Allergy / Prevention of allergy by viruslike nanoparticles (VNP) delivering shielded versions of major allergens in a humanized murine allergy model

Background: In highrisk populations, allergenspecific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of viruslike nanoparticles (VNP) expressing surfaceexposed or shielded allergens. Methods: Fulllength major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. Results: Viruslike nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surfaceexposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergenspecific T cells in vitro. Upon intranasal application in mice, VNP expressing surfaceexposed but not shielded allergen induced allergenspecific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergenprotected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Tregdominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. Conclusion: Viruslike nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization.DKW1248SFB F4605SFB F4609(VLID)313247

Identification of RTN1A as a marker for human dendritic cells and investigations on the cytokine influence on T cells in human skin

Die Haut ist das größte sichtbare Barriereorgan des menschlichen Körpers. Ähnlich wie starke Mauern einer Festung sind die ziegelartigen verzahnten Hautkeratinozyten dicht gepackt, um eindringende Krankheitserreger abzuschirmen, und bieten Schutz vor physikalisch-chemischen Einflüssen. Darüber hinaus wird die Haut von residenten Immunzellen wie dendritischen Zellen (DZ) und T Zellen, die wichtige immunologische Funktionen erfüllen, geschützt. Obwohl in vielen Publikationen die Funktion und Biologie von Hautimmunzellen charakterisiert worden ist, sind noch viele Fragen offen. Per Zufall entdeckten wir, dass RTN1A, ein Protein das zur Reticulon-Familie gehört, nicht wie erwartet in Hautnerven exprimiert wurde, sondern in DZ der Haut von erwachsenen Menschen. Insbesondere war die RTN1A Expression in Langerhans Zellen (LZ) der Epidermis sowie DZ der Dermis nachweisbar. Darüber hinaus konnten wir RTN1A+ DZ in anderen Organen wie Gingiva, Trachea, Tonsillen und Thymus detektieren. Im Blut fanden wir ein positives RTN1A Signal in einer Subpopulation von CD14+HLA-DR+CD11c+CD1c+ Zellen. Bei der näheren Betrachtung des räumlich-zeitlichen Expressionsmusters von RTN1A wurde festgestellt, dass es teilweise mit dem endoplasmischen Retikulum Marker Protein Disulfid Isomerase (PDI) ko-lokalisiert ist, jedoch auf der Zelloberfläche des LZ Zelllinienmodells MUTZ-3 nicht nachweisbar war. Während der MUTZ-3 Differenzierung zu LZ war die Expression von RTN1A jedoch der Expression von typischen LZ-Proteinen wie HLA-DR, CD1a und CD207 voraus. Nabelschnurblut LZ exprimierten RTN1A zusammen mit HLA-DR am Tag 7 der Differenzierung. In menschlicher fötaler Haut wurde RTN1A im Gestationsalter von 9 Wochen nachgewiesen. Bislang konnte keine konkrete Funktion von RTN1A entdeckt werden aber Untersuchungen einer möglichen Rolle von RTN1A bei der DZ-Migration, der DZ-T Zell-Interaktion oder der Antigenverarbeitung werden derzeit in unserem Labor durchgeführt. In der Haut ansässige T Zellen können an ihrem spezifischen Ort schnell auf eindringende Pathogene reagieren. IL-9 produzierende T Zellen sind ein häufig vorkommender Subtypus insbesondere in gesunder menschlicher Haut von Erwachsenen welcher verstärkt in entzündlichen Hauterkrankungen wie atopischer Dermatitis und Psoriasis nachgewiesen wurde. Diese Zellen wirken Wurminfektionen und Melanomen entgegen, spielen allerdings aber auch eine Rolle in allergischen Reaktionen sowie entzündlichen Haut- und Darmerkrankungen. Mit Hilfe einer Kulturmethode von Hautexplantaten untersuchten wir den Einfluss von Zytokinen auf T Zellen der Haut mit besonderem Fokus auf IL-9 produzierende T Zellen. Der Transkriptionsfaktor IRF4 allerdings nicht PU.1 wurde von IL-9 produzierenden T Zellen ko-exprimiert. Unter Kulturbedingungen, die die TH9 Zelldifferenzierung fördern [IL-2, IL-4 und TGF- (TH9-PC)] fanden wir signifikant mehr IL-9 produzierende T Zellen im Vergleich zu Standardbedingungen [IL-2, IL-15 (SC)]. Darüber hinaus wurde der Marker CD69 unter TH9-PC im Vergleich zu SC herunterreguliert, während CCR4 und CD103 hochreguliert wurden. Die Häufigkeit von IL-9 produzierenden T Zellen fiel nach einem Wechsel von anfänglich TH9-PC zu SC rasch ab, was auf ihre Abhängigkeit vom Zytokin Stimulus hindeutet. Im Allgemeinen produzierten IL-9 produzierende Hautzellen vernachlässigbar wenig IFN-, IL-13 oder IL-17, was die Theorie eines unabhängigen IL-9+ T Zell Subtyps unterstützt. Die RNA-Sequenzierung von ausgewanderten Hautzellen zeigte, dass CXCL8 und CXCL13 zu den am stärksten hochregulierten Genen in TH9-PC im Vergleich zu SC gehörten. In funktionellen Studien war das Überleben der neutrophilen Granulozyten durch Zugabe des Überstands von stimulierten, von der Haut stammenden T Zellen, die die Chemokine CXCL8 und CXCL13 enthielten, erhöht. Zusammenfassend lässt sich festhalten, dass Gewebszytokine einen großen Einfluss auf den Phänotyp der T Zellen in der Haut haben.The skin is the largest visible barrier organ of the human body. Similar to strong walls of a fortress, the brick like skin keratinocytes are tightly packed shielding off invading pathogens and provide protection against physico-chemical stresses. Furthermore, the skin is heavily guarded by resident immune cells such as dendritic cells (DCs) and T cells which perform important immunological functions. Although these functions and the biology of skin immune cells are well characterized, still many open questions remain. By serendipity we identified that RTN1A which belongs to the reticulon family was not expressed in adult human skin nerves as expected but instead was present in skin resident DCs. More specifically, RTN1A identified Langerhans cells (LCs) of the epidermis as well as DCs of the dermis. Furthermore, we detected RTN1A+ DCs in other organs such as the gingiva, trachea, tonsil and thymus. In the blood, a subpopulation of CD14+HLA-DR+CD11c+CD1c+ cells stained positive for RTN1A. When defining the spatio-temporal expression pattern of RTN1A, we found that it partly co-localized with the endoplasmic reticulum marker protein disulfide isomerase (PDI) but was absent on the cell surface of the LC model MUTZ-3. However, during MUTZ-3 differentiation into LCs, RTN1A expression preceded expression of typical LC proteins such as HLA-DR, CD1a and CD207. Cord blood-differentiated LCs expressed RTN1A on day 7 together with HLA-DR. In human fetal skin, RTN1A was detected at 9 weeks of estimated gestational age. Currently, the function of RTN1A still remains elusive but its contribution to DC migration, DC-T cell interaction and antigen processing is currently under investigation in our laboratory. Skin resident T cells are able to rapidly respond to invading pathogens at their specific site. Among them IL-9 producing T cells were discovered especially in healthy human skin with high frequency and are even more abundant in inflammatory skin diseases such as atopic dermatitis and psoriasis. These cells were found to fuel allergic reaction as well as inflammatory skin and bowel diseases but protect against helminth infection and melanoma. Adapting a skin explant culture method, we studied the cytokine influence on skin T cells with a special focus on IL-9 producing T cells. The transcription factor IRF4 but not PU.1 was co-expressed by IL-9 producing T cells. When TH9 cell promoting cytokine conditions [IL-2, IL-4 and TGF- (TH9-PC)] were used in skin explant cultures we found significantly higher frequencies of IL-9 producing T cells compared to standard conditions [IL-2, IL-15 (SC)]. Furthermore, CD69 was downregulated under TH9-PC compared to SC whereas CCR4 and CD103 were upregulated. The frequency of IL-9 producing T cells dropped rapidly when culture conditions were changed from initial TH9-PC to SC, suggesting their dependency on the cytokine stimulus. In general, IL-9 producing skin T cells did not co-produce IFN-, IL-13 or IL-17 supporting the theory of an independent IL-9+ T cell subset. RNA sequencing of emigrated skin T cells showed that CXCL8 and CXCL13 ranged among the most upregulated genes in TH9-PC versus SC. In functional studies neutrophil survival was increased by addition of CXCL8 and CXCL13 containing supernatant of stimulated skin-derived T cells. Taken together, our data show that the cytokine environment influences the skin T cell phenotype.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersArbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftMedizinische Universität Wien, Diss., 2019(VLID)370082

