TcpC inhibits neutrophil extracellular trap formation by enhancing ubiquitination mediated degradation of peptidylarginine deiminase 4.
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ABSTRACT:
SUBMITTER: Ou Q
PROVIDER: S-EPMC8190435 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis is a systemic small-vessel vasculitis, wherein, MPO-ANCA plays a critical role in the pathogenesis. Neutrophil extracellular traps (NETs) released from activated neutrophils are composed of extracellular web-like DNA and antimicrobial proteins, including MPO. Diverse stimuli, such as phorbol myristate acetate (PMA) and ligands of toll-like receptors (TLR), induce NETs. Although TLR-mediated NET formation can occur with preservation of living neutrophilic functions (called vital NETosis), PMA-stimulated neutrophils undergo cell death with NET formation (called suicidal NETosis). In the process of suicidal NETosis, histones are citrullinated by peptidylarginine deiminase 4 (PAD4). Since this step is necessary for decondensation of DNA, PAD4 plays a pivotal role in suicidal NETosis. Although NETs are essential for elimination of microorganisms, excessive formation of NETs has been suggested to be implicated in MPO-ANCA production. This study aimed to determine if pan-PAD inhibitors could suppress MPO-ANCA production in vivo. At first, NETs were induced in peripheral blood neutrophils derived from healthy donors (1 × 10(6)/ml) by stimulation with 20 nM PMA with or without 20 μM propylthiouracil (PTU), an anti-thyroid drug. We then determined that the in vitro NET formation was inhibited completely by 200 μM Cl-amidine, a pan-PAD inhibitor. Next, we established mouse models with MPO-ANCA production. BALB/c mice were given intraperitoneal (i.p.) injection of PMA (50 ng at days 0 and 7) and oral PTU (2.5 mg/day) for 2 weeks. These mice were divided into two groups; the first group was given daily i.p. injection of PBS (200 μl/day) (n = 13) and the other group with daily i.p. injection of Cl-amidine (0.3 mg/200 μl PBS/day) (n = 7). Two weeks later, citrullination as an indicator of NET formation in the peritoneum and serum MPO-ANCA titer was compared between the two groups. Results demonstrated that citrullination in the peritoneum was significantly reduced in the Cl-amidine-treated mice compared with the vehicle-injected control mice (38% reduction). Additionally, the serum MPO-ANCA titer of the Cl-amidine-treated mice (32.3 ± 31.0 ng/ml) was significantly lower than that in the vehicle-injected mice (132.1 ± 41.6 ng/ml). The collective findings indicate that excessive formation of NETs may be implicated in MPO-ANCA production in vivo.
| S-EPMC4896908 | biostudies-literature
Project description:ObjectiveThe release of neutrophil extracellular traps (NETs) by hyperactive neutrophils has recently been recognized to play an important role in antiphospholipid syndrome (APS). This study was undertaken to evaluate autoantibodies targeting NETs in patients with primary APS, and to determine their potential functions and clinical associations.MethodsWe measured global anti-NET activity in 76 patients with primary APS, 23 patients with systemic lupus erythematosus without antiphospholipid antibodies (aPL), 11 patients with a history of unprovoked venous thrombosis without aPL, and 44 healthy controls. The ability of APS sera to degrade NETs was also assessed.ResultsWe found markedly elevated levels of anti-NET IgG and IgM in patients with primary APS compared with healthy controls (for IgG, mean ± SD optical density 0.55 ± 0.34 versus 0.33 ± 0.17; for IgM, mean ± SD optical density 0.76 ± 0.51 versus 0.26 ± 0.23). This anti-NET activity did not correlate with levels of traditional aPL and was relatively stable over time. Mechanistically, anti-NET antibodies (especially of the IgG isotype) impaired the ability of patient sera to degrade NETs (r = 0.4, P = 0.003). Levels of anti-NET IgM inversely correlated with complement C4 (r = 0.4, P = 0.019). Clinically, anti-NET antibodies associated with certain APS clinical manifestations, and in particular recurrent venous thrombosis (odds ratio 4.3; P = 0.002). Interestingly, anti-NET antibody levels also appeared to be associated with unprovoked venous thrombosis in the general population (for IgM, mean ± SD optical density 0.67 ± 0.34 versus 0.26 ± 0.23).ConclusionOur data indicate high levels of anti-NET antibodies in patients with primary APS, which may impair NET clearance and activate the complement cascade. These findings may ultimately enable more effective risk stratification.
