ABSTRACT
Background: SARs-CoV- 2 infection recruits high numbers of neutrophils that extrude neutrophil extracellular traps (NETs), webs of extracellular DNA coated with citrullinated histones (cit-His) and antimicrobial proteins. NETs have also been shown to entrap virions, concentrate antiviral proteins, and inactivate viruses. However, when NETs are degraded, they release NET degradation products (NDPs) such as cit-His, cell-free (cf) DNA, myeloperoxidase (MPO) and neutrophil elastase (NE) that can be toxic to the host. Our group and others have found that NETs and NDPs are highly prominent in patients with severe COVID-19 and are associated with the development of respiratory failure (Figure 1). Platelet factor 4 (PF4) is a highly-positively charged, platelet-specific chemokine that aggregates polyanionic molecules like heparin and DNA. We have shown that PF4 binds to NETs, reducing the release of NDPs by preventing NET digestion by circulating nucleases. Importantly, PF4-NET complexes markedly enhance gram-positive and -negative bacterial entrapment, likely by bridging the negatively charged polyanionic phosphoribose backbone of the NET DNA scaffold to polyanionic surface molecules in the bacterial cell wall. Treatment with PF4 improved outcomes in lipopolysaccharide endotoxemia and cecal ligation and puncture models of murine sepsis. Objectives: The objective of this study was to investigate whether PF4 binding to NETs is similarly protective in SARs-CoV- 2 infection by preventing the degradation of NETs and by enhancing NET-mediated viral capture. Methods: We generated NET-lined microfluidic channels. Neutrophils were isolated from healthy human donors, adhered to fibronectin-coated channels, and incubated with phorbol myristate acetate (PMA) to induce the release of NETs. Channels were then treated with buffer alone or PF4 (100 μg/ml) to compact NETs, after which gamma-irradiated SARS-CoV- 2 (1 x 107 PFU) were infused at 2 dynes/cm2 for 1 hour. Viral particles were then labeled with SARS-CoV- 2 guinea pig antiserum and visualized with a fluorescently-labeled secondary antibody. Viral binding to NETs was quantified using confocal microscopy. Results: Similar to that seen with bacterial attachment to NETs, we observed scant viral binding to non-compact NETs. In contrast, there was abundant binding of SARs-CoV- 2 aggregates to PF4 compacted NETs (Figure 2). Conclusions: These findings demonstrate that PF4 plays a crucial role in NET-mediated viral capture and suggest that PF4-NET complexes may be part of the physiologic mechanism by which viral spread is contained in the host. Moreover, we have previously shown that an Fc-modified version of KKO, a monoclonal antibody directed against complexes of PF4 and polyanions, markedly enhanced the protective effects of PF4 in vitro and in murine models of sepsis. Therefore, we will examine whether PF4 plus modified KKO infusions are able to limit SARS-CoV- 2 viremia, preventing the pneumonitis and multi-system organ dysfunction of severe COVID-19. (Figure Presented).
ABSTRACT
Background: NETs are webs of extracellular DNA that entrap bacteria and potentially viruses. Unfortunately, NET degradation products (NDPs) are toxic, and growing evidence suggests that NDPs contribute to organ damage in COVID-19. We have previously shown that PF4, a platelet-specific chemokine, aggregates NETs, enhancing bacterial capture while protecting NETs from nuclease degradation, thereby limiting NDP release. Aims: We investigated the effect of PF4 on NET-mediated entrapment and inactivation of coronaviruses. Methods: Inactivated SARS-CoV was incubated in the absence or presence of PF4 (100 μg/ml) and confocal microscopy was used to quantify aggregate formation. NETs released by neutrophils isolated from healthy human donors, were plated on glass slides and microfluidic channels. These NETs were then incubated with PF4 (10-100 μg/ml) prior to exposure to SARs-CoV-1 or SARs-CoV-2. Some PF4-NETs were treated with KKO, a monoclonal antibody, which binds to PF4:NET complexes, improving their nuclease resistance and limiting NET toxicity. Viral-NETs adhesion was quantified with confocal microscopy and evaluated with scanning electron microscopy (SEM). A plaque forming unit assay was performed to measure the effect of PF4 on the infectivity of the SARs-related coronavirus, murine hepatitis virus (MHV)-1. Results: Coronaviruses formed aggregates with PF4 (Figure 1A) and adhered to the NET surface in the presence of PF4 as seen on confocal microscopy (Figure 1B) and SEM (Figure 1C). In SEM studies, KKO enhanced virion adhesion to PF4:NETs. PF4 treatment at physiologic concentrations limited MHV-1 infectivity (Figure 2). Conclusions: Just as with bacteria, PF4 binding enhances NETmediated capture of coronaviruses, an effect enhanced by KKO. Thus, PF4 released by activated platelets may improve virion entrapment by NETs and limit infectivity, while treatment with PF4 infusions may provide additional clinical benefit. We now plan to assess whether KKO can improve NET entrapment of SARS-CoV-2 in the presence of PF4 as a combination therapy to decrease disease severity in COVID-19.