Neutrophil extracellular traps (NET) formation is part of the neutrophil response to infections, but excessive or inappropriate NETosis may trigger the production of autoantibodies and cause organ ...damage in autoimmune disorders. Spontaneously netting neutrophils are not frequent and induction of NET in vitro by selected stimuli is necessary to investigate their structure. In the present work, the protein composition and post-translational modifications of NET produced under different stimuli have been studied by means of proteomic analysis. Neutrophils from healthy donors were stimulated by PMA, A23187, Escherichia coli LPS or untreated; after three hours, cells were washed, treated with DNase and supernatants collected for mass spectrometry. Data were analyzed by unsupervised hierarchical clustering analyses. We identified proteins contained in NETs of any source or exclusive of one stimulus: LPS-induced and spontaneous NET diverge in protein composition, while PMA- and A23187-induced NET appear more similar. Among the post-translational modifications we examined, methionine sulfoxidation is frequent especially in PMA- and LPS-induced NETs. Myeloperoxidase is the protein more extensively modified. Thus, proteomic analysis indicates that NETs induced by different stimuli are heterogeneous in terms of both protein composition and post-translational modifications, suggesting that NET induced in different conditions may have different biological effects.
New biomarkers in SLE: from bench to bedside Capecchi, Riccardo; Puxeddu, Ilaria; Pratesi, Federico ...
Rheumatology (Oxford, England),
12/2020, Letnik:
59, Številka:
Suppl5
Journal Article
Recenzirano
Odprti dostop
Biomarkers may have a diagnostic or monitoring value, or may predict response to therapy or disease course. The aim of this review is to discuss new serum and urinary biomarkers recently proposed for ...the diagnosis and management of SLE patients. Novel sensitive and specific assays have been proposed to evaluate complement proteins, 'old' biomarkers that are still a cornerstone in the management of this disease. Chemokines and lectins have been evaluated as surrogate biomarkers of IFN signature. Other cytokines like the B cell activating factor (BAFF) family cytokines are directly related to perturbations of the B cell compartment as key pathogenetic mechanism of the disease. A large number of urine biomarkers have been proposed, either related to the migration and homing of leukocytes to the kidney or to the local regulation of inflammatory circuits and the survival of renal intrinsic cells. The combination of traditional disease-specific biomarkers and novel serum or urine biomarkers may represent the best choice to correctly classify, stage and treat patients with SLE.
Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated antigens with generation of anti-citrullinated peptide/proteins antibodies (ACPA). Currently, the nature ...and source of citrullinated antigens driving the humoral autoimmune response within synovial ectopic lymphoid structures (ELS) is a crucial unknown aspect of RA pathogenesis. Here we characterised the autoreactive B-cell response of lesional B cells isolated from ELS+RA synovium.
Single synovial tissue CD19+cells were Fluorescence Activated Cell Sorting (FACS)-sorted and VH/VL Ig genes cloned to generate recombinant monoclonal antibodies (rmAbs) from patients with ELS+/ACPA+RA.
RA-rmAbs immunoreactivity analysis provided the following key findings: (1) in a chIP-based array containing 300 autoantigens and in a 'citrullinome' multiplex assay, a strong reactivity against citrullinated histones H2A/H2B (citH2A/H2B) was observed in ∼40% of RA-rmAbs, followed by cit-fibrinogen and cit-vimentin; (2) anti-citH2A/H2B-reactive RA-rmAbs (but not anti-citH2A/H2B negative) selectively recognised neutrophil extracellular traps (NETs) from peripheral blood and/or RA joint neutrophils; (3) anti-citH2A/citH2B and anti-NET immunobinding was dependent on affinity maturation and was completely abrogated following reversion of hypermutated IgVH/VL genes to germline sequences; (4) ELS+ (not ELS-) RA synovial tissues engrafted into Severe Combined ImmunoDeficiency (SCID) mice released human anti-citH2A/citH2B and anti-NET antibodies in association with the intra-graft expression of CXCL13 and lymphotoxin (LT)-β, two master regulators of ELS.
We provided novel evidence that B cells differentiated within synovial ELS in the RA joints frequent target deiminated proteins which could be generated during NETosis of RA synovial neutrophils including histones. Thus, NETs could represent a source of citrullinated antigens fuelling the ACPA autoimmune response within the RA synovium.
In neutrophils (but also in eosinophils and in mast cells), different inflammatory stimuli induce histone deimination, chromatin decondensation, and NET formation. These web-like structures that trap ...and kill microbes contain DNA, cationic granule proteins, and antimicrobial peptides, but the most abundant proteins are core histones. Histones contained in NETs have been deiminated, and arginines are converted in citrullines. While deimination is a physiological process amplified in inflammatory conditions, only individuals carrying genetic predisposition to develop rheumatoid arthritis (RA) make antibodies to deiminated proteins. These antibodies, collectively identified as anti-citrullinated proteins/peptides antibodies (ACPA), react with different deiminated proteins and display partially overlapping specificities. In this paper, we will summarize current evidence supporting the role of NETosis as critical mechanism in the breach of tolerance to self-antigens and in supporting expansion and differentiation of autoreactive cells. In fact, several lines of evidence connect NETosis with RA: RA unstimulated synovial fluid neutrophils display enhanced NETosis; sera from RA patients with Felty's syndrome bind deiminated H3 and NETs; a high number of RA sera bind deiminated H4 contained in NETs; human monoclonal antibodies generated from RA synovial B cells decorate NETs and bind deiminated histones. In RA, NETs represent on one side an important source of autoantigens bearing posttranslational modifications and fueling the production of ACPA. On the other side, NETs deliver signals that maintain an inflammatory milieu and contribute to the expansion and differentiation of ACPA-producing B cells.
