IL-17 Induces an Expanded Range of Downstream Genes in Reconstituted Human Epidermis Model

<div><p>Background</p><p>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.</p><p>Methodology/Principal Findings</p><p>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.</p><p>Conclusions/Significance</p><p>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.</p></div

Selected genes involved in keratinocyte proliferation and differentiation.

1<p>FCH, fold change;</p>2<p>FDR, false discovery rate.</p

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

<p>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.</p

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

<p>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.</p

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

<p>(<b>A</b>) 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>B</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. (<b>C</b>) Proportion of genes in IL-17-treated RHE that were differentially regulated in psoriasis (blue shaded area of (<b>A</b>)) 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. (<b>D</b>) The average change in expression (log₂FCH) of RHE+IL-17 genes toward recovery with ixekizumab, etanercept, or placebo at 2 weeks.</p

Clinical photographs of two representative patients with disease relapse upon cessation of efalizumab treatment.

<p>(<b>A</b>) Baseline lesions (left), response to efalizumab (center) and a marked erythematous psoriatic reaction 5 weeks after ceasing treatment (right). (<b>B</b>) Baseline photographs showing upper leg psoriatic plaques (left) which responded to efalizumab at week 12 (center) and a relapse in the same location (right). Note the inflammatory nature of the lesions during relapse.</p

GSEA analysis of published gene sets.

a<p>Size, number of genes in gene set.</p>b<p>ES, enrichment score.</p>c<p>NES, normalized enrichment score.</p>d<p>CS, connectivity score.</p>e<p>REF, reference for gene set.</p

Genomic characterization of relapsed psoriasis lesions.

<p>(<b>A</b>) Venn diagram showing DEGs for different comparisons, week 12 versus LS; week 12 versus relapse; relapse versus LS. There were no unique DEGs in relapse compared to LS skin. (<b>B</b>) Scatter plot showing an excellent correlation between the “treatment effect” and “relapse effect”. (<b>C</b>) Number of genes improved by treatment and reversed by relapse. Red blocks were increased DEGs, green blocks were decreased DEGs.</p

Increased inflammatory myeloid DCs in relapsed lesions.

<p>(<b>A</b>) Representative immunohistochemistry and (<b>B</b>) counts of CD11c⁺ cells per mm in non-lesional skin (NL), lesional skin (LS), and in the index lesional plaque at weeks 2, 6 and 12 and time of disease relapse. (<b>C</b>) The numbers of CD1c⁺ cells did not change with treatment or relapse. (<b>D</b>) CD83⁺ mature DCs followed the same pattern as CD11c⁺ DCs. (<b>E</b>) There were many inflammatory myeloid DCs in the relapsed lesions, shown by immunofluorescence as CD11c⁺ cells (red) that were not co-expressing CD1c (and thus were not yellow), and (<b>F</b>) quantified as CD11c⁺ minus CD1c⁺ cells. (<b>G</b>) Inflammatory CD11c⁺ DCs were co-expressing TNFSF10/TRAIL. (<b>H</b>) TNF- and iNOS-producing DCs (TIP-DCs) were found in relapsed lesions. CD11c⁺ cells (red) co-expressing (<b>I</b>) CD14 and (<b>J</b>) CD16 (both green). Cells that co-express the two markers in a similar location are yellow in color. A white line denotes the dermo-epidermal junction. Bar is 100 µm.</p

Enrollment flowchart.

<p>Enrollment flowchart.</p