Involvement of cGAS/STING Signaling in the Pathogenesis of Candida albicans Keratitis: Insights From Genetic and Pharmacological Approaches

Abstract

Purpose: Fungal keratitis (FK) is an invasive corneal infection associated with significant risk to vision. Although the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) signaling pathway has been recognized for its role in defending against viral infections, its involvement in FK still remains largely unclear. This study sought to elucidate the contribution of the cGAS/STING signaling pathway to the pathogenesis of FK.

Methods: The expression of cGAS/STING signaling components was assessed in a murine model of Candida albicans keratitis through RNA sequencing, western blot analysis, immunofluorescence staining, and real-time PCR. Both genetic (utilizing Sting1^gt/gt mice) and pharmacological (using C176) interventions were employed to inhibit STING activity, allowing for the evaluation of resultant pathogenic alterations in FK using slit-lamp examination, clinical scoring, hematoxylin and eosin (H&E) staining, fungal culture, and RNA sequencing. Subconjunctival administration of the NOD-like receptor protein 3 (NLRP3) inflammasome inhibitor MCC950 was performed to evaluate FK manifestations following STING activity blockade. Furthermore, the impact of the STING agonist diABZI on FK progression was investigated.

Results: Compared to uninfected corneas, those infected with C. albicans exhibited increased expression of cGAS/STING signaling components, as well as its elevated activity. Inhibiting cGAS/STING signaling exacerbated the advancement of FK, as evidenced by elevated clinical scores, augmented fungal load, and heightened inflammatory response, including NLRP3 inflammasome activation and pyroptosis. Pharmacological inhibition of the NLRP3 inflammasome effectively mitigated the exacerbated FK by suppressing STING activity. Conversely, pre-activation of STING exacerbated FK progression compared to the PBS control, characterized by increased fungal burden and reinforced inflammatory infiltration.

Conclusions: This study demonstrates the essential role of the cGAS/STING signaling pathway in FK pathogenesis and highlights the necessity of its proper activation for the host against FK.

Fungal keratitis (FK) presents an intractable ocular infectious disease, often leading to corneal ulcer, perforation, and even permanent blindness.¹ FK incidence ranges widely, from 6% to 56%, across different regions.^2,3 Accumulating evidence points to agricultural ocular trauma, postoperative corneal infections, prolonged use of antibiotics and contact lenses, and misuse of glucocorticoids as primary risk factors for FK.^4–6 Diabetes was also reported to be responsible for a higher incidence of FK with worse prognosis.⁷ Currently, frequent application of topical antifungal drugs (e.g., voriconazole, amphotericin B) is the major clinical strategy for treating FK.⁸ However, the existing antifungal therapies remain limited in efficacy due to issues such as drug insensitivity and resistance,^9,10 compounded by the presence of various ocular barriers.^11,12 Therefore, a comprehensive understanding of the pathogenesis of FK is crucial for the development of novel antifungal strategies. 

The innate immune system serves as the initial defense mechanism against fungal infections.^13,14 Various studies have reported the significance of innate immune cells, such as neutrophils and macrophages, in the pathogenesis of FK.^15–17 Activation of the innate immune system primarily occurs through the recognition of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) by pattern-recognition receptors (PRRs) during fungal infections,¹³ and several PRRs, including C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and Toll-like receptors (TLRs), have been identified as playing pivotal roles in FK pathogenesis.¹⁸ The increased innate immune response often accompanies adaptive immunity, particularly involving Th17 cells and Th1 cells, in combating fungal infections.^14,19,20 However, the inflammatory cascades triggered by innate and adaptive immunity during fungal infections represent a double-edged sword. Although they effectively eliminate fungal pathogens, excessive cytokine production can lead to tissue damage.^21,22 

cGAS/STING signaling, consisting of the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), has been reported to recognize both host and pathogen cytoplasmic DNA, thereby modulating the innate immune response through the TANK-binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) axis and nuclear factor kappa B (NF-κB) p65 activation, respectively (Fig. 1C).^23,24 Numerous studies have also revealed the crosstalk network of this cytosolic DNA-sensing pathway with the NOD-like receptor protein 3 (NLRP3) inflammasome and pyroptosis, contributing to inflammatory cascade response and disease progression.^25–27 Moreover, the cGAS/STING signaling pathway is implicated in various physiological and pathological conditions, including viral infections^28–31 and bacterial infection.^32–35 Additionally, accumulating evidence suggests a close association between cGAS/STING signaling and fungal infections, such as those caused by Candida albicans³⁶ and Aspergillus fumigatus.³⁷ Although the involvement of cGAS/STING signaling in several ocular diseases, such as, ischemia-induced retinopathy,³⁸ Mooren's ulcer,³⁹ and dry eye disease,⁴⁰ has been investigated, its precise role in the pathogenesis of FK remains entirely uncertain. 

