Aggregation of Enveloped Viruses By Platelet Factor 4 (PF4) and Neutrophil Extracellular Traps (NETs): Implications for the Anti-Viral Properties of NETs

Abstract

As part of the innate immune response, neutrophils release neutrophil extracellular traps (NETs), webs of DNA coated with histones that ensnare and kill bacteria. A growing body of literature suggests that NETs can also capture and inactivate enveloped viruses. The ability of NETs to limit the systemic spread of pathogens occurs at the expense of collateral organ damage induced when high concentrations of NETs are digested by DNases to release toxic NET degradation products (NDPs) including cell free DNA and histones. Our group has shown that the positively charged platelet-specific chemokine (PF4, CXCL4), compacts NETs by forming tetramers that electrostatically cross-bind two polyanionic fragments of DNA, similarly to the way it aggregates heparans. PF4 complexed to NETs induces NET resistance to nuclease digestion, preventing the release of toxic NDPs while enhancing NET-bacterial capture by bridging anionic DNA to the negatively charged microbial surface. PF4 has also been shown limit pulmonary injury and enhance survival in mice infected with PR8, a mouse-adapted H1N1 influenza virus. Our group and others have found that both PF4 and NETs are elevated in the setting of SARS-CoV-2, leading us to hypothesize that they may act synergistically to combat the proliferation of enveloped viruses. To investigate the effect of PF4 on the pathogenicity of enveloped viruses, we studied PR8 and murine hepatitis virus (MHV)-1, a betacoronavirus that induces severe pulmonary disease. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) were used to assess the ability of PF4 and human-derived NETs to aggregate MHV-1 and PR8 virions. ChAdOx1, a replication-deficient adenovirus was used as a non-enveloped viral control. The DLS and SEM studies showed that PF4 (5-20 µg/ml) aggregated MHV-1 and PR8, forming complexes with an average diameter of 500nm, the width of 4-5 virions (Figure 1). In contrast, PF4 did not aggregate non-enveloped adenovirus. DLS studies showed that PF4 does not form complexes with coronavirus spike protein. However, the anti-PF4-polyanion antibody KKO, bound to PF4-viral complexes, leading to an additional 50% increase in viral aggregate size, an effect not seen with an isotype control antibody TRA (Figure A). These results suggest that PF4 binds to polyanions on the viral envelop via electrostatic interaction, much in the same way that it binds to heparans and DNA. We preformed subsequent DLS studies in which MHV-1 virions were incubated with both intact and digested NETs, and viral-NET aggregation was only observed in the presence of PF4. To determine if PF4 aggregates virus in vivo, IHC studies were performed on lungs harvested from MHV-1-infected A/J mice inoculated intranasally with 5 x 103 PFU of virus and sacrificed at 48h. The lungs were prepared for immunohistochemistry (IHC), and labeled with antibodies directed against PF4 and the MHV-1 nucleocapsid protein. We calculated the average Pearson's coefficient in 4 randomly selected 20x confocal images of the lungs of MHV-1 infected animals (n=4 mice, 16 images analyzed) using the FIJI JaCoP colocalization plugin. We observed co-localization of PF4 and MHV-1 within the cytoplasm of a subset of cells (r= 0.70225, p<0.0001), suggesting that PF4-MHV-1 aggregation occurs in vivo (Figure B) Lastly, the plaque forming unit assay was used to measure the effect of PF4 on MHV-1 infectivity of L2 cells. We found that incubating MHV-1 with PF4 (0-100µg/ml), leads to a 50% decrease in infectivity(n=5, p=0.023). Our studies demonstrate that PF4 binds to and aggregates MHV-1 and PR8 in vitro likely through electrostatic interactions with the viral envelop rather than adhesion to the viral spike protein. These results also suggest that PF4-mediated aggregation enhances viral capture by NETs to form NETs:PF4:viral complexes that may limit viral spread. IHC studies of lungs from MHV-1 infected mice demonstrate that PF4 colocalizes with the virus, suggesting that aggregation also occurs in vivo. In ongoing studies, we are investigating whether PF4 and NETs act synergistically to reduce coronavirus and influenza infectivity and enhance immune recognition and subsequent clearance of viral aggregates. These findings will help to define the anti-viral properties of NETs and may generate insights relevant to the treatment of coronavirus, influenza, and other enveloped viruses. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal