Integrative Keratinocyte Responses to TWEAK with IL-13 and IL-22 Reveal Pathogenic Transcriptomes Associated with Atopic Dermatitis

To the Editor

Atopic dermatitis (AD) is characterized by dysregulated activity of keratinocytes, and IL-13 and IL-22 are considered drivers of disease in part through stimulating these cells (Facheris et al., 2023, Kim et al., 2019, Humeau et al., 2022). Whether these cytokines act independently or with other inflammatory molecules is not clear. Previously, we reported that mice deficient in the cytokine TWEAK (tumor necrosis factor-related weak inducer of apoptosis; TNFSF12) were resistant to developing experimental AD, with its receptor Fn14 (TNFRSF12A) prominently expressed on keratinocytes (Sidler et al., 2017). We also showed that deletion of Fn14 only in keratinocytes protected mice from developing maximal AD phenotypes, and therapeutic blocking of TWEAK was as effective as blocking IL-13 in reducing AD symptoms (Gupta et al., 2023). Gene set enrichment analysis from lesional skin samples has further suggested that there is a strong association of AD with TWEAK and Fn14 activity (Sidler et al., 2017), and TWEAK and/or its receptor were found upregulated in AD skin lesions (Chen et al., 2011, Liu et al., 2020, Zimmermann et al., 2011).

Here, using a transcriptomic approach, we further reinforce the contention that the TWEAK-Fn14 axis is central to AD. Bulk RNA-seq was performed in normal human epidermal keratinocytes stimulated with TWEAK, IL-13, or IL-22. Broad transcriptional changes were induced by all cytokines (Figure S1A). 185 of the transcripts induced by TWEAK were present in a transcriptome of 2895 genes described to be upregulated in AD skin lesions (Table S1A, (Bangert et al., 2021, He et al., 2021), compared to 121 and 101 induced by IL-13 and IL-22, respectively (Figure 1A and 1B, Figure S1B-C, Table S2). Gene ontology (GO) analysis revealed that the TWEAK upregulated genes were related to inflammatory activities (Figure S1D, Table S3). The 2895 gene AD transcriptome was further divided it into 1568 AD-specific genes and 1327 genes shared between AD and psoriasis (Table S1B-C). TWEAK upregulated 55 AD-specific transcripts compared to 130 that were shared between AD and psoriasis (Figure 1A-B, Table S4A-B). IL-13 and IL-22 drove induction of 60 and 32 genes unique to AD, and 61 and 69 genes common to both diseases, respectively (Figure 1A-B, Table S4A-B). The TWEAK-induced AD-specific transcripts (Figure 1C selected, Figure S1E total) included those related to epidermal barrier function or hyperplasia (CYP27B1, SPINK5, IL24, C1R, FUT4, SACS), extracellular matrix (ADAMTS4, BCL3, MMP10), and inflammation (TNF, PDGFB, TLR1, IL32, ID2). TWEAK-induced genes common to AD and psoriasis (Figure 1D selected, Figure S1F total) included others associated with impaired epidermal barrier function or hyperplasia (SERPINB1, S100A8/S100A9, CYP24A1, SPRR2D), pro-inflammatory cytokines (IL36G, IL23A), chemokines (CXCL5, CXCL10, CXCL11, CXCL16, CCL5, CCL20), more general inflammation (MMP9, MMP19, ADAM8, VIM, CD40, ICAM1, ITGAM, IL6ST, TNFRSF14, CSF1, CSF2), and NF-κB signaling (RELB, NFKB2, MAP3K14, BIRC3, TRAF3).

Figure 1. TWEAK induces inflammatory activity in human keratinocytes relevant for AD.

(A-D) RNA-seq of human keratinocytes with triplicate cell cultures stimulated with human TWEAK, IL-13, or IL-22. (A-B) Venn diagram of upregulated transcripts that are AD specific (A) or common to both AD and psoriasis (B). (C-D) Heatmaps of a select subset of TWEAK upregulated transcripts that are also found upregulated in the skin lesions of AD patients categorized either as AD-specific transcripts (C) or commonly upregulated in AD and psoriasis (D). (E) Heatmaps showing the z-score transformed values of mean percentage of keratinocytes that were positive (i.e., have non-zero gene expression in single-cell data) for genes associated with TWEAK activity (left) and the average expression for each gene (right) from healthy controls, untreated AD patients, or dupilumab-treated AD patients. AD; Atopic dermatitis.