Figure 1.

 

FK activates the cGAS/STING signaling pathway. (A) GSEA shows the significant enrichment of major PRR signaling pathways in the corneas of the FK group compared to the Nor group. An enrichment score (ES) > 0 and the ES peak appear in the FK positively correlated region (red area), indicating a significant upregulation of the enriched gene set. (B) Heatmap displaying the DEGs involved in the cytosolic DNA-sensing pathway. Red indicates high expression, and blue denotes low expression within the group. (C) A schematic view of the cGAS/STING signaling pathway. (D) Expression levels of cGAS and STING in corneas, with or without infection, determined by western blotting. Upper panels show western blotting bands, and lower panels depict relative expression quantified by Image J. (E) Immunofluorescence staining revealed the expression of cGAS (green) and STING (green) in corneal samples under different conditions. Nuclear DNA was counterstained using DAPI (blue). Scale bar: 50 µm. (F) Phosphorylation levels of TBK1 and IRF3 assessed by western blotting. Upper panels represent western blotting bands, while lower panels show relative expression analyzed by Image J. (G) Immunofluorescence staining demonstrating phosphorylated TBK1 (green) and IRF3 (green) in corneal samples. Nuclear DNA is indicated by DAPI staining (blue). Scale bar: 50 µm. (H) Quantitative RT-PCR analysis of IFN-β, IL-1β, IL-6, and CXCL10 levels in corneal tissues from different groups. NES, normalized enrichment scores; FDR, false-positive detection rate. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

C. albicans has been reported to be one of the main pathogens of FK,^41,42 and a murine C. albicans keratitis model has been widely used to investigate the pathogenesis and treatment of FK.^43,44 Recently, with the wide use of contact lenses and the abuse of glucocorticoids and antibiotics, C. albicans keratitis has become a worldwide problem, and its incidence is on the rise.⁴⁵ In this study, we established a C. albicans keratitis model to explore the involvement of cGAS/STING signaling in the pathogenesis of FK. Transcriptomic analysis revealed a significant enrichment of the cytosolic DNA–sensing pathway in corneal tissues affected by FK, including Sting1. Through genetic and pharmacological interventions, we demonstrated that blocking STING activity exacerbated FK development, as evidenced by increased fungal burden and heightened inflammatory response. Moreover, targeting the NLRP3 inflammasome with MCC950 significantly reversed the exacerbated FK resulting from STING activity inhibition. Conversely, pharmacological activation of cGAS/STING signaling also worsened FK progression. These findings underscore the nuanced modulatory role of cGAS/STING signaling in FK pathogenesis, whereby both enhancing and inhibiting its activity can promote disease progression. 

Materials and Methods

Animals

Sting1^gt/gt mice with a C57BL/6J background (strain #017537) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA).⁴⁶ The wild-type (WT) adult C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). All of the animals were housed in a specific pathogen-free facility at the Eye Institute of Shandong First Medical University (Qingdao, China). The animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the protocol was approved by the Animal Investigation Committee of the Eye Institute of Shandong First Medical University (approval number 62). 

C. albicans Preparation and Establishment of Murine FK

The standard C. albicans strain CMCC(F)98001 was cultured according to the Shandong Eye Institution Biosafety Code. Briefly, C. albicans was cultured in Sabouraud Dextrose Agar Medium (Thermo Fisher Scientific, Waltham, MA, USA). Single colonies were inoculated in a YPD Broth Medium (Hopebio, Qingdao, China) and cultured for 24 hours in a shaker at 37°C. After washing and centrifugation, the C. albicans was then incubated in Sabouraud Dextrose Broth Medium for 24 hours in a shaker at 37°C. Blastospores were harvested and suspended in phosphate-buffered saline (PBS), and the concentration was adjusted to 1 × 10⁸ CFU/mL for subsequent experiments. 

The FK model was established as per a previous protocol.⁴⁷ Briefly, 6- to 8-week-old female WT and Sting1^gt/gt mice were randomly divided into groups (n = 6/group) and anesthetized using 0.6% pentobarbital sodium anesthetic solution (50 mg/kg) and proparacaine hydrochloride. A suspension of blastospores (3 × 10⁶ CFU/mL, 2 µL) was injected into the corneal stroma using a 33-gauge needle (Hamilton Company, Reno, NV, USA). The sham-infected group received an equivalent volume of PBS. 

