Although analysis pipelines have been developed to use RNA-seq to identify long non-coding RNAs (lncRNAs), inference of their biological and pathological relevance remains a challenge. As a result, most transcriptome studies of autoimmune disease have only assessed protein-coding transcripts.
We used RNA-seq data from 99 lesional psoriatic, 27 uninvolved psoriatic, and 90 normal skin biopsies, and applied computational approaches to identify and characterize expressed lncRNAs. We detect 2,942 previously annotated and 1,080 novel lncRNAs which are expected to be skin specific. Notably, over 40% of the novel lncRNAs are differentially expressed and the proportions of differentially expressed transcripts among protein-coding mRNAs and previously-annotated lncRNAs are lower in psoriasis lesions versus uninvolved or normal skin. We find that many lncRNAs, in particular those that are differentially expressed, are co-expressed with genes involved in immune related functions, and that novel lncRNAs are enriched for localization in the epidermal differentiation complex. We also identify distinct tissue-specific expression patterns and epigenetic profiles for novel lncRNAs, some of which are shown to be regulated by cytokine treatment in cultured human keratinocytes.
Together, our results implicate many lncRNAs in the immunopathogenesis of psoriasis, and our results provide a resource for lncRNA studies in other autoimmune diseases.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0570-4) contains supplementary material, which is available to authorized users.
To increase our understanding of psoriasis, we utilized RNA-seq to assay the transcriptomes of lesional psoriatic and normal skin. We sequenced polyadenylated RNA-derived cDNAs 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 co-expression network analysis (WGCNA) revealed multiple modules of coordinately expressed epidermal differentiation genes, overlapping significantly with genes regulated by the long non-coding 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 down-regulated in psoriatic biopsies, most likely due to expansion of the epidermal compartment. These results demonstrate the power of WGCNA to elucidate gene regulatory circuits in psoriasis, and emphasize the influence of tissue architecture in both differential expression and co-expression analysis.
skin; inflammation; immunology; cytokine; dermatology; psoriasis; transcriptome; network analysis
The Atonal Homolog 7 (ATOH7) gene has been implicated in association studies with optic nerve head diameter size. Hence, we screened optic nerve hypoplasia (ONH) patient DNA samples from Australia, France, and the United States for sequence variants in the ATOH7 gene using Sanger sequencing.
Sanger sequencing of the ATOH7 gene was performed on 34 affected individual DNA samples. Sequencing was also carried out in 3 unaffected family members to confirm segregation of identified single nucleotide variations.
Seven sequence variations were identified in ATOH7. No disease-causing sequence changes in the ATOH7 gene was discovered in the ONH patient samples.
Mutations within the ATOH7 gene are not implicated in the pathogenesis of optic nerve hypoplasia in our patient cohort.
Genome-scale studies of psoriasis have been used to identify genes of potential relevance to disease mechanisms. For many identified genes, however, the cell type mediating disease activity is uncertain, which has limited our ability to design gene functional studies based on genomic findings.
We identified differentially expressed genes (DEGs) with altered expression in psoriasis lesions (n = 216 patients), as well as candidate genes near susceptibility loci from psoriasis GWAS studies. These gene sets were characterized based upon their expression across 10 cell types present in psoriasis lesions. Susceptibility-associated variation at intergenic (non-coding) loci was evaluated to identify sites of allele-specific transcription factor binding.
Half of DEGs showed highest expression in skin cells, although the dominant cell type differed between psoriasis-increased DEGs (keratinocytes, 35%) and psoriasis-decreased DEGs (fibroblasts, 33%). In contrast, psoriasis GWAS candidates tended to have highest expression in immune cells (71%), with a significant fraction showing maximal expression in neutrophils (24%, P < 0.001). By identifying candidate cell types for genes near susceptibility loci, we could identify and prioritize SNPs at which susceptibility variants are predicted to influence transcription factor binding. This led to the identification of potentially causal (non-coding) SNPs for which susceptibility variants influence binding of AP-1, NF-κB, IRF1, STAT3 and STAT4.
