Filaggrin status modulates gene transcription, DNA damage and epidermal barrier repair during clinical response to methotrexate in atopic eczema

Background: Methotrexate (MTX) is an effective treatment for atopic eczema (AE); however, the mechanism of action remains incompletely understood. MTX is known to induce keratinocyte differentiation, which can occur following DNA damage triggered by external stimuli. Objectives: We sought to investigate the mechanism of action of MTX in AE using a combination of in vitro models and study of AE skin during the early phases of therapy with MTX. Specifically, we investigated the relationship between MTX-induced DNA damage, keratinocyte differentiation, barrier function, filaggrin status and disease activity. Methods: Lesional and non-lesional skin from two AE cohorts and in vitro human 3-D epidermal organoids (living epidermal equivalents – [LEEs]) were used to assess response to MTX. Markers of late keratinocyte differentiation and response to purine/pyrimidine nucleosides were assessed. Keratinocyte DNA damage following MTX was evaluated through measurement of DNA damage markers and transcriptomic analysis. Results: MTX restored barrier function and induced filaggrin and loricrin expression in AE skin and LEE, through a mechanism inhibited by thymidine. MTX-induced DNA damage and DNA damage response signalling pathways in LEE and AE skin which were upstream of keratinocyte differentiation. There was an interaction between gene expression, disease severity (EASI) and FLG status (heterozygotes versus wildtype, e.g. LGALS7, SPINK7). Conclusions: MTX induces key epidermal barrier proteins through thymidine-dependent DNA damage in the absence of inflammatory cells. Findings were associated with improved disease outcomes in AE skin. Overall design: We performed a combined bulk RNAseq analysis from 44 samples across 11 AE subjects (lesional and non-lesional skin). Skin punch biopsies from lesional and non-lesional skin were collected at week 0 (baseline), week 2 (visit 1 (V1)) and week 12 (visit 2 (V2)) following MTX initiation.