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. 2026 Mar 31;17:1715595. doi: 10.3389/fimmu.2026.1715595

IFI204-STING drives protective innate immunity against gangrenous Clostridium perfringens infection via regulation of NLRP3 signaling

Ming-Yue Zhang 1,†, Jia-Qi Li 1,†, Qian Xu 1,†, Zi-Jian Zhuang 1, Jing Liang 1, Ao-Bo He 1, Shu-Xin Zhang 1, Yi Zhao 1, Xue Chen 1, Zhen-Yu Li 1, Ping Sheng 1, Yang Liu 2, Shui-Xing Yu 1,*,‡
PMCID: PMC13076142  PMID: 41988177

Abstract

Inflammasomes are an essential component of the innate immune response against pathogen infections. However, the molecular mechanism regulating the inflammasome signaling in response to gangrenous Clostridium perfringens (C. perfringens) infection remains elusive. We herein report the unexpected discovery that IFI204 (the murine homolog of human IFI16)–dependent STING protects against C. perfringens gas gangrene via enhancing NLRP3 inflammasome signaling. In the C. perfringens gas gangrene model, compared with wild-type (WT) mice, Sting deficiency (Sting-/-) mice displayed an increased susceptibility to C. perfringens soft tissue infection, with more bacterial colonization, severe muscle damage, and higher mortality rates. Obviously, Sting deficiency leads to the defect of inflammasome signaling activation and bacterial killing and clearance. STING promotes inflammasome signaling activation in an IFI204-dependent manner. Crucially, the IFI204-STING axis enhances NLRP3 inflammasome signaling activation, which, in turn, facilitates pathogen elimination and host defense. Our findings highlight STING acts as a positive regulator in defense of C. perfringens infection and present it as a potential target for anti-infection drug development.

Keywords: Clostridium perfringens, gas gangrene, Ifi204, innate immune, NLRP3 inflammasome, STING

Introduction

C. perfringens can cause explosive fulminant necrotizing infections and is a common cause of gas gangrene or muscle necrosis (1, 2), posing a significant problem to both animal husbandry and human health (3). The key feature of these diseases is that they are mediated by the production of potent toxins and extracellular enzymes (4, 5). At least 20 toxins have been reported as produced by C. perfringens, of which α and θ toxins are essential for gas gangrene (6–8). Hitherto, traumatic injury contributes to 70% of gas gangrene cases, of which 80% are caused by C. perfringens (9, 10). However, the treatment recommendation for severe C. perfringens gas gangrene is still radical amputation or the use of high doses of antibiotics, a combination of penicillin with clindamycin or carbapenem (11–13). Presently, with the emergence of drug-resistant strains and growing scientific concern over the resistance spectrum, the novel preventive and therapeutic strategies for the control of C. perfringens infections have become more urgent.

Host innate immunity is critical for detecting invading pathogens, triggering anti-infection defenses, and facilitating pathogen clearance (14–16). Sensing and categorizing pathogen invaders via a majority of germline-encoded pattern recognition receptors, including NOD-like receptors, cytosolic DNA sensors, toll-like receptors, C-type lectin receptors, and RIG-I like receptors, which in turn initiates appropriate immune responses that mobilize defense mechanisms (17–20). Concerning C. perfringens, TLR2, TLR4, AIM2, and NLRP3, were involved in C. perfringens recognition (21–24). Also, we provided evidence indicating that the free fatty acid receptor GPR120 and the necroptosis effector MLKL contribute to host protection against C. perfringens infection by enhancing NLRP3 inflammasome signaling and extracellular trap formation (12, 25). Therefore, deeper research on innate immune mechanisms of host defense is expected to open new avenues for therapeutic interventions of pathogenic infectious diseases.

STING, also named TMEM173, STING1, ERIS, MITA, or MPYS, is a pivotal cyclic dinucleotide sensor (26–28) and adaptor protein for various DNA sensors such as IFI204 (IFI204, the murine homolog of human IFI16), cGAS, DDX41, and ZBP1 (29–31). In past decades, emerging evidence shows STING is associated with health and disease by regulating innate immune responses, inflammatory signaling, cell death, and host-pathogen interaction (32–35). As an endoplasmic reticulum-associated transmembrane protein, STING can be directly initiated by microbial cyclic dinucleotides (including cyclic-di-GMP and cyclic-di-AMP) or the second messenger cGAMP, a process that participates in inflammation signaling and immune response by producing IFNs and pro-inflammatory mediators. Indeed, it was previously reported that the effect of STING on pathogenic infectious diseases is controversial, which is not only beneficial to host protection against S. aureus, M. tuberculosis, S. pneumoniae, P. aeruginosa, Brucella, and L. monocytogenes (36–41) but also detrimental to the host during S. aureus cutaneous infection may depend on diverse pathogens and different infectious models (42). However, there is little knowledge regarding the biological implications of STING in response to C. perfringens infection.

