Dectin-1 and NF-κB signaling pathways involve PGLYRPs induction in HCECs exposed to HKCA.

<p>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).</p

Human corneal epithelial cells (HCECs) produce PGLYRPs in response to live or heat-killed <i>Candida albicans</i> (HKCA).

<p>Primary HCECs were exposed to live <i>C</i>. <i>albicans</i> 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 <i>C</i>. <i>albicans</i> 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.</p

Dectin-1 expression in corneal epithelial tissue and primary cultured HCECs.

<p>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.</p

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.

<p>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).</p

Perostin expression in human limbal epithelial cells (HLECs) at different growth stages.

<p><b>(A).</b> Reverse transcription-quantitative real-time polymerase chain reaction displayed the expression levels (relative fold of mRNA) of periostin by 50% and 70% confluent HLECs compared with 100% confluent cells. <b>(B).</b> Representative images showing periostin, integrin β1, TCF4 and p63 immunofluorescent staining (green) with propidium iodide (PI, red) counterstaining in HLECs at different growth stages (50%, 70% and 100% confluence). <b>(C).</b> Percentages of periostin-, integrin β1-, TCF4- and p63-positive cells in 50%, 70% and 100% confluent cultures. Data shown as mean ± standard deviation, n = 3; *p<.05; **p<.01.</p

ST2 was expressed by human corneal epithelium.

<p><b>A.</b> Representative images showing ST2 localization in ex vivo donor corneal tissues without or with exposure to IL-33 (10 ng/ml) by immunohistochemical staining with isotype IgG as a negative control. <b>B.</b> Immunohistochemical images showing ST2 protein in primary HCECs without or with exposure to IL-33, with isotype IgG as a negative control. Magnification 400×. <b>C.</b> The mRNA expression of ST2 by HCECs exposed to IL-33 in time course and dose response.</p

Representative immuno-fluorescent staining profiles for corneal epithelial phenotype.

<p>Corneal epithelial markers, including the differentiation markers, keratin 3 (K3), and connexin 43 (Cx43), as well as progenitor markers, EGFR, nuclear p63 and integrin β1, were expressed by HLE regenerated on human feeder of Hs68 fibroblasts; Hoechst 33342 was used for nuclear counterstaining.</p

Human primer sequences used for RT-PCR.

<p>Human primer sequences used for RT-PCR.</p

IL-33 induced inflammatory mediators in HCECs with time course and dose response.

<p>The expression of inflammatory cytokines (TNF-α, IL-1β and IL-6) and chemokine IL-8 were measured by RT-qPCR for mRNA (<b>A</b> & <b>B</b>) and by ELISA for protein levels in culture supernatants (<b>C</b>). Results shown are mean ± SD of four independent experiments. *p<0.05; **p<0.01, n = 4.</p

Dynamic change of periostin and other stem cell markers during limbal epithelial regeneration.

<p><b>(A).</b> Representative images of immunofluorescent staining of periostin, integrin β1, TCF4 and p63 showing their dynamic alternation at different time points after wound. <b>(B).</b> Percentages of periostin-, integrin β1-, TCF4- and p63-positive cells at different time points in in vitro wound healing model. Data were shown as the mean ± standard deviation, *p<.05, **P<.01, n = 3.</p