STAT6-mediated keratitis and blepharitis: a novel murine model of ocular atopic dermatitis

Abstract

Purpose: Atopic dermatitis (AD) is a common inflammatory disease that can affect the eye, resulting in ocular pathologies, including blepharitis, keratitis, and uveitis; however, the pathogenic mechanisms underlying the ocular manifestations of AD are not well understood.

Methods: In the present study, we characterized the ocular pathologies that develop in the Stat6VT mouse model of AD. We examined the cytokine profile of the eyelid lesions, measured the behavioral response, and documented the treatment response to topical steroids.

Results: Our results show that Stat6VT mice spontaneously developed blepharitis, keratitis, and uveitis similar to that observed in patients with AD. Histologic findings of allergic inflammation in affected eyelids in this model include the presence of a lymphocyte-predominant infiltrate and tissue eosinophilia in the dermis. Gene expression analysis of affected eyelid tissue by quantitative PCR revealed increased amounts of mRNAs for the Th2 cytokines IL-4, IL-5, and IL-13. In addition, increased eyelid scratching was seen in Stat6VT mice with blepharitis. Topical treatment with the corticosteroid clobetasol reduced eyelid inflammation, tissue eosinophilia, and Th2 cytokine expression.

Conclusions: The development of AD-like ocular pathologies in this model supports the idea that in humans, AD-associated disease of the eye may be driven by Th2-mediated inflammation and demonstrates that the Stat6VT mouse may be a useful system in which to further investigate pathogenesis of and treatment strategies for blepharitis and other ocular diseases that develop in association with AD.

Keywords: STAT6; atopic blepharitis; ocular atopic dermatitis.

Figure 1

Clinical features of ocular inflammation in Stat6VT mice. (A) Photographs showing blepharitis in Stat6VT mouse (right: arrow denotes lid edema) compared with unaffected WT control (left). Scale: 5 mm. (B) Cumulative incidence of blepharitis in Stat6VT mice (n = 20, 24 respectively for Stat6VT and WT). (C) Slit-lamp photographs of WT and Stat6VT mice showing diffuse corneal neovascularization in Stat6VT mouse (arrow). Scale: 500 μm. (D) Behavioral assay with video recording determined the rate of grooming versus lid scratching over a 30-minute period in both WT and Stat6VT transgenic mice (n = 5, 7 respectively of WT and Stat6VT). Counts were obtained by two independent observers; *P < 0.05).

Figure 2

Histological analysis of eyelid and corneal tissue from WT and Stat6VT mice. (A) Samples were fixed stained with H&E, original magnification ×400 (Inset: arrow indicates idicates eosinophil). (B) Meibomian glands of WT and Stat6VT transgenic mice. Scale bar: 250 μm. (C) Subepithelial cellular infiltration in Stat6VT mice. Scale bar: 250 μm. (D) Bar graph represents quantification of cellular infiltrate in WT and Stat6VT mice (*P < 0.05, paired Student's t-test). Representative of the average for 10 high-power fields examined and three independent experiments by two observers. (E) Quantitation of eosinophils in WT and Stat6VT mice (*P < 0.05, paired Student's t-test). Representative of average of 10 high power fields from three to five mice examined and three independent experiments. (F) Histologic staining of corneal tissue in WT and Stat6VT mice (arrow, intrastromal neovascularization). Scale bar: 30 μm. (G) Anterior chamber inflammation with loss of Descemete's membrane (arrowhead). Scale: 50 μm.

Figure 3

Increased Th2 cytokine levels in eyelids of Stat6VT mice with blepharitis. (A) RNA was isolated from eyelids of WT and Stat6VT mice with active lesions and cytokine mRNA (IL-4, IL-5, IL-10, IL-13, TSLP) was analyzed using quantitative PCR. Results are the average of three independent experiments performed in triplicate, from five to seven mice per group. (B) Total protein from eyelids of WT and Stat6VT transgenic mice were subjected to SDS-PAGE; immunoblot analysis was performed using anti-IL4 and –β-actin. β-actin was used as a loading control. Bar graph represents the average densitometry of three samples normalized to β-actin (*P < 0.05, Student's t-test; representative of three independent experiments).

Figure 4

Treatment of eyelids with a topical corticosteroid reverses blepharitis and restores Th2 imbalance in Stat6VT mice. (A) Clobetasol (50 mg) was applied topically daily for 7 consecutive days on both WT and Stat6VT transgenic mice. Upper eyelid thickness at midline was measured on days 0, 4, and 7 (* and **P < 0.05, two-tailed Student's t-test). Inset photographs shown for appearance of blepharitis phenotype in the mouse before (left, D0) and 7 days after (right, D7) initiating treatment with clobetasol ointment. Scale: 25 μm. (Representative of the average for three independent experiments by two observers.) (B) Eosinophil counts in the subdermal tissue were performed for both WT and Stat6VT mice treated with either vehicle or clobetasol. Representative of the average for 10 high-power fields examined and three independent experiments. (C) RNA isolated from eyelids of WT and Stat6VT mice treated with vehicle or clobetasol was used to quantify IL-4 transcripts (normalized to GAPDH). Representative of the average from three independent experiments (*P < 0.05, two-tailed Student's t-test).