Hacat keratinocytes

The antibodies used in this study are listed in Supplemental Table 1. The following cell lines were used: HaCaT keratinocytes (Addex Bio) and THP-1 (American Type Culture Collection [ATCC], TIB-202). The recombinant HSV-2 strain 333, which encodes the β-gal bene (HSV-2/Gal) under CMV promoter control, inserted between virus UL3 and UL4 genes, was provided by Patricia Spear and Richard Longnecker (Northwestern University, Evanston, Illinois, USA) (31 (link)). The stem of influenza hemagglutinin (Flu-HA) strain H1 1999 NC was expressed using the vector VRC-3925 (32 (link)) in 293F cells and was provided by M. Gray and L. Stamatatos (Fred Hutchinson Cancer Research Center, Seattle, Washington, USA). The HSV-2 proteins gB2, gC2, and gH2/gL2 were provided by G. Cohen and R. Eisenberg (University of Pennsylvania, Philadelphia, Pennsylvania, USA). The HSV-2 proteins UL19ud, UL25, and gD2 (33 (link)) were provided by Immune Design Corp.

HaCaT keratinocytes were purchased from AddexBio Technologies (San Diego, CA, USA) and cultured at 37 °C in 5% CO₂ in high-glucose Dulbecco modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Gibco). For developing in vitro barrier dysfunction models, TNF-α/IFN-γ treatment and UVB irradiation were used. First, 6 × 10⁵ HaCaT cells/well were seeded in six-well plates and cultured until confluence. Then, the cells were rinsed with PBS, covered with a thin PBS layer, exposed to 10 mJ/cm² UVB irradiation for 10 s, and further incubated in DMEM for 48 h for developing the UVB-irradiated skin dysfunction models. A CL-1000M UV crosslinker (UVP; Upland, CA, USA), with a UV peak at 302 nm, was used for UVB irradiation. On the contrary, the medium was replaced with DMEM containing 10 ng/mL TNF-α or IFN-γ (R&D Systems; Minneapolis, MN, USA) and further cultured for 48 h for developing the inflamed skin dysfunction models. When required, the cells were treated with three different CHS (BOC sciences; Shirley, NY, USA) or sandacanol (Glentham Life Sciences; Wiltshire, UK) concentrations for 48 h.

RBL-2H3 mast cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and HaCaT keratinocytes were purchased from AddexBio technologies (San Diego, CA, USA). RBL-2H3 and HaCaT cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; 56 °C for 30 min; Gibco, Grand Island, NY, USA) and 1% 100 unit/mL penicillin–streptomycin (Gibco, Grand Island, NY, USA). The cells were grown at 37 °C in a 5% CO₂ atmosphere incubator (Sanyo, Osaka, Japan).