| S-EPMC7722115 | biostudies-literature
Project description:BackgroundTriptolide (PG490), as a triterpene dicyclic oxide has been reported to increase the generation of reactive oxygen species (ROS) and nitric oxide (NO) and induce apoptosis of RAW 264.7 cells in a dose-dependent manner. The activity of death NETs plays an important role in anti-bacterial processes in the human body. This study aimed to investigate the effect of triptolide (PG490) on neutrophil extracellular traps (NETs) formation.MethodsAfter isolating peripheral blood neutrophils from healthy volunteers, cells were incubated with PG490 to observe and detect the level of NETs and detect the level of reactive oxygen species (ROS). The cells were cultured, stained and analyzed by fluorescence microscopy.ResultsCompared with the 12-myristate-13-acetate (PMA) group, the average fluorescence intensity of SYTOX Green in the PG490 + PMA group, as detected by a multifunctional microplate reader, was significantly decreased. Intracellular ROS were labeled by fluorescence, with fluorescence intensity then measured by multifunctional microplate reader and flow cytometry. The results showed that compared with the control group, the fluorescence intensity of the PMA group was significantly increased, while there was no significant difference between PMA group and PG490 + PMA group.ConclusionsThe production of NETs is inhibited by PG490 in vitro, which is not associated with the level of cellular ROS. This suggests that PG490in Tripterygium wilfordii Hook F can suppress related diseases.
| S-EPMC8506553 | biostudies-literature
Project description:In periodontitis, polymorphonuclear leucocytes (PMNs) are activated. They entrap and eliminate pathogens by releasing neutrophil extracellular traps (NETs). Abnormal NET degradation is part of a pro-inflammatory status, affecting co-morbidities such as cardiovascular disease. We aimed to investigate the ex vivo NET degradation capacity of plasma from periodontitis patients compared to controls (part 1) and to quantify NET degradation before and after periodontal therapy (part 2). Fresh NETs were obtained by stimulating blood-derived PMNs with phorbol 12-myristate 13-acetate. Plasma samples from untreated periodontitis patients and controls were incubated for 3 h onto freshly generated NETs (part 1). Similarly, for part 2, NET degradation was studied for 91 patients before and 3, 6 and 12 mo after non-surgical periodontal therapy with and without adjunctive systemic antibiotics. Finally, NET degradation was fluorospectrometrically quantified. NET degradation levels did not differ between periodontitis patients and controls, irrespective of subject-related background characteristics. NET degradation significantly increased from 65.6 ± 1.7% before periodontal treatment to 75.7 ± 1.2% at 3 mo post periodontal therapy, and this improvement was maintained at 6 and 12 mo, irrespective of systemic usage of antibiotics. Improved NET degradation after periodontitis treatment is another systemic biomarker reflecting a decreased pro-inflammatory status, which also contributes to an improved cardiovascular condition.
| S-EPMC7903525 | biostudies-literature
Project description:ObjectiveMacrophages are immune cells, capable to remodel the extracellular matrix, which can harbor extracellular DNA incorporated into neutrophil extracellular traps (NETs). To study the breakdown of NETs we studied the capability of macrophage subsets to degrade these structures in vitro and in vivo in a murine thrombosis model. Furthermore, we analyzed human abdominal aortic aneurysm samples in support of our in vitro and in vivo results. Approach and Results: Macrophages were seeded onto blood clots or isolated NETs and polarized. All macrophages were capable to degrade NETs. For initial breakdown, macrophages relied on extracellular deoxyribonucleases. Proinflammatory polarization enhanced NET degradation. The boost in degradation was because of increased macropinocytosis, as inhibition by imipramine diminished their NET breakdown. Inhibition of macropinocytosis in a murine thrombosis model led to increased NET burden and reduced thrombus resolution in vivo. When analyzing abdominal aortic aneurysm samples, macrophage density furthermore corresponded negatively with the amount of local NETs in the intraluminal thrombi as well as in the vessel wall, as increased macrophage density was associated with a reduction in NET burden.ConclusionsWe provide evidence that macrophages degrade NETs by extracellular predigestion and subsequent uptake. Furthermore, we show that proinflammatory macrophages increase NET degradation through enhanced macropinocytosis, priming them for NET engulfment. Based on our findings, that inhibition of macropinocytosis in mice corresponded to increased NET amounts in thrombi and that local macrophage density in human abdominal aortic aneurysm is negatively associated with surrounding NETs, we hypothesize, that macrophages are able to degrade NETs in vivo.