In this study, we deliberated whether STING is involved in host defense against C. perfringens gas gangrene. The collected data here demonstrate that the active STING promotes NLRP3 inflammasome signaling-mediated bacterial killing and clearance. Activated STING downstream of IFI204 confers host resistance to gangrenous C. perfringens infection. These findings reveal a scaffolding role of STING in enhancing protective innate immunity against C. perfringens, which provides further insight into host-pathogen interaction mechanisms and innovative strategies targeting pathogenic infectious diseases.

Materials and methods

Experimental animals and cells

Ifi204 deficiency (Ifi204-/-) and Sting-/- mice were gifts from Dr. Yong-Jun Yang (Jilin University, China). Nlrp3 deficiency (Nlrp3-/-) and C57BL/6J (WT) mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). The mice were maintained in a pathogen-free environment at the Laboratory Animal Center of Inner Mongolia University, and all experiments adhered to Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (43) and were conducted according to experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at Inner Mongolia University ([2022] 071). Murine macrophages (bone marrow–derived macrophages, BMDMs) were isolated from the femurs of 6- to 8-week-old mice, cultured, and matured in a certain medium, RPMI1640 (Gibco, #31800-022), fetal bovine serum (Gibco, #A31608-02), and L929 cell–conditioned supernatant supplemented with penicillin/streptomycin (Gibco, #15140-122) as previously described (12).

Gas gangrene model

The C. perfringens gas gangrene model was established as previously reported (25), and the experiments complied with the manual of the care and use of laboratory animals published by the US National Institutes of Health. In brief, age- and sex-matched mice were maintained under general anesthesia with isoflurane and were intramuscularly challenged with 1 × 107 CFUs of log-phase pathogenic C. perfringens strain CP1 (a clinical isolate, NCBI GenBank: MW440585). At 24 hp.i., infected mice were euthanized by IV injection with sodium pentobarbital, and homogenized muscle, liver, and spleen tissues were plated onto BHI agar plates (Haibo Biotechnology, #HB8297-5) for bacterial loads (CFUs/g) to be enumerated. For the survival study, age- and sex-matched mice were intramuscularly infected with 5 × 107 CFUs of log-phase pathogenic C. perfringens strain CP1, and the mortality of mice were monitored. The pathology scoring criteria of gas gangrene were determined according to the severity of blackening, limping, grip, stiffness, malaise, deformation, and swelling (each item scores from 0 to 3) as previously described (43).

Inflammasome activation

Matured BMDMs were pre-primed with LPS (500 ng/ml, 4h, Invitrogen, #tlrl-3pelps) and then incubated/infected with ATP (5 mM, 30 min, Sigma, #A2383), C. perfringens strain CP1 (NCBI GenBank: MW440585) or strain ATCC13124 (MOI = 20, 90 min). Cell samples were obtained for ELISA, immunostaining, and immunoblotting detections.

Bacterial killing assessment

LPS-primed mature BMDMs were pre-treated with recombinant IL-18 (rIL-18, 1 ng/ml, 1h, Novoprotein, #CK06) or equal PBS prior to challenge with C. perfringens strain CP1 (NCBI GenBank: MW440585) or strain ATCC13124 (MOI = 5, 6h). The cell supernatants were harvested and plated on BHI agar plates, and bacterial loads (CFUs/ml) were counted.

RNA sequencing

LPS-primed matured BMDMs were infected with C. perfringens strain CP1 (NCBI GenBank: MW440585) or strain ATCC13124 (MOI = 20) for 90 min. The total cell RNA was then extracted, examined, generated double-stranded cDNA, and submitted for sequencing on an Illumina NovaSeq 6000 at Shanghai Personal Biotechnology Co., Ltd., China. The differentially expressed genes were estimated by calculating the fold change (log2 fold change > 1) and p-value (< 0.05).

Histology and immunofluorescence

The infected leg muscle was aseptically harvested and fixed in 4% buffered formalin solution (Macklin, #P804536), and sections were stained with H&E (Solarbio, #SL7050-500) or immunofluorescence staining. Immunostaining, including muscle sections or stimulated BMDMs, was performed using anti-STING (Proteintech, #19851-1-AP), anti-F4/80 (BioLegend, #123119), anti-Gr-1 (Abcam, #ab196436), anti-IFI204 (Abcam, #ab307201), anti-ASC (Adipogen, #A29151803f), Alexa Fluor488–conjugated anti-rabbit IgG (Invitrogen, #ab150077), Alexa Fluor594–conjugated anti-mouse IgG (Invitrogen, #ab150116) antibodies, and DAPI (Solarbio, #C0065).

