C5aR inhibition of nonimmune cells suppresses inflammation and maintains epithelial integrity in SARS-CoV-2–infected primary human airway epithelia

Abstract

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.

Potent SARS-CoV-2-Specific T Cell Immunity and Low Anaphylatoxin Levels Correlate With Mild Disease Progression in COVID-19 Patients

“…Furthermore, treatment with monoclonal antibodies against anaphylatoxin C5a receptor 1 (C5aR1) prevented C5a-mediated recruitment and activation of human myeloid cells and inhibited acute lung injury in in vivo experiments (47). Indeed, our group demonstrated that targeting receptors for C3a and C5a from nonimmune cells prevented lung inflammation and tissue damage in a 3D respiratory tissue model (27).…”

“…We and others identified a link between complement regulation and cytokine overexpression in SARS-CoV-2 infected tissues, since complement deposition and high anaphylatoxin serum levels have been reported in patients with severe or critical disease (25). Also in murine models it has been previously shown that, during infection with different human coronaviruses, depletion or blockage of complement component 3 (C3) in SARS-CoV infection and complement component 5 (C5) in MERS-CoV infection resulted in milder disease severity (26,27).…”

ColdZyme Maintains Integrity in SARS-CoV-2-Infected Airway Epithelia

“…Elevated anaphylatoxin levels resulted in downmodulation of regulators of complement activation such as CD55 and CD46 and changes in injury markers on HAE cells (27). As illustrated in our highly differentiated, pseudostratified 3D models, extremely high levels of C3a are secreted from the airway epithelium upon interaction with SARS-CoV-2 (20), which was significantly downmodulated by applying ColdZyme. Both C3a and C5a comprise important effector molecules attracting, activating, and regulating components of innate and adaptive immunity (29).…”

“…Further, ColdZyme completely antagonized not only binding of SARS-CoV-2 but also infection and concomitant virusinduced tissue damage and local complement activation as indicators for innate immune activation. Exacerbation of injury by local complement activation in the airway epithelium was already shown for SARS-CoV infection (20,26). Additionally, increased anaphylatoxin levels (C3a, C5a) in plasma and lung homogenates have been implicated in the pathogenesis of various lung conditions including cystic fibrosis and idiopathic pulmonary fibrosis (27,28).…”

“…After the incubation period, the tissue models were fixed and stained for immunofluorescence (IF) analyses using Alexa 594-labeled antibodies against the SARS-CoV-2 spike 1 (S1) and nucleocapsid (N) proteins to detect virus binding, Hoechst stain for nuclei, wheat germ agglutinin (WGA) for glycocalyx, and complement component C3 as marker for innate immune activation of NHBE cells. Here, intracellular (IC) C3 was used as an indicator for tissue damage during SARS-CoV-2 infection of NHBE cells, since we recently found that infection in primary airway epithelial cells was accompanied by extensive induction of IC C3 and secretion of the anaphylatoxin C3a from HAE cells (20). NHBE tissue models illustrated an excessive binding and uptake of SARS-CoV-2 (red), already detectable when imaging the overview of the whole Transwell filter containing the fully differentiated epithelium, going along with concomitant activation of IC C3 (green) (Fig.…”