A Potential Link Between TSLP/TSLPR/STAT5 And TLR2/MyD88/NFκB-p65 In Human Corneal Epithelial Cells For Aspergillus Fumigatus Tolerance

Background:Fungal keratitis is a serious eye infection, with an increasing incidence in recent years. Corneal ulcer caused by fungal infections is one of the central reasons lead to blindness. The cornea cells play an important role in innate immunity. And corneal cells are not traditional immune cells, but certain receptors on cells can quickly identify fungi and resist the invasion, playing a role in immunity. Thymic stromal lymphocytes hormone, a new kind of Th2 cytokines mediated hypersensitivity disease associated. Some recent findings suggested that TSLP might be involved in infections, and TSLP was detected to be expressed in human corneal epithelium through TLR/NFκB signal path. However its functional role in response to A. fumigatus infection in HCECs is still unclear.Objective:To investigate the crosstalk between TSLP/TSLPR/STAT5 and TLR2/MyD88/NFκB-p65 in inflammatory responses triggered by Aspergillus fumigatus in human telomerase-immortalized human corneal epithelial cells (THCEs).Methods:Telomerase-immortalized human corneal epithelial cells were seeded at 1× 105 cells per well in six-well plates, and THCEs were cultured with A. fumigatus hyphae (106 pieces/ml) for various periods (1h,3h,6h,12h,24h,48h), then PCR was performed to assess the mRNA levels of TSLP at 12h of A. fumigatus stimulation, TSLPR and IL-7Ra for various periods (1h,3h,6h,12h,24h,48h). After THCEs were stimulated with A. fumigatus for 12h, Western blot was performed to determine levels of TSLPR and p-STAT5, and Immunofluorescence was peformed to detect the expression and localization of p-STAT5. THCEs were treated with zymosan at 10 mg/ml for various periods (1h,3h,6h,12h,24h,48h). RT-PCR was performed to assess TSLP expression at 12h of zymosan treatment and TSLPR mRNA expression for various periods (1h,3h,6h,12h,24h,48h). ELISA was performed to measure concentration of TSLP in the supernatant after THCEs were stimulated with A. fumigatus for 12h. Western blot was performed to assess TSLPR and p-STAT5 protein level after zymosan treatment for 48h. THCEs were challenged by A. fumigatus hyphea for 12h with or without TLR2 monoclonal antibody, then RT-PCR and ELISA was performed to assess TSLP expression, and TSLPR, total STAT5 and p-STAT5 was detected by Western blot analysis. The mRNA levels of MyD88 at various periods (30min, 1h,3h and 6h) were assayed by RT-PCR, and the protein level of NFKB-p65 was detected by Western blot after rTSLP treatment for 3h. THCEs were cultured with A. fumigatus hyphae pre-transfered with or without TSLP siRNA. The mRNA levels of MyD88 at various periods (30min, 1h,3h,6h) were assayed by RT-PCR, and the protein level of NFκB-p65 was detected by western blot after A. fumigatus hyphae treatment for 3h.Results:It was demonstrated that signaling molecules in TSLP/TSLPR/STAT5 pathway were increased in THCEs challenged with A. fumigatus hyphea. Zymosan treatment induced the increased expression of TSLPR and phosphorylation of p-STAT5 in THCEs. In addition, inhibition of TLR2 with monoclonal antibody prevented elevation of signaling molecules in the TSLP/TSLPR/STAT5 pathway upon A. fumigatus challenge. rTSLP stimulates expression of MyD88 and NFκB-p65. Inhibition of TSLP with small interfering RNA caused an impaired response of TLR2 downstream signaling pathway upon A. fumigatus challenge.Conclusions:THCEs represent a novel target of TSLP, TSLP/TSLPR/STAT5 signaling plays an important role in response to A. fumigatus infection in THCEs, and TLR2 downstream signaling molecules up regulate TSLP/TSLPR/STAT5 signaling as well as TSLP downstream signaling molecules up regulate TLR2/MyD88/NFκB-p65 signaling in this phenomenon.