MS4A7's role in controlling immune cell responses and inflammation after spinal cord injury through the cGAS-STING-NLRP3 pathway

In this study, female C57BL/6J mice, aged 8 weeks, were used as the experimental model for SCI. All mice were housed in a temperature‐controlled facility (22°C ± 2°C) under a strict 12‐h light–dark cycle, with ad libitum access to food and water. The housing environment was carefully regulated to minimize external stressors that could influence post‐injury recovery. Mice were also housed in standard cages with appropriate enrichment to reduce variability in locomotor function assessments. All animal experiments were conducted in accordance with institutional animal care guidelines and approved by the Ethic Committee of Zhengzhou University. For the SCI procedure, a contusion injury was induced at the thoracic (T10) level of the spinal cord using the impactor system from a height of 6.5 cm with a successful rate of 95% (5 g, RWD, Shenzhen, Guangdong, China). Post‐injury, animals were monitored weekly for signs of recovery, including motor function and allodynia. All experiments were designed and reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

BV2 cells, a widely used mouse‐derived microglial cell line, were selected for this study due to their well‐characterized polarization responses and ease of manipulation in vitro [24, 25]. BV2 microglial cells were cultured in DMEM (Dulbecco's Modified Eagle Medium, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Servicebio) in a humidified incubator at 37°C with 5% CO₂. For all experiments, cells were passaged at 80%–90% confluence. To ensure consistency across experiments, cells were seeded at a uniform density of 2 × 10⁶ cells per well in 6‐well plates or 1 × 10⁵ cells per well in 24‐well plates and allowed to adhere for 24 h. Following adherence, cells were treated with lipopolysaccharide (LPS, 1 μg/mL) and adenosine 5′‐triphosphate (ATP, 5 mM) for 24 h to induce an inflammatory response or vehicle (PBS) as control. All experiments were performed in triplicate and repeated independently.

Hindlimb motor function was assessed using the Basso mouse scale (BMS) at baseline (prior to surgery) and on postoperative Days 1, 3, 7, 14, 21, and 28, as described by Basso et al. [26]. The BMS scoring system evaluates locomotor recovery in mice, assigning scores ranging from 0 to 9, where 0 represents complete absence of ankle movement and 9 denotes full functional recovery. Scoring was performed by two independent, blinded observers to minimize bias and ensure consistency in the evaluation process. The final score for each mouse was determined by averaging the scores given by both assessors.

Hindlimb function, trunk stability, and body angle were assessed using the Louisville swim scale (LSS) prior to injury and at 7, 14, 21, and 28 days post‐SCI, as described by Smith et al. [27]. The LSS assigns scores ranging from 0 to 15, with 0 indicating no hindlimb movement and 15 representing normal hindlimb movement. To ensure acclimatization to the swimming environment, all mice underwent pre‐training sessions before the assessment. Scoring was conducted by two independent, blinded observers to enhance reliability, and the average score for each mouse was calculated and used for statistical analysis.

Footprint analysis was conducted at 7, 14, 21, and 28 days post‐SCI following the protocol described by de Medinaceli et al. [28]. Nontoxic paint was applied to the plantar surfaces of the limbs, with red for the forelimbs and green for the hindlimbs. Mice were then placed in a confined walkway lined with paper to record their footprints. Trials were repeated if a mouse turned around before completing the walkway. Quantitative parameters, including stride length (distance from the start to the endpoint of the hind paw), angle of rotation (angle between the third toe and the stride line), and interlimb coordination (distance between ipsilateral forelimb and hindlimb placement), were analyzed. Each mouse underwent three trials per time point, and the average value for each parameter was used for analysis.

The formalin pain test was performed to evaluate pain behavior in mice [29]. Mice were first acclimatized to the testing environment by placing them individually in a transparent cylindrical jar (9.5 cm in diameter and 10 cm in height) for 5 min. Following acclimation, 20 μL of 2% formalin solution was injected subcutaneously into the plantar surface of the right hind paw. Pain responses were assessed in two phases: the acute phase (0–10 min post‐injection), during which the number of instances of paw licking was recorded, and the inflammatory phase (10–30 min post‐injection), during which the cumulative duration of paw licking was measured to reflect the inflammatory response of the surrounding tissue. This method provided a quantitative assessment of pain‐related behavior induced by formalin injection.

The writhing test was conducted to assess nociceptive responses [30]. This test involved the intraperitoneal injection of irritant substances to induce visceral pain, characterized by acute, tonic, and diffuse nociceptive sensations mediated by spinal and supraspinal pathways. In this study, writhes were induced by administering 0.1 mL/10 g body weight of 0.6% acetic acid intraperitoneally. Five minutes post‐injection, the nociceptive response was evaluated by recording the number of abdominal writhes displayed by the animals over a 5‐min observation period. This method provided quantitative data on pain‐related behavior elicited by chemical stimulation of the peritoneal cavity.

The paw withdrawal threshold (PWT) was assessed using the von Frey filament (VFF) test to evaluate mechanical nociception. Mice were individually placed on a mesh floor in transparent plastic boxes and allowed to acclimate. A series of VFFs with ascending forces (ranging from 0.02 to 1.4 g) were applied to the plantar surface of the hindpaw. Each filament was applied for 5–8 s, and the test was conducted five times with a 5‐min interval between stimulations. The PWT was determined as the lowest force that elicited a withdrawal response in at least three out of five stimulations. Withdrawal reflexes were identified by observable behaviors, including ipsilateral rear leg vibration, paw withdrawal, nibbling, or vocalization. This method provided a quantitative measure of mechanical pain sensitivity.

The Hargreaves test was conducted to assess thermal nociception. Mice were placed in individual plastic boxes on a glass platform maintained at 30°C (IITC model 400, Woodland Hills, USA) to acclimate. A radiant heat source (Ugo Basile, Italy) with an intensity of 50 W was directed at the plantar surface of the hindpaw through the glass bottom. The paw withdrawal latency (PWL) was automatically recorded as the time from heat application to paw withdrawal. To prevent tissue damage, the maximum cutoff latency was set at 25 s. Each paw was tested at 15‐min intervals, and the average PWL was calculated from 3 to 6 trials per paw. This procedure provided a precise measure of thermal nociceptive sensitivity.

Immunofluorescence staining was performed to assess the localization and expression of specific proteins in tissue samples. Tissue sections were obtained from spinal cord or other relevant tissues, depending on the experimental model, and were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature. Following fixation, sections were washed with PBS and permeabilized using 0.3% Triton X‐100 in PBS for 10 min to allow for antibody penetration. Non‐specific binding sites were blocked by incubating the sections in 5% bovine serum albumin (BSA) for 1 h at room temperature. Primary antibodies targeting specific proteins were diluted in blocking buffer and applied to the sections overnight at 4°C. After primary antibody incubation, sections were washed three times in PBS, followed by incubation with appropriate secondary antibodies for 1 h at room temperature in the dark. Nuclei were counterstained with 4',6‐diamidino‐2‐phenylindole (DAPI) to visualize cell nuclei. Then sections were washed again with PBS and mounted on glass slides using a fluorescence mounting medium. The fluorescence signals were visualized and captured using a confocal microscope (LSM710, Zeiss, Oberkochen, Baden‐Würburg, Germany).

Total RNA was extracted from the relevant tissue (spinal cord or cultured cells) using the RNeasy Plus Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. Spinal cord tissues from the lesion epicenter (approximately 0.3 cm) were collected following euthanasia of the mice. The tissues were homogenized for RNA isolation. Total RNA was extracted using Trizol reagent (Takara, Shanghai, China) according to the manufacturer's protocol. Reverse transcription of RNA into complementary DNA (cDNA) was performed using a reverse transcription kit (R122‐01, Vazyme, Nanjing, China). Real‐time polymerase chain reaction (RT‐qPCR) was carried out using AceQ qPCR SYBR Green Master Mix (Q111‐02, Vazyme) on an RT‐PCR machine (Applied Biosystems, Carlsbad, CA, USA). The cycling conditions were as follows: an initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s, with a final step at 65°C for 5 s. Primer sequences used in the experiments are listed in Table. All reactions were performed in triplicate to ensure accuracy and reproducibility. S1

In this study, the enzyme‐linked immunosorbent assay (ELISA) was employed to quantify the concentration of specific biomarkers in samples. Briefly, 96‐well plates were coated with 100 μL of capture antibody diluted in buffer and incubated overnight at 4°C. After blocking with 1% BSA in PBS for 1 h at room temperature, 100 μL of sample or standard (prepared according to the manufacturer's instructions) was added to each well. Plates were incubated for 2 h at room temperature, followed by washing with PBS containing 0.05% Tween 20. A biotinylated detection antibody was then added to each well and incubated for 1 h. After washing, 100 μL of horseradish peroxidase‐conjugated streptavidin was added and incubated for 30 min. Absorbance was measured using a microplate reader. All assays were performed in duplicate, and results were compared with a standard curve.

Lactate dehydrogenase (LDH) release was assessed to measure cytotoxicity in BV2 cells. BV2 cells were seeded in 96‐well plates at a density of approximately 1 × 10⁴ cells per well. LDH activity in the cell culture supernatants was quantified using the LDH assay kit (Servicebio, G1610‐100 T). The working solution was prepared by mixing the substrate mixture, iodine‐nitrotetrazolium substrate solution, dilution buffer, and enzyme solution in the volume ratios specified by the manufacturer's protocol. The working solution was then added to the wells containing the supernatants. Following incubation at 37°C for 30 min, the optical density (OD) of each well was measured at 490 nm using a microplate reader. The results provided a quantitative measure of LDH release, indicating the extent of cell membrane damage.

BV2 cells were seeded in 24‐well plates at a density of approximately 2 × 10⁵ cells per well and allowed to adhere. Cells were then stained with propidium iodide (PI, red) and DAPI to assess cell viability and nuclear morphology. Staining was performed by incubating the cells with the dyes for 30 min at room temperature, protected from light. After staining, the cells were visualized and imaged using a confocal microscope to evaluate the distribution and intensity of PI and DAPI signals. This method provided qualitative and quantitative insights into cell death and nuclear integrity.

The publicly available dataset GSE93561↗ was utilized to identify differentially expressed genes (DEGs) between normal (sham group) and injured (SCI group) spinal cord tissues. Gene expression profiles were retrieved and analyzed. DEGs were identified by comparing gene expression levels between the sham and SCI groups using a threshold of |log2 fold change| > 1 and an adjusted p‐value < 0.05. A heatmap was generated to visualize the clustering patterns of upregulated and downregulated genes across the groups. To further highlight the gene expression changes, a volcano plot was constructed, pinpointing genes with significant differential expression.

Data analysis was performed by investigators blinded to the group assignments to ensure objectivity. Statistical analyses were carried out using GraphPad Prism (Version 10.0.3; GraphPad Software, Boston, MA, USA). To assess the distribution of continuous variables, the Shapiro–Wilk test was applied. If the data followed a normal distribution, comparisons between two groups were performed using Student's t‐test, and comparisons among multiple groups were conducted using one‐way analysis of variance (ANOVA) followed by Tukey's post hoc test. For non‐normally distributed data, the Mann–Whitney U test was used for two‐group comparisons, and the Kruskal‐Wallis test followed by Dunn's post hoc test was applied for multiple‐group comparisons. Statistical significance was set at p < 0.05. Data are presented as mean ± standard deviation (SD), and graphical representations were prepared to illustrate the analyzed outcomes.

