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GATA3 gene silencing in activated T cells displays a promising option to early-on undermine pathological pathways in the disease formation of allergic asthma. The central transcription factor of T helper 2 (Th2) cell cytokines IL-4, IL-5, and IL-13 plays a major role in immune and inflammatory cascades underlying asthmatic processes in the airways. Pulmonary delivery of small interfering RNAs (siRNA) to induce GATA3 knockdown within disease related T cells of asthmatic lungs via RNA interference (RNAi) presents an auspicious base to realize this strategy, however, still faces some major hurdles. Main obstacles for successful siRNA delivery in general comprise stability and targeting issues, while in addition the transfection of T cells presents a particularly challenging task itself. In previous studies, we have developed and advanced an eligible siRNA delivery system composed of polyethylenimine (PEI) as polycationic carrier, transferrin (Tf) as targeting ligand and melittin (Mel) as endosomolytic agent. Resulting Tf-Mel-PEI polyplexes exhibited ideal characteristics for targeted siRNA delivery to activated T cells and achieved efficient and sequence-specific gene knockdown in vitro. In this work, the therapeutic potential of this carrier system was evaluated in an optimized cellular model displaying the activated status of asthmatic T cells. Moreover, a suitable siRNA sequence combination was found for effective gene silencing of GATA3. To confirm the translatability of our findings, Tf-Mel-PEI polyplexes were additionally tested ex vivo in activated human precision-cut lung slices (PCLS). Here, the formulation showed a safe profile as well as successful delivery to the lung epithelium with 88% GATA3 silencing in lung explants. These findings support the feasibility of Tf-Mel-PEI as siRNA delivery system for targeted gene knockdown in activated T cells as a potential novel therapy for allergic asthma., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Viral infections are common causes of asthma exacerbations. To model these processes ex vivo, human precision-cut lung slices (PCLSs) can be used. Here we describe the infection of human PCLSs with the human influenza virus. We then provide methods to quantify the virus and reveal its localization within infected PCLSs and study consequences of infection, including cell death and production of cytokines, chemokine, and mucus. We also describe the stimulation of PCLSs with immune mediators such as pro-inflammatory tumor necrosis factor α (TNF-α). These models are useful to investigate mechanisms of virally induced asthma exacerbations and study modes of action and efficacy of antiviral and/or anti-inflammatory drugs., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Asthma is a heterogeneous syndrome substantiating the urgent requirement for endotype-specific biomarkers. Dysbalance of fibrosis and fibrolysis in asthmatic lung tissue leads to reduced levels of the inflammation-protective collagen 4 (COL4A3)., Objective: To delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response., Methods: The serological COL4A3 degradation marker C4Ma3 (Nordic Bioscience, Denmark) and serum cytokines were measured in the ALLIANCE cohort (paediatric cases/controls: n=134/n=35; adult cases/controls: n=149/n=31). Exacerbation of allergic airway disease in mice was induced by sensitising to ovalbumin (OVA), challenge with OVA aerosol and instillation of poly(cytidylic-inosinic). Fulacimstat (chymase inhibitor; Bayer) was used to determine the role of mast cell chymase in COL4A3 degradation. Patients with cystic fibrosis (n=14) and cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA; n=9) as well as patients with severe allergic uncontrolled asthma (n=19) were tested for COL4A3 degradation. Omalizumab (anti-IgE) treatment was assessed using the Asthma Control Test., Results: Serum levels of C4Ma3 were increased in asthma in adults and children alike and linked to a more severe, exacerbating allergic asthma phenotype. In an experimental asthma mouse model, C4Ma3 was dependent on mast cell chymase. Serum C4Ma3 was significantly elevated in cystic fibrosis plus ABPA and at baseline predicted the success of the anti-IgE therapy in allergic, uncontrolled asthmatics (diagnostic OR 31.5)., Conclusion: C4Ma3 levels depend on lung mast cell chymase and are increased in a severe, exacerbating allergic asthma phenotype. C4Ma3 may serve as a novel biomarker to predict anti-IgE therapy response., Competing Interests: Conflict of interest: M. Weckmann reports grants from Federal Ministry for Education and Research (BMBF), University of Lübeck (E42-2012 and JC01-2016) and German Academic Exchange Service (56266000), during the conduct of the study. Conflict of interest: T. Bahmer has nothing to disclose. Conflict of interest: J.M. Sand is a full-time employee of Nordic Bioscience. Conflict of interest: S. Rank Rønnow is employed by Nordic Bioscience, and has received grants from Innovation Foundation, during the conduct of the study. Conflict of interest: M. Pech has nothing to disclose. Conflict of interest: C. Vermeulen has nothing to disclose. Conflict of interest: A. Faiz has nothing to disclose. Conflict of interest: D.J. Leeming is a stockholder and full-time employee of Nordic Bioscience A/S. Conflict of interest: M.A. Karsdal is a stockholder and full-time employee of Nordic Bioscience. Conflict of interest: L. Lunding has nothing to disclose. Conflict of interest: B.G.G. Oliver has nothing to disclose. Conflict of interest: M. Wegmann has nothing to disclose. Conflict of interest: G. Ulrich-Merzenich reports grants for research and meeting attendance from Novartis AG, during the conduct of the study; and has a patent EPC 18185472.0-1118 pending. Conflict of interest: U.R. Juergens has nothing to disclose. Conflict of interest: J. Duhn has nothing to disclose. Conflict of interest: Y. Laumonnier has nothing to disclose. Conflict of interest: O. Danov has nothing to disclose. Conflict of interest: K. Sewald has nothing to disclose. Conflict of interest: U. Zissler reports grants and personal fees from Federal Ministry for Education and Research of Germany, during the conduct of the study. Conflict of interest: M. Jonker has nothing to disclose. Conflict of interest: I. König has nothing to disclose. Conflict of interest: G. Hansen is a consultant for Novartis and Sanofi. Conflict of interest: E. von Mutius received consultancy fees from European Commission, Tampereen Yliopisto, University of Edinburgh, Nestec S.A., University of Veterinary Medicine, Vienna, Chinese University of Hongkong, Research Center Borstel – Leibniz Lung Center, OM Pharma S.A., Pharmaventures Ltd, Peptinnovate Ltd, Turun Yliopisto, Helsingin Yliopisto, Chinese University of Hongkong, Imperial College London, Universiteit Utrecht, Universität Salzburg, Österreichische Gesellschaft f. Allergologie u. Immunologie, HiPP GmbH & Co KG; received fees for speaking from Japanese Society of Pediatric Allergy and Clinical Immunology (JSPACI), The American Academy of Allergy Asthma and Immunology, British Society for Immunology, Medical University of Vienna, Schweizerisches Institiut für Allergie- und Asthmaforschung, Howard Hughes Medical Institute, University Hospital Erlangen, Margaux Orange, Deutsche Akademie der Naturforscher Leopoldina e.V., Hannover Medical School, American Thoracic Society, Inc., European Academy of Allergy and Clinical Immunology, Mundipharma Deutschland GmbH & Co. KG, DOC Congress SRL, ITÄ-Suomen Yliopisto, Interplan – Congress, Meeting & Event Management AG, INC, Ökosoziales Forum Oberösterreich, Imperial College London, WMA Kongress GmbH, University Hospital rechts der Isar, European Respiratory Society, HAL Allergie GmbH, PersonalGenomes.org, Nestlé Deutschland AG, Universitätsklinikum Aachen, SIAF – Swiss Institute of Allergy and Asthma Research, Deutsche Pharmazeutische Gesellschaft e.V., Verein zur Förderung der Pneumologie am Krankenhaus Großhansdorf e.V., Pneumologie Developpement, Mondial Congress & Events GmbH & Co. KG, Volkswagen Stiftung, Boehringer Ingelheim International GmbH, Hanson Wade Ltd, DSI Dansk Borneastma Center; received author honoraria from Elsevier Ltd, Springer-Verlag GmbH, Schattauer GmbH, Georg Thieme Verlag, Springer Medizin Verlag GmbH; is on the editorial board of the New England Journal of Medicine; and has a patent Application number LU101064, Barn dust extract for the prevention and treatment of diseases pending; a patent Publication number EP2361632: Specific environmental bacteria for the protection from and/or the treatment of allergic, chronic inflammatory and/or autoimmune disorders with royalties paid to Protectimmun GmbH; a patent Publication number EP 1411977: Composition containing bacterial antigens used for the prophylaxis and the treatment of allergic diseases licensed to Protectimmun GmbH; a patent Publication number EP1637147: Stable dust extract for allergy protection licensed to Protectimmun GmbH; and a patent Publication number EP 1964570: Pharmaceutical compound to protect against allergies and inflammatory diseases licensed to Protectimmun GmbH. Conflict of interest: O. Fuchs is a consultant for Menarini and Vifor; has received speaker's fees from Vertex, aha! Allergy