The CCR6/CCL20 axis mediates Th17 cell migration to the ocular surface in dry eye disease

Abstract

Purpose: Th17 cells are believed to be the primary effector cells in the pathogenesis of dry eye disease (DED). However, the mechanisms by which Th17 cells migrate from the lymphoid tissues to the ocular surface are unknown. The purpose of this study was to investigate the role of the C-C chemokine receptor 6/C-C chemokine ligand 20 (CCR6/CCL20) chemokine axis in mediating Th17 cell migration in DED.

Methods: DED was induced by housing C57BL/6 mice in a low-humidity environment supplemented with scopolamine treatment. Th17 cell expression of CCR6 was evaluated using flow cytometry and ocular surface expression of CCL20 was measured using PCR and ELISA assays. CCL20 neutralizing antibody was administered subconjunctivally to DED mice and disease severity, including the frequency of conjunctival Th17 cells, was evaluated.

Results: CCR6 is preferentially expressed by Th17 cells in both normal and DED mice and DED significantly upregulates ocular surface expression of CCL20. Disruption of CCR6/CCL20 binding with CCL20 neutralizing antibody decreases T-cell migration in vitro and reduces Th17 cell infiltration of the conjunctiva when administered in vivo, significantly improving clinical signs of DED. These changes were accompanied by a decrease in ocular surface inflammatory cytokine levels and corneal CD11b+ cell frequencies. Treatment also significantly reduced the generation of Th17 cells.

Conclusions: Local neutralization of CCL20 decreases Th17 cell infiltration of the ocular surface in DED, leading to improvement in clinical signs of disease. This suggests that CCR6/CCL20 interactions direct Th17 cell migration in DED and that disruption of this axis may be a novel therapeutic approach to this condition.

Keywords: CCL20; CCR6; Th17 cell; cell migration; dry eye.

Figures

Figure 1

(a) DED was induced by housing mice in a low-humidity–controlled environment and administering scopolamine hydrobromide. CFS, the principal clinical measure of corneal epitheliopathy, was used to confirm disease induction. At days 3, 7, and 12, DED mice (solid line) exhibit a significant increase in CFS scores compared with normal, room-air mice (dashed line) (n = 10 mice per group, *P < 0.001; representative data from one of three experiments are shown). (b) Representative flow cytometry histograms showing the generation of Th17 cells (top) and Th1 cells (bottom) in the draining LNs of DED mice. Frequencies are of the representative plot. (c) The frequency of Th1 and Th17 cells in the LNs of normal (white bars) and DED mice (gray bars) at day 12 are compared. There is a statistically significant increase in Th17 cell frequency but not Th1 cell frequency following induction of DED (n = 9–10 mice per group, *P = 0.01; averages are of three experiments).

Figure 2

(a) IL-17A+ (Th17) and IFN-γ+ (Th1) CD4+ cells were evaluated for CCR6 expression by comparing the MFI of each subset to isotype control (gray). Th17 cells (dark line) isolated from the draining LNs of both normal and DED mice express approximately twice as much CCR6 as their Th1 cell counterparts (light line). Representative histograms shown. (b) In both normal (left) and DED (right) mice, Th17 cells (gray bar) express a significantly higher level of CCR6 than that of Th1 cells (white bar) (Normal: P = 0.002, DED: P = 0.005, n = 9–10 mice per group; averages are from one experiment of three). (c) Representative histograms showing the expression of CCR6 by Th17 cells in the LNs and conjunctiva in both normal and DED mice. (MFIs are of the representative histogram.) (d) CCR6 MFI on Th17 cells remains constant between normal and DED conditions (n = 9–10 mice per group; representative data from one of three experiments are shown).

Figure 3

CCL20 messenger RNA (mRNA) transcript levels are upregulated in DED in both cornea (a) and conjunctiva (b). CCL20 protein production increases in DED in both cornea (c) and conjunctiva (d), as measured by ELISA (n = 4–6 mice per group, *P < 0.05, **P < 0.005; data from one experiment of two are shown).

Figure 4

CD4+ T cells isolated from the draining lymph nodes of DED mice were placed in the upper well of an 8-μm transwell filter and cultured with cornea and conjunctiva (lower well) from normal or DED mice. After 2 hours, migrated cells in the lower well were counted. Significantly more CD4+ cells migrated when DED tissue was included in culture compared with normal tissue (P = 0.04). Treatment with CCL20 neutralizing antibody decreased the number of migrated CD4+ cells compared with both isotype control antibody treated (P = 0.003) and untreated DED groups (P = 0.001) (n = 5 mice per group, *P < 0.05, **P < 0.005; data from one of two experiments are shown).

Figure 5

(a) Experimental design of CCL20 neutralization experiment. Following the induction of DED, mice were randomly assigned to receive anti-CCL20 neutralizing antibody or control antibody every other day for 4 days via subconjunctival injection. (b) Corneal epitheliopathy was evaluated by scoring CFS at days 0, 3, 7, and 10. At day 10, mice receiving subconjunctival anti-CCL20 neutralizing antibody (solid line) showed a statistically significant reduction in CFS score compared with isotype control treated mice (dashed line). Mice were blindly scored according to the National Eye Institute CFS grading system (n = 10 eyes per group, *P < 0.001; data from one of two experiments are shown).

Figure 6

(a) Conjunctival flow cytometry samples were gated on CD4 then IL-17A to quantify the frequency of Th17 cells reaching the ocular surface. Conjunctiva from DED mice receiving anti-CCL20 antibody demonstrates a decreased frequency of Th17 cells compared with mice receiving isotype control antibody (P = 0.002) and untreated DED mice (P = 0.005) (*P < 0.005, n = 5 mice per group; representative data from one of two experiments are shown). (b) Representative micrographs of whole-mount cornea immunostaining (×40). Corneas from normal, DED treated with anti-CCL20 antibody, and DED treated with isotype control antibody mice were stained with anti-CD11b antibody (green) and DAPI (blue). (c) Treatment with anti-CCL20 antibody significantly decreased the number of CD11b+ cells in peripheral cornea compared with isotype control antibody (three corneas per group were each evaluated in four distinct peripheral areas; *P < 0.05, **P ≤ 0.001, NS, not statistically significant). (d) Real-time PCR analysis of conjunctiva showing expression levels of IL-6, IL-23, MMP-3, TNF-α, and IFN-γ. (e) Real-time PCR analysis of cornea showing expression levels of IL-6, IL-23, MMP-3, and TNF-α (n = 4–6 eyes per group, *P < 0.05, **P ≤ 0.001). White bars: DED mice treated with isotype control antibody. Gray bars: DED mice treated with anti-CCL20 antibody. Results are normalized to mRNA levels in normal mice.

Figure 7

Th17 cells in the draining lymph nodes of DED mice were quantified by flow cytometry following treatment with CCL20 neutralizing antibody or control antibody. DED mice generate fewer Th17 cells when treated with anti-CCL20 antibody as compared with both isotype control antibody treatment (P = 0.01) and no treatment (P = 0.01) (n = 5 mice per group, *P < 0.05, **P < 0.005; representative data from one of two experiments are shown).