Treatment

To determine the effect of cGAS/STING signaling on FK development, the infected mice were subconjunctivally injected with STING inhibitor C176⁴⁸ (8-mM, 5 µL;, MedChem Express, Shanghai, China) or agonist diABZI⁴⁹ (4-µM, 5µL; MedChem Express) on pre-infection day 2 and post-infection day 1, respectively. The same volume of vehicle (10% dimethyl sulfoxide [DMSO], 20% sulfobutylether-β-cyclodextrin [SBE-β-CD], and 70% saline) was used as control. The development of FK was evaluated using a slit lamp (SL-D701; Topcon, Tokyo, Japan) on post-infection days 1, 2, and 3, and the corneal thickness was measured on post-infection day 3 using an optical coherence tomography (OCT) scanner (RTVue XR; Optovue, Fremont, CA, USA). 

To investigate the effect of the NLRP3 inflammasome on FK after treatment with or without the STING inhibitor C176 or the agonist diABZI, NLRP3 inflammasome inhibitor MCC950 (500-µM, 5 µL; MedChem Express) was administered on pre-infection day 2 and post-infection day 1, respectively. A vehicle solution of PBS was administered in the same volume as control. The severity of FK was assessed using slit-lamp examination and OCT imaging, as previously described. 

Clinical Scoring

The severity of FK was assessed daily using slit-lamp examination and was evaluated utilizing a previously established 12-point scoring system.⁵⁰ This scoring system primarily considers three parameters: the extent and density of corneal opacity, as well as surface regularity. Each parameter is scored on a scale of 0 to 4. Scoring of slit-lamp images was conducted twice by three clinicians using a double-blind method based on the scoring criteria, and the mean of the scores was calculated for inclusion in the results. 

Colony Forming Unit Assay

The fungal burden in infected corneas from different groups was quantified by counting colony-forming units (CFUs) on post-infection day 2. Corneal samples were collected and homogenized in 500 µL of PBS. Serial dilutions of 50-µL aliquots were then inoculated onto Sabouraud medium plates in triplicate and incubated at 37°C for 24 hours. The fungal burden in each sample was determined based on the visible fungal colonies and the dilution factor. 

Quantitative Real-Time Polymerase Chain Reaction

On day 3 post-infection, corneal samples (n = 3/group, in pools of 3) were collected, and total RNA was extracted using an isolation kit (TransGen Biotech, Beijing, China). Then, the RNA was reverse-transcribed into cDNA using a cDNA synthesis kit (Vazyme, Nanjing, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on an ABI Prism 7500 system (Applied Biosystems, Waltham, MA, USA) using ChamQ Universal SYBR qPCR Mix (Vazyme). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized as an internal reference, and the relative expression levels were analyzed using the 2^−ΔΔCt method. The primer sequences are listed in Table 1. 

Table 1.

 

Primers Used for Real-Time PCR

 

Immunoblotting

Corneal samples from different groups (n = 3/group, in pools of 3) were harvested on day 3 post-infection and lysed in radioimmunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China) supplemented with 1% phenylmethylsulfonyl fluoride (PMSF) reagent (Solarbio) and phosphatase inhibitors (CWBio, Jiangsu, China). The extracted proteins were subsequently separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). Following blocking with 5% BSA (Solarbio), the PVDF membrane was incubated with primary antibodies and horseradish peroxidase–conjugated goat anti-rabbit antibodies (Immunoway, Jiangsu, China). Target proteins were visualized using an enhanced chemiluminescence reagent kit (EMD Millipore) on a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). Relative quantification was determined by densitometry image analysis using Image J (National Institutes of Health, Bethesda, MD, USA). The primary antibodies used are listed in Table 2. 

Table 2.

 

Antibodies Used for This Study

 

Immunofluorescence Staining

The collected eyeballs on post-infection day 3 were embedded in optimal cutting temperature compound (Sakura, Torrance, CA, USA) and sectioned into 7-µm slices (n = 3/group). After fixation with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and permeabilization with 0.2% Triton X-100, the slices were incubated with primary antibodies at 4°C overnight. Then, the slices were incubated with Invitrogen Alexa Fluor 488–conjugated Goat anti-Rabbit IgG or Alexa Fluor 594–conjugated Goat anti-Rabbit IgG secondary antibodies (Thermo Fisher Scientific), and FITC anti-Mouse CD3, FITC anti-Mouse F4/80, or FITC anti-Mouse Ly-6G/Ly-6C (Gr-1) antibodies at room temperature in the dark for 2 hours. Then, 4′,6-diamidino-2-phenylindole (DAPI) was used to counterstain the nucleus. Images were captured using an Echo Revolve fluorescence microscope (Echo, San Diego, CA, USA). The primary antibodies used are listed in Table 2. 

Hematoxylin and Eosin Staining

To assess the pathological changes in the corneas of the different groups, hematoxylin and eosin (H&E) staining was performed. Briefly, on day 3 post-infection, the collected eyeballs and corneas from the different groups (n = 5/group) were fixed and embedded in paraffin, followed by sectioning with an ultramicrotome. The corneal slides were then H&E stained and observed using an optical microscope (Eclipse E800; Nikon, Tokyo, Japan). 

