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Gene profiling and signaling pathways of Candida albicans keratitis.

ABSTRACT: To compare the global gene expression patterns in uninfected and fungus-infected mouse corneas at the onset of Candida albicans keratitis.Fungal keratitis was generated by scarifying the corneal epithelium of BALB/c mice followed by topical inoculation with Candida albicans. Corneal infection was allowed to progress for one day, and total RNA was then extracted from excised corneas. Microarray was performed to detect 45,102 murine genes and processed to identify genetic regulation of signaling pathways. Selected genes encoding interleukins (IL), chemokine ligands, and other cytokines were confirmed by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR).Compared to mock-inoculated control eyes, genetic microarray analysis of Candida albicans keratitis showed that 3,977 genes (8.8%) changed at least twofold and 1,672 genes (3.7%) changed at least fourfold. Hierarchical clustering identified that upregulated genes affected immune and inflammatory responses, intercellular signaling, and cellular proliferation. Pathways having more than 20% of their genes significantly upregulated signaled leukocyte extravasation, increased interleukin production, and affected toll-like receptors. Upregulated transcript levels for IL-1beta and IL-6 were confirmed by real-time RT-PCR.Host gene expression during the initial stage of Candida albicans keratitis involves pathways contributing to acute inflammation mediated by interleukins and other signals of leukocyte recruitment. This murine study confirms the involvement of innate immunity in the cornea during the initiation of Candida albicans keratitis.

SUBMITTER: Yuan X

PROVIDER: S-EPMC2562425 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Gene profiling and signaling pathways of Candida albicans keratitis.

Yuan Xiaoyong X Mitchell Bradley M BM Wilhelmus Kirk R KR

Molecular vision 20080926

<h4>Purpose</h4>To compare the global gene expression patterns in uninfected and fungus-infected mouse corneas at the onset of Candida albicans keratitis.<h4>Methods</h4>Fungal keratitis was generated by scarifying the corneal epithelium of BALB/c mice followed by topical inoculation with Candida albicans. Corneal infection was allowed to progress for one day, and total RNA was then extracted from excised corneas. Microarray was performed to detect 45,102 murine genes and processed to identify g ...[more]

PMID: 18843377

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Project description:PurposeFungal 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.MethodsAn 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.ResultsCompared 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.ConclusionIn 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.

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Gene profiling and signaling pathways of Candida albicans keratitis.

Publications

Gene profiling and signaling pathways of Candida albicans keratitis.

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