These findings underscore the role of innate immunity in psoriasis and highlight neutrophils as a cell type linked with pathogenetic mechanisms. Assignment of candidate cell types to genes emerging from GWAS studies provides a first step towards functional analysis, and we have proposed an approach for generating hypotheses to explain GWAS hits at intergenic loci.
AP-1; Fibroblast; GWAS; Keratinocyte; Microarray; Neutrophil; TNFRSF9; Transcription factor
Shading is known to produce vivid perceptions of depth. However, the influence of specular highlights on perceived shape is unclear: some studies have shown that highlights improve quantitative shape perception while others have shown no effect. Here we ask how specular highlights combine with Lambertian shading cues to determine perceived surface curvature, and to what degree this is based upon a coherent model of the scene geometry. Observers viewed ambiguous convex/concave shaded surfaces, with or without highlights. We show that the presence/absence of specular highlights has an effect on qualitative shape, their presence biasing perception toward convex interpretations of ambiguous shaded objects. We also find that the alignment of a highlight with the Lambertian shading modulates its effect on perceived shape; misaligned highlights are less likely to be perceived as specularities, and thus have less effect on shape perception. Increasing the depth of the surface or the slant of the illuminant also modulated the effect of the highlight, increasing the bias toward convexity. The effect of highlights on perceived shape can be understood probabilistically in terms of scene geometry: for deeper objects and/or highly slanted illuminants, highlights will occur on convex but not concave surfaces, due to occlusion of the illuminant. Given uncertainty about the exact object depth and illuminant direction, the presence of a highlight increases the probability that the surface is convex.
A primary goal of the human visual system is to reconstruct the three-dimensional structure of the environment from two-dimensional retinal images. This process is under-determined: an infinite number of combinations of shape, material properties and illumination conditions could give rise to any single image. Rather than determining the true three-dimensional scene in a deductive manner, the visual system must make its ‘best guess’ based on the image, probabilistic models of image formation, and the stored probability of various scene configurations. For example, the visual system appears to assume that convex surfaces are more common than concave ones, biasing perception toward convex surfaces when the image is ambiguous. Here we identify a new probabilistic cue for surface shape: a shape with a visible specular highlight is more likely to be convex than one without. Highlights occur when light is reflected in a mirror-like way from glossy surfaces such as polished marble or metal. Due to the geometry of reflection, however, highlights are more likely to be occluded on concave objects. We show that the human visual system makes use of this constraint: shape perception is biased toward convex surfaces when highlights are apparent.
The IL12B gene encodes the common p40 subunit of IL-12 and IL-23, cytokines with key roles in Th1 and Th17 biology, respectively, and genetic variation in this region significantly influences risk of psoriasis. Here, we demonstrate that a psoriasis-associated risk haplotype at the IL12B locus leads to increased expression of IL12B by monocytes and correlated with increased serum levels of IL-12, IFN-γ and the IFN-γ induced chemokine, CXCL10. In contrast, serum IL-23 levels were decreased in risk carriers when compared with non-carriers. We further demonstrate that IL-12 is increased in psoriatic skin and that risk carriers manifest a skewing of the inflammatory network toward stronger IFN-γ responses. Taken together, our data demonstrate that the risk variant in IL12B associates with its increased expression and predisposes to stronger Th1 polarization through deviation of the local inflammatory environment toward increased IL-12/IFN-γ at the expense of IL-23/IL-17 responses.
Atopic dermatitis is a common inflammatory skin disease with a strong heritable component. Pathogenetic models consider keratinocyte differentiation defects and immune alterations as scaffolds1, and recent data indicate a role for autoreactivity in at least a subgroup of patients2. With filaggrin (FLG) a major locus causing a skin barrier deficiency was identified3. To better define risk variants and identify additional susceptibility loci, we densely genotyped 2,425 German cases and 5,449 controls using the Immunochip array, followed by replication in 7,196 cases and 15,480 controls from Germany, Ireland, Japan and China. We identified 4 new susceptibility loci for atopic dermatitis and replicated previous associations. This brings the number of atopic dermatitis risk loci reported in individuals of European ancestry to 11. We estimate that these susceptibility loci together account for 14.4% of the heritability for atopic dermatitis.