RNA Sequencing and Analysis

To comprehensively analyze the pathological alterations in FK corneas with the different treatments, RNA sequencing (RNA-seq) and analysis were conducted by OE Biotech (Qingdao, China). Briefly, total RNA from the samples was extracted using TRIzol. RNA purity and quantification were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and RNA integrity was evaluated using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Following library construction, RNA-seq was performed on the Illumina NovaSeq 6000 platform (Thermo Fisher Scientific), generating paired-end reads. Clean reads were mapped to the reference genome using the HISAT2 program. Fragments per kilobase of transcript per million mapped reads (FPKM) values were calculated for each gene, and read counts for each gene were obtained using htseq-count. 

Principal component analysis (PCA) was conducted using R 3.2.0 to assess sample clustering. Differentially expressed genes (DEGs) were identified using DESeq2, with a threshold of q < 0.05 and fold change > 2 or fold change < 0.5. Gene Ontology (GO) analysis and Gene Set Enrichment Analysis (GSEA) were performed to elucidate the function of DEGs in FK progression. GSEA was utilized to determine whether predefined biological processes were enriched, with enriched pathways sorted based on their normalized enrichment scores (NES), satisfying |NES| > 1, P < 0.05, and false discovery rate < 0.25 as filtering criteria. The corresponding signal-to-noise ratio for each gene is represented as a gray area. 

Statistical Analysis

Each experiment was conducted independently at least three times, and the data are presented as mean ± standard deviation (SD). Statistical analysis was performed using an unpaired two-tailed Student's t-test to compare differences between two groups. For comparisons involving multiple groups, one-way analysis of variance (ANOVA) with Bonferroni post hoc corrections was applied. P < 0.05 was considered statistically significant. 

Results

Increased cGAS/STING Signaling Activity During FK Development

To better understand the pathogenesis of FK, we performed RNA-seq analysis. PCA revealed notable differences in gene expression profiles between infected corneas (FK) and uninfected corneas (Nor) (Supplementary Fig. S1A). Following rigorous quality control procedures, a total of 4663 DEGs were identified in FK corneas, comprised of 1680 downregulated genes and 2983 upregulated genes (Supplementary Fig. S1B). GSEA revealed significant enrichment of several PRR signaling pathways in FK corneas, including the CLR signaling pathway, NLR signaling pathway, TLR signaling pathway, and cytosolic DNA-sensing pathway (Fig. 1A). Notably, these pathways have been extensively implicated in FK, particularly the TLR signaling, CLR signaling, and NLR signaling pathways.¹⁶ 

Subsequently, we focused on addressing the role of the cytosolic DNA-sensing pathway in FK development. A heatmap depicted a cluster of DEGs associated with this signaling pathway, notably including Sting1 (Fig. 1B), suggesting the potential involvement of cGAS/STING signaling in FK progression. The major components of cGAS/STING signaling in infected corneal tissues were analyzed, including cGAS, STING, phosphorylated TBK1, and IRF3 (Fig. 1C). Immunoblotting analysis revealed higher expression levels of cGAS and STING in infected corneas compared to untreated corneas (Fig. 1D). Immunofluorescence staining further confirmed the elevated expression of cGAS and STING in infected samples, predominantly localized in the corneal epithelium and infiltrated immune cells (Fig. 1E). Additionally, increased phosphorylation of TBK1 and IRF3 was observed in infected corneas compared to untreated samples, as determined by western blotting (Fig. 1F), with a similar pattern observed in immunofluorescence staining, particularly evident in corneal epithelial cells and infiltrated immune cells (Fig. 1G). Double-immunofluorescence staining revealed the co-localization of STING with various immune cells, including F4/80⁺ macrophages, Ly6G⁺ neutrophils, and CD3⁺ lymphocytes (Supplementary Fig. S2A), suggesting the involvement of these cells in the STING signaling pathway. Moreover, several downstream genes of the cGAS/STING signaling pathway exhibited higher expression levels in infected corneas compared to untreated corneas, including interferon beta (IFN-β), interleukin-1β (IL-1β), IL-6, and C-X-C motif chemokine ligand 10 (CXCL10) (Fig. 1H), indicating increased activity of the cGAS/STING signaling pathway in FK. 

