Congenital deficiency reveals critical role of ISG15 in skin homeostasis.

Ulcerating skin lesions are manifestations of human ISG15 deficiency, a type I interferonopathy. However, chronic inflammation may not be their exclusive cause. We describe two siblings with recurrent skin ulcers that healed with scar formation upon corticosteroid treatment. Both had a homozygous nonsense mutation in the ISG15 gene, leading to unstable ISG15 protein lacking the functional domain. We characterized ISG15-/- dermal fibroblasts, HaCaT keratinocytes, and human induced pluripotent stem cell-derived vascular endothelial cells. ISG15-deficient cells exhibited the expected hyperinflammatory phenotype, but also dysregulated expression of molecules critical for connective tissue and epidermis integrity, including reduced collagens and adhesion molecules, but increased matrix metalloproteinases. ISG15-/- fibroblasts exhibited elevated ROS levels and reduced ROS scavenger expression. As opposed to hyperinflammation, defective collagen and integrin synthesis was not rescued by conjugation-deficient ISG15. Cell migration was retarded in ISG15-/- fibroblasts and HaCaT keratinocytes, but normalized under ruxolitinib treatment. Desmosome density was reduced in an ISG15-/- 3D epidermis model. Additionally, there were loose architecture and reduced collagen and desmoglein expression, which could be reversed by treatment with ruxolitinib/doxycycline/TGF-ß1. These results reveal critical roles of ISG15 in maintaining cell migration and epidermis and connective tissue homeostasis, whereby the latter likely requires its conjugation to yet unidentified targets.

Free human DNA attenuates the activity of antimicrobial peptides in atopic dermatitis.

BACKGROUND: The high susceptibility of AD patients to microbial skin infections has been attributed to a deficient antimicrobial peptide (AMP) expression, which is contradicted by a growing amount of recent studies clearly demonstrating that AMP expression is not impaired in lesional skin of AD patients. The reasons for the high susceptibility of AD patients to microbial infections are still unknown. METHODS: The influence of self-DNA on the antimicrobial activity of RNase 7, LL-37, and hBD2 has been investigated using antibacterial and antiviral assays. The amount of self-DNA on skin has been analyzed by skin rinsings and subsequent quantification using dsDNA assays. DNA source was identified by qPCR. RESULTS: Complex formation of the AMPs with self-DNA significantly impaired their antibacterial activity against Staphylococcus aureus and their antiviral activity against HSV-1. The inhibition of the antibacterial activity was dependent on the DNA concentration but not on the length of the DNA molecules. Of note, we detected significant higher amounts of cell-free self-DNA in skin rinses taken from lesional AD skin compared to skin rinses from non-lesional skin and from normal skin of healthy donors. Consequently, rinse solution from AD lesional skin prevented antibacterial activity of LL-37. CONCLUSION: Our study indicates that extracellular self-DNA is released in considerable amounts in AD skin lesions and AMP-self-DNA-complex formation leads to a significant loss of antibacterial and antiviral activity in atopic dermatitis. Studies on strategies to reduce the amount of extracellular DNA in AD are needed to identify possible methods relevant in clinical settings.

Eczema herpeticum in atopic dermatitis.

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases leading to pruritic skin lesions. A subset of AD patients exhibits a disseminated severe HSV infection called eczema herpeticum (EH) that can cause life-threatening complications. This review gives an overview of the clinical picture, and characteristics of the patients as well as the diagnosis and therapy of EH. A special focus lies on the pathophysiological hallmarks identified so far that predispose for EH. This aspect covers genetic aberrations, immunological changes, and environmental influences displaying a complex multifactorial situation, which is not completely understood. Type 2 skewing of virus-specific T cells in ADEH+ patients has been implicated in immune profile abnormalities, along with impaired functions of dendritic cells and natural killer cells. Furthermore, aberrations in interferon pathway-related genes such as IFNG and IFNGR1 have been identified to increase the risk of EH. IL-4, IL-25, and thymic stromal lymphopoietin (TSLP) are overexpressed in EH, whereas antimicrobial peptides like human ß-defensins and LL-37 are reduced. Concerning the epidermal barrier, single nucleotide polymorphisms (SNPs) in skin barrier proteins such as filaggrin were identified in ADEH+ patients. A dysbalance of the skin microbiome also contributes to EH due to an increase of Staphylococcus aureus, which provides a supporting role to the viral infection via secreted toxins such as α-toxin. The risk of EH is reduced in AD patients treated with dupilumab. Further research is needed to identify and specifically target risk factors for EH in AD patients.