Whole-genome expression profiling has been used to characterize molecular-level differences between psoriasis lesions and normal skin. Pathway analysis, however, is complicated by the fact that expression profiles have been derived from bulk skin biopsies with RNA derived from multiple cell types.
We analyzed gene expression across a large sample of psoriatic (PP) and uninvolved/normal (PN) skin biopsies (n = 215 patients). We identified 1975 differentially expressed genes, including 8 associated with psoriasis susceptibility loci. To facilitate pathway analysis, PP versus PN differences in gene expression were analyzed with respect to 235 gene modules, each containing genes with a similar expression pattern in keratinocytes and epidermis. We identified 30 differentially expressed modules (DEMs) biased towards PP-increased or PP-decreased expression. These DEMs were associated with regulatory axes involving cytokines (e.g., IFN-γ, IL-17A, TNF-α), transcription factors (e.g., STAT1, NF-κB, E2F, RUNX1) and chromatin modifiers (SETDB1). We identified an interferon-induced DEM with genes encoding anti-viral proteins (designated “STAT1-57”), which was activated in psoriatic epidermis but repressed following biologic therapy. Genes within this DEM shared a motif near the transcription start site resembling the interferon-stimulated response element (ISRE).
We analyzed a large patient cohort and developed a new approach for delineating epidermis-specific pathways and regulatory mechanisms that underlie altered gene expression in psoriasis. Our findings highlight previously unrecognized “transcription circuits” that can provide targets for development of non-systemic therapies.
Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor with high mortality rates. Merkel cell polyomavirus (MCPyV), identified in the majority of MCC, may drive tumorigenesis via viral T antigens. However, mechanisms underlying pathogenesis in MCPyV-negative MCC remain poorly understood. To nominate genes contributing to pathogenesis of MCPyV-negative MCC, we performed DNA microarray analysis on 30 MCCs. MCPyV status of MCCs was determined by PCR for viral DNA and RNA. 1593 probe-sets were differentially expressed between MCPyV-negative and -positive MCC, with significant differential expression defined as at least 2-fold change in either direction and p-value of ≤ 0.05. MCPyV-negative tumors showed decreased RB1 expression, whereas MCPyV-positive tumors were enriched for immune response genes. Validation studies included immunohistochemistry demonstration of decreased RB protein expression in MCPyV-negative tumors and increased peritumoral CD8+ T lymphocytes surrounding MCPyV-positive tumors. In conclusion, our data suggest that loss of RB1 expression may play an important role in tumorigenesis of MCPyV-negative MCC. Functional and clinical validation studies are needed to determine whether this tumor suppressor pathway represents an avenue for targeted therapy.
Psoriasis vulgaris (PsV) and psoriatic arthritis (PsA) are inter-related disorders, with PsA representing a disease within a disease. From an epidemiological perspective, the genetic contributions of PsV and PsA are now well documented. HLA-C is firmly established as a PsV/PsA gene, with HLA-Cw*0602 as a major risk allele. Fine mapping studies within the MHC region in PsV and PsA have identified novel loci that are independent of the HLA-Cw6 allele. Recent genome-wide association scans have led to a substantial increase in the number of candidate genes reaching genome-wide significance in PsV and PsA cohorts. Most of these genes can be grouped into an integrated pathogenic model of PsV/PsA comprised of distinct signaling networks affecting skin barrier function (LCE3, DEFB4, GJB2), innate immune responses involving NFkB and interferon signaling (TNFAIP3, TNIP1, NFKBIA, REL, FBXL19, TYK2, NOS2), and adaptive immune responses involving CD8 T-lymphocytes and IL-23/IL-17-mediated lymphocyte signaling (HLA-C, IL12B, IL23R, IL23A, TRAF3IP2, ERAP1). Further development of a global genetic risk score and inclusion of potential gene/gene and gene/environment interactions will likely enhance the predictive value of recently identified genetic variants.