Blocking cGAS/STING Signaling Exacerbated the Development of FK

To investigate the role of cGAS/STING signaling in the pathogenesis of FK, we employed a pharmacological approach to examine the effect of cGAS/STING signaling on FK. Subconjunctival injection of C176 was utilized to inhibit STING activity.⁵¹ Comparative analysis revealed that infected corneas treated with C176 exhibited more severe keratitis symptoms compared to vehicle-treated infected corneas, characterized by increased opacity and more pronounced corneal ulceration (Fig. 2A). Correspondingly, clinical scores based on clinical symptoms were higher in C176-treated infected corneas compared to vehicle-treated infected corneas (Fig. 2B). Histopathological analysis revealed significant loss of corneal architecture and increased inflammatory infiltration in C176-treated infected corneas (Fig. 2C). OCT examination demonstrated greater increases in corneal thickness in C176-treated infected corneas compared to vehicle-treated infected corneas (Figs. 2C, 2D). Importantly, C176-treated infected corneas exhibited a significantly higher fungal burden than vehicle-treated infected corneas (Fig. 2E). Furthermore, mRNA levels of pro-inflammatory cytokines, including IFN-β, IL-1β, IL-6, and CXCL10, were significantly elevated in C176-treated infected corneas compared to vehicle-treated infected corneas (Fig. 2F). These findings collectively indicate that cGAS/STING signaling is essential for controlling FK. 

Figure 2.

 

Pharmacologically blocking STING activity aggravated FK progression. (A) Slit-lamp examination of infected corneas with or without C176 treatment. (B) Clinical scores depicting the severity of FK in corneas subjected to different treatments. (C) Representative H&E images and OCT photographs of corneas from various treatment groups. Upper panel shows H&E images, and the lower panel shows OCT photographs. Scale bar: 50 µm. (D) Corneal thickness measured by OCT across different groups. (E) Fungal burden assessed by CFU counting. (F) Transcriptional levels of IFN-β, IL-1β, IL-6, and CXCL10 in different groups determined by qRT-PCR. Vehicle+C. albicans, infected corneas group; C176+C. albicans, infected corneas following C176 treatment group. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

To further determine the effect of cGAS/STING signaling on the pathogenesis of FK, we utilized Sting1^gt/gt mice to establish the FK model and monitored them using slit-lamp examination at 1, 2, and 3 days post-infection (dpi). Although infected WT mice exhibited symptoms of corneal opacity and stromal edema (Fig. 3A), Sting1^gt/gt mice displayed more severe keratitis symptoms, characterized by larger and denser gray–white infiltration, as well as visible corneal ulceration (Fig. 3A). Consistent with these observations, clinical scores in infected Sting1^gt/gt mice were significantly higher than in infected WT mice at 1, 2, and 3 dpi, respectively (Fig. 3B). On post-infection day 2, Sting1^gt/gt mice showed a significantly higher fungal burden compared to infected WT mice (Fig. 3C). Moreover, the phosphorylation levels of TBK1 and IRF3 in infected Sting1^gt/gt corneas were notably lower than in infected WT samples (Fig. 3D), suggesting decreased cGAS/STING signaling activity in Sting1^gt/gt corneas following infection. Collectively, the results obtained from the genetic approach support the conclusions drawn from the pharmacological approach. 

Figure 3.

 

Sting1^gt/gt mice showed the exacerbated FK. (A) Representative slit-lamp images showing the corneas of WT and Sting1^gt/gt mice post-infection with C. albicans. (B) Clinical scores indicating the severity of FK in WT and Sting1^gt/gt mice. (C) Fungal burdens assessed in infected corneas across different groups, quantified by CFUs. (D) Phosphorylation levels of TBK1 and IRF3 in infected corneal samples among different groups as determined by western blotting. The right panels display western blotting bands, and the left panels depict relative expression analyzed by Image J. WT+C. albicans, WT mice infected with C. albicans; Sting1^gt/gt+C. albicans, Sting1^gt/gt mice infected with C. albicans. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Transcriptomic Signatures Revealed the Elevated Inflammatory Cascades in FK After C176 Administration

To comprehensively characterize the global pathological alterations in FK samples in the presence of C176, we conducted RNA-seq analysis. Comparative analysis revealed a distinctly different gene expression pattern in corneal samples treated with C176 compared to FK samples (Supplementary Fig. S3A). In C176-treated samples, 5906 DEGs were identified, including 2064 upregulated genes and 3842 downregulated genes (Supplementary Fig. S3B). Biological process (BP) analysis of the top 10 GO terms associated with upregulated DEGs revealed enrichment of inflammation- and immunity-related pathways, including inflammatory response, immune response, positive regulation of IL-6 production, IL-1β production, and cellular response to IL-17 (Fig. 4A). Cellular component (CC) analysis indicated enrichment of various complexes related to inflammation and immunity among the upregulated genes, such as the NLRP1 inflammasome complex, mast cell granule, NLRP3 inflammasome complex, and secretory granule (Fig. 4B). Molecular function (MF) analysis revealed that the top 10 terms associated with upregulated genes were closely linked to inflammation and immune response, including cytokine activity, lipopolysaccharide binding, and chemokine activity (Fig. 4C). 

Figure 4.

