Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro

The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5–16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

Activated Platelets Autocrine 5-Hydroxytryptophan Aggravates Sepsis-Induced Acute Lung Injury by Promoting Neutrophils Extracellular Traps Formation

Excessive neutrophil extracellular trap (NET) formation is an important contributor to sepsis-induced acute lung injury (ALI). Recent reports indicate that platelets can induce neutrophil extracellular trap formation. However, the specific mechanism remains unclear. Tph1 gene, which encodes the rate-limiting enzyme for peripheral 5-hydroxytryptophan (5-HT) synthesis, was knocked out in mice to simulate peripheral 5-HT deficiency. Cecal ligation and puncture (CLP) surgery was performed to induce sepsis. We found that peripheral 5-HT deficiency reduced NET formation in lung tissues, alleviated sepsis-induced lung inflammatory injury, and reduced the mortality rate of CLP mice. In addition, peripheral 5-HT deficiency was shown to reduce the accumulation of platelets and NETs in the lung of septic mice. We found that platelets from wild-type (WT), but not Tph1 knockout (Tph1−/−), mice promote lipopolysaccharide (LPS)-induced NET formation. Exogenous 5-HT intervention increased LPS-induced NET formation when Tph1−/− platelets were co-cultured with WT neutrophils. Therefore, our study uncovers a mechanism by which peripheral 5-HT aggravated sepsis-induced ALI by promoting NET formation in the lung of septic mice.

Neutrophil Extracellular Traps Caused by Gut Leakage Trigger the Autoimmune Response in Nonobese Diabetic Mice

Increased formation of neutrophil extracellular traps (NETs) is associated with gut leakage in type 1 diabetes (T1D). To explore the mechanism of how enteropathy exacerbated by NETs triggers pancreatic autoimmunity in T1D, we carried out a correlation analysis for NET formation with gut barrier functions and autoimmunity in nonobese diabetic (NOD) mice. Inducing chronic colitis or knocking out of peptidyl arginine deiminase type 4 (PAD4) in NOD mice were used to further study the effect of NET formation on the progression of T1D. Microbial alterations in Deferribacteres and Proteobacteria, along with the loss of gut barrier function, were found to be associated with increased endotoxin and abnormal formation of NETs in NOD mice. Both DSS-induced colitis and knockout of PAD4 in NOD mice indicated that PAD4-dependent NET formation was involved in the aggravation of gut barrier dysfunction, the production of autoantibodies, and the activation of enteric autoimmune T cells, which then migrated to pancreatic lymph nodes (PLNs) and caused self-damage. The current study thus provides evidence that PAD4-dependent NET formation is engaged in leaky gut triggering pancreatic autoimmunity and suggests that either degradation of NETs or inhibition of NET formation may be helpful for innovative therapeutic interventions in T1D.

Formation of neutrophil extracellular traps requires actin cytoskeleton rearrangements

Neutrophils are important effector cells in the host defense against invading micro-organisms. One of the mechanisms they employ to eliminate pathogens is the release of neutrophil extracellular traps (NETs). Although NET release and subsequent cell death known as NETosis have been intensively studied, the cellular components and factors determining or facilitating the formation of NETs remain incompletely understood. Using various actin polymerization and myosin II modulators on neutrophils from healthy individuals, we show that intact F-actin dynamics and myosin II function are essential for NET formation when induced by different stimuli, i.e. phorbol 12-myristate 13-acetate, monosodium urate crystals and Candida albicans. The role of actin polymerization in NET formation could not be explained by the lack of reactive oxygen species production or granule release, which were normal or enhanced under the given conditions. Neutrophils from patients with very rare inherited actin polymerization defects by either ARPC1B- or MKL1-deficiency also failed to show NETosis. We found that upon inhibition of actin dynamics there is a lack of translocation of NE to the nucleus, which may well explain the impaired NET formation. Collectively, our data illustrate the essential requirement of an intact and active actin polymerization process, as well as active myosin II to enable the release of nuclear DNA by neutrophils during NET formation.