psoriasis; psoriatic arthritis; genes; genome-wide association scans
EphA2 is a receptor tyrosine kinase (RTK) that triggers keratinocyte differentiation upon activation and subsequently down-regulation by ephrin-A1 ligand. The objective for this study was to determine if the EphA2/ephrin-A1 signaling axis was altered in psoriasis, an inflammatory skin condition where keratinocyte differentiation is abnormal. Microarray analysis of skin biopsies from psoriasis patients revealed increased mRNA transcripts for several members of this RTK family in plaques, including the EphA1, EphA2 and EphA4 subtypes prominently expressed by keratinocytes. Of these, EphA2 showed the greatest up-regulation, a finding that was confirmed by quantitative RT-PCR, IHC analysis and ELISA. In contrast, psoriatic lesions exhibited reduced ephrin-A ligand immunoreactivity. Exposure of primary keratinocytes induced to differentiated in high calcium or a 3-dimensiosnal raft culture of human epidermis to a combination of growth factors and cytokines elevated in psoriasis increased EphA2 mRNA and protein expression while inducing S100A7 and disrupting differentiation. Pharmacological delivery of a soluble ephrin-A1 peptidomimetic ligand led to a reduction in EphA2 expression and ameliorated proliferation and differentiation in raft cultures exposed to EGF and IL-1α. These findings suggest that ephrin-A1-mediated down-regulation of EphA2 supports keratinocyte differentiation in the context of cytokine perturbation.
Psoriasis lesions are characterized by large-scale shifts in gene expression. Mechanisms that underlie differentially expressed genes (DEGs), however, are not completely understood. We analyzed existing datasets to evaluate genome-wide expression in lesions from 163 psoriasis patients. Our aims were to identify mechanisms that drive differential expression and to characterize heterogeneity among lesions in this large sample.
We identified 1233 psoriasis-increased DEGs and 977 psoriasis-decreased DEGs. Increased DEGs were attributed to keratinocyte activity (56%) and infiltration of lesions by T-cells (14%) and macrophages (11%). Decreased DEGs, in contrast, were associated with adipose tissue (63%), epidermis (14%) and dermis (4%). KC/epidermis DEGs were enriched for genes induced by IL-1, IL-17A and IL-20 family cytokines, and were also disproportionately associated with AP-1 binding sites. Among all patients, 50% exhibited a heightened inflammatory signature, with increased expression of genes expressed by T-cells, monocytes and dendritic cells. 66% of patients displayed an IFN-γ-strong signature, with increased expression of genes induced by IFN-γ in addition to several other cytokines (e.g., IL-1, IL-17A and TNF). We show that such differences in gene expression can be used to differentiate between etanercept responders and non-responders.
Psoriasis DEGs are partly explained by shifts in the cellular composition of psoriasis lesions. Epidermal DEGs, however, may be driven by the activity of AP-1 and cellular responses to IL-1, IL-17A and IL-20 family cytokines. Among patients, we uncovered a range of inflammatory- and cytokine-associated gene expression patterns. Such patterns may provide biomarkers for predicting individual responses to biologic therapy.
AP-1; Etanercept; IL-17; IL-20; Inflammation; Keratinocyte; Microarray; TNF; T-cell; Transcription factor
To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of three genome-wide association studies (GWAS) and two independent datasets genotyped on the Immunochip, involving 10,588 cases and 22,806 controls in total. We identified 15 new disease susceptibility regions, increasing the number of psoriasis-associated loci to 36 for Caucasians. Conditional analyses identified five independent signals within previously known loci. The newly identified shared disease regions encompassed a number of genes whose products regulate T-cell function (e.g. RUNX3, TAGAP and STAT3). The new psoriasis-specific regions were notable for candidate genes whose products are involved in innate host defense, encoding proteins with roles in interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C), and NF-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense.
