key: cord-0771554-8srzlxk8 authors: Zuo, Y.; Yalavarthi, S.; Navaz, S.; Hoy, C.; Shi, H.; Harbaugh, A.; Gockman, K.; Zuo, M.; Madison, J. A.; Kanthi, Y.; Knight, J. S. title: Autoantibodies stabilize neutrophil extracellular traps in COVID-19 date: 2021-04-05 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2021.03.31.21254692 sha: 456aed9b7fcae4fb8862b6ad914a50076ac06ca6 doc_id: 771554 cord_uid: 8srzlxk8 The release of neutrophil extracellular traps (NETs) by hyperactive neutrophils is recognized to play an important role in the thromboinflammatory milieu inherent to severe presentations of COVID-19. At the same time, a variety of functional autoantibodies have been observed in individuals with severe COVID-19 where they likely contribute to immunopathology. Here, we aimed to determine the extent to which autoantibodies might target NETs in COVID-19 and, if detected, to elucidate their potential functions and clinical associations. We measured global anti-NET activity in 171 individuals hospitalized with COVID-19 alongside 48 healthy controls. We found high anti-NET activity in the IgG and IgM fractions of approximately 40% and 50% of patients, respectively. There was a strong correlation between anti-NET IgG and anti-NET IgM, with high anti-NET antibody levels in general associating with circulating markers of NETs such as myeloperoxidase-DNA complexes and calprotectin. Clinically, anti-NET antibodies tracked with impaired oxygenation efficiency and elevated levels of circulating D-dimer. Furthermore, patients who required mechanical ventilation had higher levels of anti-NET antibodies than those who did not require oxygen supplementation. Mechanistically, anti-NET antibodies of the IgG isotype impaired the ability of DNases in healthy serum to degrade NETs. In summary, these data reveal high levels of anti-NET antibodies in individuals hospitalized with COVID-19, where they likely impair NET clearance and thereby potentiate SARS-CoV-2 mediated thromboinflammation. While it has been more than a year since the initial outbreak, coronavirus disease 2019 (COVID-19)-caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-remains a global health challenge with alarming death tolls (1) . Many survivors of COVID-19 continue to suffer from post-acute sequelae of the infection and the cause of these long-term symptoms remains unknown (2) (3) (4) . Severe, acute COVID-19 is characterized by a thromboinflammatory state driven by a complex interplay between innate and adaptive immune responses (1) . This state manifests clinically as acute respiratory distress syndrome, and, in some patients, widespread thrombotic microangiopathy. Activated neutrophils-and, in particular, neutrophil extracellular traps (NETs)-continue to receive significant attention as drivers of SARS-CoV-2-mediated thromboinflammation. NETs are an extracellular meshwork of DNA, histones, and microbicidal proteins released from activated neutrophils via a cell death program called NETosis. Neutrophils presumably deploy NETs to trap and kill pathogens (5) ; however, NETs may also be key players in the pathophysiology of thromboinflammatory diseases such as cancer, systemic lupus erythematosus, antiphospholipid syndrome (APS), and-based on recent work-COVID-19 (6) (7) (8) . Indeed, our group and others have described high levels of NETs circulating in the blood of hospitalized COVID-19 patients, where they correlate with disease severity (6, (9) (10) (11) (12) . We have also found that neutrophil hyperactivity at the time of hospital admission predicts a more severe hospital course (13) , and that NET levels are especially high in patients who experience thrombotic complications (14) . Another hallmark of COVID-19 is the development of autoantibodies against a variety of selfantigens, particularly among COVID-19 patients with severe disease (15) (16) (17) (18) . Many of those autoantibodies appear to perturb normal immune function while influencing disease severity and progression. For example, anti-type I interferon antibodies attenuate a presumably protective immune response against SARS-CoV-2 and thereby exacerbate disease (19) . Autoantibodies against annexin A2 and other immunomodulatory proteins are also associated with severe COVID-19 (20, 21) . Furthermore, work by our group found that many hospitalized COVID-19 patients developed antiphospholipid antibodies routinely found in APS, an