SCI is characterized by neuroinflammation, and targeting excessive inflammatory responses is a useful method for treating SCI. Therefore, we tended to figure out the potential genes that play pivotal roles in modulating inflammation. The dataset GSE93561↗ was used to analyze the differentially expressed genes between normal and injured spinal cord tissues, in which a mice SCI model was established to investigate the mechanism of SCI progression [31]. The heatmap illustrates the differential expression of genes between the sham group and the SCI group, showing distinct clusters of upregulated and downregulated genes (Figure 1A). Among the differentially expressed genes, an inflammation modulation‐associated gene, MS4A7, is significantly upregulated in SCI conditions compared to sham controls. The volcano plot highlights the differential expression profile of genes, with MS4A7 prominently upregulated, confirming MS4A7 as a candidate gene of interest in the SCI group due to its substantial upregulation (Figure 1B). Semiquantitative mRNA levels of MS4A7 demonstrated a significant increase in MS4A7 mRNA levels in BV2 cells treated with lipopolysaccharide (LPS) and ATP compared to untreated controls (Ctrl), suggesting that MS4A7 is responsive to inflammatory stimuli (Figure 1C). MS4A7 protein levels and localization were visualized using immunofluorescence. Under control conditions, weak expression of MS4A7 was observed, while treatment with LPS + ATP significantly enhanced MS4A7 expression (Figure 1D,E). RT‐qPCR reveals significantly elevated MS4A7 mRNA levels in the SCI group compared to sham control, confirming transcriptional upregulation of MS4A7 in response to SCI (Figure 1F). Immunostaining for IBA1, a microglia marker, shows no significant difference in overall IBA1 fluorescence intensity between sham and SCI groups (Figure 1G,H). The level of iNOS was dramatically raised in the SCI group, indicating pro‐inflammatory activation, and the MS4A7 IF intensity was also remarkably upregulated after injury (Figure 1G,I,J). The results collectively demonstrate that MS4A7 is significantly upregulated in response to SCI and inflammatory stimuli (LPS + ATP), highlighting MS4A7 as a potential regulator of the inflammatory response in microglia and a candidate for further investigation in SCI‐related pathophysiology.

Spinal cord is the central nervous system, and the injury in T10 often results in impaired locomotor function and sensitized pain sensation. The results of BMS score showed that both SCI + shNC and SCI + shMS4A7 groups showed dramatically lowered BMS score compared to the sham‐operated groups (Sham + shNC and Sham + shMS4A7) at Day 1, indicating normal locomotion without SCI and successful SCI model establishment (Figure 2A). In addition, in the SCI + shMS4A7 group, the recovery of BMS scores was significantly restored compared to the SCI + shNC group, suggesting that MS4A7 knockdown promoted motor functional recovery after SCI (Figure 2A). Swimming ability is also an important way for assessing mice locomotor function. The SCI + shMS4A7 mice displayed improved limb movement and body coordination compared to the SCI + shNC group, demonstrating the critical role of MS4A7 in regulating post‐SCI motor function (Figure 2B). Swimming scores, an indicator of motor recovery, show that SCI + shMS4A7 mice had significantly poorer swimming performance compared to SCI + shNC mice (Figure 2B,C). Gait patterns visualized through foot‐printing reveal disrupted hindlimb placement and irregular stride patterns in SCI + shNC mice compared to the SCI + shMS4A7 group (Figure 2). The relative angle of rotation analysis indicates a significant increase in coordination and stability in SCI + shMS4A7 mice compared to the SCI + shNC group, emphasizing the impact of MS4A7 knockdown on rotational movements after SCI (Figure 2E). Relative hindlimb coordination analysis demonstrates that SCI + shMS4A7 mice exhibited substantial improvement in inter‐limb coordination compared to the SCI + shNC group (Figure 2F). The results of relative stride length show that SCI + shMS4A7 mice had a longer stride length compared to SCI + shNC mice (Figure 2G). What is more, pain‐related behaviors were evaluated. SCI significantly reduced PWL, and the SCI + shMS4A7 group showed significantly restored PWL compared to the SCI + shNC group, suggesting that MS4A7 knockdown improved thermal hyperalgesia (Figure 2H). The PWT analysis indicates a significant raise in mechanical nociceptive thresholds in SCI + shMS4A7 mice compared to SCI + shNC, suggesting an improvement of mechanical hyperalgesia upon MS4A7 knockdown (Figure 2I). In the writhing test, a marker of visceral pain, SCI + shMS4A7 mice exhibited a significantly lower number of writhes compared to SCI + shNC, demonstrating that MS4A7 knockdown alleviates pain perception after SCI (Figure 2J). The time spent licking in a formalin test was significantly decreased in the SCI + shMS4A7 group compared to SCI + shNC, indicating a decrease in pain‐related behaviors due to MS4A7 knockdown (Figure 2K). These findings collectively highlight the critical role of MS4A7 in modulating functional recovery, gait stability, and pain‐related behaviors following SCI. Knockdown of MS4A7 significantly improves locomotor recovery and alleviates mechanical and visceral pain, suggesting that MS4A7 may influence inflammatory or neural repair processes post‐SCI.

