scholarly journals In Vivo Transmigrated Human Neutrophils Are Highly Primed for Intracellular Radical Production Induced by Monosodium Urate Crystals

2020 ◽  
Vol 21 (11) ◽  
pp. 3750 ◽  
Author(s):  
Lisa Davidsson ◽  
Agnes Dahlstrand Rudin ◽  
Felix Peter Sanchez Klose ◽  
Alicia Buck ◽  
Lena Björkman ◽  
...  
Keyword(s):  
Neutrophil Extracellular Trap ◽  
Human Neutrophils ◽  
Ros Production ◽  
Monosodium Urate ◽  
Intracellular Compartments ◽  
Blood Neutrophils ◽  
Msu Crystals ◽  
Urate Crystals

Gout is an inflammatory disease caused by monosodium urate (MSU) crystals. The role of neutrophils in gout is less clear, although several studies have shown neutrophil extracellular trap (NET) formation in acutely inflamed joints of gout patients. MSU crystals are known to induce the production of reactive oxygen species (ROS) and NET formation in neutrophils isolated from blood, but there is inconclusive knowledge on the localization of ROS production as well as whether the ROS are required for NET formation. In this report we demonstrate that MSU crystals activate human neutrophils to produce ROS exclusively in intracellular compartments. Additionally, in vivo transmigrated neutrophils derived from experimental skin chambers displayed markedly increased ROS production as compared to resting blood neutrophils. We also confirmed that MSU stimulation potently induced NET formation, but this response was not primed in in vivo transmigrated neutrophils. In line with this we found that MSU-triggered NET formation was independent of ROS production and proceeded normally in neutrophils from patients with dysfunctional respiratory burst (chronic granulomatous disease (CGD) and complete myeloperoxidase (MPO) deficiency). Our data indicate that in vivo transmigrated neutrophils are markedly primed for oxidative responses to MSU crystals and that MSU triggered NET formation is independent of ROS production.

scholarly journals Why Does Hyperuricemia Not Necessarily Induce Gout?

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 280
Author(s):  
Wei-Zheng Zhang
Keyword(s):  
Metabolic Disease ◽  
Uric Acid ◽  
Antioxidant Properties ◽  
Monosodium Urate ◽  
Therapeutic Regime ◽  
Gout Flare ◽  
Msu Crystals ◽  
Gout Flares

Hyperuricemia is a risk factor for gout. It has been well observed that a large proportion of individuals with hyperuricemia have never had a gout flare(s), while some patients with gout can have a normuricemia. This raises a puzzle of the real role of serum uric acid (SUA) in the occurrence of gout flares. As the molecule of uric acid has its dual effects in vivo with antioxidant properties as well as being an inflammatory promoter, it has been placed in a delicate position in balancing metabolisms. Gout seems to be a multifactorial metabolic disease and its pathogenesis should not rely solely on hyperuricemia or monosodium urate (MSU) crystals. This critical review aims to unfold the mechanisms of the SUA role participating in gout development. It also discusses some key elements which are prerequisites for the formation of gout in association with the current therapeutic regime. The compilation should be helpful in precisely fighting for a cure of gout clinically and pharmaceutically.

scholarly journals POS0133 MONOSODIUM URATE CRYSTALS REDUCE HUMAN LIGAMENT CELLS VIABILITY THROUGH INCREASE OF ROS PRODUCTION

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 278.1-278
Author(s):  
Y. Huang ◽  
Y. Liu ◽  
Q. Huang ◽  
W. Deng ◽  
T. W. Li
Keyword(s):  
Cell Migration ◽  
Annexin V ◽  
Cell Counting ◽  
Ros Production ◽  
Monosodium Urate ◽  
Migration Ability ◽  
Monosodium Urate Crystals ◽  
Migration Assay ◽  
Msu Crystals ◽  
Urate Crystals

