Infectious and inflammatory stimuli induce the release of neutrophil extracellular traps (NETs), webs of cell‐free (cf) DNA complexed with histones and antimicrobial proteins, that capture and kill ...pathogens. Despite their protective role in the initial stages of sepsis, excessive NET release accompanied by NET degradation, leads to the release of NET degradation products (NDPs), including cfDNA, histones, and myeloperoxidase that injure the microvasculature. Murine studies have shown that clearance or neutralization of NDPs improves outcomes, demonstrating that NETs have a causal link to disease and are not merely biomarkers. Recently, elevated NDPs have been associated with disease severity in sepsis and coronavirus disease 2019, raising further interest in targeting NETs. Many propose eliminating NETs, either by preventing their release, or by degrading them. However, NET inhibition may impede the innate immune response and is difficult to achieve in rapid‐onset conditions such as sepsis. On the other hand, approaches that accelerate NET degradation have met with mixed results in murine studies, raising the concern thatstrategy may liberate NET‐captured pathogens while increasing circulating levels of harmful NDPs. Alternative NET‐directed strategies include therapies that neutralize, sequester, or remove NDPs from the circulation. Others propose modifying released NETs to decrease their capacity to induce collateral tissue damage while enhancing their ability to capture microorganisms. Synthetic NETs have also been designed to combat antibiotic‐resistant organisms. Although it is still in its infancy, the field of NET‐targeted therapeutics is advancing rapidly and may soon find application in the treatment of sepsis and other inflammatory disorders.
Abstract
BACKGROUND
We have previously shown that higher concentrations of plasma cell free DNA (cfDNA), both at baseline and following chemoradiotherapy, are associated with worse survival in GBM. ...Because only scant amounts of GBM-derived cfDNA shed into the peripheral circulation, we sought to determine the predominant source of prognostic plasma cfDNA in these patients and hypothesized that this may help unravel the mechanism underlying the association of high cfDNA and survival.
METHODS
Plasma was obtained for 126 patients prior to resection of newly diagnosed GBM. A subset of 46 plasma samples had ccfDNA extracted, enzymatically converted, whole genome amplified, and analyzed on the Illumina Epic array for deconvolution of cfDNA methylation patterns. Based on these results demonstrating neutrophils as the predominant cfDNA source, we performed ELISA for citrullinated-histone 3 (citH3) in plasma from 113 of the patients as a specific marker of NETosis, a pro-tumor process of programmed neutrophil cell death during which DNA is released in web-like structures called neutrophil extracellular traps (NETs).
RESULTS
Deconvolution of cfDNA methylation patterns revealed that neutrophils were the dominant source of cfDNA (median percentage of cfDNA derived from neutrophils = 46% , range 17-67%) and correlated with baseline plasma cfDNA concentration (n = 46, r = 0.35, p = 0.017). Additionally, cfDNA concentration was significantly correlated with the concentration of citH3 (n = 113, r = 0.60, p < 0.0001), which was higher in GBM patients compared to 26 healthy controls (p < 0.0001).
CONCLUSION
Our findings suggest that NETosis may be the dominant source of plasma cfDNA, which we have previously shown to be highly prognostic in patients with GBM. Additional studies interrogating the tumor microenvironment for NETosis in GBM tumors are ongoing. These results lay the foundation for further exploration of NETosis as a therapeutic target and underscore the emerging importance of neutrophil biology in GBM.
-Thalassemia (AT) is one of the most commonly occurring inherited hematological diseases. However, few treatments are available, and allogeneic bone marrow transplantation (BMT) is the only ...available therapeutic option for patients with severe AT. Research into AT has remained limited due to a lack of adult mouse models, with severe AT typically resulting in in utero lethality. By using a lipid nanoparticle (LNP) targeting the receptor CD117 and delivering a Cre mRNA (mRNACreLNPCD117), we were able to delete floxed -globin genes at high efficiency in hematopoietic stem cells (HSC) ex vivo. These cells were then engrafted in the absence or presence of a novel α-globin expressing lentiviral vector (ALS20I). Myeloablated mice transplanted with mRNACreLNPCD117-treated HSC showed a complete knockout of -globin genes. They demonstrated a phenotype characterized by the synthesis of hemoglobin H (-tetramers, or HbH), aberrant erythropoiesis, and abnormal organ morphology, culminating in lethality approximately eight weeks following engraftment. Mice receiving mRNACreLNPCD117-treated HSC with at least one copy of ALS20I survived long-term with normalization of erythropoiesis, decreased the production of HbH, and ameliorated the abnormal organ morphology. Furthermore, we tested ALS20I in erythroid progenitors derived from -globin-KO CD34+ and cells isolated from patients with both deletional and non-deletional HbH disease, demonstrating improvement in -globin/-globin mRNA ratio and reduction in the formation of HbH by HPLC. Our results demonstrate the broad applicability of LNP for disease modeling, characterization of a novel severe mouse model of AT, and the efficacy of ALS20I for treating AT.
Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular ...traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately downstream of nascent venous thrombi, after which neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulate into the thrombi. Using a microfluidic system, we found that PF4 binds to NETs, leading them to become compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, which further protect them from nuclease digestion. In HIT mice, inhibition of NET formation through Padi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivation did affect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activation contributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelial adhesion and neutrophil clot infiltration, where incorporated PF4-NET-HIT antibody complexes lead to thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophil chemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release should be explored as strategies to prevent venous thrombosis in HIT.
When stimulated by infection or inflammation, neutrophils expel NETs, decondensed chromatin coated with histones and antimicrobial proteins that ensnares pathogens but also damages host tissue. ...Platelet factor 4 (PF4, CXCL4) is a CXC chemokine stored in platelet alpha-granules and released in high concentrations during platelet activation. Tetrameric PF4 has a very high affinity for polyanionic molecules, including DNA, and we have found that PF4 binds and physically compacts NETs, causing them to have increased resistance to endonuclease digestion. Our group has also observed that PF4 expression leads to enhanced survival in a murine model of sepsis. Based on these findings, we chose to investigate whether PF4-mediated NET compaction is protective in endotoxemia. To study PF4-NET interactions, we developed a microfluidic assay in which neutrophils were adhered to fibronectin-coated channels and then stimulated to release NETs with phorbol myristate acetate(PMA). NETs were visualized by staining with the fluorescent nucleic acid stain SYTOX. Changes in NET morphology and fluorescence were quantified in the presence of varying PF4 concentrations. DNase I was then infused through these channels and the extent of digestion was measured. These experiments showed that the presence of PF4 led to NET compaction and decreased NET degradation following DNase infusion. We then performed in vitro studies examining NET-endothelial interactions in which isolated neutrophils were stimulated to release NETs, incubated with buffer alone or buffer containing PF4, and flowed through human endothelial umbilical vein cell (HUVEC) lined microfluidic channels that had been stimulated with tumor necrosis factor (TNF) α. EC viability was assessed 24-hours post NET exposure and revealed that the presence of PF4 protected HUVECs from NET-induced damage. To further investigate PF4-NET interactions in endotoxemia, we conducted in vivo studies using PF4-deficient mice (mPF4-/-) and wildtype (WT) controls injected with lipopolysaccharide (LPS). Plasma NET markers cell free DNA (cfDNA), citrullinated histones (cit-His), and myeloperoxidase (MPO) were quantified via ELISA and Western blot at various time points following LPS injection. mPF4-/- mice were also implanted with PF4-containing osmotic pumps and the NET markers were also assessed following LPS exposure. These experiments revealed that compared to WT mice, LPS injected mPF4-/- mice had significantly higher plasma levels of NET components, including cfDNA, cit-His and MPO. When mPF4-/- mice were implanted with PF4-releasing osmotic pumps prior to LPS injection, they had plasma NET component levels comparable to those observed in WT mice. Based on the results of our in vitro and in vivo studies, we propose that PF4 infusion compacts NETs, decreasing their susceptibility to DNAse lysis, and preventing the release of toxic NET degradation products (NDPs) such as cfDNA and cit-His. We posit that PF4-mediated sequestration of NDPs prevents endothelial cell damage in the HUVEC-lined microfluidic model. We believe that the results of our studies in mPF4-/- mice demonstrate that PF4 has a similarly protective effect in vivo, decreasing NET lysis and reducing NDP generation. These findings suggest that in sepsis, the stabilization rather than the lysis of NETs may be therapeutic. Further investigation should be performed to determine if treatment with PF4 or other small positively-charged proteins such as protamine sulfate that can sequester NDPs, may be beneficial the treatment of sepsis.
No relevant conflicts of interest to declare.