Common and different roles of IL-4 and IL-13 in skin allergy and clinical implications.

PURPOSE OF REVIEW: This review summarizes the mode of action of IL-4 and IL-13 in skin allergy, upcoming therapeutics and depicts key outcomes of the latest clinical trials. RECENT FINDINGS: Atopic dermatitis is considered to be one of the most common inflammatory skin disease in industrialized countries. Accompanied by strong pruritus, atopic dermatitis has a significant impact on quality of life in severely affected individuals. Aside from unspecific immunosuppressant medications, therapeutics targeting the key cytokines IL-4 and IL-13 and their downstream mediators are under development or have been approved just recently with outstanding potential. SUMMARY: The recent development of several biologics and small compounds has the potential to revolutionize the treatment of atopic dermatitis, and applying this set of state-of-the-art drugs will provide a unique chance to gain insights into this skin disorder, patient subgroups, and key inflammatory mediators.

Molecular Regulation of Acute Tie2 Suppression in Sepsis.

OBJECTIVES: Tie2 is a tyrosine kinase receptor expressed by endothelial cells that maintains vascular barrier function. We recently reported that diverse critical illnesses acutely decrease Tie2 expression and that experimental Tie2 reduction suffices to recapitulate cardinal features of the septic vasculature. Here we investigated molecular mechanisms driving Tie2 suppression in settings of critical illness. DESIGN: Laboratory and animal research, postmortem kidney biopsies from acute kidney injury patients and serum from septic shock patients. SETTING: Research laboratories and ICU of Hannover Medical School, Harvard Medical School, and University of Groningen. PATIENTS: Deceased septic acute kidney injury patients (n = 16) and controls (n = 12) and septic shock patients (n = 57) and controls (n = 22). INTERVENTIONS: Molecular biology assays (Western blot, quantitative polymerase chain reaction) + in vitro models of flow and transendothelial electrical resistance experiments in human umbilical vein endothelial cells; murine cecal ligation and puncture and lipopolysaccharide administration. MEASUREMENTS AND MAIN RESULTS: We observed rapid reduction of both Tie2 messenger RNA and protein in mice following cecal ligation and puncture. In cultured endothelial cells exposed to tumor necrosis factor-α, suppression of Tie2 protein was more severe than Tie2 messenger RNA, suggesting distinct regulatory mechanisms. Evidence of protein-level regulation was found in tumor necrosis factor-α-treated endothelial cells, septic mice, and septic humans, all three of which displayed elevation of the soluble N-terminal fragment of Tie2. The matrix metalloprotease 14 was both necessary and sufficient for N-terminal Tie2 shedding. Since clinical settings of Tie2 suppression are often characterized by shock, we next investigated the effects of laminar flow on Tie2 expression. Compared with absence of flow, laminar flow induced both Tie2 messenger RNA and the expression of GATA binding protein 3. Conversely, septic lungs exhibited reduced GATA binding protein 3, and knockdown of GATA binding protein 3 in flow-exposed endothelial cells reduced Tie2 messenger RNA. Postmortem tissue from septic patients showed a trend toward reduced GATA binding protein 3 expression that was associated with Tie2 messenger RNA levels (p < 0.005). CONCLUSIONS: Tie2 suppression is a pivotal event in sepsis that may be regulated both by matrix metalloprotease 14-driven Tie2 protein cleavage and GATA binding protein 3-driven flow regulation of Tie2 transcript.

GATA3 regulates FLG and FLG2 expression in human primary keratinocytes.

