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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 Sting1gt/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 is a relatively common ocular disease requiring positive medical management combined with surgical intervention. Interleukin-17 (IL-17) was reported to promote the activation and mobilization of neutrophile granulocyte to foci of inflammation. This study investigated the effect of IL-17 production from Th17 cells on the progression of fungal keratitis. A mouse model of fungal keratitis induced by Candida albicans was successfully constructed to detect infiltration of inflammatory cells in corneal tissues by hematoxylin-eosin (HE) staining and immunohistochemistry. Fungal load capacity of mouse cornea was also detected. The regulatory role of IL-17 in fungal keratitis with the involvement of CX43 was investigated with the relevant expression of inflammatory factors detected and activation of vascular endothelial cells assessed. Furthermore, in vivo experiment was also performed to confirm the role of CX43 in keratitis. Mice with fungal keratitis showed increased level of inflammatory cytokines and infiltration of inflammatory cells. Silencing IL-17 in Th17 cells and overexpressing CX43 could inhibit the activation of vascular endothelial cells. Besides, CX43 knockdown in vivo alleviated fungal keratitis in mice. The possible mechanism of the above findings could be IL-17 inhibiting the level of CX43 through the AKT signaling pathway. Taken together, IL-17 could inhibit the occurrence and development of fungal keratitis by suppressing CX43 expression through the AKT signaling pathway. Therefore, this study provides a potential target for the treatment of fungal keratitis.

Purpose: ISG15, a di-ubiquitin-like protein, is critical for controlling certain viral and bacterial infections. We sought to determine if ISG15 plays a role in corneal innate immunity against Candida albicans (C. albicans) using a C57BL/6 (B6) mouse model of human fungal keratitis., Methods: Scarified corneas of adult B6 mice were pretreated with TLR5 ligand flagellin and then inoculated with C. albicans. The expression of ISG15 and other genes involved in ISG15 conjugation (ISGylation) was determined by real-time PCR. ISG15 expression and distribution in infected corneas were assessed by immunohistochemistry. ISGylation was examined by Western blotting. siRNA knockdown and recombinant ISG15 were used to elucidate the effects of ISG15 on controlling fungal keratitis by clinical scoring, fungal number plate counting, ELISA cytokine determination, and polymorphonuclear leukocytes (PMN) infiltration measurement., Results: Heat-killed C. albicans induced expression of ISG15, and hBD2 was markedly enhanced by flagellin-pretreatment in cultured human primary corneal epithelial cells (CECs). In vivo, C. albicans infection induced the expression of ISG15, ISGylation-associated genes (UBE1L, UBCH8, and HERC5), and ISGylation in mouse CECs, all of which were enhanced by flagellin-pretreatment. siRNA knockdown of ISG15 increased keratitis severity, dampened flagellin-induced protection, and greatly suppressed the expressions of ISGylation enzymes, IFN-γ, but not CXCL2 in B6 mouse CECs. Recombinant ISG15, on the other hand, enhanced corneal innate immunity against C. albicans and suppressed infection-induced IL-1β, but not IL-Ra expression. ISG15 alone induced the expression of IL-1Ra, CXCL10, and CRAMP in mouse CECs. ISG15 was upregulated and secreted in cultured human CECs in response to challenge in a type 1 IFN-dependent manner., Conclusions: Our data, for the first time, demonstrate that ISG15 acts as an immunomodulator in the cornea and plays a critical role in controlling fungal keratitis.