Microglia plays a pivotal role in modulating neuroinflammation in SCI. BV2 cells are a type of microglial cell derived from C57/BL6 murine [32]. To assess whether MS4A7 could affect microglia polarization, immunofluorescence staining of Arg1 (indicative of M2 polarization) and iNOS (indicative of M1 polarization) demonstrates the effects of MS4A7 silencing. The IF intensity of both Arg1 and iNOS was raised after the treatment of LPS + ATP (Figure 3A–C), indicating that inflammation was triggered and the anti‐inflammation mechanism was also activated. However, the downregulation of MS4A7 through siMS4A7 further elevated the level of Arg1 but lowered the level of iNOS (Figure 3A–C), showing that MS4A7 downregulation not only suppressed the M1 polarization but promoted the M2 polarization. Furthermore, the protein levels of M1 and M2 polarization indicators were detected through ELISA. The results showed that secreted levels of ARG1 and IL‐10 (anti‐inflammatory cytokines) were elevated in the LPS + ATP + siMS4A7 group, highlighting a shift toward an anti‐inflammatory, M2‐polarized state (Figure 3D,E). Inflammatory cytokines IL‐1β and TNF‐α were significantly reduced in the LPS + ATP + siMS4A7 group compared to the LPS + ATP group (Figure 3F,G), supporting the regulatory role of MS4A7 in suppressing inflammation. Additionally, the upregulation of mRNA levels of Arg1, CD206, and IL‐10, and the downregulation of mRNA levels of IL‐1β, iNOS, and TNF‐α confirm the promotion of M2 polarization (Figure 3H–M). To verify the role of MS4A7 in regulating microglia polarization in mice, the levels of polarization indicators such as Arg1 and iNOS were assessed. The fluorescence intensity of IBA1does not significantly differ between groups, indicating similar levels of microglial activation across conditions. However, the intensity of Arg1 is significantly increased in the SCI + shMS4A7 group compared to SCI + shNC, while iNOS intensity is reduced, consistent with enhanced M2 polarization (Figure 3N–Q). What is more, the mRNA levels of anti‐inflammatory cytokines Arg1, CD206, and IL‐10 are upregulated in SCI + shMS4A7 tissues, while pro‐inflammatory markers IL‐1β, iNOS, and TNF‐α are significantly reduced when compared to those in the SCI + shNC group (Figure 3R–W). These results collectively demonstrate that silencing MS4A7 promotes M2 microglial polarization, indicating that MS4A7 might play a critical regulatory role in modulating microglial polarization and inflammatory responses in SCI.

To further confirm the role of MS4A7 in promoting microglial M2 polarization in neuroinflammation conditions, we overexpressed MS4A7 in BV2 cells with or without LPS + ATP treatment. Overexpression of MS4A7 (OE‐MS4A7) significantly reduced the mRNA expression of M2 macrophage markers, including Arg1, CD206, and IL‐10, in BV2 cells stimulated with LPS and ATP (Figure 4A–C). This suggests a suppression of the M2 (anti‐inflammatory) phenotype by OE‐MS4A7. Conversely, OE‐MS4A7 significantly raised the mRNA levels of pro‐inflammatory cytokines, such as IL‐1β, iNOS, and TNF‐α, compared to LPS and ATP treatment alone, indicating a promotion of the M1 (pro‐inflammatory) phenotype (Figure 4D–F). Representative IF images illustrate decreased Arg1 expression and increased iNOS expression in BV2 cells treated with LPS + ATP + OE‐MS4A7 compared to LPS + ATP alone, further confirming the polarization toward the M1 phenotype (Figure 4G–I). Furthermore, the secreted levels of M1/2‐associated cytokines were detected. The M2‐associated cytokines ARG1 and IL‐10 were significantly reduced in the LPS + ATP + OE‐MS4A7 group compared to LPS + ATP alone, indicating suppressed anti‐inflammatory activity (Figure 4J,K). The levels of pro‐inflammatory cytokines IL‐1β and TNF‐α were significantly elevated in the OE‐MS4A7 group, demonstrating promotion of the inflammatory response (Figure 4L,M). Thus, the above results suggest a pivotal role for MS4A7 in regulating microglia polarization‐associated inflammation, potentially offering therapeutic insights for neuroinflammation in SCI.

