Phospholipase Cγ2 is critical for Ca2+ flux and cytokine production in anti-fungal innate immunity of human corneal epithelial cells

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. 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).

PLCγ2 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.

The 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).

We 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. Conclusions: PLCγ2 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.