Proinflammatory cytokines measurements

Cytokines and chemokines in the supernatants of homogenized muscle tissues or stimulated BMDMs were detected by ELISA according to the manufacturer’s instructions. The mouse IL-1β (Cat#MLB00C), mouse TNF-α (Cat#MTA00B), mouse IL-6 (Cat#PM6000B), and mouse KC (Cat#PMKC00B) ELISA kits were purchased from R&D Systems.

Immunoblotting

The total lysates or supernatants of stimulated BMDMs or homogenized muscle tissues were separated by SDS-PAGE and transferred to a PVDF membrane. Immunoblotting was performed using anti-STING (Proteintech, #19851-1-AP), anti-Caspase-1 (Adipogen, #A28881708), anti-NLRP3 (AdipoGen, #AG-20B-0014), anti-IFI204 (Abcam, #ab307201), and anti-GAPDH (Proteintech, #60004-1-Ig).

Statistical analysis

Quantification and statistical analysis were performed using GraphPad Software (La Jolla, CA), with a significance threshold set at p < 0.05 (*p < 0.05 or **p < 0.01). Error bars represent the mean ± standard deviation (SD). An unpaired Student’s t test was utilized for comparisons between two groups, while analysis of variance followed by Dunnett’s, Tukey’s, or log-rank test was employed for multiple group comparisons.

Results

Sting deficiency leads to a defect in bacterial clearance following gangrenous C. perfringens infection

To investigate the possible involvement of STING in the host response to C. perfringens soft tissue infection, a C. perfringens gas gangrene model was initially established. Littermate WT control mice and Sting-/- mice were intramuscularly infected with 1 × 107 CFUs of gangrenous C. perfringens strain CP1 (NCBI GenBank: MW440585). At 24h p.i., bacterial loads were assessed. Notably, significantly increased bacterial counts were detected in the muscle, liver, and spleen of infected Sting-/- mice compared to infected WT counterparts (Figure 1a). In accordance with this, infected Sting-/- mice also exhibited severely impaired muscle architecture, suggesting that Sting-/- mice are more susceptible to gangrenous C. perfringens infection (Figure 1b). To gain additional evidence of the protective role of STING against gangrenous C. perfringens infection, the survival rate of WT and Sting-/- mice was monitored following the intramuscular administration of 5 × 107 CFUs of gangrenous C. perfringens. The results showed that all of the Sting-/- mice had died at 48h p.i. along with obvious clinical symptoms, including limping, swelling, blackening, grip loss, deformation, intense stiffness, and malaise (Figures 1c, d). Thus, these data demonstrate that STING contributes to host protection against gangrenous C. perfringens infection.

Figure 1.

Figure with four panels comparing WT and Sting knockout mice following infection. Panel a shows three bar graphs indicating higher bacterial burden in muscle, liver, and spleen for Sting knockout mice compared to WT. Panel b includes histological micrographs, where Sting knockout muscle infected with CP1 shows significant tissue damage relative to PBS controls and WT. Panel c displays a Kaplan-Meier survival curve with reduced survival in Sting knockout mice post-infection compared to WT. Panel d is a stacked bar chart summarizing higher pathological scores, including blackening, limping, and swelling, in Sting knockout mice.

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STING contributes to protecting against pathogenic C. perfringens soft tissue infection. Age- and sex-matched WT and Sting-/- mice (n = 10 each group) were intramuscularly challenged with log-phase C. perfringens. (a) Bacterial loads in the muscle, liver, and spleen were enumerated (1 × 107 CFUs, at 24h p.i.). (b) Representative HE staining of the leg muscle tissue (1 × 107 CFUs, at 24h p.i., magnification, ×400). (c, d) Survival and cumulative gross pathology scores of infected mice (5 × 107 CFUs). Graphs are means ± standard deviation (SD) from data pooled ten (a, c, d) biological replicates. Data were considered significant when **p < 0.01.

Sting deficiency attenuates inflammasome signaling activation during pathogenic C. perfringens soft tissue infection

To further illustrate the potential immunological role of STING in defense of gangrenous C. perfringens, inflammatory response was initially determined by protein levels in vivo. Obviously, despite increased infiltration, including macrophages and neutrophils (PMNs), and elevated KC expression (Figures 2a, b), the IL-1β production was significantly attenuated in the muscles of Sting-/- mice compared to WT controls following pathogenic C. perfringens soft tissue infection (Figure 2c), whereas TNF-α release was comparable between both genotypes (Figure 2d). Moreover, inflammasome-dependent Caspase-1 activation was also significantly inhibited in the muscles of infected Sting-/- mice (Figure 2e; Supplementary Figure 1). These results revealed that Sting deficiency results in aberrant inflammation and impaired inflammasome signaling in coping with gangrenous C. perfringens infection.

