Abstract
BackgroundFungal keratitis (FK) is a sight-threatening disease, accounting for a significant portion with its complex presentation, suboptimal efficacy of the existing therapies and uncontrollable excessive innate inflammation. Phospholipase C-γ2 (PLCγ2) is a non-receptor tyrosine kinase that plays an important role at the early period of innate immunity. This study aimed to identify the role of PLCγ2 in Dectin-1-mediated Ca2+ Flux and its effect on the expression of proinflammatory mediators at the exposure to Aspergillus fumigatus (A. fumigatus) hyphae antigens in human corneal epithelial cells (HCECs).MethodsThe HCECs were preincubated with or without different inhibitors respectively before A. fumigatus hyphae stimulation. Intracellular calcium flux in HCECs and levels of PLCγ2 and spleen-tyrosine kinase (Syk) were detected by fluorescence imaging and Western Blotting. The expression of proinflammatory mediators was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsWe demonstrated that an intracellular Ca2+ flux in HCECs was triggered by A. fumigatus hyphae and could be reduced by pre-treatment with PLCγ2-inhibitor U73122. A. fumigatus hyphae induced PLCγ2 phosphorylation was regulated by Dectin-1 via Syk. Furthermore, PLCγ2-deficient HCECs showed a drastic impairment in the Ca2+ signaling and the secretion of IL-6, CXCL1 and TNF-α.ConclusionsPLCγ2 plays a critical role for Ca2+ Flux in HCECs stimulated by A. fumigatus hyphae. Syk acts upstream of PLCγ2 in the Dectin-1 signaling pathway. The expressions of proinflammatory mediators induced by A. fumigatus are regulated by the activation of Dectin-1-mediated PLCγ2 signaling pathway in HCECs.
Highlights
Fungal keratitis (FK) is a sight-threatening disease, accounting for a significant portion with its complex presentation, suboptimal efficacy of the existing therapies and uncontrollable excessive innate inflammation
The data demonstrated that IL-6, CXCL1 and TNF-α mRNA expression and protein secretion were induced by hyphae in a timeand dose-dependent manner in human corneal epithelial cells (HCECs)
Phospholipase C-γ2 (PLCγ2) could be activated by A. fumigatus and induced by the engagement of Dectin-1 in HCECs We stimulated HCECs with A. fumigatus hyphae, and the data showed that the stimulation led to the activation of PLCγ2 as indicated by their phosphorylation status
Summary
Fungal keratitis (FK) is a sight-threatening disease, accounting for a significant portion with its complex presentation, suboptimal efficacy of the existing therapies and uncontrollable excessive innate inflammation. Phospholipase C-γ2 (PLCγ2) is a non-receptor tyrosine kinase that plays an important role at the early period of innate immunity. FK is a sight threatening disorder associated with multiple risk factors, such as ocular surface disease, extended wear contact lenses, and traumatic ocular surface accidents [1], presenting a therapeutic challenge due to the lack of effective antifungal agents and uncontrollable excessive innate inflammation. Excessive innate inflammatory response could damage normal corneal epithelial cells whilst slowing down the pathological progress of FK [2]. Cytoplasmic Ca2+ flux has been demonstrated to play an important role in innate immune response. Cytoplasmic Ca2+ flux is one of the hallmarks of B cell receptor (BCR) signaling [8, 9], in
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