A single previous study has demonstrated significant association of psoriasis with copy number of beta-defensin genes, using DNA from psoriasis cases and controls from Nijmegen and Erlangen. In this study we attempted to replicate that finding in larger new cohorts from Erlangen (N = 2017) and Michigan (N = 5412), using improved methods for beta-defensin copy number determination based on the paralog ratio test (PRT), and enhanced methods of analysis and association testing implemented in the CNVtools resource. We demonstrate that the association with psoriasis found in the discovery sample is maintained after applying improved typing and analysis methods (p = 5.5 × 10−4, OR = 1.25). We also find that the association is replicated in 2616 cases and 2526 controls from Michigan, although at reduced significance (p = 0.014), but not in new samples from Erlangen (1396 cases and 621 controls, p = 0.38). Meta-analysis across all cohorts suggests a nominally significant association (p = 6.6 × 10−3/2 × 10−4) with an effect size (OR = 1.081) much lower than found in the discovery study (OR = 1.32). This reduced effect size and significance on replication is consistent with a genuine but weak association.
We have shown that autocrine proliferation of human keratinocytes (KC) is strongly dependent upon amphiregulin (AREG), whereas blockade of heparin-binding EGF-like growth factor (HB-EGF) inhibits KC migration in scratch wound assays. Here we demonstrate that expression of soluble HB-EGF (sHB-EGF) or full-length transmembrane HB-EGF (proHB-EGF), but not proAREG, results in profound increases in KC migration and invasiveness in monolayer culture. Coincident with these changes, HB-EGF significantly decreases mRNA expression of several epithelial markers including keratins 1, 5, 10, and 14, while increasing expression of markers of cellular motility including SNAI1, ZEB1, COX-2 and MMP1. Immunostaining revealed HB-EGF-induced expression of the mesenchymal protein vimentin and decreased expression of E-cadherin as well as nuclear translocation of β-catenin. Suggestive of a trade-off between KC motility and proliferation, overexpression of HB-EGF also reduced KC growth by more than 90%. We also show that HB-EGF is strongly induced in regenerating epidermis after partial thickness wounding of human skin. Taken together, our data suggest that expression of HB-EGF in human KC triggers a migratory and invasive phenotype with many features of epithelial-mesenchymal transition (EMT), which may be beneficial in the context of cutaneous wound healing.
Heparin-binding EGF-like growth factor; epidermal growth factor receptor; epithelial-mesenchymal transition; wound healing; invasion
In 1912, Max Wertheimer published his paper on phi motion, widely recognized as the start of Gestalt psychology. Because of its continued relevance in modern psychology, this centennial anniversary is an excellent opportunity to take stock of what Gestalt psychology has offered and how it has changed since its inception. We first introduce the key findings and ideas in the Berlin school of Gestalt psychology, and then briefly sketch its development, rise, and fall. Next, we discuss its empirical and conceptual problems, and indicate how they are addressed in contemporary research on perceptual grouping and figure-ground organization. In particular, we review the principles of grouping, both classical (e.g., proximity, similarity, common fate, good continuation, closure, symmetry, parallelism) and new (e.g., synchrony, common region, element and uniform connectedness), and their role in contour integration and completion. We then review classic and new image-based principles of figure-ground organization, how it is influenced by past experience and attention, and how it relates to shape and depth perception. After an integrated review of the neural mechanisms involved in contour grouping, border-ownership, and figure-ground perception, we conclude by evaluating what modern vision science has offered compared to traditional Gestalt psychology, whether we can speak of a Gestalt revival, and where the remaining limitations and challenges lie. A better integration of this research tradition with the rest of vision science requires further progress regarding the conceptual and theoretical foundations of the Gestalt approach, which will be the focus of a second review paper.