Figure 2.

Panel a presents histological images comparing immune cell infiltration in heart tissues stained for F4/80 and Gr-1 between wild-type and Sting knockout mice. Panel b shows a bar graph with significantly elevated KC levels in Sting knockout compared to wild-type mice. Panel c shows a bar graph indicating significantly higher IL-1β in wild-type versus Sting knockout mice. Panel d provides a bar graph with similar TNF-α levels in both groups. Panel e displays a western blot comparing pro-Caspase-1, Caspase-1 p20, and GAPDH protein levels in wild-type and Sting knockout samples with and without CP1 treatment.

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STING activates inflammasome signaling following gangrenous C. perfringens infection. Age- and sex-matched WT and Sting-/- mice were intramuscularly stimulated with 1×107 CFUs log-phase C. perfringens at 24 h p.i. (a) Representative infected muscle sections. F4/80 (IHC, a macrophages marker) and Gr-1 (IHC, a neutrophil marker) stained brown (magnification, ×400). (b–d) Secreted KC, IL-1β, and TNF-α in the homogenate supernatant of infected muscle were determined by ELISA. (e) Cleaved Caspase-1 in the muscle tissue lysate was examined by immunoblotting. Graphs are means ± standard deviation (SD) from data pooled five (b–d) biological replicates. Data were considered significant when **p < 0.01.

STING activates NLRP3 inflammasome signaling in response to pathogenic C. perfringens

Since immunohistochemical staining showed that STING expression was mainly located in the infiltrated inflammatory cells of the infected muscles during gangrenous C. perfringens infection (Figure 3a), and STING expression was strikingly up-regulated by bacteria administration in BMDMs (Figure 4g; Supplementary Figure 3), we then further substantiate the inflammatory pathways in macrophages in vitro. In line with in-vivo findings, the inflammasome signaling was significantly suppressed in infected Sting-/- BMDMs compared to infected WT BMDMs when exposed to pathogenic C. perfringens, observed by restricted Caspase-1 activation and impaired IL-1β release, while TNF-α production was little affected (Figures 3b–d; Supplementary Figure 2). Importantly, NLRP3 expression was markedly attenuated in Sting-/- BMDMs versus WT BMDMs (Figure 3d; Supplementary Figure 2). These results demonstrated that Sting deficiency leads to the defect of NLRP3 inflammasome signaling activation following pathogenic C. perfringens challenge.

Figure 3.

Panel a contains immunohistochemical images comparing STING expression in WT and Sting knockout tissue, showing reduced staining in knockout. Panel b presents a bar graph of IL-1β secretion with WT displaying higher levels than knockout under various treatments. Panel c shows TNF-α secretion levels, with WT slightly higher but not statistically significant. Panel d presents Western blots for pro-Caspase-1, Caspase-1 p20, NLRP3, and GAPDH in cell supernatants and lysates, comparing responses to treatments in WT versus Sting knockout.

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STING promotes NLRP3 inflammasome signaling activation in host macrophages. Age- and sex-matched WT and Sting-/- mice were intramuscularly infected with 1×107 CFUs log-phase C. perfringens at 24h p.i. (a) Representative infected muscle sections. STING (IHC) stained brown (magnification, ×400). LPS-primed WT and Sting-/- BMDMs were exposed to indicated concentrations/dosages of ATP (5mM, 30min) or C. perfringens (MOI = 20, 90 min). (b, c) Secreted IL-1β and TNF-α in the culture supernatant were measured by ELISA. (d) Cleaved Caspase-1 in culture supernatants (Sup.) or in cell lysates (Lys.), as well as NLRP3 in cell lysates were analyzed by immunoblotting. Graphs are means ± standard deviation (SD) from data pooled five (b, c) biological replicates. Data were considered significant when **p < 0.01.

Figure 4.

Seven scientific panels display results from gene expression and pathway analysis experiments: (a, b) two volcano plots show significant gene expression changes with green, red, and blue dots; (c, d) two pathway enrichment bubble plots depicting size and color corresponding to gene counts and p-values for specific signaling pathways; (e) a clustered heatmap shows differential gene expression across groups with color gradient; (f) immunofluorescence microscopy images compare wild-type and knockout cells with cell nuclei stained in blue and green signal; (g) immunoblot analysis presents bands for proteins in wild-type and knockout samples following various treatments, showing loading controls and target proteins.

