Sylviane Lambert

Genome-wide association study identifies a psoriasis susceptibility locus at TRAF3IP2.

Psoriasis is a multifactorial skin disease characterized by epidermal hyperproliferation and chronic inflammation, the most common form of which is psoriasis vulgaris (PsV). We present a genome-wide association analysis of 2,339,118 SNPs in 472 PsV cases and 1,146 controls from Germany, with follow-up of the 147 most significant SNPs in 2,746 PsV cases and 4,140 controls from three independent replication panels. We identified an association at TRAF3IP2 on 6q21 and genotyped two SNPs at this locus in two additional replication panels (the combined discovery and replication panels consisted of 6,487 cases and 8,037 controls; combined P = 2.36 × 10−10 for rs13210247 and combined P = 1.24 × 10−16 for rs33980500). About 15% of psoriasis cases develop psoriatic arthritis (PsA). A stratified analysis of our datasets including only PsA cases (1,922 cases compared to 8,037 controls, P = 4.57 × 10−12 for rs33980500) suggested that TRAF3IP2 represents a shared susceptibility for PsV and PsA. TRAF3IP2 encodes a protein involved in IL-17 signaling and which interacts with members of the Rel/NF-κB transcription factor family.

Enhanced meta-analysis and replication studies identify five new psoriasis susceptibility loci.

Psoriasis is a chronic autoimmune disease with complex genetic architecture. Previous genomewide association studies (GWAS) and a recent meta-analysis using Immunochip data have uncovered 36 susceptibility loci. Here, we extend our previous meta-analysis of European ancestry by refined genotype calling and imputation and by the addition of 5,033 cases and 5,707 controls. The combined analysis, consisting of over 15,000 cases and 27,000 controls, identifies five new psoriasis susceptibility loci at genomewide significance (p < 5 × 10−8). The newly identified signals include two that reside in intergenic regions (1q31.1 and 5p13.1) and three residing near PLCL2 (3p24.3), NFKBIZ (3q12.3), and CAMK2G (10q22.2). We further demonstrate that NFKBIZ is a TRAF3IP2–dependent target of IL-17 signaling in human skin keratinocytes, thereby functionally linking two strong candidate genes. These results further integrate the genetics and immunology of psoriasis, suggesting new avenues for functional analysis and improved therapies.

The EGF receptor ligand amphiregulin controls cell division via FoxM1

Epidermal growth factor receptor (EGFR) is central to epithelial cell physiology, and deregulated EGFR signaling has an important role in a variety of human carcinomas. Here we show that silencing of the EGF-related factor amphiregulin (AREG) markedly inhibits the expansion of human keratinocytes through mitotic failure and accumulation of cells with ⩾4n DNA content. RNA-sequencing-based transcriptome analysis revealed that tetracycline-mediated AREG silencing significantly altered the expression of 2331 genes, 623 of which were not normalized by treatment with EGF. Interestingly, genes irreversibly upregulated by suppression of AREG overlapped with genes involved in keratinocyte differentiation. Moreover, a significant proportion of the irreversibly downregulated genes featured upstream binding sites recognized by forkhead box protein M1 (FoxM1), a key transcription factor in the control of mitosis that is widely dysregulated in cancer. The downregulation of FoxM1 and its target genes preceded mitotic arrest. Constitutive expression of FoxM1 in AREG knockdown cells normalized cell proliferation, reduced the number of cells with ⩾4n DNA content and rescued expression of FoxM1 target genes. These results demonstrate that AREG controls G2/M progression and cytokinesis in keratinocytes via activation of a FoxM1-dependent transcriptional program, suggesting new avenues for treatment of epithelial cancer.

Erlotinib-induced skin inflammation is IL-1 mediated in KC-Tie2 mice and human skin organ culture

Epidermal growth factor receptor (EGFR) is the prototypical member of the ErbB network comprised of four transmembrane receptor tyrosine kinases (EGFR/ErbB1, ErbB2, ErbB3, and ErbB4). These receptors bind EGF and heregulin family ligands in a dynamic, feedback-regulated fashion, activating multiple signal transduction pathways and ultimately affecting many cellular functions (Avraham and Yarden, 2011; Wilson et al., 2009). ErbB receptors and their ligands are overexpressed in a wide spectrum of hyperplastic epithelial disorders including psoriasis and cancers. Numerous monoclonal antibodies and receptor tyrosine kinase inhibitors have been designed to block their activation (Yarden and Pines, 2012), and are highly effective for treating colorectal and non-small cell lung cancers. However, skin toxicity is frequently observed in patients undergoing therapy with EGFR inhibitors (EGFRIs) (Lacouture, 2006), including papular and pustular eruptions of the face and trunk, nail changes, dry skin, itch, and hair loss (Lacouture and Lai, 2006). These side-effects are the major dose-limiting toxicity of EGFRI therapy (Boone et al., 2007) and emphasize the importance of understanding the mechanisms involved.

Transgenic expression of human amphiregulin in mouse skin: inflammatory epidermal hyperplasia and enlarged sebaceous glands.

