Background Excessive inflammation triggered by a hitherto undescribed mechanism is a hallmark of severe SARS-CoV-2 infections and is associated with enhanced pathogenicity and mortality. Objective Complement hyper activation promotes lung injury and was observed in patients suffering from MERS-CoV, SARS-CoV-1 and SARS-CoV-2 infections. Therefore, we investigated the very first interactions of primary human airway epithelial cells upon exposure to SARS-CoV-2 in terms of complement C3-mediated effects. Methods For this, we used highly differentiated primary human 3D tissue models infected with SARS-CoV-2 patient isolates. Upon infection, viral load, viral infectivity, intracellular complement activation, inflammatory mechanisms and tissue destruction were analyzed by real-time RT-PCR, high content screening, plaque assays, luminex analyses and TEER measurements. Results Here we show that primary normal human bronchial and small airway epithelial cells respond to SARS-CoV-2 infection by an inflated local C3 mobilization. SARS-CoV-2 infection resulted in exaggerated intracellular complement activation and destruction of the epithelial integrity in monolayer cultures of primary human airway cells and highly differentiated, pseudostratified, mucus-producing, ciliated respiratory tissue models. SARS-CoV-2-infected 3D cultures secreted significantly higher levels of C3a and the pro-inflammatory cytokines IL-6, MCP-1, IL-1α and RANTES. Conclusion Crucially, we illustrate here for the first time, that targeting the anaphylotoxin receptors C3aR and C5aR in non-immune respiratory cells can prevent intrinsic lung inflammation and tissue damage. This opens up the exciting possibility in the treatment of COVID-19.

To study interactions of airborne pathogens, e.g. Aspergillus (A.) fumigatus with upper and lower respiratory tract epithelial and immune cells, we set up a perfused 3D human bronchial and small airway epithelial cell system. Culturing of normal human bronchial or small airway epithelial (NHBE, SAE) cells under air liquid interphase (ALI) and perfusion resulted in a significantly accelerated development of the lung epithelia associated with higher ciliogenesis, cilia movement, mucus-production and improved barrier function compared to growth under static conditions. Following the accelerated differentiation under perfusion, epithelial cells were transferred into static conditions and antigen-presenting cells (APCs) added to study their functionality upon infection with A. fumigatus. Fungi were efficiently sensed by apically applied macrophages or basolaterally adhered dendritic cells (DCs), as illustrated by phagocytosis, maturation and migration characteristics. We illustrate here that perfusion greatly improves differentiation of primary epithelial cells in vitro, which enables fast-track addition of primary immune cells and significant shortening of experimental procedures. Additionally, co-cultured primary DCs and macrophages were fully functional and fulfilled their tasks of sensing and sampling fungal pathogens present at the apical surface of epithelial cells, thereby promoting novel possibilities to study airborne infections under conditions mimicking the in vivo situation.

T cells play a fundamental role in the early control and clearance of many viral infections of the respiratory system. In SARS-CoV-2-infected individuals, lymphopenia with drastically reduced CD4+ and CD8+ T cells correlates with Coronavirus disease 2019 (COVID-19)-associated disease severity and mortality. In this study, we characterized cellular and humoral immune responses induced in patients with mild, severe and critical COVID-19. Peripheral blood mononuclear cells of 37 patients with mild, severe and critical COVID-19 and 10 healthy individuals were analyzed by IFNγ ELISpot and multi-color flow cytometry upon stimulation with peptide pools covering complete immunodominant SARS-CoV-2 matrix, nucleocapsid and spike proteins. In addition SARS-CoV-2 antibody levels, neutralization abilities and anaphylatoxin levels were evaluated by various commercially available ELISA platforms. Our data clearly demonstrates a significantly stronger induction of SARS-CoV-2 specific CD8+ T lymphocytes and higher IFNγ production in patients with mild compared to patients with severe or critical COVID-19. In all patients SARS-CoV-2-specific antibodies with similar neutralizing activity were detected, but highest titers of total IgGs were observed in critical patients. Finally, elevated anaphylatoxin C3a and C5a levels were identified in severe and critical COVID-19 patients probably caused by aberrant immune complex formation due to elevated antibody titers in these patients. Crucially, we provide a full picture of cellular and humoral immune responses of COVID-19 patients and prove that robust polyfunctional CD8+ T cell responses concomitant with low anaphylatoxin levels correlate with mild infections. In addition, our data indicates that high SARS-CoV-2 antibody titers are associated with severe disease progression.