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.

Inhibition of PAD4 mediated neutrophil extracellular traps prevents fibrotic osseointegration failure in a tibial implant murine model

Aims Aseptic loosening is a leading cause of uncemented arthroplasty failure, often accompanied by fibrotic tissue at the bone-implant interface. A biological target, neutrophil extracellular traps (NETs), was investigated as a crucial connection between the innate immune system’s response to injury, fibrotic tissue development, and proper bone healing. Prevalence of NETs in peri-implant fibrotic tissue from aseptic loosening patients was assessed. A murine model of osseointegration failure was used to test the hypothesis that inhibition (through Pad4-/- mice that display defects in peptidyl arginine deiminase 4 (PAD4), an essential protein required for NETs) or resolution (via DNase 1 treatment, an enzyme that degrades the cytotoxic DNA matrix) of NETs can prevent osseointegration failure and formation of peri-implant fibrotic tissue. Methods Patient peri-implant fibrotic tissue was analyzed for NETs biomarkers. To enhance osseointegration in loose implant conditions, an innate immune system pathway (NETs) was either inhibited ( Pad4-/- mice) or resolved with a pharmacological agent (DNase 1) in a murine model of osseointegration failure. Results NETs biomarkers were identified in peri-implant fibrotic tissue collected from aseptic loosening patients and at the bone-implant interface in a murine model of osseointegration failure. Inhibition ( Pad4-/- ) or resolution (DNase 1) of NETs improved osseointegration and reduced fibrotic tissue despite loose implant conditions in mice. Conclusion This study identifies a biological target (NETs) for potential noninvasive treatments of aseptic loosening by discovering a novel connection between the innate immune system and post-injury bone remodelling caused by implant loosening. By inhibiting or resolving NETs in an osseointegration failure murine model, fibrotic tissue encapsulation around an implant is reduced and osseointegration is enhanced, despite loose implant conditions. Cite this article: Bone Joint J 2021;103-B(7 Supple B):135–144.

Identification of monoclonal antibodies against human renal glomerular endothelial cells in lupus nephritis that induce endothelial interferon-alpha production

Background The pathogenesis of lupus nephritis (LN) remains not fully understood. In this study, we aimed to explore the pathogenic roles of autoantibodies against human renal glomerular endothelial cells (HRGEC) in LN patients. Methods The serum levels of anti-HRGEC antibodies in systemic lupus erythematosus (SLE) patients without LN and LN patients were determined by cell-based enzyme-linked immunosorbent assay (ELISA). Monoclonal IgG anti-HRGEC antibodies were subsequently generated from LN patients. The binding activities of these monoclonal antibodies to HRGEC, their cross-reactivity with double-stranded DNA (dsDNA), and the ability to activate HRGEC were further evaluated. Results LN patients had higher serum levels of IgG anti-HRGEC antibodies than SLE patients without LN and healthy controls. Four monoclonal IgG anti-HRGEC antibodies (LN1–4) were obtained; LN1 and LN2 were IgG3 while LN3 and LN4 were IgG1. Among these monoclonal antibodies, LN1–3 were cross-reactive with dsDNA. The functional assays showed that compared with IgG1/IgG3 isotype controls, LN3 had an effect on HRGEC to enhance interleukin (IL)-6 production, LN4 could enhance IL-8 and monocyte chemoattractant protein (MCP)-1 production, and LN1–3 possessed the ability to induce interferon (IFN)-α production by HRGEC. Moreover, the removal of DNA on the HRGEC surface by DNAse 1 did not interpose the binding of LN1–3 to HRGEC and the effects of LN1–3 on IFN-α induction by HRGEC. Conclusions Some IgG anti-HRGEC antibodies in LN patients had the ability to enhance endothelial proinflammatory cytokine (IL-6, IL-8, and MCP-1) production, and some could induce the DNA-independent production of IFN-α by HRGEC.

