195 N/TERTs replicate primary keratinocytes in barrier formation and viral infectivity

Abstract

Atopic dermatitis (AD) is a skin disease characterized by an impaired epidermal barrier and increased susceptibility to cutaneous viral infections. We have previously observed that barrier disruption (caused by our tight junction disrupting peptide [TJDP®]), enhances vaccinia virus (VV) infection of primary human foreskin keratinocytes (PHFK). To evaluate the importance of AD-relevant tight junction proteins in epidermal viral infections, we sought to identify an epidermal cell line that we could genetically manipulate. We tested N/TERT cells (immortalized PHFK) to determine whether they faithfully recapitulated primary cells in both our barrier and VV infection models. We found that N/TERTs developed a robust barrier, as measured by transepithelial electrical resistance (TEER), with peak values of 300–400 ohms/cm2 observed 4-5 days after treatment with high Ca+2 (1.8mM) containing media. After exposure to our TJDP®, N/TERTs exhibited a substantial reduction in barrier compared to media controls (e.g. TEER reduction: 46% ± 13 and 68% ± 11 at Days 2 and 3, respectively; n=3). We previously observed that PHFK are more susceptible to VV when treated with TJDP® during differentiation. With N/TERTs, we observed an increase in VV susceptibility as measured by % change in plaque number (179% ± 64 at Day 2; n=3) and % of the monolayer infected (27% ± 24 vs 42% ± 20 at Day 2 for media compared to TJDP® treated samples, n=3). These observations suggest that N/TERTs behave similarly to PHFK in both our infection and barrier experiments. Future studies will focus on determining how reduction in AD-relevant TJ barrier proteins (Claudin 1, 4, 23, occludin, and zonula occludens 1) using the CRISPR/Cas9 system impacts the susceptibility of N/TERTs to viral infections (VV and herpes simplex virus).