Psoriasis is a TH17-driven inflammatory disease affecting a significant proportion of the world population. The molecular consequences of IL-17 signaling in the skin are only partially understood. Therefore, we evaluated the IL-17A effects on organotypic 3-dimensional skin models and observed that IL-17A interfered with keratinocyte differentiation. In agreement with this phenotype, IL-17A repressed the expression of many genes encoding structural proteins. Moreover, genes encoding anti-microbial peptides were induced, resulting in a strengthening of the chemical barrier. Finally, we observed enhanced expression of the three IL-36 cytokines IL-36α, β and γ. We found that IL-36γ was secreted from keratinocytes in an inactive form and that neutrophilic proteases, including elastase, were capable of activating this cytokine. Functionally and similar to IL-17A, truncated IL-36 cytokines interfered with keratinocyte differentiation in 3D models. The molecular analysis revealed strong cooperative effects of IL-17A and IL-36 cytokines in regulating target genes, which was dependent on the proteolytic activation of the latter. Together these findings suggest an amplification cycle that can be initiated by IL-17A, involving IL-36 cytokines and immune cell derived proteases and resulting in active IL-36 cytokines which synergize with IL-17A. This amplification cycle might be relevant for a persistent psoriatic phenotype.

Atopic dermatitis, a chronic inflammatory skin disease with increasing prevalence, is closely associated with skin barrier defects. A cytokine related to disease severity and inhibition of keratinocyte differentiation is IL-31. To identify its molecular targets, IL-31–dependent gene expression was determined in three-dimensional organotypic skin models. IL-31–regulated genes are involved in the formation of an intact physical skin barrier. Many of these genes were poorly induced during differentiation as a consequence of IL-31 treatment, resulting in increased penetrability to allergens and irritants. Furthermore, studies employing cell-sorted skin equivalents in SCID/NOD mice demonstrated enhanced transepidermal water loss following s.c. administration of IL-31. We identified the IL-1 cytokine network as a downstream effector of IL-31 signaling. Anakinra, an IL-1R antagonist, blocked the IL-31 effects on skin differentiation. In addition to the effects on the physical barrier, IL-31 stimulated the expression of antimicrobial peptides, thereby inhibiting bacterial growth on the three-dimensional organotypic skin models. This was evident already at low doses of IL-31, insufficient to interfere with the physical barrier. Together, these findings demonstrate that IL-31 affects keratinocyte differentiation in multiple ways and that the IL-1 cytokine network is a major downstream effector of IL-31 signaling in deregulating the physical skin barrier. Moreover, by interfering with IL-31, a currently evaluated drug target, we will have to consider that low doses of IL-31 promote the antimicrobial barrier, and thus a complete inhibition of IL-31 signaling may be undesirable.

Background Janus kinase (JAK) inhibition may be a promising new treatment modality for inflammatory (skin) diseases. However, little is known about direct effects of kinase inhibitors on keratinocyte differentiation and function as well as skin barrier formation. Objective Our aim was to address the direct impact of kinase inhibition of the JAK1/3 pathways by tofacitinib on keratinocyte immune function and barrier formation in atopic dermatitis (AD) and psoriasis. Methods 3D skin equivalents of both diseases were developed and concurrently pretreated with tofacitinib. To induce AD, 3D skin equivalents were stimulated with recombinant human IL‐4 and IL‐13. Psoriasis‐like conditions were induced by incubation with IL‐17A, IL‐22 and tumour necrosis factor α (TNFα). The activation of signal transducer and activator of transcription (STAT)1, STAT3 and STAT6 was assessed by Western blot analysis. Microarray analysis and quantitative real‐time PCR were used for gene expression analysis. Results Tofacitinib pretreatment preserved epidermal morphology and reduced STAT3 and STAT6 phosphorylation of AD‐like and STAT3 phosphorylation of psoriasis‐like culture conditions in 3D skin models compared to sham‐controls. Filaggrin expression was fully maintained in the AD‐like models, but only partially in psoriasis‐like conditions after pretreatment with tofacitinib. In addition, tofacitinib upregulated DSC1, FLG and KRT1. Using gene expression analysis, downregulation of POSTN and IL24 was observed in AD‐like conditions, whereas downregulation of IL20 and IL1B was observed in psoriasis‐like conditions. Conclusion JAK1/3 inhibition counteracted cytokine‐induced AD‐ and psoriasis‐like epidermal morphology and enhanced keratinocyte differentiation in 3D skin models. This effect was more pronounced in the AD‐like models compared to the psoriasis‐like 3D skin models.

