Circulating apoptotic microparticles in SLE patients drive the activation of DC subsets and prime neutrophils for NETosis (original) (raw)
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Scandinavian Journal of Immunology, 2013
Microparticles (MPs) are small membrane‐bound vesicles with potent biological activities that can promote the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus (SLE). These particles contain diverse cellular components and are shed from cells during apoptosis or activation. MPs can drive inflammation and autoimmunity by multiple mechanisms reflecting their content of bioactive molecules and ability to engage multiple receptor systems simultaneously. In the rheumatoid joint, particles can stimulate synovitis via their display of cytokines, chemokines, complement and angiogenesis factors. In SLE, particles can serve as an important source of extracellular nuclear molecules to signal ‘danger’ and form pathogenic immune complexes. Future studies will define the pathways by which particles promote pathogenesis, strategies to block their activity and their utility as biomarkers to assess disease activity and the response to therapy.
Journal of immunology (Baltimore, Md. : 1950), 2012
Ongoing inflammation including activation of the complement system is a hallmark of systemic lupus erythematosus (SLE). Antimicrobial neutrophil extracellular traps (NETs) are composed of secreted chromatin that may act as a source of autoantigens typical for SLE. In this study, we investigated how complement interacts with NETs and how NET degradation is affected by complement in SLE patients. We found that sera from a subset of patients with active SLE had a reduced ability to degrade in vitro-generated NETs, which was mostly restored when these patients were in remission. Patients that failed to degrade NETs had a more active disease and they also displayed lower levels of complement proteins C4 and C3 in blood. We discovered that NETs activated complement in vitro and that deposited C1q inhibited NET degradation including a direct inhibition of DNase-I by C1q. Complement deposition on NETs may facilitate autoantibody production, and indeed, Abs against NETs and NET epitopes were...
Unique protein signature of circulating microparticles in systemic lupus erythematosus
Arthritis & Rheumatism, 2013
Objective. To characterize the unique qualities of proteins associated with circulating subcellular material in systemic lupus erythematosus (SLE) patients compared with healthy controls and patients with other chronic autoimmune diseases. Methods. Using differential centrifugation and high-sensitivity nano-liquid chromatography tandem mass spectrometry, we systematically profiled proteins of microparticles (MPs) from SLE patients (n ؍ 12), systemic sclerosis (SSc) patients (n ؍ 6), and rheumatoid arthritis (RA) patients (n ؍ 6), as well as healthy controls (n ؍ 12). Results. We identified 531 unique proteins and showed that the differences between healthy controls and patients with SLE with regard to the abundance of 248 proteins were highly statistically significant. Almost half of the proteins that were increased by >2-fold were complement proteins and Ig (increased by 100-4,000 times). MP Ig and complement loads also distinguished SLE from RA and SSc and correlated strongly with clinical SLE severity. Subsets of microtubule proteins, fibronectin, 14-3-3, and desmosomal proteins as well as ficolin 2 and galectin 3 binding protein were also highly increased. In SLE MPs, levels of cytoskeletal, mitochondrial, and organelle proteins, including lysosome-associated membrane protein 1 and transforming growth factor 1, were decreased. Conclusion. The data show that SLE patients have increased numbers of MPs that are heavily tagged for removal and fewer MPs with normal protein composition. SLE MPs are unique and specific proteins that represent novel leads for our understanding of SLE and for the development of new treatments of the disease. The autoimmune disease systemic lupus erythematosus (SLE) is characterized by DNA/RNA-Tolllike receptor-dependent activation of innate immunity, with increased expression of type I interferon (IFN) response genes, autoantibody production resulting from loss of tolerance, and chronic, severe, and fluctuating morbidity due to thrombophilia and immune complex (IC)-mediated inflammatory damage of multiple tissues and organs. In SLE, the normal noninflammatory handling of ICs and material released from dead or dying cells is defective and instead is