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STING facilitates NLRP3 inflammasome signaling activation in an IFI204-dependent manner. LPS-primed BMDMs were exposed to indicated dosages of log-phase C. perfringens (MOI = 20, 90 min). Macrophages were harvested for RNA extraction and subjected to RNA sequencing. (a, b) Volcano plot (C. perfringens group vs. PBS group). (c, d) The top significantly upregulated KEGG pathways in C. perfringens-stimulated macrophages. (e) Top significantly regulated genes in C. perfringens-triggered macrophages. LPS-primed BMDMs were exposed to indicated dosages of log-phase C. perfringens (MOI = 20, 90 min). (f) IFI204 expression was assessed by immunofluorescence (magnification, ×400). (g) Cleaved Caspase-1 in culture supernatants (Sup.) or in cell lysates (Lys.), as well as STING and IFI204 in cell lysates were measured by immunoblotting.

STING promotes NLRP3 inflammasome-mediated bacterial killing dependent on IFI204 signaling

To identify which defenses and networks may be associated with STING-mediated NLRP3 inflammasome signaling activation, we conducted RNA sequencing (RNA-seq) analysis to obtain genome expression differences in BMDMs infected by C. perfringens. Based on the threshold criterion of log2 Fold Change > 1 and p < 0.05 significance, the analysis screened 2,499 upregulated and 2,526 downregulated genes in C. perfringens strain ATCC13124-stimulated macrophage, and 2,538 upregulated and 2,622 downregulated genes in C. perfringens strain CP1-stimulated macrophages (Figures 4a, b). KEGG enrichment analysis further highlighted the upregulation of the NOD‐like receptor signaling pathway, NF-kappa B signaling pathway, and cytosolic DNA-sensing pathway following C. perfringens stimulation (Figures 4c, d). As shown by the cluster heatmap of differential genes related to innate immune responses, C. perfringens infection promoted or enhanced the induction of many genes compared to uninfected controls, including Ifi204, Nlrp3, Caspase-1, Gsdmd, and Cgas (Figure 4e). Given that IFI204 (the murine homolog of human IFI16), a STING pathway regulator, has been previously indicated to sense pathogen infection, we set out to evaluate the functional role of IFI204 in response to pathogenic C. perfringens. Interestingly, immunofluorescence and Western blot analysis confirmed that the expression of the IFI204 protein level was remarkably upregulated by pathogenic C. perfringens challenge in BMDMs (Figures 4f, g; Supplementary Figure 3). Exhilaratingly, the induction of the STING expression was significantly abrogated in infected Ifi204-/- BMDMs compared to infected WT BMDMs (Figure 4g; Supplementary Figure 3), indicating that IFI204 may be involved in STING-mediated protective responses.

Subsequently, we asked if IFI204 regulated STING-mediated NLRP3 inflammasome signaling. Similarly to that of infected Sting-/- BMDMs, Caspase-1 cleavage, ASC speckles formation, and mature IL-1β secretion were abolished in infected Ifi204-/- BMDMs, although IL-6 production was little affected (Figures 4g, 5a–d; Supplementary Figure 3). Crucially, Sting deficiency, Ifi204 deficiency, and Nlrp3 deficiency impaired the bacterial killing capacity of BMDMs, and exogenous rIL-18 administration could rescue the defect of bacterial killing in Sting-/- BMDMs, Ifi204-/- BMDMs, and Nlrp3-/- BMDMs (Figure 5e). Indeed, our previous studies have reported that NLRP3 inflammasome signaling activation is beneficial to strengthening bacterial control and host defense following pathogenic C. perfringens infection (12, 25). These findings suggested that IFI204 is a crucial regulator of STING signaling for activating antibacterial innate immunity.

Figure 5.

Panel a shows three rows of fluorescence microscopy images labeled WT, Ifi204-/-, and Sting-/-, comparing NT, LPS+ATCC13124, and LPS+CP1 conditions, with cell nuclei stained blue and other cellular structures indicated in red. Panel b is a bar graph showing the percentage of cells with ASC specks for WT, Ifi204-/-, and Sting-/- groups under LPS+ATCC13124 and LPS+CP1, with significant differences indicated by asterisks. Panel c is a bar graph measuring IL-1β (pg/mL) for WT and Ifi204-/- across NT, LPS+ATCC13124, and LPS+CP1, with significant differences. Panel d is a bar graph showing IL-6 (pg/mL) for WT and Ifi204-/- under all conditions. Panel e is a bar graph comparing bacterial survival fold changes across WT, Ifi204-/-, Sting-/-, Nlrp3-/-, rIL-18-Ifi204-/-, rIL-18-Sting-/-, and rIL-18-Nlrp3-/- for LPS+ATCC13124 and LPS+CP1, with multiple significant differences marked.

