Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility (original) (raw)
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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.
NETosis as Source of Autoantigens in Rheumatoid Arthritis
Frontiers in Immunology, 2016
NETosis was discovered as a new function of neutrophils and thoroughly investigated as an important mechanism in the protection against bacterial, fungal, and parasitic infections (1). When the size of microorganisms is excessive for phagocytosis (2), neutrophils activate an alternative pathway leading to the extrusion of decondensed chromatin fibers containing histones as well as antimicrobial granular and cytoplasmic proteins (3). NETs are released during a form of cell death, distinct from necrosis and apoptosis, which requires reactive oxygen species (ROS) produced by NADPH oxidase. Recent data however challenge the prevalent view of NETosis as a cellular suicide. An early NETosis has been described, which occurs rapidly after exposure to microbial specific molecular
Arthritis & Rheumatology, 2015
Objective. In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA-protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. Methods. Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by Western blotting, mass spectrometry, immunofluorescence staining, and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and those with OA. Results. Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. Conclusion. Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA.
Immuno, 2022
Polymorphonuclear neutrophils (PMN) are the most abundant leucocytes in the circulation in humans. They represent a heterogeneous population exerting diverse functions through several activities. Usually described as typical pro-inflammatory cells, immunomodulatory properties of PMNs have been reported. Among others, once activated and depending on the stimulus, PMNs expel neutrophil extracellular traps (NET) in the extracellular space. NETs are complexes made of DNA and granule proteins representing an innate immune mechanism fighting infections. Nevertheless, an excess of NET formation might be involved in the development of inflammatory or autoimmune responses. Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are two chronic, inflammatory, autoimmune diseases of unknown etiology and affecting mostly women. Several abnormal or non-classical functions of PMNs or PMN sub-populations have been described in SLE and RA. Particularly, NETs have been suggested to trigger ...
Autoimmunity reviews, 2015
The pathogenesis of many autoimmune diseases is initially based on a redundant or prolonged activation of the innate immune system. It was suggested that an excessive activation of the innate immunity is often the result of a chronic inflammatory process in the organism. This inflammation can be induced by exogenous and endogenous alarm factors, or alarmins. We believe that the recently discovered neutrophil extracellular traps, or NETs, completely meet the criteria of alarmins. This review summarizes current knowledge concerning the general characteristics of NETs, their antimicrobial properties, and their role in the development of chronic inflammatory processes that underlie the pathogenesis of psoriasis and atherosclerosis. Studies on the NETosis can provide the foundation for developing new diagnostic methods and effective treatment of chronic inflammatory and autoimmune diseases.
Neutrophil gene expression in rheumatoid arthritis
Pathophysiology, 2005
There is now a growing awareness that infiltrating neutrophils play an important role in the molecular pathology of rheumatoid arthritis. In part, this arises from the fact that neutrophils have potent cytotoxic activity, but additionally from the fact that inflammatory neutrophils can generate a number of cytokines and chemokines that can have a direct influence on the progress of an inflammatory episode. Furthermore, the molecular properties of inflammatory neutrophils are quite different from those normally found in the circulation. For example, inflammatory neutrophils, but not blood neutrophils, can express cell surface receptors (such as MHC Class II molecules and Fc␥RI) that dramatically alter the way in which these cells can interact with ligands to modulate immune function. Cytokine/chemokine expression and surface expression of these novel cell surface receptors is dependent upon the neutrophil responding to local environmental factors to selectively up-regulate the expression of key cellular components via signalling pathways coupled to transcriptional activation. However, major changes in the expression levels of some proteins are also regulated by post-translational modifications that alter rates of proteolysis, and hence changes in the steady-state levels of these molecules.