NETs Enhance STING To Promote Surgical Adhesion

Background Postoperative adhesion (PA) following abdominal surgery may cause bowel obstruction, chronic pain, infertility, or even death. Knowledge of adhesion biology is limited, and preventive agents in clinical trials have failed to achieve efficacy. Results In the present study, we showed that neutrophils accumulate in the injured peritoneum at early stage of PA, and neutrophils within the ischemic buttons undergo cell death and form neutrophil extracellular traps (NETs) that contribute to PA. Neutrophil depletion reduces adhesion burden at 7 days after adhesion induction. Peptidylarginine deiminase 4 (PAD4), an essential enzyme for NET formation, is increased in ischemic buttons. Degradation of NETs by DNase 1 and suppression of NET formation by pharmacologic inhibition of PAD4 alleviated adhesion burden, collogen deposition and fibrosis formation. Mechanistically, administration of DNase I and PAD inhibitor reduces STING-mediated inflammatory response. STING deficiency attenuates adhesion burden, collogen deposition, and α-SMA production in the adhesive tissues at 7 days after surgery. Conclusions Collectively, our findings reveal NETs/STING signaling as a therapeutic target to prevent PA.

Deoxyribonuclease 1 Q222R single nucleotide polymorphism and long-term mortality after acute myocardial infarction

Upon activation, neutrophils release neutrophil extracellular traps (NETs), which contribute to circulating DNA burden and thrombosis, including ST-segment elevation myocardial infarction (STEMI). Deoxyribonuclease (DNase) 1 degrades circulating DNA and NETs. Lower DNase activity correlates with NET burden and infarct size. The DNase 1 Q222R single nucleotide polymorphism (SNP), impairing DNase 1 function, is linked with myocardial infarction. We assessed whether the Q222R SNP is connected to increased NET burden in STEMI and influences long-term outcomes. We enrolled 711 STEMI patients undergoing primary percutaneous coronary intervention (pPCI), and 1422 controls. Genotyping was performed for DNase 1 Q222R SNP. DNase activity, double-stranded (ds)DNA and citrullinated histone H3 were determined in culprit site and peripheral plasma during pPCI. The association of the Q222R variant on cardiovascular and all-cause mortality was assessed by multivariable Cox regression adjusted for cardiovascular risk factors. Homozygous Q222R DNase 1 variant was present in 64 (9.0%) STEMI patients, at the same frequency as in controls. Patients homozygous for Q222R displayed less DNase activity and increased circulating DNA burden. In overall patients, median survival was 60 months. Homozygous Q222R variant was independently associated with cardiovascular and all-cause mortality after STEMI. dsDNA/DNase ratio independently predicted cardiovascular and all-cause mortality. These findings highlight that the Q222R DNase 1 SNP is associated with increased NET burden and decreased compensatory DNase activity, and may serve as an independent risk factor for poor outcome after STEMI.

Renal Dnase1 expression is regulated by FGF23 but loss of Dnase1 does not alter renal phosphate handling

Fibroblast growth factor 23 (FGF23) is a bone-derived endocrine hormone that regulates phosphate and vitamin D metabolism. In models of FGF23 excess, renal deoxyribonuclease 1 (Dnase1) mRNA expression is downregulated. Dnase-1 is an endonuclease which binds monomeric actin. We investigated whether FGF23 suppresses renal Dnase-1 expression to facilitate endocytic retrieval of renal sodium dependent phosphate co-transporters (NaPi-IIa/c) from the brush border membrane by promoting actin polymerization. We showed that wild type mice on low phosphate diet and Fgf23−/− mice with hyperphosphatemia have increased renal Dnase1 mRNA expression while in Hyp mice with FGF23 excess and hypophosphatemia, Dnase1 mRNA expression is decreased. Administration of FGF23 in wild type and Fgf23−/− mice lowered Dnase1 expression. Taken together, our data shows that Dnase1 is regulated by FGF23. In 6-week-old Dnase1−/− mice, plasma phosphate and renal NaPi-IIa protein were significantly lower compared to wild-type mice. However, these changes were transient, normalized by 12 weeks of age and had no impact on bone morphology. Adaptation to low and high phosphate diet were similar in Dnase1−/− and Dnase1+/+ mice, and loss of Dnase1 gene expression did not rescue hyperphosphatemia in Fgf23−/− mice. We conclude that Dnase-1 does not mediate FGF23-induced inhibition of renal tubular phosphate reabsorption.