To explore the role of amphiregulin in inflammatory epidermal hyperplasia, we overexpressed human AREG (hAREG) in FVB/N mice using a bovine K5 promoter. A construct containing AREG coding sequences flanked by 5′ and 3′ untranslated region sequences (AREG‐UTR) led to a >10‐fold increase in hAREG expression compared to an otherwise‐identical construct containing only the coding region (AREG‐CDR). AREG‐UTR mice developed tousled, greasy fur as well as elongated nails and thickened, erythematous tail skin. No such phenotype was evident in AREG‐CDR mice. Histologically, AREG‐UTR mice presented with marked epidermal hyperplasia of tail skin (2.1‐fold increase in epidermal thickness with a 9.5‐fold increase in Ki‐67+ cells) accompanied by significantly increased CD4+ T‐cell infiltration. Dorsal skin of AREG‐UTR mice manifested lesser but still significant increases in epidermal thickness and keratinocyte hyperplasia. AREG‐UTR mice also developed marked and significant sebaceous gland enlargement, with corresponding increases in Ki‐67+ cells. To determine the response of AREG‐UTR animals to a pro‐inflammatory skin challenge, topical imiquimod (IMQ) or vehicle cream was applied to dorsal and tail skin. IMQ increased dorsal skin thickness similarly in both AREG‐UTR and wild type mice (1.7‐ and 2.2‐fold vs vehicle, P < 0.001 each), but had no such effect on tail skin. These results confirm that keratinocyte expression of hAREG elicits inflammatory epidermal hyperplasia, and are consistent with prior reports of tail epidermal hyperplasia and increased sebaceous gland size in mice expressing human epigen.

Dual Role of Act1 in Keratinocyte Differentiation and Host Defense: TRAF3IP2 Silencing Alters Keratinocyte Differentiation and Inhibits IL-17 Responses

TRAF3IP2 is a candidate psoriasis susceptibility gene encoding Act1, an adaptor protein with ubiquitin ligase activity that couples the IL-17 receptor to downstream signaling pathways. We investigated the role of Act1 in keratinocyte responses to IL-17 using a tetracycline inducible short hairpin RNA targeting TRAF3IP2. Tetracycline exposure for 7 days effectively silenced TRAF3IP2 mRNA and Act1 protein, resulting in 761 genes with significant changes in expression (495 down, 266 up; >1.5-fold, P < 0.05). Gene ontology analysis showed that genes affected by TRAF3IP2 silencing are involved in epidermal differentiation, with early differentiation genes (KRT1, KRT10, DSC1, DSG1) being down-regulated and late differentiation genes (SPRR2, SPRR3, LCE3) being up-regulated. AP1 binding sites were enriched upstream of genes up-regulated by TRAF3IP2 silencing. Correspondingly, nuclear expression of FosB and Fra1 was increased in TRAF3IP2-silenced cells. Many genes involved in host defense were induced by IL-17 in a TRAF3IP2-dependent fashion. Inflammatory differentiation conditions (serum addition for 4 days postconfluence) markedly amplified these IL-17 responses and increased basal levels and TRAF3IP2 silencing-dependent up-regulation of multiple late differentiation genes. These findings suggest that TRAF3IP2 may alter both epidermal homeostasis and keratinocyte defense responses to influence psoriasis risk.

The psoriasis-protective TYK2 I684S variant impairs IL-12 stimulated pSTAT4 response in skin-homing CD4+ and CD8+ memory T-cells

Tyrosine kinase 2 (TYK2) belongs to the Janus kinase (JAK) family of tyrosine kinases, which transmit signals from activated cytokine receptors. GWAS have consistently implicated TYK2 in psoriasis susceptibility. We performed an in-depth association analysis of TYK2 using GWAS and resequencing data. Strong genetic association of three nonsynonymous variants in the exonic regions of the TYK2 gene (rs34536443, rs12720356, and rs2304256) were found. rs12720356 encoding I684S is predicted to be deleterious based on its location in the pseudokinase domain. We analyzed PBMCs from 29 individuals representing the haplotypes containing each of the significantly associated signals. STAT4 phosphorylation was evaluated by phospho-flow cytometry after CD3/CD28 activation of cells followed by IL-12 stimulation. Individuals carrying the protective I684S variant manifested significantly reduced p-STAT4 levels in CD4 + CD25 + CD45RO+ (mean Stimulation Index (S.I.) 48.08, n = 10) and CD8 + CD25 + CD45RO + cells (S.I. 55.71, n = 10), compared to controls homozygous for the ancestral haplotype (S.I. 68.19, n = 10 (p = 0.002) and 76.76 n = 10 (p = 0.0008) respectively). Reduced p-STAT4 levels were also observed in skin-homing, cutaneous lymphocyte associated antigen (CLA)-positive CD4 and CD8 cells from I684S carriers. No significant changes in p-STAT4 for the psoriasis-associated variant rs34536443 was found. These data establish the functional significance of the TYK2 I684S variant in psoriasis susceptibility.

The Act1 D10N missense variant impairs CD40 signaling in human B-cells

The TRAF3IP2 gene resides within one of at least 63 psoriasis susceptibility loci and encodes Act1, an adapter protein involved in IL-17 receptor and CD40 signaling pathways. TRAF3IP2 is distinctive (among <10% of candidate susceptibility genes) in that a strongly disease-associated variant encodes a missense SNP predicted to be functionally relevant (SNP rs33980500 C/T encoding Act1 pD10N). As assessed by flow cytometry, Act1 protein was expressed at the highest levels in monocytes, with lower levels in T-cells and B-cells. However, monocytes, T-cells and B-cells failed to respond to IL-17A stimulation of PBMC, as measured by flow cytometric determination of NF-κB phospho-p65. As an alternative stimulus, we treated PBMCs with trimerized recombinant human CD40L and assessed p65, p38 and Erk phosphorylation in CD19+ B-cells as a function of D10N genotype. The increase of phosphorylated p65, p38, and Erk was well-correlated across individuals, and CD40L-induced phosphorylation of p65, p38, and Erk was significantly attenuated in B-cells from Act1 D10N homozygotes, compared to heterozygotes and nullizygotes. Our results indicate that the Act1 D10N variant is a relevant genetic determinant of CD40L responsiveness in human B-cells, with the risk allele being associated with lower B-cell responses in an acute signaling context.