Centre Switzerland, Menarini, Novartis, Medical Tribune Switzerland, German Society of Paediatric Allergology and ALK; and has received travel support from Milupa/Nutricia, Stallergenes Greer and Bencard. Conflict of interest: A-M. Dittrich has nothing to disclose. Conflict of interest: B. Schaub reports grants from DFG, BMBF and EU, outside the submitted work. Conflict of interest: C. Happle has nothing to disclose. Conflict of interest: K.F. Rabe received grants from Boehringer Ingelheim and personal fees from AstraZeneca, Novartis, Sanofi, Regeneron, Roche and Chiesi Pharmaceuticals. Conflict of interest: M. van de Berge reports grants paid to university from GlaxoSmithKline, AstraZeneca and Genentech, outside the submitted work. Conflict of interest: J.K. Burgess reports grants from National Health and Medical Research Council, Australia, University of Groningen and European Union, during the conduct of the study. Conflict of interest: M.V. Kopp reports grants from Federal Ministry of Research and Education (BMBF), during the conduct of the study; personal fees from ALK-Abello, Allergopharma, Boehringer Ingelheim, Chiesi, Glaxo, Infectopharm, Meda, Sanofi-Aventis, Leti Pharma, Novartis and Vertex, outside the submitted work., (Copyright ©The authors 2021. For reproduction rights and permissions contact permissions@ersnet.org.)

Human rhinovirus (RV) is a major risk factor for chronic obstructive pulmonary disease (COPD) and asthma exacerbations. The exploration of RV pathogenesis has been hampered by a lack of disease-relevant model systems. We performed a detailed characterization of host responses to RV infection in human lung tissue ex vivo and investigated whether these responses are disease relevant for patients with COPD and asthma. In addition, impact of the viral replication inhibitor rupintrivir was evaluated. Human precision-cut lung slices (PCLS) were infected with RV1B with or without rupintrivir. At Days 1 and 3 after infection, RV tissue localization, tissue viability, and viral load were determined. To characterize host responses to infection, mediator and whole genome analyses were performed. RV successfully replicated in PCLS airway epithelial cells and induced both antiviral and proinflammatory cytokines such as IFNα2a, CXCL10, CXCL11, IFN-γ, TNFα, and CCL5. Genomic analyses revealed that RV not only induced antiviral immune responses but also triggered changes in epithelial cell-associated pathways. Strikingly, the RV response in PCLS was reflective of gene expression changes described in patients with COPD and asthma. Although RV-induced host immune responses were abrogated by rupintrivir, RV-triggered epithelial processes were largely refractory to antiviral treatment. Detailed analysis of RV-infected human PCLS and comparison with gene signatures of patients with COPD and asthma revealed that the human RV PCLS model represents disease-relevant biological mechanisms that can be partially inhibited by a well-known antiviral compound and provide an outstanding opportunity to evaluate novel therapeutics.

Background: Asthma is a widespread, multifactorial chronic airway disease. The influence of regulatory B cells on airway hyperreactivity (AHR) and remodeling in asthma is poorly understood., Objective: Our aim was to analyze the role of B cells in a house dust mite (HDM)-based murine asthma model., Methods: The influence of B cells on lung function, tissue remodeling, and the immune response were analyzed by using wild-type and B-cell-deficient (μMT) mice and transfer of IL-10-proficient and IL-10-deficient B cells to μMT mice., Results: After HDM-sensitization, both wild-type and μMT mice developed AHR, but the AHR was significantly stronger in μMT mice, as confirmed by 2 independent techniques: invasive lung function measurement in vivo and examination of precision-cut lung slices ex vivo. Moreover, airway remodeling was significantly increased in allergic μMT mice, as shown by enhanced collagen deposition in the airways, whereas the numbers of FoxP3 + and FoxP3 - IL-10-secreting regulatory T cells were reduced. Adoptive transfer of IL-10-proficient but not IL-10-deficient B cells into μMT mice before HDM-sensitization attenuated AHR and lung remodeling. In contrast, FoxP3 + regulatory T cells were equally upregulated by transfer of IL-10-proficient and IL-10-deficient B cells., Conclusion: Our data in a murine asthma model illustrate a central role of regulatory B cells in the control of lung function and airway remodeling and may support future concepts for B-cell-targeted prevention and treatment strategies for allergic asthma., (Copyright © 2020. Published by Elsevier Inc.)