 

Transcriptomic analysis revealed significant pathological alterations in infected corneas in the presence of C176. (A–C) GO analysis of upregulated DEGs in infected corneas after C176 treatment, including enriched BP terms (A), CC terms (B), and MF terms (C). (D) Enrichment of BPs in the downregulated DEGs of infected corneas after C176 treatment. (E) Heatmap displaying DEGs related to positive regulation of IFN-β production. (F) GSEA revealed significant enrichment in positive regulation of IFN-β production and cellular response to IFN-β in the C176 group compared to the FK group. The ES < 0 and ES peaks appear in the C176 negative correlation region (blue area), indicating significant downregulation of the enriched gene set. In E and F, FK indicates the Vehicle+C.albicans group, and C176 represents the C176+C. albicans group.

In contrast, the BPs enriched among the downregulated DEGs in infected corneal samples after C176 treatment were functionally associated with IFN signaling, particularly cellular response to IFN-β, positive regulation of IFN-β production, response to type I IFN, and positive regulation of the type I interferon-mediated signaling pathway (Fig. 4D). We have depicted the genes implicated in the positive regulation of IFN-β production in a heatmap (Fig. 4E). Additionally, GSEA revealed a significant downregulation of positive regulation of IFN-β production and cellular response to IFN-β following C176 intervention (Fig. 4F). These findings underscore the detrimental outcomes of FK after inhibiting STING activity. 

C176 Administration Fueled FK Through Activation of the NLRP3 Inflammasome

Although our findings demonstrated that blocking cGAS/STING signaling exacerbates the progression of FK, the underlying mechanism remains unclear. Previous studies have suggested that type I IFN inhibits inflammasome activation and IL-1β production,^52,53 implying potential hyperactivation of the inflammasome following cGAS/STING signaling inhibition. GSEA revealed a significant enrichment of pyroptosis in infected corneal tissues following C176 intervention (Fig. 5A). The heatmap illustrates pyroptosis-related DEGs in corneal samples after C176 treatment, notably including NLRP3, Casp1, Pycard, and Gsdma (Fig. 5B). Furthermore, western blot analysis showed upregulated expression of NLRP3 and the bioactivated form of Casp1, gasdermin D (GSDMD), and IL-1β (Fig. 5C), indicating elevated NLRP3 inflammasome activity in C176-treated FK corneas. These findings suggest a potential mechanism whereby inhibition of cGAS/STING signaling promotes inflammasome activation and pyroptosis, contributing to the exacerbation of FK. 

Figure 5.

 

Blocking the NLRP3 inflammasome alleviated the severity of FK caused by C176 treatment. (A) GSEA demonstrated the significant enrichment of pyroptosis. ES peaks appeared in the C176-positive correlation region (red area), indicating that the enriched gene set was significantly upregulated in the C176 group of corneas compared to the FK group. (B) Heatmap displaying representative DEGs related to pyroptosis. In A and B, FK designates the Vehicle+ C. albicans group, and C176 represents the C176+C. albicans group. (C) Protein levels of NLRP3, Casp-1 (bioactive form), GSDMD (bioactive form), and IL-1β (bioactive form) in infected corneal samples after C176 intervention determined by western blot. The upper panel shows western blotting bands, and the lower panel shows the relative expression quantified by Image J. (D) Representative slit-lamp images of FK corneas receiving the different treatments. (E) Clinical scores demonstrating alleviated FK symptoms after co-treatment with C176 and MCC950. (F) Representative OCT images of corneas from the different treatment groups. (G) Corneal thickness quantified by OCT. Nor, uninfected group; Vehicle+C. albicans, infected group; C176+C. albicans, infected group following C176 treatment; C176+MCC950+C. albicans, infected group following C176 and MCC950 treatment. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

To determine the role of the NLRP3 inflammasome in the pathogenesis of C176-treated infected corneas, we utilized MCC950 to inhibit NLRP3 inflammasome signaling. Compared with the FK group, MCC950 treatment alone resulted in more severe FK pathology, as evidenced by higher clinical scores and fungal loads (Supplementary Fig. S4). These results suggest the necessity of NLRP3 inflammasome activation for fungal clearance. In contrast, compared with the C176 group, pharmacological targeting of the NLRP3 inflammasome by MCC950 significantly ameliorated the exacerbated FK symptoms induced by C176 (Fig. 5D). This was characterized by reduced corneal opacity and edema, as well as decreased clinical scores (Fig. 5E). Additionally, OCT analysis revealed a much thinner corneal thickness in the C176+MCC950 group compared to the C176 group (Figs. 5F, 5G). Taken together, these findings suggest that the severity of FK following C176 treatment is at least partially attributed to the hyperactivation of the NLRP3 inflammasome. 