Background:Ligament destruction is a frequent complication of gout and is strongly associated with tophi. Ligament fibroblasts are important cellular mediators of ligament remodeling. None of study has paid attention to the effects of monosodium urate (MSU) crystals on ligament fibroblasts.Objectives:The study aims to investigate the effects and mechanism of MSU crystals on ligament fibroblasts.Methods:MSU crystals were added to human ligament fibroblasts(HLFs) cultures or primary ligament cells cultures. Cell counting kit-8 (CCK-8) assay, cell migration assay, Annexin V-FITC/PI assay were conducted. Reactive Oxygen Species(ROS) was tested by ROS Assay Kit.Results:The higher concentrations of MSU crystals (0.5-1mg/mL) reduced the viability of HLFs or primary ligament cells after 24 h as assessed by CCK8 assays, with a further reduction in viability observed at the 48 h time point. When observed under light microscopy, HLFs cultured with MSU crystals (0.5mg/mL) appeared unhealthy with fewer cells present. The cell migration ability of HLFs was decreased significantly on MSU crystals (0.5mg/mL). According to the result of Annexin V-FITC/PI assay, the survival rate of HLFs on MSU crystals (0.5mg/mL) was lower than that of 0.25mg/ml and 0 mg/ml at 72h. ROS assay results showed that the production of ROS increased as the concentrations of MSU crystals increased.Conclusion:MSU crystals inhibit human ligament cells viability through the increase of ROS production. It may contribute to disordered ligament remodeling in gout patients with ligament destruction.References:[1]Ashika Chhana, et al. Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state. Arthritis Res Ther. 2018, 20(1): 208.Figure 1.MSU crystals reduce human ligament fibroblasts and primary human ligament cells viability over time. A: CCK-8 assay; B: Observation of HLFs morphology; C: Annexin V-FITC/PI assay.Disclosure of Interests:None declared

scholarly journals Structural basis for a complex I mutation that blocks pathological ROS production

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhan Yin ◽  
Nils Burger ◽  
Duvaraka Kula-Alwar ◽  
Dunja Aksentijević ◽  
Hannah R. Bridges ◽  
...  
Keyword(s):  
Point Mutation ◽  
Complex I ◽  
Structural Changes ◽  
Ischemia Reperfusion ◽  
Single Point ◽  
Structural Basis ◽  
Single Point Mutation ◽  
Ros Production

AbstractMitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia–reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the “deactive” state, usually formed only after prolonged inactivity. Despite its tendency to adopt the “deactive” state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

scholarly journals The AP2 clathrin adaptor protein complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans

2015 ◽  
Vol 26 (10) ◽  
pp. 1887-1900 ◽  
Author(s):  
Steven D. Garafalo ◽  
Eric S. Luth ◽  
Benjamin J. Moss ◽  
Michael I. Monteiro ◽  
Emily Malkin ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Nerve Cord ◽  
Ventral Nerve Cord ◽  
Secretory Pathway ◽  
Adaptor Protein ◽  
Intracellular Compartments ◽  
Clathrin Adaptor ◽  
Strength And Plasticity

Regulation of glutamate receptor (GluR) abundance at synapses by clathrin-mediated endocytosis can control synaptic strength and plasticity. We take advantage of viable, null mutations in subunits of the clathrin adaptor protein 2 (AP2) complex in Caenorhabditis elegans to characterize the in vivo role of AP2 in GluR trafficking. In contrast to our predictions for an endocytic adaptor, we found that levels of the GluR GLR-1 are decreased at synapses in the ventral nerve cord (VNC) of animals with mutations in the AP2 subunits APM-2/μ2, APA-2/α, or APS-2/σ2. Rescue experiments indicate that APM-2/μ2 functions in glr-1–expressing interneurons and the mature nervous system to promote GLR-1 levels in the VNC. Genetic analyses suggest that APM-2/μ2 acts upstream of GLR-1 endocytosis in the VNC. Consistent with this, GLR-1 accumulates in cell bodies of apm-2 mutants. However, GLR-1 does not appear to accumulate at the plasma membrane of the cell body as expected, but instead accumulates in intracellular compartments including Syntaxin-13– and RAB-14–labeled endosomes. This study reveals a novel role for the AP2 clathrin adaptor in promoting the abundance of GluRs at synapses in vivo, and implicates AP2 in the regulation of GluR trafficking at an early step in the secretory pathway.

scholarly journals Mast Cell Tryptase Potentiates Neutrophil Extracellular Trap Formation

2021 ◽  
pp. 1-14
Author(s):  
Gunnar Pejler ◽  
Sultan Alanazi ◽  
Mirjana Grujic ◽  
Jeremy Adler ◽  
Anna-Karin Olsson ◽  
...  
Keyword(s):  
Neutrophil Extracellular Trap ◽  
Human Neutrophils ◽  
Functional Communication ◽  
Extracellular Milieu ◽  
Extracellular Traps ◽  
Inflammatory Conditions ◽  
Histone 3 ◽  
Activated Neutrophils ◽  
Core Histones