Gestalt; grouping principles; figure-ground organization; neural mechanisms; vision science
Psoriatic arthritis (PsA) is a chronic inflammatory musculoskeletal disease affecting up to 30% of psoriasis vulgaris (PsV) cases and approximately 0.25% to 1% of the general population. To identify common susceptibility loci, we performed a meta-analysis of three imputed genome-wide association studies (GWAS) on psoriasis, stratified for PsA. A total of 1,160,703 SNPs were analyzed in the discovery set consisting of 535 PsA cases and 3,432 controls from Germany, the United States and Canada. We followed up two SNPs in 1,931 PsA cases and 6,785 controls comprising six independent replication panels from Germany, Estonia, the United States and Canada. In the combined analysis, a genome-wide significant association was detected at 2p16 near the REL locus encoding c-Rel (rs13017599, P=1.18×10−8, OR=1.27, 95% CI=1.18–1.35). The rs13017599 polymorphism is known to associate with rheumatoid arthritis (RA), and another SNP near REL (rs702873) was recently implicated in PsV susceptibility. However, conditional analysis indicated that rs13017599, rather than rs702873, accounts for the PsA association at REL. We hypothesize that c-Rel, as a member of the Rel/NF-κB family, is associated with PsA in the context of disease pathways that involve other identified PsA and PsV susceptibility genes including TNIP1, TNFAIP3 and NFκBIA.
The TNFAIP3 gene has been associated with psoriasis, rheumatoid arthritis, type 1 diabetes mellitus, systemic lupus erythematosus and celiac disease. TNFAIP3 encodes A20, a TNF-α-inducible zinc finger protein thought to limit NF-κB mediated immune responses. In this study we report association of response of psoriasis to TNF blockers with two TNFAIP3 SNPs (rs2230926 in exon 3 and rs610604 in intron 6) and their haplotypes. Treatment response was self-evaluated using a 0–5 visual analog scale in 433 psoriasis patients who received TNF blockers. Confirmation was sought in 199 psoriasis and psoriatic arthritis patients from Toronto who were followed prospectively. Response variables were dichotomized separately in the two cohorts, yielding similar proportions of good responses. While significant associations were observed only for the Michigan cohort, fixed-effects meta-analysis retained significant association between dosage of the G allele of rs610604 and good combined response to all TNF blockers (OR = 1.50, pcorr = 0.050) and etanercept (OR = 1.64, pcorr = 0.016). The rs2230926 T–rs610604 G haplotype was similarly associated. By demonstrating an association with therapeutic response, these results provide a clinically relevant functional correlate to the recently described genetic association between psoriasis and TNFAIP3.
psoriasis; TNFAIP3; tumor necrosis factor; pharmacogenetics
A multicenter meta-analysis including data from 9389 psoriasis patients and 9477 control subjects was performed to investigate the contribution of the deletion of genes LCE3C and LCE3B, involved in skin barrier defense, to psoriasis susceptibility in different populations. The study confirms that the deletion of LCE3C and LCE3B is a common genetic factor for susceptibility to psoriasis in European populations [OROverall = 1.21 (1.15–1.27)], and for the first time directly demonstrated the deletion's association with psoriasis in [Chinese OR = 1.27 (1.16–1.34); Mongolian OR = 2.08 (1.44–2.99)] populations. The analysis of the HLA-Cw6 locus showed significant differences in the epistatic interaction with the LCE3C and LCE3B deletion in at least some European populations, indicating epistatic effects between these two major genetic contributors to psoriasis. The study highlights the value of examining genetic risk factors in multiple populations to identify genetic interactions, and indicates the need of further studies to understand the interaction of the skin barrier and the immune system in susceptibility to psoriasis.
