Background: Activated eosinophils cause major pathology in stable and exacerbating asthma; however, they can also display protective properties like an extracellular antiviral activity. Initial murine studies led us to further explore a potential intracellular antiviral activity by eosinophils. Methods:To follow eosinophil-virus interaction, respiratory syncytial virus (RSV) and influenza virus were labeled with a fluorescent lipophilic dye (DiD). Interactions with eosinophils were visualized by confocal microscopy, electron microscopy, and flow cytometry. Eosinophil activation was assessed by both flow cytometry and ELISA. In a separate study, eosinophils were depleted in asthma patients using anti-IL-5 (mepolizumab), followed by a challenge with rhinovirus-16 (RV16).Results: DiD-RSV and DiD-influenza rapidly adhered to human eosinophils and were internalized and inactivated (95% in ≤ 2 hours) as reflected by a reduced replication in epithelial cells. The capacity of eosinophils to capture virus was reduced up to 75% with increasing severity of asthma. Eosinophils were activated by virus in vitro and in vivo. In vivo this correlated with virus-induced loss of asthma control. Conclusions:This previously unrecognized and in asthma attenuated antiviral property provides a new perspective to eosinophils in asthma. This is indicative of an imbalance between protective and cytotoxic properties by eosinophils that may underlie asthma exacerbations. K E Y W O R D S CD69, exacerbation, influenza, rhinovirus_16, RSVThis is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Background. Influenza virus triggers severe exacerbations of asthma for which no adequate treatment is available. It is known that IL-33 levels correlate with exacerbation severity, but its role in the immune-pathogenesis of exacerbations has remained elusive. Objective. We hypothesized that IL-33 is necessary to drive asthma exacerbations. We intervened with the IL-33 cascade and sought to dissect its role, also in synergy with TSLP, in airway inflammation, anti-viral activity and lung function. We aimed to unveil the major source of IL-33 in the airways and IL-33-dependent mechanisms that underlie severe asthma exacerbation. Methods: Mild asthmatic patients were experimentally infected with rhinovirus. Mice were chronically exposed to house dust mite (HDM) extract and then infected with influenza to resemble key features of exacerbations in humans. Interventions included anti-IL-33-receptor ST2 and/or anti-TSLP. Results. We identified bronchial ciliated cells and Type-II alveolar cells as a major local source of IL-33 during virus-driven exacerbation in humans and mice, respectively. By blocking ST2 we demonstrated that IL-33 and not TSLP was necessary to drive exacerbations. IL-33 enhanced AHR and airway inflammation by suppressing innate and adaptive anti-viral responses and by instructing epithelial cells and dendritic cells (DCs) of HDM-sensitized mice to dampen IFN-β expression and prevent the Th1-promoting DCs phenotype. IL-33 also boosted luminal NETosis and halted cytolytic anti-viral activities, but did not affect the Th2-response. Conclusion: Interventions targeting the IL-33/ST2 axis could prove an effective acute, shortterm therapy for virus-induced asthma exacerbation.

Mepolizumab failed to prevent activation of remaining eosinophils and changed RV16-induced immune responses in mild asthma. Although these latter effects likely are due to attenuated eosinophil numbers, we cannot exclude a role for basophils. Clinical trial registration available at www.clinicaltrials.gov, ID NCT01520051.