GATA3 is a transcription factor with an important role in atopic diseases because of its role in the differentiation of Th2 lymphocytes. Moreover, GATA3 is expressed in keratinocytes and has a role in keratinocyte differentiation and the establishment of the epidermal barrier. In this study, we investigated the role of GATA3 in keratinocytes in the context of epidermal barrier integrity under inflammatory skin conditions. When analysing skin samples from atopic dermatitis and psoriasis patients or healthy controls, we detected decreased expression of GATA3 in the stratum spinosum and stratum granulosum of atopic dermatitis and psoriasis patients when compared to healthy controls. Our cell cultures experiments revealed that a downregulation in GATA3 by shRNA leads to a significant reduction of filaggrin mRNA under atopic dermatitis-like conditions in keratinocytes. Overexpression of GATA3 in keratinocytes reversed this effect and significantly upregulated filaggrin and, furthermore, filaggrin-2 mRNA expression. Our results demonstrate that GATA3 is involved in the regulation of filaggrin and filaggrin-2 expression during inflammatory conditions in the skin. Thus, GATA3 may be of special importance for the establishment and maintenance of an intact epidermal barrier, especially in atopic dermatitis.

Allergen-induced asthmatic responses modified by a GATA3-specific DNAzyme.

BACKGROUND: The most prevalent phenotype of asthma is characterized by eosinophil-dominated inflammation that is driven by a type 2 helper T cell (Th2). Therapeutic targeting of GATA3, an important transcription factor of the Th2 pathway, may be beneficial. We evaluated the safety and efficacy of SB010, a novel DNA enzyme (DNAzyme) that is able to cleave and inactivate GATA3 messenger RNA (mRNA). METHODS: We conducted a randomized, double-blind, placebo-controlled, multicenter clinical trial of SB010 involving patients who had allergic asthma with sputum eosinophilia and who also had biphasic early and late asthmatic responses after laboratory-based allergen provocation. A total of 40 patients could be evaluated; 21 were assigned to receive 10 mg of SB010, and 19 were assigned to receive placebo, with each study drug administered by means of inhalation once daily for 28 days. An allergen challenge was performed before and after the 28-day period. The primary end point was the late asthmatic response as quantified by the change in the area under the curve (AUC) for forced expiratory volume in 1 second (FEV1). RESULTS: After 28 days, SB010 attenuated the mean late asthmatic response by 34%, as compared with the baseline response, according to the AUC for FEV1, whereas placebo was associated with a 1% increase in the AUC for FEV1 (P=0.02). The early asthmatic response with SB010 was attenuated by 11% as measured by the AUC for FEV1, whereas the early response with placebo was increased by 10% (P=0.03). Inhibition of the late asthmatic response by SB010 was associated with attenuation of allergen-induced sputum eosinophilia and with lower levels of tryptase in sputum and lower plasma levels of interleukin-5. Allergen-induced levels of fractional exhaled nitric oxide and airway hyperresponsiveness to methacholine were not affected by either SB010 or placebo. CONCLUSIONS: Treatment with SB010 significantly attenuated both late and early asthmatic responses after allergen provocation in patients with allergic asthma. Biomarker analysis showed an attenuation of Th2-regulated inflammatory responses. (Funded by Sterna Biologicals and the German Federal Ministry of Education and Research; ClinicalTrials.gov number, NCT01743768.).

IL-27 acts as a priming signal for IL-23 but not IL-12 production on human antigen-presenting cells.