acquired autoimmune thrombophilia (15) . Mechanistically, these antibodies promote pathogenic NETs formation and accelerate thrombosis in vivo. Anti-NET activity in COVID-19. Utilizing a unique ELISA platform that we developed ( Figure 1A ), we measured anti- NET IgG and IgM antibodies in 171 patients hospitalized with COVID-19 alongside 48 healthy controls. The clinical characteristics of these patients are described in Supplementary Table 1 . Elevated levels of anti-NET IgG and IgM were detected in patients with COVID-19 as compared with healthy controls (Figure 1B-C) . Sixty-seven COVID-19 patients (39%) had high anti-NET IgG activity, while 86 (50%) had high anti-NET IgM activity (each based on a threshold set at 2 standard deviations above the control mean). We also noted a strong correlation between anti-NET IgG and anti-NET IgM (r=0.51, p<0.0001, revealed that one function of anti-NET antibodies in patients with lupus (23) and APS (22) is to impair NET degradation. Here, we asked whether antibodies purified from COVID-19 sera with high anti-NET activity might impact the ability of serum DNases to degrade NETs. We selected four COVID-19 patients with high anti-NET IgG and purified their total IgG fractions. These were tested alongside IgG pooled from healthy controls ( Figure 4A) . Indeed, control serum supplemented with COVID-19 patient IgG significantly impaired NET degradation as compared with control serum supplemented with healthy control IgG ( Figure 4B ). In summary, anti-NET IgG from COVID-19 patients impairs the ability of serum to degrade NETs. In COVID-19, NETs may be directly induced by SARS-CoV-2 virus (1, 24) . They may also be triggered indirectly via activated platelets and prothrombotic autoantibodies (1, 15) . Once formed, NETs exert direct cytotoxic effects against pulmonary epithelium resulting in alveolar damage and fibrosis (1) . They can also injure endothelial cells leading to microvascular damage and thrombotic microangiopathy in lungs, kidneys, and heart (1). Here, we explored the hypothesis that dysfunctional NET clearance may also contribute to COVID-19 pathogenesis. SARS-CoV-2 appears to have a unique relationship with the immune system. It evades host immune surveillance during early infection leading to high viral loads in some patients (1) . As a result, the body then mounts a compensatory hyper-immune response in pursuit of viral clearance. This is characterized by the presence of a lupus-like peripheral B cell compartment in which naïve B cells take an extrafollicular route to becoming antibody-producing cells, and in doing so bypass the normal tolerance checkpoints against autoimmunity provided by the germinal center (25) . While this strategy may quickly produce a large amount of virusneutralizing antibodies, it also sets the stage for the de novo production of various pathogenic autoantibodies. NETs appear to elicit autoantibody production in systemic autoimmune diseases such as lupus, rheumatoid arthritis, and ANCA-associated vasculitis (26) . For example, it has been suggested that increased NET formation, the presence of anti-NET antibodies, and impaired NET clearance all associate with disease activity and organ damage in lupus (26) . Our group has found something similar in individuals with primary APS (22) . Here, we found that high levels of anti-NET IgG and IgM are present in patients hospitalized with COVID-19. Those anti-NET antibodies not only impaired the intrinsic ability of serum DNases to clear NETs, but also All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 5, 2021. ; https://doi.org/10.1101/2021.03.31.21254692 doi: medRxiv preprint associated with impaired respiratory status and disease severity. It is possible that these anti-NET antibodies are important orchestrators of an imbalance between NET formation and clearance that perpetuates COVID-19 thromboinflammation. While the ongoing vaccination campaign is working towards reducing COVID-19 incidence and mortality, millions of survivors of COVID-19 infection continue to suffer from longer-term symptoms of the disease. Certainly, diverse and functional autoantibodies produced during COVID-19 infection are a plausible contributor to the post-COVID syndrome. Intriguingly, one recent study observed that among nine COVID-19 survivors, five developed chronic "long-haul" symptoms and all five had potentially pathological autoantibodies (27) . We have previously observed durable anti-NET IgG for up to four years among some APS patients (22) . The data presented here suggest the presence of another functional autoantibody in COVID-19, and the persistence and potential long term consequences of these antibodies warrant further investigation. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Table 1 Human neutrophil purification. Human neutrophils were isolated as we have done and described previously (22) . NETs were generated with PMA stimulation as described previously (28) . The plate was washed 5 more times with wash buffer and was developed with 3,3′,5,5′-All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 5, 2021. ; https://doi.org/10.1101/2021.03.31.21254692 doi: medRxiv preprint Tetramethylbenzidine (TMB) substrate (Invitrogen). The reaction was stopped by 2N sulfuric acid solution and the absorbance was measured at a wavelength of 450 nm using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). Each sample was tested against a corresponding control in which no NETs antigen was plated. This created an individual background value for each sample, which was subtracted to obtain the final result. Purification of human IgG fractions. IgG was purified from COVID-19 or control sera as we have done previously (15) . Immunofluorescence microscopy. 1x10 5 healthy control neutrophils were seeded onto 0.001% poly-L-lysine coated coverslips as described previously (22) . To induce NET formation, neutrophils were incubated in serum-free RPMI media supplemented with L-glutamine and stimulated with 40 nM PMA for 2 hours at 37°C and 5% CO2. Following stimulation, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, followed by overnight blocking in 10% fetal bovine serum (FBS) in PBS (blocking buffer). For protein staining, fixed cells were incubated with either control or COVID serum (diluted to 10%) and polyclonal antibody to neutrophil elastase (Abcam Ab21595) diluted 1:100 in blocking buffer for 1 hour at 4°C, followed by fluorochrome-conjugated DyLight594 anti-human IgG (Thermo Fisher) and FITC-conjugated secondary (Southern Biotech) for 1 hour at 4°C. Nuclear DNA was detected with Hoechst 33342. Coverslips were mounted with Prolong Gold Antifade (Thermo Fisher) and images were collected with a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). Quantification of S100A8/A9 (calprotectin). Calprotectin was measured with the Human S100A8/S100A9 Heterodimer DuoSet ELISA (DY8226-05, R&D Systems) according to the manufacturer's instructions (13) . Myeloperoxidase-DNA complexes were measured as has been previously described (6, 29) . NET degradation assay. PMA-stimulated NETs were degraded as previously described with minor modifications (30) . See the Supplementary Materials for a detailed description. Statistical analysis. Normally-distributed data were analyzed by 2-sided t test and skewed data were analyzed by Mann-Whitney test. Comparisons of more than two groups were analyzed by All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 5, 2021. ; for 4 hours at 37°C and 5% CO2. Following incubation, the culture media was gently aspirated and washed once with 1x PBS. NETosis was quantified by incubating the cells for 30 minutes at 37°C and 5% CO2 with SYTOX Green (Thermo Fisher) diluted in PBS to a final concentration of 1 mM. PBS was gently aspirated and fresh 1x PBS was carefully added to each well. Fluorescence was quantified at excitation and emission wavelengths of 504/523 nm using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). To assess NET degradation, the PBS was gently aspirated from each well and NETs were incubated for 90 minutes (at 37°C and 5% CO2) with healthy control serum diluted to 5% in nuclease buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 mM CaCl2, and 50 mM NaCl) and supplemented with COVID or control IgG at a final concentration of 500ug/ml. Each sample was tested in triplicate, with MNase (10 U/ml) (Thermo Fisher)-treated wells serving as positive control. Following the 90-minute incubation, supernatant was discarded and 1x PBS was added to each well. To quantify residual NETs in each well, SYTOX fluorescence was re-measured at excitation and emission wavelengths of 504/523 nm using a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 5, 2021. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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