The clinical features of psoriasis, characterized by sharply demarcated scaly erythematous plaques, are typically so distinctive that a diagnosis can easily be made on these grounds alone. However, there is great variability in treatment response between individual patients, and this may reflect heterogeneity of inflammatory networks driving the disease. In this study, whole-genome transcriptional profiling was used to characterize inflammatory and cytokine networks in 62 lesional skin samples obtained from patients with stable chronic plaque psoriasis. We were able to stratify lesions according to their inflammatory gene expression signatures, identifying those associated with strong (37% of patients), moderate (39%) and weak inflammatory infiltrates (24%). Additionally, we identified differences in cytokine signatures with heightened cytokine-response patterns in one sub-group of lesions (IL-13-strong; 50%) and attenuation of these patterns in a second sub-group (IL-13-weak; 50%). These sub-groups correlated with the composition of the inflammatory infiltrate, but were only weakly associated with increased risk allele frequency at some psoriasis susceptibility loci (e.g., REL, TRAF3IP2 and NOS2). Our findings highlight variable points in the inflammatory and cytokine networks known to drive chronic plaque psoriasis. Such heterogeneous aspects may shape clinical course and treatment responses, and can provide avenues for development of personalized treatments.
Skin aging is associated with intrinsic processes that compromise the structure of the extracellular matrix while promoting loss of functional and regenerative capacity. These processes are accompanied by a large-scale shift in gene expression, but underlying mechanisms are not understood and conservation of these mechanisms between humans and mice is uncertain.
We used genome-wide expression profiling to investigate the aging skin transcriptome. In humans, age-related shifts in gene expression were sex-specific. In females, aging increased expression of transcripts associated with T-cells, B-cells and dendritic cells, and decreased expression of genes in regions with elevated Zeb1, AP-2 and YY1 motif density. In males, however, these effects were contrasting or absent. When age-associated gene expression patterns in human skin were compared to those in tail skin from CB6F1 mice, overall human-mouse correspondence was weak. Moreover, inflammatory gene expression patterns were not induced with aging of mouse tail skin, and well-known aging biomarkers were in fact decreased (e.g., Clec7a, Lyz1 and Lyz2). These unexpected patterns and weak human-mouse correspondence may be due to decreased abundance of antigen presenting cells in mouse tail skin with age.
Aging is generally associated with a pro-inflammatory state, but we have identified an exception to this pattern with aging of CB6F1 mouse tail skin. Aging therefore does not uniformly heighten inflammatory status across all mouse tissues. Furthermore, we identified both intercellular and intracellular mechanisms of transcriptome aging, including those that are sex- and species-specific.
IL-1F6, IL-1F8 and IL-1F9 and the IL-1R6(RP2) receptor antagonist IL-1F5 constitute a novel IL-1 signaling system that is poorly characterized in skin. To further characterize these cytokines in healthy and inflamed skin, we studied their expression in healthy control (NN), uninvolved psoriasis (PN) and psoriasis plaque (PP) skin using QRT-PCR and immunohistochemistry. Expression of IL-1F5, -1F6, -1F8, and -1F9 were increased 2-3 orders of magnitude in PP versus PN skin, which was supported immunohistologically. Moreover, treatment of psoriasis with etanercept led to significantly decreased IL-1F5, -1F6, -1F8 and -1F9 mRNAs, concomitant with clinical improvement. Similarly increased expression of IL-1F5, -1F6, -1F8 and -1F9 was seen in the involved skin of two mouse models of psoriasis. Suggestive of their importance in inflamed epithelia, IL-1α and TNF-α induced IL-1F5, -1F6, -1F8, and -1F9 transcript expression by normal human keratinocytes. Microarray analysis revealed that these cytokines induce the expression of anti-microbial peptides and matrix metalloproteins by reconstituted human epidermis. In particular, IL-1F8 increased mRNA expression of HBD2, HBD3 and CAMP and protein secretion of HBD2 and HBD3. Collectively, our data suggest important roles for these novel cytokines in inflammatory skin diseases and identify these peptides as potential targets for antipsoriatic therapies.