Seeing the wood for the trees: the forgotten role of neutrophils in rheumatoid arthritis
Immunology today, 1997
n rheumatoid arthritis (RA), the hall spectrum of cellular and humoral elements of the immune system are activated and co-ordinately contribute to disease pathology. The disease is primarily associated with inflammation within peripheral joints and the destruction of joint tissues and structures. Of particular importance is the degradation of articular cartilage, which initially results in loss of function and subsequently exposes underlying bone. Thus, understanding the mechanisms that lead to cartilage erosion will be of benefit for the design of new therapeutic regimens. RA is often considered a T-cell-driven dis-Neutrophils infiltrate diseased joints in rheumatoid arthritis in huge numbers and possess considerable potential to inflict the tissue damage that is characteristic of this disease. HozoeveJ; these cells are commonly overlooked by immunologists seeking new zoays to treat disease progression. In this article, Steven Edwards and Maurice Hallett evaluate the role of neutrophils in disease pathology zoith the aim of re-azoakening interest in this important cell. found at later stages within the synovial fluid of joints. Furthermore, T cells have little or no potential to induce cartilage degradation directly• The cell with the greatest capacity to inflict damage within diseased joints is the neutrophil. Yet, over the past ten years, the focus of attention has drifted away from this cell as a potential therapeutic target. Some animal models have indicated that joint destruction may occur in the absence of a contribution from neutrophils, but conclusions drawn from these animal models are limited because they do not accurately reflect the molecular properties of human RA. This article reexamines the role of the neutrophil as a mediator of tissue ease; however, although these cells probably play an important role in the early stages of the disease, few T-cell-derived cytokines are
Neutrophils: Novel key players in Rheumatoid Arthritis. Current and future therapeutic targets
Autoimmunity reviews, 2018
Rheumatoid Arthritis (RA) is a complex systemic autoimmune disease in which various cell types are involved. Among them, neutrophils have been recognized as important players in the onset and the progression of RA. The pathogenic role of neutrophils in RA lies in the alteration of several processes, including increased cell survival and migratory capacity, abnormal inflammatory activity, elevated oxidative stress and an exacerbated release of neutrophil extracellular traps. Through these mechanisms, neutrophils can activate other immune cells, thus perpetuating inflammation and leading to the destruction of the cartilage and bone of the affected joint. Given the considerable contribution of neutrophils to the pathophysiology of RA, several studies have attempted to clarify the effects of various therapeutic agents on this subtype of leukocyte. To date, recent studies have envisaged the role of new molecules on the pathogenic profile of neutrophils in RA, which could represent novel ...
Neutrophil extracellular Traps and its implications in inflammation: An Overview
In addition to physical barriers, neutrophils are considered a part of the first line of immune defense. They can be found in the bloodstream, with a lifespan of 6-8 h, and in tissue, where they can last up to 7 days. The mechanisms that neutrophils utilize for host defense are phagocytosis, degranulation, cytokine production, and, the most recently described, neutrophil extracellular trap (NET) production. NETs are DNA structures released due to chromatin decondensation and spreading, and they thus occupy three to five times the volume of condensed chromatin. Several proteins adhere to NETs, including histones and over 30 components of primary and secondary granules, among them components with bactericidal activity such as elastase, myeloperoxidase, cathepsin G, lactoferrin, pentraxin 3, gelatinase, proteinase 3, LL37, peptidoglycan-binding proteins, and others with bactericidal activity able to destroy virulence factors. Three models for NETosis are known to date. (a) Suicidal NETosis, with a duration of 2-4 h, is the best described model. (b) In vital NETosis with nuclear DNA release, neutrophils release NETs without exhibiting loss of nuclear or plasma membrane within 5-60 min, and it is independent of reactive oxygen species (ROS) and the Raf/MERK/ERK pathway. (c) The final type is vital NETosis with release of mitochondrial DNA that is dependent on ROS and produced after stimuli with GM-CSF and lipopolysaccharide. Recent research has revealed neutrophils as more sophisticated immune cells that are able to precisely regulate their granular enzymes release by ion fluxes and can release immunomodulatory cytokines and chemokines that interact with various components of the immune system. Therefore, they can play a key role in autoimmunity and in autoinflammatory and metabolic diseases. In this review, we intend to show the two roles played by neutrophils: as a first line of defense against microorganisms and as a contributor to the pathogenesis of various illnesses, such as autoimmune, autoinflammatory, and metabolic diseases.