Neutrophils Significantly Enhance Pro-Inflammatory Cytokine Release from Airway Epithelial Cells in Response to SARS-CoV-2 Infection

RATIONALE: Novel coronavirus infectious disease (COVID-19) is caused by the severe acute respiratory distress syndrome related coronavirus 2 (SARS-CoV-2). Patients with underlying respiratory disease are considered at increased risk of contracting severe COVID-19. Many respiratory diseases including chronic obstructive pulmonary disease, asthma, and cystic fibrosis, have elevated inflammation driven by recruitment of neutrophils, known as neutrophilia, and contributes to disease pathogenesis. In response to viral infection, neutrophils are the first, and predominant, leukocyte recruited to the respiratory tract. Neutrophils release inflammatory mediators as part of the acute inflammatory response and contribute to pathogen clearance through virus internalization and killing, secretion of antimicrobial peptides and extracellular traps. It remains uncertain how critical the neutrophil response is in SARS-CoV-2 and to what extent this contributes to the cytokine response of the airways in COVID-19. We hypothesized that neutrophils are responsible for elevated inflammatory responses in patients who progress to severe COVID-19. Methods: To investigate, we developed a co-culture model of neutrophilic airways to study SARS-CoV-2 infection, combining primary human neutrophils and airway epithelial cells differentiated at the air-liquid interface. These cultures, with and without neutrophils, were infected with live SARS-CoV-2 and the inflammatory responses analyzed. In addition, extensive analysis of ACE2, neutrophils and inflammatory response, was performed comparing our models and COVID-19 patient and non-infected lung tissues. Results: ACE2 protein, the receptor binding to the S-protein of SARS-CoV-2, was predominantly expressed in submucosal glands, ciliated cells and in alveolar type 2 cells in human lung tissues. Surprisingly, substantial co-localization of ACE2 was also observed with neutrophil elastase and CD15 expressing neutrophils infiltrating the airways. Analysis of mono-and co-cultures of airway epithelial cells and neutrophils in the presence or absence of SARS-CoV-2 infection indicated significant increases in the expression of squamous epithelium associated cytokeratins, damage associated molecular patterns (DAMPS) and a downregulation of interleukin 8 (IL-8) specifically in the co-culture models. Airway supernatants from the basolateral and apical surfaces were profiled. In the neutrophil co-cultures significant increases in IFNγ, IL-1β, IL-6 and TNFα were observed in response to SARS-CoV-2 infection. Conclusions: In conclusion, we demonstrate that co-culture with neutrophils has a significant impact on SARS-CoV-2 infection, changing the inflammatory response and upregulating gene expression patterns associated with tissue remodeling and tissue damage. This study highlights the need to study SARS-CoV-2 infection in more complex, tissue-level models to fully understand the mechanisms driving the severe inflammatory response and the long-term consequences of infection.