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STING and Ifi204 deficiency prevents ASC oligomerization and results in the defect of NLRP3 inflammasome-mediated bacterial killing. LPS-primed WT, Sting-/-, Ifi204-/-, and Nlrp3-/- BMDMs were exposed to indicated dosages of log-phase C. perfringens (MOI = 20, 90 min). (a, b) ASC was determined by immunofluorescence (magnification, ×400), and the percentage of positive cells containing ASC speckles was quantified. (c, d) Secreted IL-1β and IL-6 in the culture supernatant were measured by ELISA. (e) LPS-primed WT, Sting-/-, Ifi204-/-, and Nlrp3-/- BMDMs were incubated with rIL-18 (1,000 pg/ml) or PBS for 1h prior to were exposed to indicated dosages of log-phase C. perfringens (MOI = 5, 6h). The bacterial killing was assessed. Graphs are means ± standard deviation (SD) from data pooled five (b–e) biological replicates. Data were considered significant when *p < 0.05 or **p < 0.01.

IFI204 is required for STING-mediated host defense

To further identify the underlying signaling mechanisms that IFI204 regulates STING-NLRP3 signaling-driven host defense, Ifi204-/- mice were utilized to characterize the effect of IFI204 on pathogenic C. perfringens gas gangrene. Excitedly, in line with Sting-/- mice, Ifi204-/- mice are more susceptible to gangrenous C. perfringens infection compared with WT counterparts, demonstrated by boosted bacterial numbers observed in muscle, liver, and spleen (Figure 6a), indicating that Ifi204 deficiency is also detrimental to bacterial clearance. Correspondingly, Ifi204-/- mice displayed higher mortality rates, obvious clinical symptoms, and severely impaired muscle architecture (Figures 6b–d), suggesting that IFI204 is essential for host protection against pathogenic C. perfringens soft tissue infection.

Figure 6.

Panel a displays three bar charts comparing bacterial burden in muscle, liver, and spleen among wild type, Ifi204 knockout, and Sting knockout mice, showing higher loads in knockouts. Panel b presents a Kaplan-Meier survival curve indicating reduced survival in knockout mice versus wild type. Panel c is a stacked bar chart of pathological scores, with symptoms color-coded, showing higher scores in knockouts. Panel d contains histological images of muscle tissue from the three mouse groups treated with PBS or CP1, highlighting increased pathological changes in knockout samples.

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Ifi204 deficiency is insufficient to drive bacterial clearance and host defense against gangrenous C. perfringens infection. Age- and sex-matched WT, Sting-/-, and Ifi204-/- mice (n = 10 each group) were intramuscularly challenged with log-phase C. perfringens. (a) Bacterial loads in the muscle, liver, and spleen were enumerated (1×107 CFUs, at 24h p.i.). (b, c) Survival and cumulative gross pathology scores of infected mice (5 × 107 CFUs). (d) Representative H&E staining of the leg muscle tissue (1 × 107 CFUs, at 24h p.i., magnification, ×400). Graphs are means ± standard deviation (SD) from data pooled ten (a–c) biological replicates. Data were considered significant when **p < 0.01.

Subsequently, to determine if IFI204-mediated inflammasome activation was involved in protection against C. perfringens infection. Obviously, Ifi204 deficiency dramatically abrogated inflammasome-dependent Caspase-1 activation and aggravated pathological damage in the muscle (Figures 7a, b; Supplementary Figure 4). Meanwhile, infected Ifi204-/- mice and infected Sting-/- mice administered with rIL-18 exhibited strongly decreased loads of gangrenous C. perfringens in the muscle, liver, and spleen, and ameliorated pathological damage in the muscle (Figures 7b, c). Nearly the same results can be found in the results of the rIL-18–treated macrophages (Figure 5e), indicating that Ifi204 and Sting deficiency lead to a defect in bacterial killing and clearance by impairing NLRP3 inflammasome activation. Collectively, our findings suggest that STING plays a scaffolding role in enhancing the IFI204-STING-NLRP3 signaling pathway that confers protective innate immunity against gangrenous C. perfringens infection.

Figure 7.

Panel a shows a Western blot comparing expression of pro-caspase-1, caspase-1 p20, and GAPDH in wild-type and Ifi204 knockout samples with and without CP1 treatment. Panel b contains histological images of muscle tissue from Ifi204 and Sting knockout mice treated with either PBS-CP1 or rIL-18-CP1, showing cellular structure and inflammation. Panel c includes three bar graphs for muscle, liver, and spleen, comparing bacterial burden in Sting and Ifi204 knockout mice (n equals 5 per group) treated with either PBS or rIL-18, with significant reduction observed in rIL-18 groups.