Phagocyte extracellular traps in children with neutrophilic airway inflammation

Childhood lung infection is often associated with prominent neutrophilic airway inflammation and excess production of proteases such as neutrophil elastase (NE). The mechanisms responsible for this inflammation are not well understood. One potentially relevant pathway is the production of extracellular traps by neutrophils (NETs) and macrophages (METs). The aim of this study was to measure NET and MET expression in children and the effect of deoxyribonculease (DNase) 1 and alpha-1 antitrypsin (AAT) on this process.We studied 76 children (median age of 4.0 years) with cystic fibrosis (CF) or chronic cough who underwent investigational bronchoscopy. NETs, METs and NE activity in bronchoalveolar lavage (BAL) samples were measured using confocal microscopy and functional assays. The effects of DNase 1 and AAT on NET/MET expression and NE activity were examined in vitro.Both subject groups had airway neutrophilia with prominent BAL production of NETs with NE co-expression; the mean %±standard error of the mean of neutrophils expressing NETs in the CF group was 23.3±2.8% and in the non-CF group was 28.4±3.9%. NET expression was higher in subjects who had detectable NE activity (p≤0.0074). The percentage of macrophages expressing METs in the CF group was 10.7±1.2% and in the non-CF group was 13.2±1.9%. DNase 1 decreased NET/MET expression (p<0.0001), but increased NE activity (p≤0.0137). The combination of AAT and DNase 1 reduced NE activity (p≤0.0049).We observed prominent extracellular trap formation in symptomatic children with and without CF. This innate inflammatory response was down-regulated by a combination of currently available therapeutics.

Neutrophil extracellular traps promote tPA-induced brain hemorrhage via cGAS in mice with stroke

Intracerebral hemorrhage associated with thrombolytic therapy with tissue plasminogen activator (tPA) in acute ischemic stroke continues to present a major clinical problem. Here, we report that infusion of tPA resulted in a significant increase in markers of neutrophil extracellular traps (NETs) in the ischemic cortex and plasma of mice subjected to photothrombotic middle cerebral artery occlusion. Peptidylarginine deiminase 4 (PAD4), a critical enzyme for NET formation, is also significantly upregulated in the ischemic brains in tPA-treated mice. Blood-brain barrier (BBB) disruption following ischemic challenge in an in vitro model of BBB was exacerbated after exposure to NETs. Importantly, disruption of NETs by DNase 1 or inhibition of NET production by PAD4 deficiency restored tPA-induced loss of BBB integrity and consequently decreased tPA-associated brain hemorrhage after ischemic stroke. Furthermore, either DNase 1 or PAD4 deficiency reversed tPA-mediated upregulation of the DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS). Administration of cGAMP after stroke abolished DNase 1-mediated downregulation of the STING pathway and type I interferon (IFN) production, and blocked the antihemorrhagic effect of DNase 1 in tPA-treated mice. We also show that tPA-associated brain hemorrhage after ischemic stroke was significantly reduced in cGas-/- mice. Collectively, these findings demonstrate that NETs significantly contribute to tPA-induced BBB breakdown in ischemic brain, and suggest that targeting NETs or cGAS may ameliorate thrombolytic therapy for ischemic stroke by reducing tPA-associated hemorrhage.

Structural basis for polarized elongation of actin filaments

Actin filaments elongate and shorten much faster at their barbed end than their pointed end, but the molecular basis of this difference has not been understood. We use all-atom molecular dynamics simulations to investigate the properties of subunits at both ends of the filament. The terminal subunits tend toward conformations that resemble actin monomers in solution, while contacts with neighboring subunits progressively flatten the conformation of internal subunits. At the barbed end the terminal subunit is loosely tethered by its DNase-1 loop to the third subunit, because its monomer-like conformation precludes stabilizing contacts with the penultimate subunit. The motions of the terminal subunit make the partially flattened penultimate subunit accessible for binding monomers. At the pointed end, unique contacts between the penultimate and terminal subunits are consistent with existing cryogenic electron microscopic (cryo-EM) maps, limit binding to incoming monomers, and flatten the terminal subunit, which likely promotes ATP hydrolysis and rapid phosphate release. These structures explain the distinct polymerization kinetics of the two ends.