JAK1/3 inhibition preserves epidermal morphology in full-thickness 3D skin models of atopic dermatitis and psoriasis.

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.

Molecular effects of fractional ablative erbium:YAG laser treatment with multiple stacked pulses on standardized human three-dimensional organotypic skin models.

The molecular changes in gene expression following ablative laser treatment of skin lesions, such as atrophic scars and UV-damaged skin, are not completely understood. A standardized in vitro model of human skin, to study the effects of laser treatment on human skin, has been recently developed. Therefore, the aim of the investigation was to examine morphological and molecular changes caused by fractional ablative erbium:YAG laser treatment on an in vitro full-thickness 3D standardized organotypic model of human skin. A fractional ablative erbium:YAG laser was used to irradiate organotypic human 3D models. Laser treatments were performed at four different settings using a variety of stacked pulses with similar cumulative total energy fluence (60 J/cm2). Specimens were harvested at specified time points and real-time PCR (qRT-PCR) and microarray studies were performed. Frozen sections were examined histologically. Three days after erbium:YAG laser treatment, a significantly increased mRNA expression of matrix metalloproteinases and their inhibitors (MMP1, MMP2, MMP3, TIMP1, and TIMP2), chemokines (CXCL1, CXCL2, CXCL5, and CXCL6), and cytokines such as IL6, IL8, and IL24 could be detected. qRT-PCR studies confirmed the enhanced mRNA expression of IL6, IL8, IL24, CXCLs, and MMPs. In contrast, the mRNA expression of epidermal differentiation markers, such as keratin-associated protein 4, filaggrin, filaggrin 2, and loricrin, and antimicrobial peptides (S100A7A, S100A9, and S100A12) as well as CASP14, DSG2, IL18, and IL36ß was reduced. Four different settings with similar cumulative doses have been tested (N10%, C10%, E10%, and W25%). These laser treatments resulted in different morphological changes and effects on gene regulations. Longer pulse durations (1000 µs) especially had the strongest impact on gene expression and resulted in an upregulation of genes, such as collagen-1A2, collagen-5A2, and collagen-6A2, as well as FGF2. Histologically, all treatment settings resulted in a complete regeneration of the epidermis 3 days after irradiation. Fractional ablative erbium:YAG laser treatment with a pulse stacking technique resulted in histological alterations and shifts in the expression of various genes related to epidermal differentiation, inflammation, and dermal remodeling depending on the treatment setting applied. A standardized in vitro 3D model of human skin proved to be a useful tool for exploring the effects of various laser settings both on skin morphology and gene expression during wound healing. It provides novel data on the gene expression and microscopic architecture of the exposed skin. This may enhance our understanding of laser treatment at a molecular level.

Dexpanthenol modulates gene expression in skin wound healing in vivo.

Topical application of dexpanthenol is widely used in clinical practice for the improvement of wound healing. Previous in vitro experiments identified a stimulatory effect of pantothenate on migration, proliferation and gene regulation in cultured human dermal fibroblasts. To correlate these in vitro findings with the more complex in vivo situation of wound healing, a clinical trial was performed in which the dexpanthenol-induced gene expression profile in punch biopsies of previously injured and dexpanthenol-treated skin in comparison to placebo-treated skin was analyzed at the molecular level by Affymetrix® GeneChip analysis. Upregulation of IL-6, IL-1ß, CYP1B1, CXCL1, CCL18 and KAP 4-2 gene expression and downregulation of psorasin mRNA and protein expression were identified in samples treated topically with dexpanthenol. This in vivo study might provide new insight into the molecular mechanisms responsible for the effect of dexpanthenol in wound healing and shows strong correlations to previous in vitro data using cultured dermal fibroblasts.

Ultraviolet B radiation and reactive oxygen species modulate interleukin-31 expression in T lymphocytes, monocytes and dendritic cells.

BACKGROUND: Interleukin (IL)-31 is a novel Th2 T-cell cytokine that induces pruritus and dermatitis in transgenic mice. While enhanced mRNA expression of this cytokine is detected in skin samples of inflammatory skin diseases, the regulation of IL-31 expression is poorly understood. OBJECTIVES: To assess the effects of ultraviolet (UV) B radiation and H2O2 on IL-31 mRNA and protein expression in skin and different peripheral blood mononuclear cells (PBMCs). METHODS: The effects of UVB radiation and H2O2, as a prototypic reactive oxygen species, on IL-31 mRNA and protein expression were analysed in various inflammation-related cells and murine skin tissue. RESULTSTreatment of cells with UVB radiation and H2 O2 strongly induced IL-31 mRNA and protein expression in human PBMCs and in the skin of SKH-1 mice. Following exposure to UVB or H2O2, we observed increased expression of IL-31 mRNA in T cells, monocytes, macrophages, and immature and especially mature dendritic cells. H2O2 treatment but not UVB radiation led to a moderate upregulation of IL-31 mRNA expression in epidermal keratinocytes and dermal fibroblasts. Pretreatment of T lymphocytes with the MAPK p38 inhibitor SB203580 or the MEK1 inhibitor U0126 reduced the stimulatory effect of H2O2. These experiments suggest that p38 is involved in the regulation of IL-31 expression in human skin. CONCLUSIONS: Our studies reveal that UVB and reactive oxygen species stimulate the expression of IL-31 in PBMCs and skin, especially in T cells, monocytes and monocyte-derived dendritic cells.

Enzyme-linked immunosorbent assay for detection of peptide-specific human antidesmoplakin autoantibodies.

BACKGROUND: Autoantibodies directed against desmoplakin (Dp) I and II have recently been characterized in a subset of patients with severe erythema multiforme (EM), a recurrent inflammatory skin disease with a broad spectrum of clinical manifestations. These autoantibodies recognize a peptide epitope localized within the extreme end of the carboxy terminal domain of Dp responsible for the assembly of keratin filaments to the desmosomal plaque. Using dot blot analysis with overlapping synthetic peptides, the binding epitope YSYSYS has been identified. OBJECTIVES: To establish an enzyme-linked immunosorbent assay (ELISA) for detection of peptide-specific anti-Dp autoantibodies in sera of patients with EM. METHODS: A synthetic peptide containing the respective amino acid sequence was used as matrix for ELISA plates. Serum samples from patients with known EM and peptide-specific anti-Dp autoantibodies verified by immunoblotting, immunoprecipitation and epitope mapping were used. RESULTS: Establishing an index value of 42.0, 25 of 25 serum samples from five patients with peptide-specific anti-Dp autoantibodies were positive in the ELISA. From control sera, none of 31 bullous disease sera and only one (1.2%) of 83 normal human sera were positive. CONCLUSIONS: These data show that the ELISA presented in this study represents a sensitive and highly specific tool for the detection of peptide-specific anti-Dp autoantibodies in patients with EM.