Overactivation of cGAS/STING Signaling Promoted FK Progression

Although we have explored the impact of inhibiting cGAS/STING signaling on FK, the role of hyperactivation of cGAS/STING signaling in FK development remains unclear. To address this question, we utilized the STING agonist diABZI. Surprisingly, compared with infected corneas treated with PBS, corneas treated with diABZI displayed more severe symptoms, characterized by increased opacity and higher clinical scores (Figs. 6A, 6B). Histological analysis via H&E staining revealed a significant loss of corneal integrity and increased infiltration of immune cells into corneas in the diABZI-treated group compared to the PBS-treated group (Fig. 6C). OCT analysis also indicated thicker corneas in the diABZI-treated group than in the PBS-treated group (Figs. 6C, 6D). Moreover, corneas treated with diABZI exhibited significantly lower fungal clearance ability, as evidenced by higher CFU loads compared to PBS-treated corneas (Fig. 6E). Additionally, the transcriptional levels of pro-inflammatory cytokines (IFN-β, IL-1β, IL-6, and CXCL10) were significantly increased in diABZI-treated infected corneas compared to PBS-treated samples (Fig. 6F). In summary, these results demonstrate that hyperactivation of cGAS/STING signaling exacerbates FK and decreases the fungicidal potential of corneas. 

Figure 6.

 

Over-activation of cGAS/STING signaling using diABZI promoted FK progression. (A) Representative slit-lamp images of corneas from different groups. (B) Clinical scores indicating more severe FK symptoms in the diABZI-treated group than in the untreated group. (C) Representative H&E images and OCT scans of corneas from different groups. The upper panel shows H&E images; the lower panel shows OCT photographs. Scale bar: 50 µm. (D) Corneal thickness measured by OCT in the different groups. (E) Fungal burden quantified by counting CFUs. (F) Transcriptional levels of IFN-β, IL-1β, IL-6, and CXCL10 in the different groups as determined by qRT-PCR. Nor, uninfected group; PBS+C. albicans, infected group; diABZI+C. albicans, infected group with diABZI treatment. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Next, we investigated whether the overactivation of STING by diABZI during fungal infection affected NLRP3 inflammasome activity. Immunoblotting revealed the upregulation of NLRP3 expression and the bioactivated forms of Casp-1, GSDMD, and IL-1β in the corneas of the diABZI-treated group of mouse corneas (Supplementary Fig. S5A), indicating elevated NLRP3 inflammasome activity in the corneas of diABZI-treated FK mice. Subsequently, we assessed the pathogenesis of diABZI-treated FK by blocking the NLRP3 inflammasome with MCC950. Interestingly, the combination of MCC950 and diABZI resulted in more severe keratitis symptoms, as evidenced by higher clinical scores and fungal loads (Supplementary Fig. S5). These findings contrasted with the results obtained from FK mice co-treated with C176 and MCC950. 

Discussion

The role of cGAS/STING signaling is well documented in combating viruses and cancer by inducing IFN-I production and inflammation.^54,55 Utilizing a murine C. albicans keratitis model, we observed an upregulation of cGAS/STING signaling components along with heightened activity. Inhibiting cGAS/STING signaling by targeting STING exacerbated FK progression, mechanistically linked to NLRP3 inflammasome activation. Conversely, activating cGAS/STING signaling by targeting STING also worsened FK development, leading to increased fungal burden. Overall, our findings underscore the importance of proper cGAS/STING signaling activation in mitigating FK progression. 

As one of the most important components of the innate immunity system, the cGAS/STING signaling axis detects both exogenous and endogenous DNA, initiating an innate immune response characterized by IFN-I production via IRF3 phosphorylation and pro-inflammatory cytokine release through NF-κB activation.^29,56 This signaling pathway plays vital roles in various physiological and pathological processes, including viral infection,^28,29 autoimmune diseases,^56,57 and cellular senescence.^58,59 Recent evidence has also implicated cGAS and STING in the pathogenesis of fungal infections, yielding differing outcomes.^36,37,60 Our study revealed that both inhibiting and activating STING activity exacerbated C. albicans keratitis, contrary to previous findings involving an Aspergillus fumigatus keratitis model targeting cGAS activity.⁶⁰ We hypothesize that such disparity could be attributed to differences in fungal pathogens and target proteins. 

The primary function of cGAS/STING signaling is the induction of IFN-I production, which has found broad applications in antiviral and antitumor therapies.^29,56,61 Our findings demonstrated that genetic or pharmacological blockade of STING activity exacerbated C. albicans keratitis, whereas targeting the NLRP3 inflammasome with MCC950 significantly ameliorated clinical and pathological manifestations. Gringhuis et al.⁶² have shown that the IFN-I response is crucial for the development of non-pathogenic TH17 cells during fungal infections. Although non-pathogenic TH17 cells contribute to anti-infection responses, pathogenic TH17 cells can lead to tissue inflammation and damage.^63–65 Additionally, several studies have highlighted the importance of Dectin-induced IFN-β production in combating C. albicans infections.^66,67 These results suggest that the IFN-I response contributes to fungal clearance. Furthermore, IFNs-I have been reported to inhibit IL-1β production and inflammasome activity,^52,53 indicating that the absence of IFNs-I may lead to heightened inflammation mediated by the NLRP3 inflammasome. These findings likely explain the exacerbated C. albicans keratitis observed after STING inhibition, although further investigations are necessary to elucidate the exact pathogenic mechanisms. 

