Th17-associated cytokines IL-17 and IL-23 in inflamed skin of Darier disease patients as potential therapeutic targets

Abstract

Darier disease (DD) is a rare, inherited multi-organ disorder associated with mutations in the ATP2A2 gene. DD patients often have skin involvement characterized by malodorous, inflamed skin and recurrent, severe infections. Therapeutic options are limited and inadequate for the long-term management of this chronic disease. The aim of this study was to characterize the cutaneous immune infiltrate in DD skin lesions in detail and to identify new therapeutic targets. Using gene and protein expression profiling assays including scRNA sequencing, we demonstrate enhanced expression of Th17-related genes and cytokines and increased numbers of Th17 cells in six DD patients. We provide evidence that targeting the IL-17/IL-23 axis in a case series of three DD patients with monoclonal antibodies is efficacious with significant clinical improvement. As DD is a chronic, relapsing disease, our findings might pave the way toward additional options for the long-term management of skin inflammation in patients with DD.

Fig. 1. Enhanced expression of Th17-related genes and cytokines in the skin of patients with Darier disease (DD).

a UMAP analysis (upper panel) and percentage of different cell types (lower panel) determined by scRNA-seq analysis of skin samples from four DD patients (lesional skin, N = 4, 3746 cells). Results were compared to publicly available scRNA-seq data of psoriasis (PSO, lesional skin, N = 3, 11417 cells) and healthy controls (HC, N = 3, 12817 cells). b Heat map of differentially expressed (DE) genes expressed as Z-score (DD vs HC, fold change > 1.5) as determined by NanoString nCounter analysis using the Immunology_V2 panel in skin of untreated DD patients (N = 5), healthy controls (N = 2) and psoriasis patients (N = 2). c Biocarta pathways most enriched in DD patients as compared to HC, as determined by NanoString nCounter and Gene Set Enrichment Analysis. Pathways are ordered according to their normalized enrichment scores. Bars represent -log10 of False Discovery Rates (FDR) (See Suppl. Table 2 for complete table). d Relative mRNA expression (qRT-PCR) of Th1, Th2, and Th17-associated cytokines normalized to housekeeping gene ACTB and relative to HC. Bars represent means, error bars represent standard deviations. p-values were calculated using 2-way ANOVA with Dunnett multiple comparison correction (all against HC). e Protein expression of Th17-related cytokines in the skin of HC and DD patients as determined by bead-based immunoassays (LegendPlex analysis). p-values were calculated using 2-way ANOVA. Error bars represent standard deviation.

Fig. 2. Increased numbers of Th17 cells in lesional skin of DD patients.

a Sum of IL17A/F normalized expression in different cell types of healthy controls (HC, N = 3, 12817 cells), Darier Disease patients (DD, N = 4, 3746 cells) and Psoriasis patients (PSO, N = 3, 11417 cells) skin samples as determined by scRNA-seq analysis. Significance bars represent results of one-tailed student’s T-test of IL17A/F expression between T helper and non-T helper cells within one group (blue p-value for HC, red p-value for DD, beige p-value for PSO), without adjustment for multiple comparisons. b Representative multi-color immunofluorescence (IF) images of tissue micro array sections stained with OPAL technique for IL-23A, CD4 and DAPI in skin samples of DD patients (N = 7), atopic dermatitis patients (AD, N = 3) and PSO (N = 3). CD4 positive T cells are marked in red, IL-23A positive cells are marked in green, DAPI-stained nuclei are marked in blue. Isotype controls are shown in Suppl. Fig. 5. c Quantification of IL-23A positive cells as determined by mean fluorescence intensity of multi-color immunofluorescence stained TMA sections (skin samples: DD (N = 7, red), PSO (N = 3, beige), AD (N = 3, blue)). d Representative multi-color immunofluorescence (IF) images of tissue micro array sections stained with OPAL technique for IL-17A, CD4 and DAPI in skin samples of DD patients (N = 7), atopic dermatitis patients (AD, N = 3) and PSO (N = 3). CD4 positive T cells are marked in red, IL-17A positive cells are marked in green, DAPI-stained nuclei are marked in blue. Isotype controls are shown in Suppl. Fig. 5. e Quantification of IL17A/CD4/DAPI triple-positive cells of multi-color immunofluorescence stained TMA sections (skin samples: DD (N = 7, red), PSO (N = 3, beige), AD (N = 3, blue)). f IL-17A production of live gated human CD3⁺CD4⁺CD45RA^-CLA⁺CD103⁺ circulating skin-resident memory T cells (cT_RM) in the blood of DD patients (red) compared to HC (blue). Flow cytometric analysis of IL-17A upon ex vivo stimulation with PMA/ionomycin and intracellular cytokine staining. p-values were calculated using unpaired, two-tailed student’s t test. Gating strategy is shown in Suppl. Fig. 7.

Fig. 3. Targeting the IL-17/IL-23 axis with monoclonal antibodies is effective in patients with Darier disease.

a Cutaneous improvement after personalized targeted antibody therapy. Clinical images of guselkumab (upper, PAT1) and secukinumab (central and lower, PAT2 and PAT9) treated patients before, 3–4 and 11–12 months after initiation of antibody therapy. b Clinical improvement of DD patients during therapy as determined by the Investigator’s Global Assessment (IGA) score.

Fig. 4. Normalization of the inflammatory gene expression profile and Th17 cell numbers in DD patients treated with anti-IL-17/IL-23 antibodies.

a, b Relative mRNA expression (qRT-PCR) of Th1, Th2, and Th17-associated cytokines normalized to housekeeping gene ACTB and relative to HC for PAT1 and PAT2. Bars represent means of different individuals; error bars represent standard deviations. c Normalization of gene expression during therapy of differentially regulated genes (DD vs HC) in DD patients determined by NanoString nCounter analysis using the Immunology_V2 panel. Genes represent DE genes expressed as Z-score (DD vs HC, fold change >1.5). d Assessment of Th17 cell counts with multiplex flow cytometry in skin samples from PAT1 before and during therapy with guselkumab. Dot plot of IL-22 and IL-17A expression of CD4⁺ T cells of PAT1 before therapy initiation (red) and during therapy (salmon, 3.5 months after therapy initiation). Gating strategy is shown in Suppl. Fig. 10. e Quantification of results in d compared to HC (blue, N = 7), PAT1 before therapy initiation (red) and PAT1 during therapy (salmon). Individual symbols represent different donors, error bars represent standard deviation.