IL-17 induces an expanded range of downstream genes in reconstituted human epidermis model

Abstract

Background: IL-17 is the defining cytokine of the Th17, Tc17, and γδ T cell populations that plays a critical role in mediating inflammation and autoimmunity. Psoriasis vulgaris is an inflammatory skin disease mediated by Th1 and Th17 cytokines with relevant contributions of IFN-γ, TNF-α, and IL-17. Despite the pivotal role IL-17 plays in psoriasis, and in contrast to the other key mediators involved in the psoriasis cytokine cascade that are capable of inducing broad effects on keratinocytes, IL-17 was demonstrated to regulate the expression of a limited number of genes in monolayer keratinocytes cultured in vitro.

Methodology/principal findings: Given the clinical efficacy of anti-IL-17 agents is associated with an impressive reduction in a large set of inflammatory genes, we sought a full-thickness skin model that more closely resemble in vivo epidermal architecture. Using a reconstructed human epidermis (RHE), IL-17 was able to upregulate 419 gene probes and downregulate 216 gene probes. As possible explanation for the increased gene induction in the RHE model is that C/CAAT-enhancer-binding proteins (C/EBP) -β, the transcription factor regulating IL-17-responsive genes, is expressed preferentially in differentiated keratinocytes.

Conclusions/significance: The genes identified in IL-17-treated RHE are likely relevant to the IL-17 effects in psoriasis, since ixekizumab (anti-IL-17A agent) strongly suppressed the "RHE" genes in psoriasis patients treated in vivo with this IL-17 antagonist.

Figure 1. IL-17-regulated C/EBPβ, human β-defensin 2, and lipocalin are expressed by terminally differentiated keratinocytes.

Immunohistochemistry for C/EBPβ (top), human β-defensin 2 (HBD2, middle), and lipocalin (LCN-2, bottom) in normal, non-lesional or lesional psoriatic skin showing predominant expression in the spinous-granular layer of the epidermis.

Figure 2. IL-17 induces a large number of genes in RHE.

Venn diagram illustrates the number of up-regulated (red) and down-regulated (green) probe-sets with the number of unique DEGs in parentheses of IL-17-treated keratinocytes, fibroblasts or RHE compared to the respective untreated conditions. U133A 2.0 arrays were used for KC and fibroblasts, while U133A Plus 2.0 arrays were used for RHE (FCH >1.5 and FDR<0.1 were used for all arrays). The additional semi-circle of RHE genes represents the probe-sets (DEGs) that were not present in the U133A 2.0 arrays.

Figure 3. Improvement of psoriasis with IL-17 blockade is associated with reduced expression of IL-17-induced RHE genes.

(A) Correlation between various gene sets and RHE gene profile response to cytokine stimulation (IL-17, IFN-γ, or IL-22) using GSEA. NES: normalized enrichment score; FDR: false discovery rate. (B) Venn diagram summarizing the number of DEGs among those in the psoriasis transcriptome or IL-17-treated RHE with improvement of at least 75% at two weeks post-ixekizumab. (C) Proportion of genes in IL-17-treated RHE that were differentially regulated in psoriasis (blue shaded area of (A)) and on the U133A 2.0 arrays (n = 95 out of 147 total DEGs which included additional genes only seen on the U133A Plus 2.0 arrays) that responded to treatment with IL-17 blockade (Ixekizumab, blue), TNF blockade (etanercept, red) or placebo (gray) at 2 weeks. Colored lines are changes in all MAD-3 psoriasis genes after both treatments. (D) The average change in expression (log₂FCH) of RHE+IL-17 genes toward recovery with ixekizumab, etanercept, or placebo at 2 weeks.