The AMP-activated protein kinase (AMPK) is a master sensor of the cellular energy status that is crucial for the adaptive response to limited energy availability. AMPK is implicated in the regulation of many cellular processes, including autophagy. However, the precise mechanisms by which AMPK controls these processes and the identities of relevant substrates are not fully understood. Using protein microarrays, we identify Cyclin Y as an AMPK substrate that is phosphorylated at Serine 326 (S326) both in vitro and in cells. Phosphorylation of Cyclin Y at S326 promotes its interaction with the Cyclin-dependent kinase 16 (CDK16), thereby stimulating its catalytic activity. When expressed in cells, Cyclin Y/CDK16 is sufficient to promote autophagy. Moreover, Cyclin Y/CDK16 is necessary for efficient AMPK-dependent activation of autophagy. This functional interaction is mediated by AMPK phosphorylating S326 of Cyclin Y. Collectively, we define Cyclin Y/CDK16 as downstream effector of AMPK for inducing autophagy.

Psoriasis is a T H 17-driven inflammatory disease affecting a significant proportion of the world population. The molecular consequences of IL-17 signaling in the skin are only partially understood. Therefore, we evaluated the IL-17A effects on organotypic 3-dimensional skin models and observed that IL-17A interfered with keratinocyte differentiation. In agreement with this phenotype, IL-17A repressed the expression of many genes encoding structural proteins. Moreover, genes encoding anti-microbial peptides were induced, resulting in a strengthening of the chemical barrier. Finally, we observed enhanced expression of the three IL-36 cytokines IL-36α, β and γ. We found that IL-36γ was secreted from keratinocytes in an inactive form and that neutrophilic proteases, including elastase, were capable of activating this cytokine. Functionally and similar to IL-17A, truncated IL-36 cytokines interfered with keratinocyte differentiation in 3D models. The molecular analysis revealed strong cooperative effects of IL-17A and IL-36 cytokines in regulating target genes, which was dependent on the proteolytic activation of the latter. Together these findings suggest an amplification cycle that can be initiated by IL-17A, involving IL-36 cytokines and immune cell derived proteases and resulting in active IL-36 cytokines which synergize with IL-17A. This amplification cycle might be relevant for a persistent psoriatic phenotype.Psoriasis, a chronic autoimmune disease of the skin, affects approximately 2% of the population. Psoriasis is associated with systemic inflammatory processes including inflammatory arthritis, inflammatory bowel disease, and metabolic syndrome 1,2 . A typical manifestation of the disease is the hyperproliferation of keratinocytes with premature differentiation. This results in incomplete cornification with retention of nuclei in the stratum corneum, referred to as parakeratosis. Functionally this correlates with increased infiltrations of immune cells due to dendritic cells, macrophages, neutrophils and different subpopulations of T cells. These cells modify the cytokine milieu and thus affect the behavior of keratinocytes resulting in altered differentiation [1][2][3]

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Psoriasis is one of the most prevalent inflammatory skin diseases and IL-17A has been identified as a key cytokine in the pathogenesis of the disease. Therefore we investigated the effects of IL-17A on the formation and the functionality of the skin barrier in human organotypic 3D skin equivalents. On the one hand we applied a novel full thickness psoriasis model implying human epidermal keratinocytes (NHEKs) and dermal fibroblasts (HDFs) from psoriatic lesions of patients and on the other hand we used control models containing cells from healthy donors. Stimulation with IL-17A revealed changes in skin morphology in both models including parakeratosis and varying epidermal thickness. Microarrays and immunohistological stainings revealed down-regulation of genes and proteins important for epidermal differentiation, including filaggrin and loricrin. In addition, we detected increased expression of several antimicrobial peptides (AMPs), including human beta defensins (hBDs) and members of the S100 calcium binding family. While the same gene clusters were deregulated upon IL-17A treatment in both models, we observed qualitative differences with some genes being deregulated more substantially in the psoriatic model. Among the IL-17A induced genes were the IL-36 cytokines. We hypothesized that these are downstream effectors of IL-17A. Indeed all isoforms of IL-36 showed alterations in NHEK differentiation and skin barrier formation and they induced the expression of AMPs as well as IL-36 cytokines themselves. In addition the effects of IL-17A were abrogated with Secukinumab, an IL-17A antibody, which was recently approved for the treatment of psoriasis. In conclusion we established full thickness 3D psoriatic skin models containing NHEKs and HDFs of psoriasis patients. In these and in control models IL-17A interfered with keratinocyte differentiation. Our results suggest that IL-36 cytokines mediate in part the IL-17A effects.

Skin microbiome has an important role as host guardian, contributing to several physiological functions including skin barrier maintenance and protection against pathogenic microorganisms. Skin microbiome varies according to geography, cultural and ethnic conditions and all these parameters interfere in skin-microorganism communication. In this study, we identified the skin microbiome of a South American group and studied its behavior when associated with a 3D skin model. Both metagenomic and transcriptomic evaluations were performed to understand the interaction between the microbiome and the skin in vitro. Altogether, our results suggest that although it is feasible to cultivate a wild type microbiome with a 3D skin model, it generates some artifacts. Curiously, these artifacts mimic some in vitro models found in the literature and raises the debate about the biological relevance of some conclusions that have been made in a fast growing field.