Fig 1.
Human corneal epithelial cells (HCECs) produce PGLYRPs in response to live or heat-killed Candida albicans (HKCA).
Primary HCECs were exposed to live C. albicans or HKCA with increasing doses (103–106 cells/ml) for 2–24 hours, using untreated cultures as normal controls. A. Dose-dependent stimulation of PGLYRPs 2–4 mRNA in HCECs by live C. albicans for 4 hours. B. The time course of PGLYRP mRNA induction in HCECs exposed to 106 cells/ml of HKCA, evaluated by RT-qPCR with GAPDH as an internal control; C. Dose-dependent stimulation of PGLYRP mRNA in HCECs by HKCA for 4 hours. Data are presented as mean ± SD, n = 5; * p< 0.05, ** p< 0.01, *** p<0.001, vs. controls.
Fig 2.
Dectin-1 expression in corneal epithelial tissue and primary cultured HCECs.
Representative images showed immunofluorescent staining of dectin-1 on human corneal (A) and limbal tissue (B), as well as in HCECs (C). D. Dose-dependent stimulation of dectin-1 mRNA in HCECs by HKCA for 4 hours. E. Total protein of HCECs treated for 48 hours was extracted with RIPA buffer for western blot with dectin-1 or β-actin antibody. F. Quantitative ratio of the dectin-1/β-actin protein, evaluated by western blotting, in HCECs with or without exposure to 106 cell/ml of HKCA. Propidium iodide (PI) was used as nuclear counterstaining (red color). Magnification: 400Х (bar = 25μm). Data are presented as mean ± SD, n = 5; * p< 0.05, ** p< 0.01, vs. controls.
Fig 3.
Dectin-1 and NF-κB signaling pathways involve PGLYRPs induction in HCECs exposed to HKCA.
A. The HCECs were exposed to 106 cells/ml HKCA with prior incubation in the absence or presence of isotype IgG (10μg/ml), dectin-1 Ab (10μg/ml), BAY11-7082 (10μM) or NF-κB activation inhibitor quinazoline (NF-κB-I, 10μM) for 1 h. Cultures treated by HKCA for 4 h were subjected to RT-qPCR to measure mRNA. B. Total protein of HCECs treated for 48 hours was extracted with RIPA buffer for western blot to examine PGLYRP-2 production. C. Protein levels of PGLYRP-2 were evaluated by western blot using β-actin as control with quantitative ratio of PGLYRP-2/β-actin. Results shown are the mean ± SD of four independent experiments; *** p<0.001, as compared with normal control; ^^ p<0.005, ^^^ p<0.001, as compared with HCECs exposed to HKCA. Magnification: 400Х (bar = 25μm).
Fig 4.
NF-κB p65 activation was induced by HKCA and inhibited by dectin-1 neutralizing antibody and NF-κB activation inhibitor quinazoline (NF-κB-I) in HCECs.
A. HCECs were exposed to HKCA (106 cells/ml) with prior incubation in the absence or presence of isotype IgG (10μg/ml), dectin-1 neutralizing Ab (10μg/ml), BAY11-7082 (10μM) or NF-κB activation inhibitor quinazoline (NF-κB-I, 10μM) for 1 h. HCECs were treated with 106 cells/ml HKCA for 48 hours in 8-chamber slides and examined by immunofluorescent staining for PGLYRPs 2–4. B. HCECs were treated for 4 hours in 8-chamber slides and were fixed in acetone for immunofluroscent staining total p65 (nuclear translocation) (green). C. The percentages of positive cells of PGLYRPs 2–4 staining in HCECs in A was quantified. D. The percentages of NF-ĸB p65 nuclear staining positive cells in B was quantified. Images are representatives from three independent experiments. Results shown are the mean ± SD of four independent experiments; *** p<0.001, as compared with normal control; ^^ p<0.005, ^^^ p<0.001, as compared with HCECs exposed to HKCA. Magnification: 400Х (bar = 25μm).
Fig 5.
In vitro anti-fungal activity of rhPGLYRP-2.
C. albicans (1×104 CFU) in 400 μL PBS were incubated without or with rhPGLYRP-2 in different concentration (1, 10 or 100 μg/ml) at 37°C for different time periods, 2h (A), 6h (B) and 24h (C). At the end of incubation, the samples were subjected to fungal culture and plate-colony counting. Results were presented as colony-forming units (CFU) of C. albicans (mean± SD) in cultures with different concentrations of rhPGLYRP-2. Each symbol represents an individual sample, and data are representative of three independent experiments.*p<0.05.