Skin; inflammation; cytokine; IL-1; psoriasis; anti-microbial peptides
Earlier studies have shown that psoriasis in Japan and Thailand is associated with two different MHC haplotypes—those bearing HLA-Cw6 and those bearing HLA-Cw1 and HLA-B46. In an independent case-control sample from Thailand, we confirmed association of psoriasis with both haplotypes. No association was seen in Thai HLA-Cw1 haplotypes lacking HLA-B46, nor was HLA-Cw1 associated with psoriasis in a large Caucasian sample. To assess whether these risk haplotypes share a common origin, we sequenced genomic DNA from a Thai HLA-Cw1-B46 homozygote across the ~300 kb MHC risk interval, and compared it to sequence of a HLA-Cw6-B57 risk haplotype. Three small regions of homology were found, but these regions share equivalent sequence similarity with one or more clearly non-risk haplotypes, and they contain no polymorphism alleles unique to all risk haplotypes. Differences in psoriasis phenotype were also observed, including lower risk of disease, greater nail involvement, and later age at onset in HLA-Cw1-B46 carriers compared to HLA-Cw6 carriers. These findings suggest locus heterogeneity at PSORS1, the major psoriasis susceptibility locus in the MHC, with HLA-Cw6 imparting risk in both Caucasians and Asians, and an allele other than HLA-Cw1 on the HLA-Cw1-B46 haplotype acting as an additional risk variant in East Asians.
Psoriasis; Human Leukocyte Antigens; human genetics; Major Histocompatibility Complex
Genome-wide association (GWA) studies have identified numerous, replicable, genetic associations between common single nucleotide polymorphisms (SNPs) and risk of common autoimmune and inflammatory (immune-mediated) diseases, some of which are shared between two diseases. Along with epidemiological and clinical evidence, this suggests that some genetic risk factors may be shared across diseases—as is the case with alleles in the Major Histocompatibility Locus. In this work we evaluate the extent of this sharing for 107 immune disease-risk SNPs in seven diseases: celiac disease, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes. We have developed a novel statistic for Cross Phenotype Meta-Analysis (CPMA) which detects association of a SNP to multiple, but not necessarily all, phenotypes. With it, we find evidence that 47/107 (44%) immune-mediated disease risk SNPs are associated to multiple—but not all—immune-mediated diseases (SNP-wise PCPMA<0.01). We also show that distinct groups of interacting proteins are encoded near SNPs which predispose to the same subsets of diseases; we propose these as the mechanistic basis of shared disease risk. We are thus able to leverage genetic data across diseases to construct biological hypotheses about the underlying mechanism of pathogenesis.
Over the last five years we have found over 100 genetic variants predisposing to common diseases affecting the immune system. In this study we analyze 107 such variants across seven diseases and find that almost half are shared across diseases. We also find that the patterns of sharing across diseases cluster these variants into groups; proteins encoded near variants in the same group tend to interact. This suggests that genetic variation may influence entire pathways to create risk to multiple diseases.
Ligands of the EGF family regulate autocrine keratinocyte proliferation and IL-1 family cytokines orchestrate epithelial defense responses. While members of both families are overexpressed in wound healing and psoriasis, their roles in regulating the innate immune functions of keratinocytes remain incompletely explored. Using sensitive assays, we found significant increases of HB-EGF, TGF-α and amphiregulin mRNA and protein in lesional psoriasis compared with uninvolved or control skin. In normal human keratinocyte (NHK) monolayers, EGFR ligands were ineffective in inducing DEFB4, S100A7, and CCL20 mRNAs and hBD-2 peptide. Combined with IL-1α, however, EGFR ligands provoked 250× more DEFB4 and CCL20 and a 9-fold rise in S100A7 mRNA relative to the EGFR ligand alone. This synergy was also reflected in secreted hBD-2 protein, both from NHK and reconstituted human epidermis. Keratinocyte differentiation was critical for these responses, as postconfluent NHK yielded mRNA and protein levels an order of magnitude greater than subconfluent cells. Differentiation also influenced signal transduction, with subconfluent cells utilizing NF-κB and postconfluent cells utilizing EGFR, MEK1/2, and p38. We propose that EGFR ligands are important modifiers of IL-1 activity, synergizing with IL-1 to stimulate epidermal production of hBD-2, S100A7 and CCL20, three of the most upregulated transcripts in psoriatic plaques.