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NLRP3 Inflammasome signaling downstream of IFI204-STING axis confers protection against gangrenous C. perfringens infection. Age- and sex-matched WT and Ifi204-/- mice were intramuscularly challenged with 1 × 107 CFUs log-phase C. perfringens at 24 h p.i. (a) Cleaved Caspase-1 in the muscle tissue lysate was examined by immunoblotting. For one group of Ifi204-/- or Sting-/- mice, rIL-18 (1 μg/mouse) was injected intraperitoneally daily starting the day before the intramuscularly infected with 1 × 107 CFUs log-phase C. perfringens at 24h p.i. (b) Representative H&E staining of the leg muscle tissue (magnification, ×400). (c) Bacterial loads in the muscle, liver and spleen were enumerated. Graphs are means ± standard deviation (SD) from data pooled five (c) biological replicates. Data were considered significant when **p < 0.01.

Discussion

C. perfringens is the most common cause of trauma-associated necrotizing gas gangrene with increasing mortality rates. Radical amputation and broad-spectrum antibiotic drugs are recommended choices for the treatment of this pathogenic infectious disease (44, 45). However, antibiotic use has been associated with adverse outcomes, including encouraging the spread of resistance and increasing the prevalence of drug-resistant strains. Given the crucial role of innate immunity in host protection against pathogenic infections, improving its response may be a potential prevention strategy for C. perfringens infections. Here, this observation highlights the importance of STING in generating host-protective innate immunity to restrict C. perfringens soft tissue infection.

STING is a central molecule to activate innate immunity during infection (46). The early innate immune response to Carbapenem-resistant A. baumannii infection is activated in a cGAS-STING-dependent manner (47). Moreover, STING is reported to be crucial for the host defense against S. aureus infection through suppressing necroptosis (36). Although the study has reported STING-dependent trained immunity contributes to host resistance to C. perfringens infection through the mTOR signaling pathway (48), the biological implications of STING-regulating innate immune response remain poorly understood. Using the gas gangrene model, we elucidated that compared with WT mice, Sting-/- mice were more susceptible to C. perfringens infection, manifested by higher bacterial loads, enhanced inflammatory cell recruitment, increased mortality, and severe damage to the muscle architecture. These results indicate that STING plays a critical protective role against C. perfringens soft tissue infections.

Subsequently, we investigated the immune response mechanisms of STING in defending against C. perfringens infection. We found increased PMN infiltration, elevated KC expression, and attenuated inflammasome signaling activation in the muscle tissue of infected Sting-/- mice relative to infected WT mice. These findings demonstrate that Sting deficiency disrupts inflammatory homeostasis in response to C. perfringens–induced gas gangrene pathogenesis. Notably, we observed that STING was predominantly expressed in inflammatory cells and significantly induced in BMDMs during C. perfringens infection. Therefore, macrophages were further explored as a model for in-vitro studies. Consistent with in-vivo data, we have found that Sting deficiency significantly impaired C. perfringens–induced NLRP3 inflammasome signaling, demonstrated by eliminated Caspase-1, reduced mature IL-1β release, and decreased NLRP3 expression. Indeed, inflammasome signaling is implicated in C. perfringens infections. Although recent evidence shows that PFO exacerbates myonecrosis through NLRP3-dependent signaling (23), our results suggest that MLKL and GPR120 promote bacterial killing and clearance, enhancing NLRP3 inflammasome signaling during C. perfringens infection (12, 25). Meanwhile, our previous report describes that the AIM2 inflammasome contributes to host protection against C. perfringens gas gangrene (12, 22). It is therefore imperative that research explore whether STING-mediated anti-infection is dependent on inflammasome signaling during pathogenic C. perfringens infection. Since IL-18 has been identified as a downstream molecule of the inflammasome signaling activation, and the administration of exogenous IL-18 has been reported to mitigate Salmonella, M. tuberculosis and C. perfringens colonization (12, 49, 50). Exhilaratingly, Sting deficiency impaired the bacterial killing capacity, and rIL-18 treatment could rescue the defect of bacterial-killing in Sting-/- BMDMs. It has been shown that STING protects against C. perfringens infection by enhancing inflammasome activation.

It was reported that STING-mediated type I IFNs are involved in the innate immune response (51). Obviously, evidence has shown that STING-mediated IFNs promote host survival via sensing S. pyogenes–derived c-di-AMP (52, 53). Moreover, STING alleviates P. aeruginosa–induced pulmonary inflammation via cGAS-STING-IFNs signaling (54). In addition to cGAS, IFI204 senses dsDNA during F. novicida infection to elicit STING activation and the type I IFN response (55). Based on these studies, to elucidate the upstream signaling of STING activation during C. perfringens infection, the transcriptome assay was performed. The results show that the expression of Ifi204 was significantly upregulated in infected BMDMs. Simultaneously, we found that Ifi204 deficiency substantially inhibited STING accumulation during C. perfringens infection, suggesting that IFI204 may be involved in STING-mediated host defense. Indeed, studies have shown that IFI204 promotes the STING-IRF3 pathway and enhances bacterial clearance during S. aureus infection (56). Obviously, similarly to Sting-/- macrophages, significant inhibited Caspase-1 activation, reduced ASC speckles formation, and attenuated bacteria-killing capacity were seen in Ifi204-/- macrophages compared with WT macrophages. Based on these findings, it implies that STING-driven NLRP3 signaling is dependent on active IFI204.