Involvement of neutrophil extracellular traps in the pathogenesis of glomerulonephritis in a case of systemic lupus erythematosus and antineutrophil cytoplasmic antibody-associated vasculitis overlap syndrome

Systemic lupus erythematosus (SLE) and antineutrophil cytoplasmic antibody-associated vasculitis (AAV) are autoimmune diseases that often cause rapidly progressive glomerulonephritis, with neutrophil extracellular traps (NETs) involved in their pathogenesis. However, the involvement of NETs in the renal damage caused by SLE/AAV overlap syndrome has not been clarified yet. In this study, we detected renal deposition of NETs in a patient with SLE/AAV overlap syndrome. In addition, a significantly increased level of NET-inducing activity was observed in the patient’s serum, which improved with treatment. On the other hand, a markedly lower level of NET degradation was observed in the patient’s serum as compared to healthy subjects’ sera, without any posttreatment changes. These findings suggest that NETs may play a role in the pathogenesis of renal injury associated with SLE/AAV overlap syndrome.

DNase 1 Protects From Increased Thrombin Generation and Venous Thrombosis During Aging: Cross‐Sectional Study in Mice and Humans

Background Human aging is associated with increased risk of thrombosis, but the mechanisms are poorly defined. We hypothesized that aging induces peroxide‐dependent release of neutrophil extracellular traps that contribute to thrombin generation and thrombosis. Methods and Results We studied C57BL6J mice and littermates of glutathione peroxidase‐1 transgenic and wild‐type mice at young (4 month) and old (20 month) ages and a healthy cohort of young (18–39 years) or middle‐aged/older (50–72 years) humans. In plasma, we measured thrombin generation potential and components of neutrophil extracellular traps (cell‐free DNA and citrullinated histone). Aged wild‐type mice displayed a significant increase in thrombin generation that was decreased in aged glutathione peroxidase‐1 transgenic mice. Both aged wild‐type and aged glutathione peroxidase‐1 transgenic mice demonstrated similar elevation of plasma cell‐free DNA compared with young mice. In contrast, plasma levels of citrullinated histone were not altered with age or genotype. Release of neutrophil extracellular traps from neutrophils in vitro was also similar between young and aged wild‐type or glutathione peroxidase‐1 transgenic mice. Treatment of plasma or mice with DNase 1 decreased age‐associated increases in thrombin generation, and DNase 1 treatment blocked the development of experimental venous thrombi in aged C57BL6J mice. Similarly, thrombin generation potential and plasma cell‐free DNA, but not citrullinated histone, were higher in middle‐aged/older humans, and treatment of plasma with DNase 1 reversed the increase in thrombin generation. Conclusions We conclude that DNase 1 limits thrombin generation and protects from venous thrombosis during aging, likely by hydrolyzing cell‐free DNA.

Innate immune deficiencies are associated with severity and poor prognosis in patients with COVID-19

COVID-19 can cause acute respiratory distress syndrome, leading to death in many individuals. Evidence of a deleterious role of the innate immune system is accumulating, but the precise mechanisms involved remain unclear. In this study, we investigated the links between circulating innate phagocytes and severity in COVID-19 patients. We performed in-depth phenotyping of neutrophil and monocyte subpopulations and measured soluble activation markers in plasma. Additionally, anti-microbial functions (phagocytosis, oxidative burst, and NETosis) were evaluated on fresh cells from patients. Neutrophils and monocytes had a strikingly disturbed phenotype, and elevated concentrations of activation markers (calprotectin, myeloperoxidase, and neutrophil extracellular traps) were measured in plasma. Critical patients had increased CD13low immature neutrophils, LOX-1 + and CCR5 + immunosuppressive neutrophils, and HLA-DRlow downregulated monocytes. Markers of immature and immunosuppressive neutrophils were strongly associated with severity. Moreover, neutrophils and monocytes of critical patients had impaired antimicrobial functions, which correlated with organ dysfunction, severe infections, and mortality. Together, our results strongly argue in favor of a pivotal role of innate immunity in COVID-19 severe infections and pleads for targeted therapeutic options.