Journal of Immunology Research / The Antiseptic Octenidine Inhibits Langerhans Cell Activation and Modulates Cytokine Expression upon Superficial Wounding with Tape Stripping

Ideal agents for the topical treatment of skin wounds should have antimicrobial efficacy without negative influence on wound healing. Octenidine (OCT) has become a widely used antiseptic in professional wound care, but its influence on several components of the wound healing process remains unclear. In the present study, we have used a superficial wound model using tape stripping on human full-thickness skin ex vivo to investigate the influence of OCT on epidermal Langerhans cells (LCs) and cytokine secretion pattern of skin cells during wound healing in a model without disruption of the normal skin structure. Histological and immunofluorescence studies showed that OCT neither altered human skin architecture nor the viability of skin cells upon 48 hours of culture in unwounded or wounded skin. The epidermis of explants and LCs remained morphologically intact throughout the whole culture period upon OCT treatment. OCT inhibited the upregulation of the maturation marker CD83 on LCs and prevented their emigration in wounded skin. Furthermore, OCT reduced both pro- and anti-inflammatory mediators (IL-8, IL-33, and IL-10), while angiogenesis and growth factor mediators (VEGF and TGF-1) remained unchanged in skin explant cultures. Our data provide novel insights into the host response to OCT in the biologically relevant environment of viable human (wounded) skin.(VLID)510715