The Role Of Srec-Ⅰ In Innate Immunity To Aspergillus Fumigatus Keratitis

Purpose: To determine the role of scavenger receptor expressed by endothelial cell-1(SREC-Ⅰ)in vitro and in a mouse model of Aspergillus fumigatus(A.fumigatus)keratitis.Methods: 1.Corneal specimens of 6 patients with FK underwent penetrating keratoplasty and 6 healthy corneal tissue samples from the rest of the peripheral donor corneas were collected to detect the expression of SREC-I protein in corneas by immunofluorescence.2.SREC-Ⅰ m RNA and protein expression were detected in both normal and A.fumigatus stimulated human corneal epithelial cells(HCECs)at different time points by RT-PCR and Western blot.3.HCECs were incubated with SREC-Ⅰ si RNA and NC si RNA with a final concentration of 200 n M for 48 h,then the cells were stimulated by A.fumigatus hyphae for8 h and 24 h.Cells were collected to detect the m RNA levels of SREC-I,LOX-1,IL-1βand TNF-α by RT-PCR and the supernatant was collected to detect the protein levels of IL-1β and TNF-α by Elisa.4.Mouse fungal keratitis(FK)model was established by intrastromal injections using the A.fumigatus conidia suspension.SREC-Ⅰ m RNA and protein expression in mice corneas were detected by RT-PCR and Western blot at different time points after infection.The eyeballs were collected for immunofluorescence at 48 h after infection.5.20 μg/10 μL SREC-Ⅰ neutralizing antibody or control Ig G were given subconjunctivally into the right eyes of mice 1 day before infection.200 μg/100 μL SREC-Ⅰ neutralizing antibody or control Ig G were injected intraperitoneally 1 hour post infection.The severity of keratitis was evaluated by clinical score at 1,3 and 5 days after infection.The SREC-I,CLCX1,LOX-1,IL-1β and TNF-α m RNA levels in mice corneas were detected by RT-PCR at 12 h p.i.and the protein levels of IL-1β and TNF-α were detected by ELISA at 24 h p.i.The eyeballs were collected for hematoxylin-eosin staining at 48 p.i.Results: 1.SREC-Ⅰ expressed in normal human corneal epithelium,and the SREC-I protein expression levels in the corneal epithelium of patients with A.fumigatus keratitis is significantly higher than that in normal corneas.2.SREC-Ⅰ expressed both in normal and A.fumigatus stimulated HCECs.SREC-Ⅰ m RNA expression levels were significantly increased at 8 h after A.fumigatus stimulation,while SREC-Ⅰ protein levels started to increase at 36 h after stimulation.3.SREC-Ⅰ si RNA treatment effectively inhibited the expression of SREC-I in HCECs.Compared with the control group,SREC-Ⅰ si RNA significantly reduced the m RNA expression of inflammatory cytokines IL-1β,TNF-α and LOX-1 in A.fumigatus stimulated HCECs.4.SREC-I was detected both in normal and A.fumigatus infected mice corneas,and the expression levels of SREC-I was significantly increased after infection.SERC-I m RNA levels were peaked at 12 h p.i.while SERC-I protein levels started to increase at 2 days p.i.5.The SREC-I neutralizing antibody treatment effectively inhibited the expression of SREC-I in A.fumigatus infected mice corneas.Compared with the control Ig G treated group,clinical score in the SREC-Ⅰblockade group was significantly lower at 3 and 5 days p.i.SREC-I neutralizing antibody significantly inhibited the m RNA levels of CXCL-1,LOX-1,IL-1β and TNF-α at 12 h p.i.,and distinctly reduced the protein levels of IL-1β and TNF-α at 48 h p.i.Conclusions: SREC-Ⅰ is a key mediator in inflammatory response induced by A.fumigatus keratitis.SREC-Ⅰ blockade could be a potential therapeutic approach for FK.