Transcriptome analysis of psoriasis in a large case-control sample: RNA-seq provides insights into disease mechanisms

Abstract

To increase our understanding of psoriasis, we used high-throughput complementary DNA sequencing (RNA-seq) to assay the transcriptomes of lesional psoriatic and normal skin. We sequenced polyadenylated RNA-derived complementary DNAs from 92 psoriatic and 82 normal punch biopsies, generating an average of ∼38 million single-end 80-bp reads per sample. Comparison of 42 samples examined by both RNA-seq and microarray revealed marked differences in sensitivity, with transcripts identified only by RNA-seq having much lower expression than those also identified by microarray. RNA-seq identified many more differentially expressed transcripts enriched in immune system processes. Weighted gene coexpression network analysis (WGCNA) revealed multiple modules of coordinately expressed epidermal differentiation genes, overlapping significantly with genes regulated by the long noncoding RNA TINCR, its target gene, staufen-1 (STAU1), the p63 target gene ZNF750, and its target KLF4. Other coordinately expressed modules were enriched for lymphoid and/or myeloid signature transcripts and genes induced by IL-17 in keratinocytes. Dermally expressed genes were significantly downregulated in psoriatic biopsies, most likely because of expansion of the epidermal compartment. These results show the power of WGCNA to elucidate gene regulatory circuits in psoriasis, and emphasize the influence of tissue architecture in both differential expression and coexpression analysis.

Figure 1. Averaged RPKM and proportion of DEGs for each module

Averaged RPKM among the genes (top panel), proportions of up- and down-regulated genes (middle panel), and the average Spearman correlation (bottom panel) for the co-expression gene modules constructed from the normal (normal; left panel) and psoriatic (psoriatic; right panel) skin samples, respectively.

Figure 2. Proportion of top signature genes in different cell populations for co-expression gene modules constructed from normal (left panel) and psoriatic (right panel) skin samples

From the top to bottom, respectively, the four panels show the proportions of signature genes for different skin cell and tissue compartments (keratinocytes, epidermis, dermis, and adipose tissue); "innate immunity" lymphocytes (gamma-delta T-cells, natural killer (NK) cells, and NK-T cells); "adaptive immunity" lymphocytes (CD4+ T-cells, CD8+ T-cells, and regulatory T-cells); myeloid-derived leukocytes (monocytes, macrophages, and dendritic cells). Asterisks (*) denote significant (ie p< 1 × 10⁻⁴) enrichment for the top 5% of cell signature genes in the corresponding module.

Figure 3. Overlap of gene co-expression modules with stratum granulosum and epidermal differentiation transcription factor target genes

The figures show the proportion of genes in each module overlapping with the genes identified in different independent studies. The top two panels depict overlap of each of the normal- and psoriasis- derived modules with stratum corneum genes identified by Toulza et al. (Toulza et al., 2007), Mattiuzzo et al. (Mattiuzzo et al., 2011), or the union of both studies. The bottom two panels depict overlap with targets of the epidermal differentiation regulators STAU1 and TINCR (Kretz et al., 2012) and with the transcription factors ZNF50 and KLF4 (Sen et al., 2012). The numbers in the parentheses after the names of the studies indicate the number of genes in the corresponding gene sets used in this analysis.

Figure 4. IL-37 is co-expressed with loricrin in the granular layer of non-lesional skin but significantly reduced in lesional psoriasis skin

(a) Immunohistochemistry revealed that while IL-37 protein (DAB, brown) is expressed in the granular layer of the epidermis of normal appearing skin it is not detected in lesional psoriasis skin. Scale bar indicates 25 µm. (b) QRT-PCR results demonstrating down-regulation of IL-37 and loricrin mRNAs in lesional psoriatic skin compared with non-lesional psoriatic skin. (c) Fluorescence immunohistochemistry showing that IL-37 (green) co-localized with loricrin (red) in the granular layer of non-lesional skin and this was undetectable in lesional skin. DAPI counterstaining of nuclei is shown in blue. Scale bar indicates 200 µm. Statistical significance as assessed by 2-tailed t-test: ** p<0.01, *** p<0.001,.