Recent studies revealed that M2‐like macrophage polarization was promoted by manipulating the proteasomal degradation of NLRP3 protein [33]. Importantly, Zhou et al. revealed that the inflammasome can be activated in an MS4A7‐dependent way [13]. Their study provided a novel insight into the mechanism of microglia polarization. Thus, we assessed whether MS4A7 could modulate the activation of NLRP3. The PI staining assay was conducted to assess cell membrane integrity as an indicator of pyroptotic cell death. The results demonstrate minimal PI‐positive cells in the Ctrl and Ctrl + si‐MS4A7 groups, while LPS + ATP significantly increased PI‐positive cells, indicating robust pyroptosis (Figure 5A,B). Knockdown of MS4A7 markedly reduced PI‐positive cells in the LPS + ATP group, suggesting that MS4A7 plays a critical role in mediating pyroptosis (Figure 5A,B). Immunofluorescence staining of NLRP3 and gasdermin D (GSDMD) was performed to examine inflammasome activation and pyroptotic signaling. The LPS + ATP condition showed pronounced NLRP3 and GSDMD activation compared to the control group (Figure 5C). However, knockdown of MS4A7 in the BV2 cells treated with LPS + ATP resulted in significantly reduced fluorescence intensities for both NLRP3 and GSDMD (Figure 5C–E). These findings indicate that MS4A7 is essential for inflammasome activation and subsequent pyroptotic signaling. The results of ELISA (IL‐1β and IL‐18) support this conclusion by showing a significant reduction in the IL‐18 level upon MS4A7 knockdown in the BV2 cells treated with LPS + ATP (Figures 3U and 5F), highlighting the downstream impact of MS4A7 on inflammasome‐mediated cytokine release. LDH release was measured as an additional marker of cell lysis and pyroptosis. The LPS + ATP group showed significantly elevated LDH levels compared to the control group, while MS4A7 knockdown reduced LDH release, reinforcing the role of MS4A7 in pyroptotic cell death (Figure 5G). The function of MS4A7 in modulating NLRP3 inflammasome and pyroptosis was further assessed through in vivo experiments. Immunofluorescence staining for NLRP3 and GSDMD reveals increased activation of these markers in the SCI + shNC group compared to the sham group, indicating that SCI induces robust pyroptosis (Figure 5H–J). Knockdown of MS4A7 (SCI + shMS4A7) significantly attenuated NLRP3 and GSDMD activation, suggesting that MS4A7 facilitates pyroptotic processes in the context of SCI (Figure 5H–J). The significant reduction in IL‐1β and IL‐18 secretion and LDH release in the SCI + shMS4A7 group further supports the role of MS4A7 in promoting inflammasome activation and pyroptosis during SCI (Figure 5K–M). The above findings collectively suggest that MS4A7 is a pivotal regulator of inflammasome activation and pyroptosis. Knockdown of MS4A7 effectively mitigates the activation of NLRP3, GSDMD, and associated pyroptotic markers in both in vitro and in vivo models.

Immunofluorescence staining showed that no significant difference was observed in the NLRP3 expression between the ctrl group and the ctrl + OE‐MS4A7 group. However, compared to the ctrl group, LPS + ATP treatment significantly increases NLRP3 levels; overexpression of MS4A7 (LPS + ATP + OE‐MS4A7) further amplifies NLRP3 expression compared to LPS + ATP alone (Figure 6A,B). Immunofluorescence of GSDMD also showed that GSDMD expression is negligible in the ctrl and ctrl + OE‐MS4A7 groups; and LPS + ATP treatment markedly increases GSDMD level, which was further elevated by MS4A7 overexpression (LPS + ATP + OE‐MS4A7) (Figure 6A,C). Propidium iodide (PI) staining (red) is used to detect membrane‐permeable cells, indicative of cell death, likely through pyroptosis. LPS + ATP treatment increases PI‐positive cells, confirming pyroptosis induction. LPS + ATP + OE‐MS4A7 further elevates PI‐positive cells compared to LPS + ATP alone, suggesting that MS4A7 overexpression exacerbates pyroptotic cell death (Figure 6D,E). The release of IL‐1β and IL‐18, pro‐inflammatory cytokines, is also assessed. LPS + ATP induces robust IL‐1β and IL‐18 secretion, with significantly higher levels in the LPS + ATP + OE‐MS4A7 group compared to LPS + ATP alone. Similarly, LPS + ATP + OE‐MS4A7 further increases LDH level when compared with the LPS + ATP group, reinforcing the role of MS4A7 in promoting pyroptosis and inflammation. The above findings demonstrate that overexpression of MS4A7 significantly enhances NLRP3 inflammasome activation and the release of inflammatory mediators (IL‐1β and IL‐8) in an LPS + ATP‐induced pyroptotic model.