IL-27 belongs to the IL-12 family of cytokines and has been described not only to support T-cell polarization along the Th1 lineage, but also to induce important anti-inflammatory responses in later phases of inflammation. We and others have previously shown that the cytokine IL-27 has an important impact on the chronic manifestation of inflammatory skin diseases. Thus, the aim of this study was to specify the effects of IL-27 on the human antigen-presenting cell (APC) subtype inflammatory dendritic epidermal cells (IDEC), which are known to play an important role in eczema. IDEC and blood-derived human macrophages were generated from human peripheral blood and stimulated with IL-27. Functional responses of the cells were analysed by intracellular cytokine staining, ELISA and FlowCytomix. IL-27 was found to be the only IL-12 family member that acts on human APC as a priming signal for IL-23 but not IL-12 production. We confirmed for macrophages that IL-27 limits lipopolysaccharide-induced IL-10 production and detected the same tendency for IDEC. Furthermore, we showed that this also applies to CD40L-induced IL-10 expression in both investigated human APC subsets. We demonstrate that IL-27 exerts pro-inflammatory effects on human APC in particular in the context of a range of bacterial-derived TLR ligands. Hence, our study builds upon the idea that IL-27 exerts a pro-inflammatory effect on innate immune and tissue-resident cells and may drive eczematous reaction - in particular in the context of bacterial superinfection - towards a chronic phase.

Development of drug delivery systems for the dermal application of therapeutic DNAzymes.

DNAzymes are potent novel drugs for the treatment of inflammatory diseases such as atopic dermatitis. DNAzymes represent a novel class of pharmaceuticals that fulfil a causal therapy by interruption of the inflammation cascade at its origin. There are two challenges regarding the dermal application of DNAzymes: the large molecular weight and the sensitivity to DNases as part of the natural skin flora. To overcome these limitations suitable carrier systems have to be considered. Nano-sized drug carrier systems (submicron emulsions, microemulsions) are known to improve the skin uptake of drugs due to their ability to interact with the skin's lipids. To protect the drug against degradation, the hydrophilic drug may be incorporated into the inner aqueous phase of carrier systems, such as water-in-oil-in-water multiple emulsions. In the present study various emulsions of pharmaceutical grade were produced. Their physicochemical properties were determined and the influence of preservation systems on stability was tested. Drug release and skin uptake studies using various skin conditions and experimental set-ups were conducted. Furthermore, cellular uptake was determined by flow cytometric analysis. The investigations revealed that the developed multiple emulsion is a suitable and promising drug carrier system for the topical application of DNAzyme.

Human primary keratinocytes show restricted ability to up-regulate suppressor of cytokine signaling (SOCS)3 protein compared with autologous macrophages.

Suppressor of cytokine signaling (SOCS)3 belongs to a family of proteins that are known to exert important functions as inducible feedback inhibitors and are crucial for the balance of immune responses. There is evidence for a deregulated immune response in chronic inflammatory skin diseases. Thus, it was the aim of this study to investigate the regulation of SOCS proteins involved in intracellular signaling pathways occurring during inflammatory skin diseases and analyze their impact on the course of inflammatory responses. Because we and others have previously described that the cytokine IL-27 has an important impact on the chronic manifestation of inflammatory skin diseases, we focused here on the signaling induced by IL-27 in human primary keratinocytes compared with autologous blood-derived macrophages. Here, we demonstrate that SOCS3 is critically involved in regulating the cell-specific response to IL-27. SOCS3 was found to be significantly up-regulated by IL-27 in macrophages but not in keratinocytes. Other STAT3-activating cytokines investigated, including IL-6, IL-22, and oncostatin M, also failed to up-regulate SOCS3 in keratinocytes. Lack of SOCS3 up-regulation in skin epithelial cells was accompanied by prolonged STAT1 and STAT3 phosphorylation and enhanced CXCL10 production upon IL-27 stimulation compared with macrophages. Overexpression of SOCS3 in keratinocytes significantly diminished this enhanced CXCL10 production in response to IL-27. We conclude from our data that keratinocytes have a cell type-specific impaired capacity to up-regulate SOCS3 which may crucially determine the course of chronic inflammatory skin diseases.

Human keratinocytes release high levels of inducible heat shock protein 70 that enhances peptide uptake.

