IL-17 disrupts corneal barrier following desiccating stress

Abstract

T helper (Th)-17 is a recently identified subtype of Th response that has been implicated in host defense and autoimmunity. We investigated whether there is evidence for a Th-17 response in human and experimental murine dry eye (DE). Gene expression in the human DE conjunctiva showed increased levels of the Th-17 inducers, interleukin (IL)-23, IL-17A, and interferon-gamma (IFN-gamma). In the murine model, we found that desiccating stress increased matrix metalloproteinase-9, Th-17-associated genes (IL-6, IL-23, transforming growth factor-beta1 and -2, IL-23R, IL-17R, IL-17A, retinoid-related orphan receptor-gammat, and CC chemokine attractant ligand-20) and IFN-gamma in cornea and conjunctiva. Furthermore, we found a significantly increased concentration of IL-17 in tears and number of IL-17-producing cells on the ocular surface. Antibody neutralization of IL-17 ameliorated experimental DE-induced corneal epithelial barrier dysfunction and decreased the expression of matrix metalloproteinases 3 and 9. Taken together, these findings suggest that IL-17 has a role in corneal epithelial barrier disruption in DE.

Figure 1

mRNA transcripts in human conjunctival epithelia from normal subjects and patients with DE (n = 17). *P < 0.05, ***P < 0.001, ND = non-detectable. DE, dry eye.

Figure 2

Flow cytometry analysis of freshly isolated cells from the cornea (CN) and conjunctiva (CJ) stained with CD4-FITC-conjugated antibody in NS control (NS), 5 days (DS5) or 10 days (DS10) of desiccating stress (DS) (n = 5 per group). Lymphocytes were gated based on characteristic light-scatter properties, single lymphocytes were gated based on forward scatter height vs. forward scatter area (FSC-A). Numbers in the quadrants indicate the percentage of cells.

Figure 3

(a–f) Laser scanning immunofluorescent confocal microscopy of the cornea (a, c, f) and conjunctiva (b, d, e) sections stained for IL-6 (a and b) or IL-23 (c and d) and CCL20 (f) (in green), with propidium iodide nuclear counterstaining (in red) in NS control (NS), 5 days (DS5) or 10 days (DS10) of desiccating stress (DS) (n = 5 per group). (b) Small inset: higher magnification of an IL-6-positive cell in the stroma of the conjunctiva in the goblet-cell-rich area. Scale bar = 50 μm. (e) Immunohistochemical staining for IL-23R positively stained cells (red) indicated by arrows in conjunctival sections of NS, DS5, and DS10 mice (n = 5 per group). Scale bar = 25 μm. (g) Mean±s.d. of levels of TGF-β1 and TGF-β2 protein in conjunctival lysates obtained from NS control (NS), 5 days (DS5) or 10 days (DS10) of desiccating stress (DS). Data are presented as pg mg ⁻¹ of three independent experiments. (h) IL-12p40 and IL-12p70 concentration in tear fluid samples measured by immunobead assay obtained from mice subjected to DS. Data are presented as mean±s.d. of three independent experiments. (i) Ratio of both subunits. *P < 0.05 DS5 or DS10 vs. NS group. IL, interleukin; TGF, transforming growth factor.

Figure 4

(a and b) Laser scanning immunofluorescent confocal microscopy of corneal (a) and conjunctival (b) sections stained for IL-17 (in green) with propidium iodide nuclear counterstaining (in red) in NS control (NS), 5 days (DS5) or 10 days (DS10) of desiccating stress (DS) (n = 5 per group). Insets: higher magnification of IL-17-positive cells in DS5 and DS10 in mid-peripheral cornea. Scale bar = 50 μm. (c and d) Mean±s.d. of three independent IL-17 ELISPOTS, showing IL-17-producing cells isolated from the cornea (CN) and conjunctiva (CJ, both in (c)) and cervical lymph nodes (CLN, in (d)) after desiccating stress. (e) IL-17 concentration in tear fluid samples obtained from mice subjected to DS measured by immunobead assay. The dotted line indicates the lower limit of detection in the assay. Data are presented as mean±s.d. of three independent experiments. CLN, cervical lymph node; IL, interleukin.

Figure 5

(a) Representative digital images in C57BL/6 (B6) mice (in (a)) used to score Oregon Green Dextran-488 (OGD) permeability (in d) among the treatment groups (NS = non-stressed controls, DS5 = desiccating stress for 5 days, DS5 + α IL-17 = DS5 treated with anti-IL-17 antibody, DS5 + IC = DS5 treated with rat antibody isotype control). (b) Laser scanning immunofluorescent confocal microscopy of cornea sections stained for MMP-9 (in green) with propidium iodide nuclear counterstaining (in red) among the treatment groups described above (n = 5 per group). (c) In situ zymography (in situ z) of central corneal sections of NS, DS5, DS5 + αIL-17, and DS5 + IC mice. The fluorescence intensity is proportional to gelatinase activity within the tissue. (d) Mean±s.d. of corneal OGD score (in gray levels) measured by the Metavue Software by two masked observers among the treatment groups in C57BL/6 and IFN-γ knockout mice (IFN-γKO) (n = 30 eyes per group). (e) MMP-3 and MMP-9 mRNA transcript levels in the corneal epithelium of C57BL/6 mice subjected to desiccating stress after treatment with either anti-IL-17 antibody or isotype control. IL, interleukin; MMP-9, matrix metalloproteinase-9.