It has been reported that NLRP3 inflammasome activation was modulated by the cGAS‐STING pathway in some diseases including intervertebral disc degeneration and acute liver injury [34, 35, 36]. Thus, we speculated that cGAS‐STING pathway might play an important role in MS4A7‐mediated NLRP3 activation. We detected the level of cytosolic dsDNA and the activation of cGAS‐STING pathway after downregulating MS4A7. Immunofluorescence for dsDNA shows significantly lowered cytosolic DNA accumulation in the LPS + ATP + siMS4A7 group when compared to the LPS + ATP + siNC group (Figure 7A). Using JC‐1 staining, we observed that MS4A7 knockdown reduced mitochondrial membrane potential (ΔΨm) under LPS and ATP stimulation, as indicated by a decrease in red fluorescence aggregates and an increase in green fluorescence monomers (Figure S1A,B). To assess the impact of MS4A7 on redox balance, we measured glutathione (GSH) levels, ATP/ADP ratio, and cellular energy metabolism indicators. Compared to the LPS + APT+siNC group, LPS + APT+siMS4A7‐treated cells exhibited a significant increase in GSH level, implying decreased oxidative stress (Figure S1C). Moreover, siMS4A7 cells with LPS + APT treatment demonstrated a higher ATP/ADP ratio and ATP concentration, suggesting restored mitochondrial bioenergetics (Figure S1D,E). Consistently, the NAD+/NADH ratio was raised in siMS4A7‐treated cells, further confirming restored metabolic function (Figure S1F). These findings collectively suggest that MS4A7 deficiency plays a crucial role in maintaining mitochondrial integrity and cellular energy metabolism, particularly under inflammatory conditions. The suppression of dsDNA in the LPS + ATP + siMS4A7 group suggests that MS4A7 is involved in promoting cytosolic DNA release during LPS + ATP‐induced stress or inflammation. Immunofluorescence for cGAS and STING shows pronounced expression in the LPS + ATP + siNC group with compared to the LPS + ATP + siMS4A7 group (Figure 7B,C). Immunofluorescence and quantitative IF intensity for cGAS and STING in SCI tissues demonstrate marked reductions in the SCI + shMS4A7 group compared to the SCI + shNC group (Figure 7D,E). This further supports MS4A7's involvement in cGAS‐STING pathway activation during SCI‐induced inflammation.

To further investigate whether NLRP3 inflammasome activation could be modulated by the cGAS‐STING pathway, diABZI (STING activator) and C‐176 (STING inhibitor) were used to treat BV2 cells and mice. Immunofluorescence for NLRP3 and GSDMD in LPS + ATP‐treated BV2 cells shows elevated NLRP3 and GSDMD in the LPS + ATP + diABZI group, with reduced activation in the LPS + ATP + C‐176 group (Figure 7F,G). The above findings demonstrated that under the condition of LPS + ATP treatment, STING activation enhanced NLRP3 inflammasome activation and inhibition of the STING pathway suppressed the inflammasome, indicating that the NLRP3 inflammasome could be regulated by the cGAS‐STING pathway. What is more, the downregulation of MS4A7 (in the LPS + ATP + diABZI+siMS4A7 group) did not significantly affect the STING activator diABZI‐induced NLRP3 activation (Figure 7F,G), indicating that STING plays an essential role in MS4A7‐activated NLRP3 inflammasome. What is more, the immunofluorescence and quantitative IF intensity for NLRP3 and GSDMD in SCI tissue reveal similar trends to the in vitro results. NLRP3 and GSDMD activation were not markedly influenced in the SCI + diABZI + shMS4A7 group when compared to that in the SCI + diABZI group, confirming that MS4A7 contributes to inflammasome activation and pyroptosis in vivo through the cGAS‐STING pathway (Figure 7H,I).