associated with a sustained type I IFN response leading to chronic inflammation (1,2). Consistent with the involvement of clearance defects in SLE pathogenesis, lupus-like disease evolves in patients with complement C1q deficiency. Defects of C1q-, DNA-, and apoptotic cell-binding molecules, such as C-reactive protein, serum amyloid P component, natural IgM, milk fat globule epidermal growth factor 8, annexins, protein S, Gas-6,  2-glycoprotein I ( 2 GPI), or deficiencies in proteins involved in the phagocytosis of ingested apoptotic material, such as the Mer receptor tyrosine kinase and DNase I, also lead to lupus-like manifestations in humans or in mice (3-9). Recent studies using flow cytometry show alterations in the numbers and types of subcellular particles circulating in SLE patients (10-12). The characterization of circulating subcellular particles is challenging because of their Supported by the Danish Rheumatism Association (grants R99-A1937 and R97-A1028), the Lundbeck Foundation, and the Novo
Frontiers in Immunology
Neutrophil Extracellular Traps (NETs) are implicated in the development of auto-immunity in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) through the externalization of intracellular neoepitopes e.g., dsDNA and nuclear proteins in SLE and citrullinated peptides in RA. The aim of this work was to use quantitative proteomics to identify and measure NET proteins produced by neutrophils from healthy controls, and from patients with RA and SLE to determine if NETs can be differentially-generated to expose different sets of neoepitopes. Ultra-pure neutrophils (>99%) from healthy individuals (n = 3) and patients with RA or SLE (n = 6 each) were incubated ± PMA (50 nM, PKC super-activator) or A23187 (3.8 µM, calcium ionophore) for 4 h. NETs were liberated by nuclease digestion and concentrated onto Strataclean beads prior to on-bead digestion with trypsin. Data-dependent LC-MS/MS analyses were conducted on a QExactive HF quadrupole-Orbitrap mass spectrometer, and label-free protein quantification was carried out using Progenesis QI. PMA-induced NETs were decorated with annexins, azurocidin and histone H3, whereas A23187-induced NETs were decorated with granule proteins including CAMP/LL37, CRISP3, lipocalin and MMP8, histones H1.0, H1.4, and H1.5, interleukin-8, protein-arginine deiminase-4 (PADI4), and α-enolase. Four proteins were significantly different between PMA-NETs from RA and SLE neutrophils (p < 0.05): RNASE2 was higher in RA, whereas MPO, leukocyte elastase inhibitor and thymidine phosphorylase were higher in SLE. For A23187-NETs, six NET proteins were higher in RA (p < 0.05), including CAMP/LL37, CRISP3, interleukin-8, MMP8; Thirteen proteins were higher in SLE, including histones H1.0, H2B, and H4. This work provides the first, direct comparison of NOX2-dependent (PMA) and NOX2-independent (A23187) NETs using quantitative proteomics, and the first direct comparison of RA and SLE NETs using quantitative proteomics. We show that it is the nature of the stimulant rather than neutrophil physiology that determines NET protein profiles in disease, since stimulation of NETosis in either a NOX2-dependent Chapman et al. Proteomic Analysis of NETs or a NOX2-independent manner generates broadly similar NET proteins irrespective of the disease background. We also use our proteomics pipeline to identify an extensive range of post-translationally modified proteins in RA and SLE, including histones and granule proteins, many of which are known targets of auto-antibodies in each disease.
Scientific Reports, 2016
Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by circulating autoantibodies and the formation of immune complexes. In these responses, the selecting self-antigens likely derive from the remains of dead and dying cells, as well as from disturbances in clearance. During cell death/activation, microparticles (MPs) can be released to the circulation. Previous MP studies in SLE have been limited in size and differ regarding numbers and phenotypes. Therefore, to characterize MPs more completely, we investigated 280 SLE patients and 280 individually matched controls. MPs were measured with flow cytometry and phenotyped according to phosphatidylserine expression (PS+/PS−), cellular origin and inflammatory markers. MPs, regardless of phenotype, are 2–10 times more abundant in SLE blood compared to controls. PS− MPs predominated in SLE, but not in controls (66% vs. 42%). Selectively in SLE, PS− MPs were more numerous in females and smokers. MP numbers dec...