BACKGROUND: The stress-inducible chaperone heat shock protein (HSP) 70 is considered a 'danger signal' if released into the extracellular environment. It has been proposed to play a role in the pathogenesis of skin diseases such as psoriasis and lupus erythematosus (LE). OBJECTIVES: The aim of this study was to decipher the role of human primary keratinocytes with regard to release and reactivity to HSP70. METHODS: We determined HSP70 and IFNγ in cell supernatants by ELISA. Uptake of labelled HSP70 or labelled peptide by human primary keratinocytes or macrophages was analysed by flow cytometry and fluorescent microscopy. RESULTS: We found that living keratinocytes are an important source of HSP70 in the skin compartment. They release considerably more HSP70 than fibroblasts, macrophages or lymphocytes. Interestingly, keratinocytes also bind and internalise HSP70/HSP70-peptide complexes. TNFα, IL-27 as well as HMGB-1 enhanced the uptake of HSP70. No difference with regard to HSP70 release or uptake was observable between keratinocytes from healthy donors or patients with cutaneous LE. Keratinocytes pulsed with HSP70-peptide complexes significantly increased IFNγ production by autologous T cells. CONCLUSIONS: Production and uptake of inducible HSP70 by keratinocytes may critically influence the chronic course of inflammatory skin diseases.

IL-27 is expressed in chronic human eczematous skin lesions and stimulates human keratinocytes.

BACKGROUND: IL-27 is produced by antigen-presenting cells early during immune responses. IL-27 has been described to support T-cell polarization along the T(H)1 lineage but also to exert important anti-inflammatory responses in later phases of inflammation in murine models. OBJECTIVE: It was the aim of this study to analyze the potential role of IL-27 in epidermal inflammatory skin responses in human subjects. METHODS: Surface receptor expression and apoptosis of human primary keratinocytes were analyzed by means of flow cytometry. Supernatants of stimulated keratinocytes were either analyzed by means of ELISA or submitted to chemotaxis assays. RT-PCR from lesional skin and phospho-specific Western blotting were performed. RESULTS: Both subunits of IL-27 were expressed in chronic lesional allergic eczematous skin, whereas the IL-27 subunit EBV-induced gene 3 was not detectable in the acute phase of eczema. Human primary keratinocytes responded to IL-27. Stimulation of keratinocytes with IL-27 resulted in activation of the signal transducer and activator of transcription 1 and 3 pathways. Major effects found for IL-27 include CXCL10 production and MHC class I upregulation. Importantly, we could demonstrate that IL-27 acts as a priming signal on keratinocytes able to amplify chemokine production and surface molecule expression when used before a second signal, such as TNF-alpha. The effects of IL-27 could not be mimicked by IL-6, IL-12, or IL-23. CONCLUSION: These results support the notion that IL-27 might act in an inflammatory, disease-maintaining manner in the epidermal compartment of patients with eczema.

Keratinocytes enriched for epidermal stem cells differ in their response to IFN-gamma from other proliferative keratinocytes.

The epidermis has a pool of adult stem cells [epidermal stem cells (ESC)]. Although the localization of ESC is well described, we lack a clear understanding of their role in perturbed conditions such as inflammation. One of the most important mediators in inflammatory skin diseases acting on keratinocytes (KCs) is interferon gamma (IFN-gamma). The assumption that ESC might generate a protected niche prompted us to investigate their response to the pro-inflammatory cytokine IFN-gamma. In this study, we isolated two populations of KCs according to their adherence ability. ESC enriched by adherence showed a higher CD29 and CD49f expression compared with other KCs. Surprisingly, surface expression of CD54 was more inducible upon IFN-gamma stimulation in short-term cultures of the ESC subpopulation. In contrary to that, a markedly lower induction of IL-18 and reduced basal production of CCL2 were observable in ESC. No differences in IFN-gamma-induced interleukin (IL)-10, CXCL10, CCL22 or transforming growth factor (TGF)beta1 secretion were detectable between the two keratinocyte subpopulations. These results suggest that ESC respond to IFN-gamma with a 'restricted' pattern of pro-inflammatory cytokines, and do not build up an anti-inflammatory microenvironment by means of TGF-beta or IL-10. Activated ESC possess the capability to interact with infiltrating lymphocytes via CD54. In conclusion, the ESC compartment might actively contribute to the immunological properties of the skin organ.