To further confirm whether NLRP3 inflammasome activation could promote microglia polarization in SCI, we treated BV2 cells and mice with MCC950 (a selective inhibitor of NLRP3, MCC950 intraperitoneal injection, 3 mg/kg/day) [37]. Immunofluorescence analysis demonstrates that compared to the LPS + ATP + OE‐MS4A7 group, the LPS + ATP + OE‐MS4A7 + MCC950 group showed no significant IF intensity values of cGAS, STING, and NLRP3 (Figure 8A–D). Similarly, IF intensity values of cGAS, STING, and NLRP3 in the LPS + ATP + diABZI +MCC950 group were also not significantly changed when compared to those in the LPS + ATP + diABZI group (Figure 8A–D). The above findings demonstrated that inhibiting NLRP3 activation via MCC950 could not influence the expression or activation of the cGAS‐STING pathway. However, as a downstream protein of NLRP3 inflammasome activation, the IF intensity of GSDMD was remarkably reduced by MCC950 with or without diABZI treatment or MS4A7 overexpression (Figure 8C,D), confirming that the NLRP3 inflammasome was suppressed by MCC950 and that the NLRP3 inflammasome was indeed activated by MS4A7 overexpression or cGAS‐STING activation. Furthermore, the indicators of polarization were detected. The IF intensity value of ARG1 in the LPS + ATP + OE‐MS4A7 + MCC950 group was remarkably elevated compared to that in the LPS + ATP + OE‐MS4A7 group; similarly, the IF intensity value of ARG1 in the LPS + ATP + diABZI + MCC950 group was remarkably elevated compared to that in the LPS + ATP + diABZI group (Figure 8E,F). However, the iNOS IF intensity was remarkably reduced in the cells treated with MCC950 (Figure 8E,F). The above results demonstrate that inhibiting NLRP3 could reverse microglial M1 polarization induced by MS4A7 overexpression or STING activation under the condition of inflammation. Then, the secretion levels of ARG1, IL‐10, TNF‐α, and IL‐1β were detected. The results showed that the levels of both ARG1 and IL‐10 were significantly raised by MCC950 when compared with other groups without MCC950 treatment (Figure 8G). The levels of TNF‐α and IL‐1β were remarkably lowered by MCC950 (Figure 8G). To further verify the role of NLRP3 in modulating microglial polarization, BV2 cells were treated with C‐176 (an inhibitor of STING) and/or NLRP3 overexpression. The results showed that NLRP3 overexpression reduced the C‐176‐induced increase in ARG1 and raised the decrease in iNOS (Figure 8H,I). In vivo studies were carried out to further verify the effect of NLRP3 inflammasome activation on microglial polarization. The IF intensity of ARG1 in both the SCI + OE‐MS4A7 + MCC950 group and the SCI + diABZI + MCC950 group was remarkably elevated compared to that in the SCI + OE‐MS4A7 group and the SCI + diABZI group; and the SCI + OE‐MS4A7 + MCC950 group and the SCI + diABZI+MCC950 group showed significantly lower iNOS IF intensity than the SCI + OE‐MS4A7 group and the SCI + diABZI group (Figure 8J,K), indicating that MCC950 treatment in SCI mice suppressed M1 polarization and promoted M2 polarization. However, no significant difference in ARG1 IF intensity was observed between the SCI + MCC950 group and the SCI + OE‐MS4A7 + MCC950 group, nor between the SCI + MCC950 group and the SCI + diABZI + MCC950 group (Figure 8J,K), confirming that NLRP3 inflammasome activation is downstream of MS4A7 and cGAS‐STING when modulating microglial polarization. In vivo experiments further confirmed the above findings, showing that NLRP3 overexpression reversed the C‐176‐induced M2 polarization (Figure 8L,M). These results demonstrate that MS4A7 exacerbates inflammation and promotes a pro‐inflammatory microglia phenotype via the cGAS‐STING‐NLRP3 axis. Pharmacological inhibition of NLRP3 effectively mitigates inflammation and favors a reparative, anti‐inflammatory phenotype by reducing iNOS expression and enhancing ARG1 expression. These findings provide a mechanistic basis for targeting the MS4A7 and cGAS‐STING‐NLRP3 axis in SCI to alleviate neuroinflammation and promote tissue repair.

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.