Neutrophil extracellular traps (NETs) are webs of decondensed nuclear DNA decorated with histones that facilitate microbial killing but also promote thrombosis. NET release is dependent on chromatin ...decondensation mediated by the enzyme peptidylarginine deiminase (PAD) 4 that is expressed predominantly in granulocyte nuclei and citrullinates histone arginine residues, reducing their positive charge, and decreasing their affinity for DNA. Mice deficient in PAD4 are incapable of NET release and have been found to be protected from deep venous thrombosis formation. We have shown that PF4 physically compacts released NETs, causing them to become resistant to DNase digestion. Moreover PF4/NETs bind to heparin-induced thrombocytopenia (HIT)-specific antibodies further stabilizes PF4/NET complexes, enhancing their endonuclease resistance. We therefore speculated that these immunogenic and longer-lasting NETs may contribute to the prothrombotic nature of HIT. To investigate this hypothesis, we used CRISPR technology to disrupt Exon 2 of the Padi4 gene in embryos obtained from mice transgenic for human CXCL4 and FCGR2A, termed “HIT mice”, that have previously been found to develop thrombocytopenia and thrombosis when exposed to the monoclonal HIT-like antibody, KKO. We studied clot formation after cremaster vessel laser injury in PAD4-/- compared toPAD4+/+ HIT mice, quantifying both neutrophil and platelet accumulation. We initially noted PAD4+/+ HIT mice treated with KKO had a significant increase in neutrophil thrombus incorporation 5 minuntes and 60 minutes following venule (but not arteriole) laser injury, compared to mice injected with the isotype control antibody, TRA 8.5±1.3(n=31) vs. 3.1±1.0(n=11), p=0.026 at 5 minutes, 20.1±2.2(n=46) vs. 7.5±1.8(n=17) p=0.002 at 60 minutes). Compared to TRA, KKO infusion also led to a significant increase in venule thrombus platelet volumes 60 minutes following laser injury (p=0.0035, Figure 1). Although KKO exposed PAD4-/- mice had similar thrombus platelet volumes and neutrophil thrombus infiltration compared to PAD4+/+ animals 5 minutes after injury, by 60 minutes, the PAD4-/- animals had significantly smaller platelet volumes (p<0.004, Figure 1) and significantly fewer incorporated neutrophils (p<0.007, Figure 1). To determine if the rise in platelet volumes and increase in neutrophil-thrombus infiltration in HIT was the result of chemokine release by activated platelets, we treated PAD4+/+ mice with the CXCR2 antagonist sch527123 prior to HIT induction. We observed that these mice had a marked reduction in the number of neutrophils in their thrombi, but no decrease in platelet volumes. These findings suggest that PAD4-mediated NET release plays a unique role in the prothrombotic nature of HIT, enhancing neutrophil recruitment while also augmenting platelet accumulation through a chemokine-independent mechanism. We therefore propose that NET release is important for the venular prothrombotic nature of HIT. Furthermore, inhibition of NET-release prevents thrombus growth more effectively than the blockade of chemokine-based neutrophil-platelet interactions and merits further investigation as a treatment strategy in HIT.
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No relevant conflicts of interest to declare.
Most children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have mild or minimal disease, with a small proportion developing severe disease or multisystem inflammatory ...syndrome in children (MIS-C). Complement-mediated thrombotic microangiopathy (TMA) has been associated with SARS-CoV-2 infection in adults but has not been studied in the pediatric population. We hypothesized that complement activation plays an important role in SARS-CoV-2 infection in children and sought to understand if TMA was present in these patients. We enrolled 50 hospitalized pediatric patients with acute SARS-CoV-2 infection (n = 21, minimal coronavirus disease 2019 COVID-19; n = 11, severe COVID-19) or MIS-C (n = 18). As a biomarker of complement activation and TMA, soluble C5b9 (sC5b9, normal 247 ng/mL) was measured in plasma, and elevations were found in patients with minimal disease (median, 392 ng/mL; interquartile range IQR, 244-622 ng/mL), severe disease (median, 646 ng/mL; IQR, 203-728 ng/mL), and MIS-C (median, 630 ng/mL; IQR, 359-932 ng/mL) compared with 26 healthy control subjects (median, 57 ng/mL; IQR, 9-163 ng/mL; P < .001). Higher sC5b9 levels were associated with higher serum creatinine (P = .01) but not age. Of the 19 patients for whom complete clinical criteria were available, 17 (89%) met criteria for TMA. A high proportion of tested children with SARS-CoV-2 infection had evidence of complement activation and met clinical and diagnostic criteria for TMA. Future studies are needed to determine if hospitalized children with SARS-CoV-2 should be screened for TMA, if TMA-directed management is helpful, and if there are any short- or long-term clinical consequences of complement activation and endothelial damage in children with COVID-19 or MIS-C.