TWEAK, IL-13, and IL-22 also downregulated many transcripts in keratinocytes (Figure S2A), and a number of these (Figure S2B-C, Table S6, Table S7) are reduced in AD skin lesions as well as psoriasis skin lesions compared to healthy skin ((He et al., 2021), Table S5A,Table S5B-C). TWEAK downregulated 35 AD specific genes (Figure S2D, S2F) and 30 common to AD and psoriasis (Figure S2E, S2G; Table S8A-B). These included SYNP02, AREG, CTGF, KLK5, WNT3, DCN, LSP1, AMOT, FLG2, F2RL1, and LAMB4, associated with epithelial integrity, tight junctions, or suppression of proliferation. Similar to the upregulated genes, some downregulated targets were shared with IL-13 and IL-22, and some unique to each cytokine (Figure S2D-E).

We also asked whether evidence of TWEAK activity might be retained in the skin of AD patients treated with the IL-13/IL-4 blocking antibody dupilumab, by analyzing keratinocyte single cell RNA-seq data (Bangert et al., 2021). While many of the 185 AD-associated transcripts induced by TWEAK in vitro (Figure 1A-B) were downregulated in keratinocytes from dupilumab-treated patients, many were still upregulated and at levels similar to or greater than those in untreated patient cells, despite this drug being clinically effective (Figure 1E, Table S13). These included transcripts for LTB, CCL5, SERPINB1, SPRR2D, VIM, and IL-32, and transcripts associated with non-canonical NF-κB signaling that is a pathway utilized by Fn14, namely BIRC3, NFKB2, and RELB.

Lastly, human keratinocytes were stimulated with TWEAK in combination with IL-13 or IL-22 (Figure 2). TWEAK with IL-13 synergistically upregulated 66 AD-relevant transcripts (Figure 2A, S3A-C, Table S9A), whereas TWEAK with IL-22 synergistically upregulated 17 transcripts (Figure 2B, S3D-F, Table S9B). These encoded cytokines and chemokines (IL23A, IL1B, TNF, CSF2, IL32, CXCL6, CXCL10 CXCL11, CCL5) and matrix-related molecules (COL4A3, COL4A4, COL6A1, MMP19, and MMP7). Synergism for TWEAK and IL-13 applied to genes primarily affected by TWEAK alone as well as genes primarily affected by IL-13 alone (Figure 2A, S3C, Table S9A). Synergism between TWEAK and IL-22 largely reflected an enhancing effect of IL-22 in amplifying gene induction triggered by TWEAK (Figure 2B, S3F, Table S9B). Similarly, TWEAK and IL-13 acted synergistically to downregulate 51 AD-relevant genes (Figure 2C, S3G-I, Table S10A), compared with 75 genes synergistically downregulated with TWEAK and IL-22 (Figure 2D, S3J-L, Table S10B). These included transcripts that TWEAK alone downregulated such as KLK5, DCN, and FLG2, with additional activity on transcripts of genes, such as KRT1, KRT77, DSG1, SPRR2A, AKR1C3, DSC1 and KLF4, on which TWEAK alone had little effect in regulating. GO analyses (Table S11, Figure S4A; Table S12, Figure S4B) showed that these synergistically downregulated genes were associated with epidermal integrity (cornification, epidermis development, keratinization, keratinocyte differentiation).

Figure 2. TWEAK combined with IL-13 or IL-22 synergistically upregulates and downregulates multiple AD-related gene transcripts in keratinocytes.

RNA-seq of human keratinocytes with triplicate cell cultures stimulated with TWEAK and IL-13 or TWEAK and IL-22. (A) Heatmap showing the expression patterns of the 66 synergistically upregulated AD genes induced by TWEAK and IL-13. (B) Heatmap showing the expression patterns of the 17 synergistically upregulated AD genes induced by TWEAK and IL-22. (C) Heatmap showing the expression patterns of the 51 AD genes synergistically downregulated by TWEAK and IL-13. (D) Heatmap showing the expression patterns of the 75 AD genes synergistically downregulated by TWEAK and IL-22. AD; Atopic dermatitis.

In conclusion, these data illustrate a strong inflammatory activity of TWEAK on inducing a number of genes in human keratinocytes relevant for AD pathogenesis, and that this cytokine has activities comparable to two recognized AD signature cytokines IL-13 or IL-22. Further transcriptomic analysis show that the combined effect of TWEAK with these other cytokines could be important to enforce the inflammatory processes triggered during the progression of AD. As this is an observational study, additional work to test causal effects, validate changes at the protein level in human tissue samples, and correlate them with expression of TWEAK in patients, will be useful. The current data together with our previous animal studies support a significant role for TWEAK in the pathogenesis of atopic dermatitis, and its expression may have implications for disease re-occurrence or for patients that might not respond to current treatments.

Data availability:

Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE214503, hosted at Gene Expression Omnibus (GSE214503). All other data associated with this study are provided in the paper or the Supplementary Materials.