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NLRP3 Inflammasome-Mediated Pyroptosis Pathway Contributes to the Pathogenesis of Candida albicans Keratitis
Huifang Lian1,2,3,4, XiaoLong Fang2,4,5, Qingyu Li1,2
1Clinical College of Ophthalmology, Tianjin Medical University, Tianjin, China.
Purpose:
Fungal keratitis is a sight-threatening corneal infection caused by fungal pathogens, and the pathogenic mechanisms have not been fully elucidated. The aim of this study was to determine whether NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated pyroptosis contributes to Candida albicans (C. albicans) keratitis and explore the underlying mechanism.
Methods:
An in vivo mouse model of C. albicans keratitis and an in vitro culture model of human corneal epithelial cells (HCECs) challenged with heat-killed C. albicans (HKCA) were established in this study. The degree of corneal infection was evaluated by clinical scoring. Gene expression was assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis or immunofluorescence staining was performed to evaluate protein expression. TdT-mediated dUTP nick end labeling (TUNEL) staining was performed to examine the pyroptotic cell death. A lactate dehydrogenase (LDH) release assay was performed to assess cytotoxicity.
Results:
Compared with the mock-infected group, we observed that the mRNA levels of NLRP3, caspase-1 (CASP1), interleukin (IL)-1β and gasdermin-D (GSDMD) in C. albicans-infected mice cornea was significantly increased. Our data also demonstrated that the protein expression of NLRP3 and the pyroptosis-related markers apoptosis-associated speck-like protein containing a CARD (ASC), cleaved CASP1, N-GSDMD, cleaved IL-1β and cleaved IL-18 as well as pyroptotic cell death were dramatically elevated in the mouse model of C. albicans keratitis. More importantly, NLRP3 knockdown markedly alleviated pyroptosis and consequently reduced corneal inflammatory reaction in C. albicans keratitis. In vitro, the presence of activated NLRP3 inflammasome and pyroptotic cell death were validated in HCECs exposed to HKCA. Furthermore, the potassium (K+) channel inhibitor glyburide decreased LDH release and suppressed NLRP3 inflammasome activation and pyroptosis in HCECs exposed to HKCA.
Conclusion:
In conclusion, the current study revealed for the first time that NLRP3 inflammasome activation and pyroptosis occur in C. albicans-infected mouse corneas and HCECs. Moreover, NLRP3 inflammasome-mediated pyroptosis signaling is involved in the disease severity of C. albicans keratitis. Therefore, This NLRP3 inflammasome-dependent pathway may be an attractive target for the treatment of fungal keratitis.
Insights
NLRP3 inflammasome activation and pyroptosis contribute to Candida albicans keratitis severity. Targeting this pathway may offer new treatments for fungal keratitis.
Area of Science:
- Ophthalmology
- Immunology
- Microbiology
Background:
- Fungal keratitis is a serious eye infection with unclear pathogenic mechanisms.
- Understanding these mechanisms is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the role of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated pyroptosis in Candida albicans keratitis.
- To explore the underlying mechanisms of NLRP3 inflammasome activation in fungal keratitis.
Main Methods:
- Established mouse and human corneal epithelial cell models of Candida albicans keratitis.
- Assessed gene and protein expression of NLRP3 inflammasome components and pyroptosis markers.
- Utilized TUNEL staining for pyroptotic cell death and LDH assay for cytotoxicity.
- Investigated the effect of NLRP3 knockdown and a potassium channel inhibitor (glyburide).
Main Results:
- NLRP3 inflammasome components (NLRP3, CASP1, IL-1β, GSDMD) and pyroptosis markers were significantly elevated in infected corneas and corneal cells.
- NLRP3 knockdown reduced pyroptosis and corneal inflammation.
- Glyburide inhibited NLRP3 inflammasome activation and pyroptosis in vitro.
- Pyroptosis and elevated NLRP3 inflammasome activation were confirmed in both in vivo and in vitro models.
Conclusions:
- NLRP3 inflammasome activation and pyroptosis are key events in Candida albicans keratitis.
- This NLRP3 inflammasome-dependent pathway significantly contributes to fungal keratitis pathogenesis.
- Targeting the NLRP3 inflammasome pathway presents a potential therapeutic strategy for fungal keratitis.