Previous research has indicated an intimate functional communication between mast cells (MCs) and neutrophils during inflammatory conditions, but the nature of such communication is not fully understood. Activated neutrophils are known to release DNA-containing extracellular traps (neutrophil extracellular traps [NETs]) and, based on the known ability of tryptase to interact with negatively charged polymers, we here hypothesized that tryptase might interact with NET-contained DNA and thereby regulate NET formation. In support of this, we showed that tryptase markedly enhances NET formation in phorbol myristate acetate-activated human neutrophils. Moreover, tryptase was found to bind vividly to the NETs, to cause proteolysis of core histones and to cause a reduction in the levels of citrullinated histone-3. Secretome analysis revealed that tryptase caused increased release of numerous neutrophil granule compounds, including gelatinase, lactoferrin, and myeloperoxidase. We also show that DNA can induce the tetrameric, active organization of tryptase, suggesting that NET-contained DNA can maintain tryptase activity in the extracellular milieu. In line with such a scenario, DNA-stabilized tryptase was shown to efficiently degrade numerous pro-inflammatory compounds. Finally, we showed that tryptase is associated with NET formation in vivo in a melanoma setting and that NET formation in vivo is attenuated in mice lacking tryptase expression. Altogether, these findings reveal that NET formation can be regulated by MC tryptase, thus introducing a novel mechanism of communication between MCs and neutrophils.

scholarly journals USP7 Inhibition Alleviates H2O2-Induced Injury in Chondrocytes via Inhibiting NOX4/NLRP3 Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Gang Liu ◽  
Qingbai Liu ◽  
Bin Yan ◽  
Ziqiang Zhu ◽  
Yaozeng Xu
Keyword(s):  
Treatment Options ◽  
Current Treatment ◽  
Pathological Process ◽  
Joint Disease ◽  
Matrix Remodeling ◽  
Ros Production ◽  
Caspase 1 ◽  
Enhanced Production

Osteoarthritis (OA), the most common form of arthritis, is a very common joint disease that often affects middle-aged to elderly people. However, current treatment options for OA are predominantly palliative. Thus, understanding its pathological process and exploring its potential therapeutic approaches are of great importance. Rat chondrocytes were isolated and exposed to hydrogen peroxide (H2O2) to mimic OA. The effects of H2O2 on ubiquitin-specific protease 7 (USP7) expression, reactive oxygen species (ROS) levels, proliferation, inflammatory cytokine release, and pyroptosis were measured. USP7 was knocked down (KD) or overexpressed to investigate the role of USP7 in OA. Co-immunoprecipitation (Co-IP) was used to study the interaction between USP7 and NAD(P)H oxidases (NOX)4 as well as NOX4 ubiquitination. NOX4 inhibitor was applied to study the involvement of NOX4 in USP7-mediated OA development. USP7 inhibitor was given to OA animals to further investigate the role of USP7 in OA in vivo. Moreover, H2O2 treatment significantly increased USP7 expression, enhanced ROS levels, and inhibited proliferation in rat chondrocytes. The overexpression of USP7 enhanced pyroptosis, ROS production, interleukin (IL)-1β and IL-18 levels, and the expression level of NLRP3, GSDMD-N, active caspase-1, pro-caspase-1, matrix metalloproteinases (MMP) 1, and MMP13, which was abolished by ROS inhibition. The USP7 KD protected rat chondrocytes against H2O2-induced injury. Co-IP results showed that USP7 interacted with NOX4, and USP7 KD enhanced NOX4 ubiquitinylation. The inhibition of NOX4 blocked the pro-OA effect of USP7. Moreover, the USP7 inhibitor given to OA animals suppressed OA in vivo. USP7 inhibited NOX4 ubiquitination for degradation which leads to elevated ROS production. ROS subsequently activates NLPR3 inflammasome, leading to enhanced production of IL-1β and IL-18, GSDMD-N-dependent pyroptosis, and extracellular matrix remodeling. Thus, UPS7 contributes to the progression of OA via NOX4/ROS/NLPR3 axis.

scholarly journals Identification and characterization of a unique subpopulation (CALLA/CD10/negative) of human neutrophils manifesting a heightened chemotactic response to activated complement

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1624-1629
Author(s):  
RT McCormack ◽  
RD Nelson ◽  
DE Chenoweth ◽  
TW LeBien
Keyword(s):  
Lymphoblastic Leukemia ◽  
Low Frequency ◽  
Chemotactic Response ◽  
Human Neutrophils ◽  
Normal Peripheral Blood ◽  
Cd10 Expression ◽  
Blood Neutrophils

We have previously demonstrated that human neutrophils synthesize the common acute lymphoblastic leukemia antigen (CALLA/CD10). To determine whether CALLA/CD10-positive and -negative neutrophils have similar or distinct functional attributes, we sorted normal peripheral blood neutrophils for CALLA/CD10 expression and compared their chemotactic ability. Surprisingly, the low-frequency (approximately 5%), CALLA/CD10- negative neutrophils displayed a dramatically heightened chemotactic response to activated complement (C') that was (a) specific for C', (b) not observed with other minor subpopulations of neutrophils, (c) not due to previous activation in vivo or in vitro, and (d) apparently not due to an increase in C5a receptors. These results underscore the concept of neutrophil heterogeneity and prompt the hypothesis that CALLA/CD10-negative neutrophils may participate in an inflammatory response to trauma involving complement activation.

