P39 Methotrexate induces epidermal DNA damage response and filaggrin in atopic eczema

Abstract

Abstract Methotrexate (MTX) is an effective treatment for atopic eczema (AE), but its mechanism of action remains undefined. Our previous work identified keratinocytes as a direct target of MTX by increasing epidermal differentiation. Evidence supports the concept of DNA damage-induced keratinocyte differentiation; however, this has not been characterized in the context of MTX. We tested the hypothesis that MTX may act therapeutically in AE by inducing DNA damage and activating downstream DNA damage response signalling pathways. Patients with AE starting MTX (initial dose 10 mg weekly) were recruited (n = 12). The FLG status was determined by DNA genotyping. Clinical data and skin biopsies from lesional and nonlesional skin were collected at week 0 (baseline), week 2 [visit 1 (V1)] and week 12 [visit 2 (V2)] following MTX initiation. γH2A.X, a marker of DNA damage, was quantified to explore the relationship between DNA damage secondary to MTX treatment and FLG status. γH2A.X and filaggrin protein epidermal immunostaining were quantified. The male-to-female ratio was 8 : 4 and median participant age was 45 years. Median Eczema Area and Severity Index (EASI) score improved from 23.5 (baseline, n = 12) to 14.6 at V1 (n = 12) and 9.0 at V2 (n = 8). Median Dermatology Life Quality Index reduced from 15 (baseline, n = 12) to 10.5 (V2, n = 8). Four patients were FLG heterozygotes and eight were FLG wild type. In combined analysis of all patients, epidermal γH2A.X increased in lesional skin at both time points [n = 6 (V1: P = 0.022; V2: P = 0.009)] and in nonlesional skin by V2 (n = 6; P = 0.021). γH2A.X increased in lesional skin at V2 in both FLG heterozygotes (P = 0.044) and wild types (P = 0.038). Epidermal filaggrin in lesional and nonlesional skin increased at V1 and V2 in all patients combined (n = 5; P = 0.029). Filaggrin increased in the nonlesional skin of FLG heterozygotes (P = 0.044) and in the lesional skin of FLG wild-type patients (P = 0.009) by V2. Transcriptomic (RNAseq) analysis was performed on skin biopsies (n = 11 patients; n = 42 samples). We explored the relationship of gene expression with severity (EASI) and FLG status. AKR1B10, IL22 and LGALS7 expression increased with disease severity. The AKR1B10 (regulates keratinocyte differentiation) and LGALS7 expression (expressed during barrier dysfunction) response with EASI was enhanced in FLG heterozygotes vs. wild-type patients. Canonical pathways were upregulated in lesional skin at V2 vs. baseline. Through epidermal immunostaining and transcriptomic gene analysis, we demonstrated evidence of DNA damage and filaggrin induction in patients with AE following MTX therapy, with associated improved disease outcomes.