As an atypical inflammasome, IFI16 is reported to play a significant role in the innate immune response. Previous studies have shown that the IFI16 inflammasome was activated by Epstein-Barr virus, Kaposi sarcoma-associated herpesvirus, and bovine viral diarrhea virus (57–59). Furthermore, IFI16 can restrict HIV-1 replication via inhibiting the host transcription of Sp1 (60). Nevertheless, little is known about its role during bacterial infection. Subsequently, we further validated the critical defense mechanism of IFI204 in C. perfringens infection. At 24h p.i., similar to Sting-/- mice, Ifi204-/- mice show higher bacterial loads, increased mortality, substantial pathological damage, and dramatically impaired Caspase-1 activation compared with WT mice. Importantly, rIL-18 administration is able to rescue the susceptibility of Ifi204-/- mice. Consistently, exogenous IL-18 treatment could rescue the defect of bacterial killing in Ifi204-/-, Sting-/-, and Nlrp3-/-macrophages. Hence, it emphasizes the essential role of IFI204 in STING-mediated NLRP3 inflammasome signaling for host defense.

In summary, we identify the evidence indicating the significance of STING in an intriguing host-protective innate immune response to restrict gangrenous C. perfringens infection. Acting as a scaffolding hub, STING mediates the IFI204-STING-NLRP3 axis to strengthen bacterial control and host defense. Thus, these findings show that targeting STING may be an option for improving the therapy of pathogenic infectious diseases in the future.

Acknowledgments

We thank all the people who made this work.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the National Natural Science Foundation of China [No. 32302886, No. 32160832], Natural Science Foundation of Inner Mongolia Autonomous Region of China [No. 2025YQ030], Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region of China [No. NJYT22104], Science and Technology Leading Talent Team in Inner Mongolia Autonomous Region of China [No. 2022LJRC0009], and National Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock (Jointly Built by the Province and Ministry) -Identification of Specific Target Points for Important Pathogens in Cattle and Sheep and Development of Novel Diagnostic Technologies [No. 2025KYPT0066].

Edited by: Jagadeesh Bayry, Indian Institute of Technology Palakkad, India

Reviewed by: Hui Wang, Lanzhou University, China

Yang Liu, Qilu Medical University, China

Abbreviations: IFI204, interferon-inducible protein 204; STING, stimulator of interferon genes; NLRP3, NOD-like receptor family pyrin domain-containing 3; IFN, interferon; RIG-I, retinoic acid inducible gene I; TLR2, toll-like receptor 2; TLR4,toll-like receptor 4; AIM2, absent in melanoma 2; GPR120, G protein–coupled receptor 120; MLKL, mixed lineage kinase-like protein; cGAS, cyclic GMP-AMP synthase; DDX41, DEAD-box RNA helicase 41; ZBP1, Z-DNA–binding protein 1; ASC, Apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain; GSDMD, gasdermin D; PFO, perfringolysin O; Sp1, factor Specificity Protein 1.

Data availability statement

All data generated or analyzed during this study are publicly available in recognized repositories. RNA-seg data has been uploaded to the NCBI SRA (PRJNA1280697). C. perfringens strain CP1, a clinical isolate of C. perfringens, specific16S rDNA gene sequence data deposited at NCBI GenBank (MW440585).

Ethics statement

The animal study was approved by Animal Welfare and Research Ethics Committee at Inner Mongolia University. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

M-YZ: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. J-QL: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. QX: Data curation, Formal analysis, Validation, Writing – review & editing. Z-JZ: Data curation, Formal analysis, Validation, Writing – review & editing. JL: Data curation, Validation, Writing – review & editing. A-BH: Data curation, Validation, Writing – review & editing. S-XZ: Data curation, Validation, Writing – review & editing. YZ: Data curation, Validation, Writing – review & editing. XC: Data curation, Validation, Writing – review & editing. Z-YL: Data curation, Validation, Writing – review & editing. PS: Data curation, Validation, Writing – review & editing. YL: Data curation, Validation, Writing – review & editing. S-XY: Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing, Project administration.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2026.1715595/full#supplementary-material

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

DataSheet1.docx (765.2KB, docx)
DataSheet2.docx (203.8KB, docx)

Data Availability Statement

All data generated or analyzed during this study are publicly available in recognized repositories. RNA-seg data has been uploaded to the NCBI SRA (PRJNA1280697). C. perfringens strain CP1, a clinical isolate of C. perfringens, specific16S rDNA gene sequence data deposited at NCBI GenBank (MW440585).

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