Purpose: This study was conducted to investigate whether flagellin, the sole ligand of Toll-like receptor-5 (TLR5), induces an innate defense that is sufficient to protect injured corneas from Candida albicans., Methods: Scarified corneas of adult B6, TLR5(-/-), Camp(-/-) (cathelicidin-related antimicrobial peptide), or PMN-depleted mice were pretreated with Pseudomonas aeruginosa flagellin or a mutant and then were inoculated with C. albicans. The corneas were compared for disease progression, cytokine and Camp expression, and PMN infiltration before and after C. albicans infection. Disease progress was recorded by digital photography and clinical scoring, cytokine levels were determined by ELISA, the levels of Camp gene product were assessed by Western blot, and PMN infiltration was measured by MPO determination and immunohistochemistry., Results: Topical application of flagellin induced profound protection against Candida keratitis in a TLR5-dependent manner. The improved disease outcome including reduced tissue inflammation and rapid functional recovery can be attributed to a marked decrease in fungal burden at the early stage of C. albicans infection in flagellin-exposed B6 mouse corneas. Although both PMN infiltration and Camp upregulation contributed to corneal innate defense against fungal infection, Camp ablation totally, and PMN depletion partially, abrogated flagellin-induced fungal clearance in B6 mouse corneas., Conclusions: Flagellin induces a strong innate defense and promotes robust resistance to C. albicans infection in the cornea. Topical flagellin or its mimetic may become a new prophylactic agent for preventing contact lens or trauma/injury-associated microbial keratitis.

Unlabelled: PURPOSE/AIM OF STUDY: To investigate the expression of endogenous antimicrobial peptides within the murine cornea during the onset and progression of posttraumatic keratomycosis caused by Candida albicans., Materials and Methods: Scarified corneas of BALB/c mice were topically inoculated with C. albicans and monitored for one week. A murine gene microarray compared the relative expression of 36 antimicrobial peptide genes in infected corneas to controls. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) determined gene expression levels for murine cathelicidin and β-defensins in normal corneas, scarified corneas, and C. albicans-infected corneas. Immunofluorescent staining localized the expression of cathelicidin in corneal sections., Results: Traumatized eyes exposed to C. albicans developed progressive corneal inflammation, with a fungal inoculum of 10(6) colony-forming units (CFU) bringing about significantly (P < 0.05) more severe corneal inflammatory disease than a 10(5) CFU inoculum. Camp, encoding a murine cathelicidin-related antimicrobial peptide, was significantly upregulated 45-fold by microarray (P = 0.0007) and 36-fold by real-time RT-PCR (P = 0.0009). Camp increased significantly (P = 0.002) more in corneas receiving the higher than the lower fungal inoculum. Cathelicidin was preferentially expressed within the stroma on the first day after fungal inoculation, and Camp expression progressively declined over one week as the amount of recoverable fungi decreased. The genetic expression of β-defensin 1 and β-defensin 2 was initially downregulated (P ≤ 0.01) at the onset of fungal keratitis then returned toward normal levels., Conclusions: The antimicrobial peptide cathelicidin rapidly increases within the inflamed murine corneal stroma after the initiation of fungal keratitis and may play a role in the host responses that follow corneal trauma and infection.

Purpose: To investigate the expression of members of the small leucine-rich proteoglycan family and related leucine-rich repeat proteins during the inception and progression of experimental keratomycosis., Methods: Scarified corneas of BALB/c mice were topically inoculated with Candida albicans and monitored daily over 1 week for corneal opacification. A murine gene microarray compared infected corneas to controls 1 day postinoculation (PI). Real-time reverse transcriptase polymerase chain reaction determined small leucine-rich proteoglycan gene levels in infected and mock-infected corneas at 1, 3, and 7 days PI and in normal corneas. Immunostaining localized keratocan protein in murine corneas., Results: Eyes with C. albicans keratitis rapidly developed corneal inflammation with opacification. Microarray showed that genes for biglycan, asporin, lumican, fibromodulin, osteomodulin, keratocan, osteoglycin, and chondroadherin were significantly (P < 0.01) downregulated more than 2-fold at the onset of fungal keratitis. By real-time reverse transcriptase polymerase chain reaction, the gene encoding keratocan was initially downregulated 137-fold and remained downregulated 2.5-fold at 1 week. Genes coding for lumican, osteomodulin, and fibromodulin were downregulated 4- to 9-fold 1 day after fungal inoculation and returned to normal levels by 3 days PI. Immunofluorescence demonstrated that keratocan was present throughout the corneal stroma of normal mice and mock-infected controls but was markedly less during early fungal keratitis., Conclusions: Transcriptional levels of keratocan and other proteoglycans decrease during the initial stages of C. albicans keratitis. Alterations in the stromal extracellular matrix may contribute to the acute inflammatory response of corneal infection.

