Lung and airway neutrophils are a hallmark of severe disease in infants with respiratory syncytial virus (RSV)-induced lower respiratory tract infections. Despite their abundance in the lungs during RSV infection of both mice and man, the role of neutrophils in viral control and in immune pathology is not clear. Here, antibody mediated neutrophil depletion was used to investigate the degree to which neutrophils impact the lung immune environment, the control of viral replication and the peak severity of disease after RSV infection of mice. Neutrophil depletion did not substantially affect the levels of inflammatory mediators such as type I interferons, IL-6, TNF-α or IL-1β in response to RSV. In addition, the lack of neutrophils did not change the viral load during RSV infection. Neither neutrophil depletion nor the enhancement of lung neutrophils by administration of the chemoattractant CXCL1 during RSV infection affected disease severity as measured by weight loss. Therefore, in this model of RSV infection, lung neutrophils do not offer obvious benefits to the host in terms of increasing anti-viral inflammatory responses or restricting viral replication and neutrophils do not contribute to disease severity.

SARS‐CoV‐2 is a newly emerged coronavirus, causing the global pandemic of respiratory coronavirus disease (COVID‐19). The type I interferon (IFN) pathway is of particular importance for anti‐viral defence and recent studies identified that type I IFNs drive early inflammatory responses to SARS‐CoV‐2. Here, we use a mouse model of SARS‐CoV‐2 infection, facilitating viral entry by intranasal recombinant Adeno‐Associated Virus (rAAV) transduction of hACE2 in wildtype (WT) and type I IFN‐signalling‐deficient ( Ifnar1 –/– ) mice, to study type I IFN signalling deficiency and innate immune responses during SARS‐CoV‐2 infection. Our data show that type I IFN signaling is essential for inducing anti‐viral effector responses to SARS‐CoV‐2, control of virus replication and to prevent enhanced disease. Furthermore, hACE2‐Ifnar1 –/– mice had increased gene expression of the chemokine Cxcl1 and airway infiltration of neutrophils as well as a reduced and delayed production of monocyte‐recruiting chemokine CCL2. hACE2 ‐ Ifnar1 –/‐ mice showed altered recruitment of inflammatory myeloid cells to the lung upon SARS‐CoV‐2 infection, with a shift from Ly6C + to Ly6C – expressing cells. Together, our findings suggest that type I IFN deficiency results in a dysregulated innate immune response to SARS‐CoV‐2 infection. This article is protected by copyright. All rights reserved

Precision-cut lung slices (PCLS) are a novel tool to study cells of the lower airways. As PCLS retain the integrity and architecture of the lung, they constitute a robust model for studying the cells of the lower respiratory tract. Use of PCLS for imaging has been previously documented; however, other applications and techniques can also be applied to PCLS to increase their use and therefore decrease the number of animals needed for each experiment. We present a detailed protocol for generating PCLS from the murine lung. We show that cultured PCLS remain viable up to at least 8 days of culture, that RNA can be isolated from the tissue, and that flow cytometry can be carried out on the cells obtained from the PCLS. Furthermore, we demonstrate that cytokines and chemokines can be detected in the culture supernatants of PCLS exposed to viruses. Overall, these protocols expand the use of PCLS, especially for infection studies.

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