1419 Pre-clinical in-vitro assays for high throughput screening of drug candidates for protection and restoration of immune privilege in alopecia areata

Anagen hair bulb immune privilege (IP) collapse is a key underlying event leading to inflammatory loss in alopecia areata (AA). Although the follicle (HF) organ culture assay provides an excellent platform for identification of IP protective/restorative drugs AA management, it inadequate high-throughput screening purposes. Thus, we aimed at developing vitro models suitable latter goal. Interferon-γ (IFNγ), cytokine driving AA, was used mimic collapse-like responses, i.e. upregulation MHC class Ia and II (MHCI/II) expression human outer root sheath (ORSK) keratinocytes epidermal (NHEK), although do not enjoy vivo. Model validation performed employing guardian, α-melanocyte stimulating hormone (αMSH), immunosuppressive drug, Tacrolimus, which known protect restore HF ex vivo, as well AA-therapeutic, JAK inhibitor Tofacitinib. Secretion IFNγ-inducible chemokine CXCL10 into medium evaluated by ELISA along with classical markers quantitative immunofluorescence qRT-PCR. As expected, IFNγ significantly increased mRNA protein MHCI MHCII, release both NHEK ORSK. Tofacitinib prevented up-regulation IP-collapse secretion cell types whilst Tacrolimus αMSH successfully MHCI/II increase exclusively In therapeutic setting, also rescued IFNγ-mediated I expression. Our data reveal that ORSK can be screen interfering IFNg signaling, however only are model testing candidate targeting other pathways.