Distinct proteome pathology of circulating microparticles in systemic lupus erythematosus
Clinical Proteomics, 2017
Background: The pathogenesis of systemic lupus erythematosus (SLE) is poorly understood but has been linked to defective clearance of subcellular particulate material from the circulation. This study investigates the origin, formation, and specificity of circulating microparticles (MPs) in patients with SLE based on comprehensive MP proteome profiling using patients with systemic sclerosis (SSc) and healthy donors (HC) as controls. Methods: We purified MPs from platelet-poor plasma using differential centrifugation of samples from SLE (n = 45), SSc (n = 38), and two sets of HC (n = 35, n = 25). MP proteins were identified and quantitated after trypsin digestion by liquid chromatography-tandem mass spectrometry. The abundance of specific proteins was compared between the groups using univariate statistics and false discovery rate correction for multiple comparisons. Specific proteins and protein ratios were explored for diagnostic and disease activity information using receiver-operating characteristic curves and by analysis of correlations of protein abundance with disease activity scores. Results: We identify and quantitate more than 1000 MP proteins and show that a subpopulation of SLE-MPs (which we propose to call luposomes) are highly specific for SLE, i.e. not found in MP preparations from HC or patients with another autoimmune, systemic disease, SSc. In SLE-MPs platelet proteins and mitochondrial proteins are significantly diminished, cytoskeletal proteins deranged, and glycolytic enzymes and apoptotic proteins significantly increased. Conclusions: Normal MPs are efficiently removed in SLE, but aberrant MPs, derived from non-lymphoid leukocytes, are less efficiently removed and abundantly produced leading to an altered MP proteome in SLE. The data suggest that an abnormal generation of MPs may partake in the pathology of SLE and that new diagnostic, monitoring, and treatment strategies targeting these processes may be advantageous.
Experimental Dermatology, 2019
Neutrophils are major players at sites of tissue inflammation and use different mechanisms to fight against bacterial infections and/ or alter the inflammatory milieu. In 2004, Brinkmann et al discovered a new mechanism of action whereby activated neutrophils release their contents in a web-like structure forming traps that bind and kill bacteria extracellularly. [1] These neutrophil extracellular traps or NETs contain DNA, histones and neutrophil proteins such as myeloperoxidase and neutrophil elastase. They can also be a major source of self-molecules implicated in autoimmunity and autoinflammation. [2] This relationship between NETosis and autoimmunity has been demonstrated in a number rheumatic diseases such as small vessel vasculitis, rheumatoid arthritis, Behçet's disease, gout and systemic lupus erythematosus. [3] Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by the presence of antinuclear and anti-double-stranded DNA autoantibodies. In addition, autoantibodies against histones are found in drug-induced lupus erythematosus. [4] Interestingly, those nuclear self-antigens are components of NETs. Numerous studies on human samples and murine models have shown that NETs contribute to the pathogenesis of systemic lupus. [5] In SLE patients, circulating neutrophils have increased ability to release NETs, and those NETs contain DNA-antimicrobial peptide (like
Frontiers in immunology, 2018
Microparticles (MPs) are vesicles derived from the plasma membrane of different cells, are considered a source of circulating autoantigens, and can form immune complexes (MPs-ICs). The number of MPs and MPs-ICs increases in patients with systemic lupus erythematosus (SLE). MPs activate myeloid cells by inducing IL-6 and TNF-α in both SLE and other diseases. Therefore, we propose that the recognition of MPs-ICs by monocytes rather that MPs may define their phenotype and contribute to the inflammatory process in patients with SLE. Thus, the aims of this study were to evaluate the association among circulating MPs-ICs from different cell sources, alterations observed in monocyte subsets, and disease activity in patients with SLE and to establish whether monocytes bind and respond to MPs-ICs. Circulating MPs and monocyte subsets were characterized in 60 patients with SLE and 60 healthy controls (HCs) using multiparametric flow cytometry. Patients had higher MP counts and frequencies of ...
Arthritis Research & Therapy, 2013
A decreased ability to degrade neutrophil extracellular traps (NETs) is seen in a subgroup of patients with systemic lupus erythematosus (SLE) and correlates with the presence of autoantibodies. Antiphospholipid syndrome (APS) can develop secondary to SLE or as a primary disease. In the current study we investigated the ability of sera from patients with APS to degrade NETs. The presence of antibodies against NETs and neutrophil remnants were also determined in the same patients.