361 Ephrin-A1 ligand strengthens tight junction function and prevents Th2 cytokine-induced barrier collapse

Abstract

Atopic dermatitis (AD) is a chronic skin disease affecting all ages at a worldwide prevalence rate up to 20%. AD is largely driven by immune signals initiated by Th2 cytokines including interleukin (IL)-4 and IL-13. However, mounting evidence points to a central role for epidermal barrier dysfunction in the pathogenesis of AD. We have shown that the epidermal tight junction (TJ) network, a critical component of the barrier, depends on the receptor tyrosine kinase EphA2. Therefore, we hypothesize that EphA2 is a positive regulator of TJs in epidermis and can be targeted to improve epidermal barrier function. In 2D keratinocyte cultures, engaging EphA2 with its ligand ephrin-A1 enhances TJ barrier function compared to control (P=0.0001), as measured by trans-epithelial electrical resistance (TEER). This enhancement is accompanied by the immunofluorescent accumulation of TJ proteins, but only small changes in protein and transcript expression. Increased TEER (P<0.05) and TJ formation are also seen in 3D human epidermal equivalents (3D HEE) after ephrin-A1 treatment. These results led us to ask if ephrin-A1 can prevent epidermal barrier defects in in vitro models of AD. Concurrent treatment with IL-4 and IL-13 induces an AD-like phenotype apparent by changes in epidermal morphology. We show that ephrin-A1 treatment in 3D human skin equivalents (3D HSE) restores keratohyalin granules and the stratum corneum which are lost in 3D HSE after cytokine treatment alone. In human explant cultures of truncal skin, IL-4+IL-13 cause epidermal spongiosis and increased epidermal thickness, effects that are abrogated by ephrin-A1. In addition to altering epidermal morphology, we find that IL-4+IL-13 reduce TEER in 3D HEE compared to controls (P<0.05) and obliterate TJs, shown by loss of occludin immunofluorescence. This cytokine-induced catastrophe of TJ function and formation was rescued by co-treatment with ephrin-A1. Taken together, these results suggest that targeting the EphA2 signaling axis not only enhances TJs, but can also restore epidermal barrier function in the face of Th2 cytokine-driven TJ disassembly.