Abstract 135: Exploration and Modulation of TREM-1 in Experimental Atherosclerosis

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Stephane Potteaux ◽  
Jeremie Joffre ◽  
Bruno Esposito ◽  
Alain Tedgui ◽  
Sebastien Gibot ◽  
...  
Keyword(s):  
Cell Types ◽  
Experimental Atherosclerosis ◽  
Soluble Form ◽  
Daily Injection ◽  
Preferential Expression ◽  
Monocyte Migration ◽  
Plaque Development ◽  
Blood Neutrophils

Under conditions of atherosclerosis, monocytes are rapidely recruited into the vessel wall where they differentiate into macrophages. Both classical and nonclassical monocytes are continuously recruited to the lesion and contribute to atherosclerosis formation. Whereas the number of circulating monocytes correlates with plaque development, decrease in their number or migration is protective. Upon differentiation, macrophages increase TLR expression, which triggers the inflammatory response. The triggering receptor expressed on myeloid cells (TREM-1) has been shown to amplify TLR-induced signals in sepsis or inflammatory bowel disease. TREM-1 is also produced in a soluble form and is a predictive factor during sever infections in humans. We addressed for the first time the role of TREM-1 in atherosclerosis. We found preferential expression of TREM-1 on blood neutrophils and nonclassical monocytes in both ApoE+/+ and ApoE-/- mice, but sTREM-1 was only detectable in plasma of ApoE-/- mice (ELISA). TREM-1 expression was significantly increased on both cell types in APOE-/- mice after 4 weeks of high fat diet (Flow cytometry). We next addressed the role of TREM-1 in atherosclerosis formation by injection of a TREM-like transcript 1-derived peptide (LR12) in 8 week-old ApoE-/- mice (daily injection for 4 weeks of LR12 or peptide scramble as control). We found that pharmaceutical inhibition of TREM-1 significantly reduced plaque formation by 30% without change in cholesterol levels. This was associated with significant reduction in macrophage accumulation after treatment with LR12 (57735,34 μm2 vs 78398,38 μm2 in controls; p=0,004). We demonstrated that LR12 treatment induced a specific rapid and sustained decrease in circulating nonclassical monocytes after 7 days of treatment. By using an in vivo pulse labeling method to quantify monocyte migration, we found that LR12 also altered nonclassical monocyte recruitment to the plaque. Taken together, these data indicate that TREM-1 expression increases in the context of atherosclerosis and that pharmacological inhibition of TREM-1 protects against plaque development through decreased number and infiltration of nonclassical monocytes.

MHO_0730 as a Surface-Exposed Calcium-Dependent Nuclease of Mycoplasma hominis Promoting Neutrophil Extracellular Trap Formation and Escape

2019 ◽  
Vol 220 (12) ◽  
pp. 1999-2008 ◽  
Author(s):  
Carla Cacciotto ◽  
Daniele Dessì ◽  
Tiziana Cubeddu ◽  
Anna Rita Cocco ◽  
Andrea Pisano ◽  
...  
Keyword(s):  
Natural Infection ◽  
Mycoplasma Hominis ◽  
Neutrophil Extracellular Trap ◽  
Host Immune System ◽  
Potent Inducer ◽  
Calcium Dependent ◽  
Relevant Role ◽  
Extracellular Trap

Abstract Mycoplasma lipoproteins play a relevant role in pathogenicity and directly interact with the host immune system. Among human mycoplasmas, Mycoplasma hominis is described as a commensal bacterium that can be associated with a number of genital and extragenital conditions. Mechanisms of M. hominis pathogenicity are still largely obscure, and only a limited number of proteins have been associated with virulence. The current study focused on investigating the role of MHO_0730 as a virulence factor and demonstrated that MHO_0730 is a surface lipoprotein, potentially expressed in vivo during natural infection, acting both as a nuclease with its amino acidic portion and as a potent inducer of Neutrophil extracellular trapsosis with its N-terminal lipid moiety. Evidence for M. hominis neutrophil extracellular trap escape is also presented. Results highlight the relevance of MHO_0730 in promoting infection and modulation and evasion of innate immunity and provide additional knowledge on M. hominis virulence and survival in the host.

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