•sC5b9 plasma levels are elevated in children with SARS-CoV-2 infection, even if they have minimal symptoms of COVID-19.•A high proportion of children with SARS-CoV-2 infection met clinical criteria for TMA.
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In response to infection and inflammation, neutrophils release NETs, histone-decorated nuclear DNA that ensnares bacteria, but also damages host tissues and promotes thrombosis. Platelet Factor 4 ...(PF4, CXCL4) is a CXC chemokine stored in high concentrations in platelet alpha-granules and released during platelet activation. Tetrameric PF4 has a very high affinity for heparans and other polyanionic molecules, including DNA. At the proper molar ratio, PF4 can form high molecular weight complexes with heparin and other heparans, and these complexes are antigenic targets for pathogenic HIT antibodies. In light of this information, we chose to investigate whether PF4 can interact with NETs and whether these complexes contribute to the prothrombotic nature of HIT. Using an ELISA assay, we confirm that PF4 binds to DNA to form HIT-like complexes just as it does with heparin following a similar bell-shaped curve of HIT antigenicity. Using immunofluorescence studies and confocal microscopy, we found that exogenous PF4 adheres readily to NET DNA. We then investigated PF4-NET interactions under intravascular flow conditions by using neutrophils isolated from healthy human donors to create NET-coated microfluidic channels through which we infused recombinant human PF4. We found that PF4 selectively adhered to extracellular NET fibers but did not bind to the surface of intact neutrophils. We next noted that PF4 infusion led to a change in NET morphology with compaction of the extracellular DNA to approximately 30% of the original area (p<0.001, N=10 per arm). KKO, a monoclonal anti-PF4-heparin antibody, bound readily to NETs following PF4 incubation, indicating that PF4-NET complexes are antigenic. Of note, KKO binding did not induce additional NET compaction. We then observed that while NETs were highly susceptible to endonuclease digestion prior to PF4 incubation, PF4-NET complexes developed resistance to endonuclease digestion. PF4-NET complex incubation with either KKO or HIT IgG isolated from patient samples further enhanced resistance to endonuclease digestion by >2-fold (p<0.001, N=2 studies, 5 NETS per study), while incubation with a polyclonal anti-PF4 antibody did not have this effect. We also show that neutrophils bind readily to endothelial cells when whole blood was flowed through microfluidic channels lined with TNFα-injured endothelium. The number of adherent neutrophils increased markedly when KKO was added to the whole blood, but no significant changes were observed when an isotype control antibody was included. These findings suggest that neutrophils may contribute to thrombosis in HIT through three sequential steps: (1) HIT-antibody activated neutrophils selectively bind to injured endothelium, increasing the numbers of localized neutrophils at sites of thrombus. (2) Subsequently released NETs are bound by PF4 and HIT antibodies, generating immunogenic complexes that are likely prothrombotic, and finally, (3) PF4 and HIT antibody binding induces resistance to endonuclease digestion leading to a prolongation of NET half-life and an increased opportunity to contribute to clot formation. We believe these data support a set of mechanisms by which neutrophils can contribute to the observed prothrombotic nature of HIT, including a novel NET stabilization process. Further in vivo mouse models will now be pursued to confirm these findings.
No relevant conflicts of interest to declare.
The human tyrosine hydroxylase gene promoter Kessler, Mark A.; Yang, Ming; Gollomp, Kandace L. ...
Brain research. Molecular brain research.,
04/2003, Letnik:
112, Številka:
1
Journal Article
Recenzirano
13.329 kilobases of the single copy human tyrosine hydroxylase (hTH) gene were isolated from a genomic library. The 5′ flanking 11 kilobases fused to the reporter green fluorescent protein (GFP) ...drove high level expression in TH+ cells of the substantia nigra of embryonic and adult transgenic mice as determined by double label fluorescence microscopy. To provide a basis for future analysis of polymorphisms and structure–function studies, the previously unreported distal 10.5 kilobases of the hTH promoter were sequenced with an average coverage of 20-fold, the remainder with 4-fold coverage. Sequence features identified included four perfect matches to the bicoid binding element (BBE, consensus: BBTAATCYV) all of which exhibited specific binding by electrophoretic mobility shift assay (EMSA). Comparison to published sequences of mouse and rat TH promoters revealed five areas of exceptional homology shared by these species in the upstream TH promoter region −2 kb to −9 kb relative to the transcription start site. Within these conserved regions (CRs I–V), potential recognition sites for NR4A2 (Nurr1), HNF-3beta, HOXA4, and HOXA5 were shared across human, mouse, and rat TH promoters.
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