Increased Circulating Cell-Free DNA in Eosinophilic Granulomatosis With Polyangiitis: Implications for Eosinophil Extracellular Traps and Immunothrombosis

BackgroundEndogenous DNA derived from nuclei or mitochondria is released into the blood circulation as cell-free DNA (cfDNA) following cell damage or death. cfDNA is associated with various pathological conditions; however, its clinical significance in antineutrophil cytoplasmic antibody-associated vasculitis (AAV) remains unclear. This study aimed to evaluate the clinical significance of cfDNA in AAV.MethodsWe enrolled 35 patients with AAV, including 10 with eosinophilic granulomatosis with polyangiitis (EGPA), 13 with microscopic polyangiitis, and 12 with granulomatosis with polyangiitis. Serum cf-nuclear DNA (cf-nDNA) and cf-mitochondrial DNA (cf-mtDNA) levels were measured by quantitative polymerase chain reaction before and after the initiation of immunosuppressive therapy. Tissue samples from EGPA patients were examined by immunofluorescence and transmission electron microscopy. The structure of eosinophil extracellular traps (EETs) and neutrophil extracellular traps (NETs) and stability against DNase were assessed in vitro. Platelet adhesion of EETs were also assessed.ResultsSerum cf-nDNA and cf-mtDNA levels were significantly higher in AAV than in healthy controls, with the highest levels in EGPA; however, serum DNase activities were comparable among all groups. cf-nDNA and cf-mtDNA decreased after treatment and were associated with disease activity only in EGPA. Blood eosinophil count and plasma D-dimer levels were significantly correlated with cf-nDNA in EGPA and cf-mtDNA. EGPA tissue samples showed lytic eosinophils and EETs in small-vessel thrombi. The structure of EETs showed bolder net-like chromatin threads in vitro and EETs showed greater stability against DNase than NETs. EETs provided a scaffold for platelet adhesion.ConclusioncfDNA was increased in EGPA, associated with disease activity. The presence of DNase-resistant EETs in small-vessel thrombi might contribute to higher concentration of cfDNA and the occurrence of immunothrombosis in EGPA.

Immunity to Sda1 Protects against Infection by Sda1+ and Sda1− Serotypes of Group A Streptococcus

Group A Streptococcus (GAS) causes a variety of diseases globally. The DNases in GAS promote GAS evasion of neutrophil killing by degrading neutrophil extracellular traps (NETs). Sda1 is a prophage-encoded DNase associated with virulent GAS strains. However, protective immunity against Sda1 has not been determined. In this study, we explored the potential of Sda1 as a vaccine candidate. Sda1 was used as a vaccine to immunize mice intranasally. The effect of anti-Sda1 IgG in neutralizing degradation of NETs was determined and the protective role of Sda1 was investigated with intranasal and systemic challenge models. Antigen-specific antibodies were induced in the sera and pharyngeal mucosal site after Sda1 immunization. The anti-Sda1 IgG efficiently prevented degradation of NETs by supernatant samples from different GAS serotypes with or without Sda1. Sda1 immunization promoted clearance of GAS from the nasopharynx independent of GAS serotypes but did not reduce lethality after systemic GAS challenge. Anti-Sda1 antibody can neutralize degradation of NETs by Sda1 and other phage-encoded DNases and decrease GAS colonization at the nasopharynx across serotypes. These results indicate that Sda1 can be a potential vaccine candidate for reduction in GAS reservoir and GAS tonsillitis-associated diseases.

S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation

Neutrophils are the most abundant innate immune cells in the circulation and they are the first cells recruited to sites of infection or inflammation. Almost half of the intracellular protein content in neutrophils consists of S100A8 and S100A9, though there has been controversy about their actual localization. Once released extracellularly, these proteins are thought to act as damage-associated molecular patterns (DAMPs), though their mechanism of action is not well understood. These S100 proteins mainly form heterodimers (S100A8/A9, also known as calprotectin) and this heterocomplex is recognized as a useful biomarker for several inflammatory diseases. We observed that S100A8/A9 is highly present in the cytoplasmic fraction of neutrophils and is not part of the granule content. Furthermore, we found that S100A8/A9 was not released in parallel with granular content but upon the formation of neutrophil extracellular traps (NETs). Accordingly, neutrophils of patients with chronic granulomatous disease, who are deficient in phorbol 12-myristate 13-acetate (PMA)-induced NETosis, did not release S100A8/A9 upon PMA stimulation. Moreover, we purified S100A8/A9 from the cytoplasmic fraction of neutrophils and found that S100A8/A9 could induce neutrophil activation, including adhesion and CD11b upregulation, indicating that this DAMP might amplify neutrophil activation.