Chemotactic cytokines mediate the recruitment of leukocytes into infected tissues. This study investigated the profile of chemokines during experimental Candida albicans keratitis and determined the effects of chemokine inhibition on leukocyte infiltration and fungal growth during murine keratomycosis. Scarified corneas of BALB/c mice were topically inoculated with C. albicans and monitored daily over one week for fungal keratitis. After a gene microarray for murine chemokines compared infected corneas to controls, real-time reverse transcription polymerase chain reaction (RT-PCR) and immunostaining assessed chemokine expression in infected and mock-inoculated corneas. An anti-chemokine antibody was then administered subconjunctivally and evaluated for effects on clinical severity, corneal inflammation, fungal recovery, and cytokine expression. Of 33 chemokine genes examined by microarray, 6 CC chemokines and 6 CXC chemokines were significantly (P<0.05) upregulated more than two-fold. Chemokine (CC-motif) ligand 3 (CCL3) was upregulated 108-fold (P=0.03) by real-time RT-PCR within one day after fungal inoculation and remained increased 28-fold (P=0.02) at one week, and its in situ expression increased in the epithelium and stroma of infected corneas. Compared to the control antibody-treated group, eyes treated with anti-CCL3 antibody showed reduced clinical severity (P<0.05), less corneal neovascularization (P=0.02), and fewer inflammatory cells infiltrating corneal tissue, but the amount of recoverable fungi was not significantly (P=0.4) affected. Anti-CCL3 treatment significantly (P=0.01) reduced the expression of tumor necrosis factor and interleukin-1beta in infected corneas. These results indicate that chemokines, especially the CC chemokine CCL3, play important roles in the acute inflammatory response to C. albicans corneal infection.

The aim of this study was to determine the retinal toxicity and intraocular pharmacokinetics of vitrectomy with fluconazole infusion in rabbit eyes and to study its efficacy in the treatment of experimental candidal endophthalmitis. The right eyes of 13 New Zealand White (NZW) rabbits were vitrectomized and infused with 20 mL of 0.2 mg/mL, 1 mg/mL, or 2 mg/mL of fluconazole. An electroretinogram (ERG) was performed on both eyes of each rabbit at different time points. The right eyes of 26 different NZW rabbits were vitrectomized and infused with 20 mL of 2 mg/mL of fluconazole. These rabbits were sacrificed, and their right eyes were enucleated at hours 2, 4, 8, and 24 after the operation, and the concentration of fluconazole in the vitreous was measured by highpressure liquid chromatography. Experimental candidal endophthalmitis was induced in the right eye of 42 other NZW rabbits. Twenty-six (26) of the eyes were then vitrectomized and infused with 20 mL of 2 mg/mL of fluconazole, and the other 16 rabbits served as control. Severity of ocular infection was graded from 0-4 at different time intervals, using an indirect ophthalmoscope. In the first group, ERG showed no significant difference between the experimental eyes and the control eyes--in all concentrations of fluconazole--for up to 3 months. In the second group, the intraocular concentration of fluconazole declined so rapidly that, as of 8 hours after operation, there was none in the vitreous cavity. In the third group, significantly less vitreous opacity was found in the treated eyes on days 3 and 6. However, the difference ceased to be apparent on day 15. Our study suggests that there is no retinal toxicity resulting from vitrectomy with a 2 mg/mL fluconazole infusion, and that it is temporally effective in the treatment of experimental candidal endophthalmitis.