Effect Of Dectin-1 / Phospholipase Cγ Signaling Pathway On The Innate Immune Phase Of Antifungal Infection In Corneal Epithelial Cells

Purpose: The study was to identify the role of Phospholipase Cγ(PLCγ)signaling in Dectin-1-mediated Ca2+ Flux and investigate its effect on the expression of inflammatory chemokines in innate immune at the early period of exposure to Aspergillus fumigatus(A. fumigatus) hyphae antigens in cultured human corneal epithelial cells(HCECs).Methods: The HCECs were preincubated with or without different inhibitors in different concentrations respectively before stimulated with A. fumigatus hyphae.The intracellular calcium flux of HCECs were assessed by fluorescence imaging. Western blot was used to detect the expression of phospholipase Cγ(PLCγ)protein and spleen-tyrosine kinase(Syk) protein. The expression of inflammatory chemokines were determined by specific human chemokine reverse transcriptase polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA).Results:We demonstrated here that stimulation of HCECs with A. fumigatus hyphae triggered an intracellular Ca2+ flux which could be decreased by pre-treatment of cells with laminarin or the PLCγ-inhibitor U73122. Interestingly,the same treatment could engage Dectin-1 and result in the phosphorylation of both PLCγ1 and PLCγ2 which both can be attenuated by pre-treatment of cells with laminarin(Dectin-1 inhibitor) and Piceatannol(syk inhibitor), and laminarin abrogated hyphae-induced phosphorylation of syk. PLCγ-deficient HCECs have drastic impairment of Ca2+ signaling and are defective in the secretion of interleukin 8(IL-8) and CXCL1.Conclusions: PLCγ plays a critical role for Dectin-1-mediated Ca2+ Flux in HCECs which stimulate with A. fumigatus hyphae, and Syk acts upstream of PLCγin the Dectin-1 signaling pathway. The expression of inflammatory chemokines induced by hyphae are regulated by the activation of Dectin-1-mediated PLCγsignaling pathways in HCECs.