Even more surprisingly, a significantly more severe FK phenotype was observed following the hyperactivation of STING using the agonist diABZI, accompanied by increased fungal burden and heightened inflammatory infiltration. Despite the protective role of IFN-I signaling in fungal infections, it has also been reported to have detrimental effects. Majer et al.⁶⁸ demonstrated that the IFN-I response accelerated lethal immunopathology via inflammatory monocytes and neutrophils in C. albicans–infected Ifnar1^−/− mice. IFN-I not only disrupted host iron homeostasis and promoted Candida glabrata infection and immune evasion,^69,70 but also impaired protective IFN-γ signaling and IL-1 signaling, leading to increased susceptibility to tuberculosis.^71,72 Therefore, these findings suggest a possible explanation for the exacerbated C. albicans keratitis observed upon activation of STING activity. Nonetheless, further investigations are required to fully elucidate these phenomena. 

NLRP3 inflammasome–mediated inflammation and pyroptosis have been reported to be closely linked with the pathogenesis of FK. Blocking the NLRP3 inflammasome and pyroptosis was proven to be beneficial for treating FK.^73–75 Some evidence has also demonstrated the protective role of the NLRP3 inflammasome in pathogen clearance during FK developmemt,⁷⁶ which supports our findings of the aggravated FK in the C. albicans infection model by inhibiting the NLRP3 inflammasome using MCC950. However, in the context of C. albicans infection, much greater NLRP3 inflammasome activity under STING inhibition or activation conditions was observed compared to without STING interventions. Even more surprisingly, blocking NLRP3 inflammasome activity under these two scenarios resulted in completely different outcomes for FK. These findings suggest a complex interaction network of cGAS/STING signaling with the NLRP3 inflammasome and pyroptosis. Growing evidence indicates that cGAS/STING signaling promotes activation of the NLRP3 inflammasome and pyroptosis, amplifying the inflammatory cascade and ultimately involving the pathological process of infectious, inflammatory, and autoimmune diseases.^26,27,77,78 However, crosstalk of cGAS/STING signaling with the NLRP3 inflammasome and pyroptosis under FK condition has not been fully explored, nor has the underlying mechanism. 

Several potential concerns should be addressed in future studies. First, although we have elucidated the critical roles of cGAS/STING signaling in the pathogenesis of C. albicans keratitis by targeting STING, further genetic approaches are necessary to determine the specific cell types influencing FK development—corneal cells or myeloid cells? Additionally, detailed investigations are required to understand the crosstalk of cGAS/STING signaling with the NLRP3 inflammasome and pyroptosis during FK progression, as well as the underlying mechanism. Moreover, in addition to the canonical STING/IRF3 signaling axis, STING has also been implicated in negatively regulating antifungal immunity and restraining the Th17 pathogenic program in an IFN-I–independent manner.^36,79 Therefore, the pivotal role of STING in FK progression via an IFN-I–independent mechanism warrants exploration in future studies. 

In conclusion, our findings highlight the essential role of cGAS/STING signaling in the host response to fungal infection. Through genetic and pharmacological interventions targeting STING activity, we have demonstrated that the delicate balance of cGAS/STING signaling is essential for protecting against C. albicans keratitis (Fig. 7). Collectively, these findings contribute to our understanding of the host cGAS/STING signaling axis in the pathogenesis of FK. 

Figure 7.

 

Schematic representation of the function of STING in fungal keratitis in C. albicans infection. C. albicans induces an increase of STING signaling expression and activity in FK corneas. Inhibition or overactivation of STING exacerbates the development of FK.

Acknowledgments

The authors thank OE Biotech (Qingdao) for assistance in the RNA-seq analysis. 

Supported by grants from the Natural Science Foundation of Shandong Province (ZR2020MH175), Taishan Scholars Program (202103185), Academic Promotion Programme of Shandong First Medical University (2019ZL001 and 2019PT002), and the Innovation Project of Shandong Academy of Medical Sciences. 

Data Availability: The data that support the findings of this study are available from the corresponding author upon reasonable request. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (GSA) of China National Center for Bioinformation (CRA014057; https://ngdc.cncb.ac.cn/gsa). 

Disclosure: S. Lyu, None; T. Zhang, None; P. Peng, None; D. Cao, None; L. Ma, None; Y. Yu None; Y. Dong, None; X. Qi, None; C. Wei, None