| S-EPMC7447175 | biostudies-literature
Project description:ObjectivesPyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome is characterised by flares of sterile arthritis with neutrophil infiltrate and the overproduction of interleukin (IL)-1β. The purpose of this study was to elucidate the potential role of neutrophil subsets and neutrophil extracellular traps (NET) in the pathogenesis of PAPA.MethodsNeutrophils and low-density granulocytes (LDG) were quantified by flow cytometry. Circulating NETs were measured by ELISA and PAPA serum was tested for the ability to degrade NETs. The capacity of NETs from PAPA neutrophils to activate macrophages was assessed. Skin biopsies were analysed for NETs and neutrophil gene signatures.ResultsCirculating LDGs are elevated in PAPA subjects. PAPA neutrophils and LDGs display enhanced NET formation compared with control neutrophils. PAPA sera exhibit impaired NET degradation and this is corrected with exogenous DNase1. Recombinant human IL-1β induces NET formation in PAPA neutrophils but not healthy control neutrophils. NET formation in healthy control neutrophils is induced by PAPA serum and this effect is inhibited by the IL-1 receptor antagonist, anakinra. NETs from PAPA neutrophils and LDGs stimulate IL-6 release in healthy control macrophages. NETs are detected in skin biopsies of patients with PAPA syndrome in association with increased tissue IL-1β, IL-8 and IL-17. Furthermore, LDG gene signatures are detected in PAPA skin.ConclusionsPAPA syndrome is characterised by an imbalance of NET formation and degradation that may enhance the half-life of these structures in vivo, promoting inflammation. Anakinra ameliorates NET formation in PAPA and this finding supports a role for IL-1 signalling in exacerbated neutrophil responses in this disease. The study also highlights other inflammatory pathways potentially pathogenic in PAPA, including IL-17 and IL-6, and these results may help guide new therapeutic approaches in this severe and often treatment-refractory condition.
| S-EPMC6728908 | biostudies-literature
Project description:Pyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome is characterized by flares of sterile arthritis with neutrophil infiltrate and the overproduction of Interleukin (IL)-1β. The purpose of this study was to elucidate the potential role of neutrophil subsets and neutrophil extracellular traps (NETs) in the pathogenesis of PAPA. The transcriptome of PAPA normal-dense neutrophils, autologous LDGs, and healthy control normal-dense neutrophils was characterized using RNASeq. The transcriptome of a PAPA skin biopsy and a healthy control skin sample was elucidated using RNASeq.
2018-10-23 | GSE117168 | GEO
Project description:Systemic lupus erythematosus (SLE) is an autoimmune disease in which patients develop autoantibodies to DNA, histones, and often to neutrophil proteins. These form immune complexes that are pathogenic and may cause lupus nephritis. In SLE patients, infections can initiate flares and are a major cause of mortality. Neutrophils respond to infections and release extracellular traps (NETs), which are antimicrobial and are made of DNA, histones, and neutrophil proteins. The timely removal of NETs may be crucial for tissue homeostasis to avoid presentation of self-antigens. We tested the hypothesis that SLE patients cannot clear NETs, contributing to the pathogenesis of lupus nephritis. Here we show that serum endonuclease DNase1 is essential for disassembly of NETs. Interestingly, a subset of SLE patients' sera degraded NETs poorly. Two mechanisms caused this impaired NET degradation: (i) the presence of DNase1 inhibitors or (ii) anti-NET antibodies prevented DNase1 access to NETs. Impairment of DNase1 function and failure to dismantle NETs correlated with kidney involvement. Hence, identification of SLE patients who cannot dismantle NETs might be a useful indicator of renal involvement. Moreover, NETs might represent a therapeutic target in SLE.
| S-EPMC2906830 | biostudies-other
Project description:Multiple myeloma is a plasma cell malignancy, which grows in the bone marrow (BM). The major population of cells in the BM is represented by neutrophils and they can form neutrophil extracellular traps (NET). Here, we investigated whether multiple myeloma cells induce NET formation and whether targeting this process would delay multiple myeloma progression. We demonstrated that murine and human multiple myeloma cells stimulate citrullination of histone H3 and NET formation by neutrophils and that this process is abrogated by pharmacological targeting of peptidylarginine deiminase 4 (PAD4) with a novel-specific small molecule inhibitor BMS-P5. Administration of BMS-P5 to multiple myeloma-bearing mice delays appearance of symptoms and disease progression. Taken together, our data demonstrate that targeting PAD4 may be beneficial for treatment of multiple myeloma.
| S-EPMC7335350 | biostudies-literature
Project description:Background and purposeUpon stimulation, neutrophils release their nuclear contents called neutrophil extracellular traps (NETs), which contain unfolded chromatin and lysosomal enzymes. NETs have been demonstrated to play a critical role in host defence, although the role of PGE2 , a bioactive substance generated in inflammatory tissues, in the formation of NETs remains unclear.Experimental approachThe effects of PGE2 , agonists and antagonists of its receptors, and modulators of the cAMP-PKA pathway on the formation of NETs were examined in vitro in isolated neutrophils and in vivo in a newly established mouse model.Key resultsPGE2 inhibited PMA-induced NET formation in vitro through EP2 and EP4 Gαs-coupled receptors. Incubation with a cell-permeable cAMP analogue, dibutyryl cAMP, or various inhibitors of a cAMP-degrading enzyme, PDE, also suppressed NET formation. In the assay established here, where an agarose gel was s.c. implanted in mice and NET formation was detected on the surface of the gel, the extent of the NET formed was inhibited in agarose gels containing rolipram, a PDE4 inhibitor, and butaprost, an EP2 receptor agonist.Conclusions and implicationsPGE2 inhibits NET formation through the production of cAMP. These findings will contribute to the development of novel treatments for NETosis-related diseases.
| S-EPMC5341226 | biostudies-literature