NLRP3 Inflammasome-Mediated Pyroptosis Pathway Contributes to the Pathogenesis of Candida albicans Keratitis

Abstract

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.

Gasdermins: New Therapeutic Targets in Host Defense, Inflammatory Diseases, and Cancer

“…The role of GSDMD-induced pyroptosis has been recently elucidated in a mouse model of Candida albicans keratitis, a sight-threatening corneal infection. Expression levels of NLRP3, caspase-1, IL-1b, and GSDMD, and pyroptotic cell death were significantly increased in Candida albicans-infected corneas (54). shRNA-mediated NLRP3 knockdown in corneas reduced inflammation and clinical scores, indicating that NLRP3 inflammasome activation is involved in the progression of Candida albicans keratitis (54).…”

“…Inflammasome activation also operates during opportunistic fungal infections caused by Aspergillus fumigatus (53) and Candida albicans (54). Although inflammasome involvement during fungal infections was already established (55,56), the role of GSDMs is still incompletely clarified.…”

“…Expression levels of NLRP3, caspase-1, IL-1b, and GSDMD, and pyroptotic cell death were significantly increased in Candida albicans-infected corneas (54). shRNA-mediated NLRP3 knockdown in corneas reduced inflammation and clinical scores, indicating that NLRP3 inflammasome activation is involved in the progression of Candida albicans keratitis (54). Further investigations are needed to elucidate the exact activation mechanisms and effector actions of GSDMs throughout fungal infections.…”

Intracellular replication ofPseudomonas aeruginosain epithelial cells requires suppression of the caspase-4 inflammasome

“…corneal epithelial cells) is more limited, and functions less-well understood [44]. One inflammasome pathway studied in some corneal diseases and cultured cells is NLRP3 [45, 46], which can detect numerous stimuli [47] including ionic flux from pore formation [48], lysosomal damaging agents [49], and bacterial RNA [50]. However, not all relevant studies verified which corneal cell type expressed NLRP3, or explicitly determined if NLRP3 was activated, versus other inflammasome pathways that could also yield mature IL-1β secretion.…”

A positive feedback cycle between the alarmin S100A8/A9 and NLRP3 inflammasome-GSDMD signalling reinforces the innate immune response in Candida albicans keratitis

“…Neutrophil-derived S100A8/A9 primed the NLRP3 in ammasome in acute myocardial infarction [24,25], and HMGB1 was necessary for hepatocyte NLRP3 in ammasome activation in liver injury following heatstroke [26]. Our previous study demonstrated that the NLRP3 in ammasome-mediated gasdermin-D (GSDMD) pathway participated in the pathogenesis of C. albicans keratitis [27]. However, whether and how NLRP3 senses the alarmin S100A8/A9 in C. albicans keratitis and connects this endogenous danger signal to in ammatory and innate immune responses remain unknown.…”