplates and then incubated at 37°C for 2 h. After washing, horse radish peroxidase working solution was added into the wells, incubating for 45 min at 37°C and washing again. Finally, the samples were incubated in chromogenic solution and the reaction was stopped with stop solution. The absorbance value of the samples was read at 450 nm and 630 nm using Synery 2 microplate reader (Agilent). The concentrations were calculated from the calibrated standard curve. Each sample was measured in triplicate.

MCC950 administration

MCC950 (Cat#5381200001, Sigma), a NLRP3 Inhibitor, was injected intraperitoneally (100 μl, 50 mg/kg) at 1 h after the onset of artery occlusion (Chen et al., 2022).

Statistical analysis

All data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed by the Prism 8 software (Graphpad). The significance of the differences in mean values between and within multiple groups was examined by one-way ANOVA followed by the Fisher's LSD test. A P-value <0.05 was considered as statistically significant.

Funding

This work was supported by grants from the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX20_2857 and KYCX22_3372), Senile Health Scientific Research Project of Jiangsu Province (LR2021026), Nantong Social Livelihood Science and Technology General Project (MS12020019), Science and Technology Planning Project of Nantong Municipality (JC2021079 and JC2020010), and Natural Science Fund for Colleges and Universities in Jiangsu Province (19KJB320002).

Conflict of interest: none declared.

Author contributions: X.L. and Y.Y. designed the study, supervised the work, interpreted the data, and wrote, revised, and final approved the manuscript. K.X. was involved in the experimental conception and acquisition, analysis, and interpretation of data. M.Q., T.S., and J.L. helped with the acquisition of data and double-blinded analysis. Z.J., Y.Z., X.W., G.W., and X.S. helped with the interpretation of data and design of the study. L.X. and B.P. helped with in vivo and in vitro model build-up. All authors read and approved the final manuscript.