Jana Zeitvogel

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.).

Expression of interleukin (IL)-1 family members upon stimulation with IL-17 differs in keratinocytes derived from patients with psoriasis and healthy donors

Background A number of studies have challenged the T‐cell‐centred pathogenetic view of psoriasis by the finding that epithelium‐expressed genes are intimately involved in the inflammatory process. Interleukin (IL)‐17 is an important inflammatory mediator in skin psoriasis.

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.

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 skins 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.

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

Abstract:  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.

Human Primary Keratinocytes Show Restricted Ability to Up-regulate Suppressor of Cytokine Signaling (SOCS)3 Protein Compared with Autologous Macrophages

Background: A disordered immune regulation contributes to chronic inflammatory skin diseases. Results: Human keratinocytes show a restricted ability to up-regulate SOCS3. Conclusion: Keratinocytes miss an important negative feedback mechanism. Significance: The failure of keratinocytes to up-regulate SOCS3 efficiently may contribute to ongoing proinflammatory epithelial responses that impact on leukocyte infiltration and thus to the maintenance of chronic skin inflammation. 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

Abstract:  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).

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

Abstract:  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‐γ). The assumption that ESC might generate a protected niche prompted us to investigate their response to the pro‐inflammatory cytokine IFN‐γ. 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‐γ 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‐γ‐induced interleukin (IL)‐10, CXCL10, CCL22 or transforming growth factor (TGF)β1 secretion were detectable between the two keratinocyte subpopulations. These results suggest that ESC respond to IFN‐γ with a ‘restricted’ pattern of pro‐inflammatory cytokines, and do not build up an anti‐inflammatory microenvironment by means of TGF‐β 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.

RNase 7 downregulates TH2 cytokine production by activated human T-cells

The antimicrobial peptide (AMP) RNase 7 is constitutively expressed in the epidermis of healthy human skin and has been found to be upregulated in chronic inflammatory skin diseases such as atopic dermatitis and psoriasis. Activated T cells in lesional skin of patients with atopic dermatitis (AD) and psoriasis (PSO) might be directly exposed to RNase 7. In addition to their antimicrobial activity, immunoregulatory functions have been published for several AMPs. In this study, we investigated immunoregulatory effects of the antimicrobial peptide RNase 7 on activated T cells.

Expression of IL-1 family members upon stimulation with IL-17 differs in keratinocytes derived from psoriasis patients and healthy donors

In the presence of IL-17 psoriasis derived keratinocytes showed a significantly higher induction of the proinflammatory members IL-1F6 and IL-1F9 compared with those from healthy individuals but not of antiinflammatory members IL-1F5, IL-1F7 or IL-1F3. Both basal, as well as IL-17 induced production of IL-1F2/IL1b and IL-1F1/IL-1a were found to be significantly lower in psoriasis keratinocytes. Conclusion