Cytokines and receptors of the IL-1 family are key mediators in innate immune and inflammatory reactions in physiological defensive conditions, but are also significantly involved in immune-mediated ...inflammatory diseases. Here, we will address the role of cytokines of the IL-1 superfamily and their receptors in neuroinflammatory and neurodegenerative diseases, in particular Multiple Sclerosis and Alzheimer's disease. Notably, several members of the IL-1 family are present in the brain as tissue-specific splice variants. Attention will be devoted to understanding whether these molecules are involved in the disease onset or are effectors of the downstream degenerative events. We will focus on the balance between the inflammatory cytokines IL-1β and IL-18 and inhibitory cytokines and receptors, in view of future therapeutic approaches.
Although the mechanisms underlying AD neurodegeneration are not fully understood, it is now recognised that inflammation could play a crucial role in the initiation and progression of AD ...neurodegeneration. A neuro-inflammatory network, based on the anomalous activation of microglial cells, includes the production of a number of inflammatory cytokines both locally and systemically. These may serve as diagnostic markers or therapeutic targets for AD neurodegeneration.
We have measured the levels of the inflammation-related cytokines and receptors of the IL-1 family in serum of subjects with AD, compared to mild cognitive impairment (MCI), subjective memory complaints (SMC), and normal healthy subjects (NHS). Using a custom-made multiplex ELISA array, we examined ten factors of the IL-1 family, the inflammation-related cytokines IL-1α, IL-1β, IL-18, and IL-33, the natural inhibitors IL-1Ra and IL-18BP, and the soluble receptors sIL-1R1, sIL-1R2, sIL-1R3, and sIL-1R4.
The inflammatory cytokines IL-1α and IL-1β, their antagonist IL-1Ra, and their soluble receptor sIL-1R1 were increased in AD. The decoy IL-1 receptor sIL-1R2 was only increased in MCI. IL-33 and its soluble receptor sIL-1R4 were also significantly higher in AD. The soluble form of the accessory receptor for both IL-1 and IL-33 receptor complexes, sIL-1R3, was increased in SMC and even more in AD. Total IL-18 levels were unchanged, whereas the inhibitor IL-18BP was significantly reduced in MCI and SMC, and highly increased in AD. The levels of free IL-18 were significantly higher in MCI.
AD is characterised by a significant alteration in the circulating levels of the cytokines and receptors of the IL-1 family. The elevation of sIL-1R4 in AD is in agreement with findings in other diseases and can be considered a marker of ongoing inflammation. Increased levels of IL-1Ra, sIL-1R1, sIL-1R4, and IL-18BP distinguished AD from MCI and SMC, and from other inflammatory diseases. Importantly, sIL-1R1, sIL-1R3, sIL-1R4, and IL-18BP negatively correlated with cognitive impairment. A significant elevation of circulating sIL-1R2 and free IL-18, not present in SMC, is characteristic of MCI and disappears in AD, making them additional interesting markers for evaluating progression from MCI to AD.
The “inflammatory hypothesis” of atherosclerosis postulates that inflammatory cell signalling drives the formation, growth and ultimately the instability of atherosclerotic plaques, setting up the ...substrate for the thrombotic response that causes myocardial damage or infarction. The recent Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) trial has been hailed as the first demonstration, ex iuvantibus, of the inflammatory hypothesis. Indeed, interleukin (IL)-1β inhibition was found to reduce cardiovascular events in patients with previous myocardial infarction and raised high-sensitivity C-reactive protein, despite no effects on the lipid profile. These results prompt a dissection of inflammatory mechanisms of atherosclerosis in order to search for specific biomarkers with prognostic value and/or therapeutic targets.
Under this respect, the IL-33/suppression of tumorigenesis 2 (ST2) pathway deserves consideration. Indeed, its elements are particularly expressed in the endothelium of arterial vessels, and the interaction between IL-33 and the ST2 receptor blunts the immune response characteristic of atherosclerosis. By contrast, soluble ST2 (sST2) acts as a decoy receptor for IL-33, thus blocking its protective effects. Despite a solid theoretical framework, no definite demonstration of an involvement of the IL-33/ST2 pathway in atherosclerosis has been provided. Therefore, further studies are warranted to verify if elements of the IL-33/ST2 pathway may be proposed as markers of plaque burden and predictors of future cardiovascular events, and to explore the potential clinical benefit of enhanced IL-33/ST2 signalling in atherosclerosis.
•Knowledge on the IL-33/ST2 pathway in atherosclerosis is limited.•The elements of the IL-33/ST2 pathway are expressed in the endothelium of arterial vessels.•IL-33 blunts the immune response characteristic of atherosclerosis.•Soluble ST2 acts as a decoy receptor for IL-33, thus blocking its protective effects.
New and alternative models for agri-food production and consumption have brought up questions regarding the effects they have on local development processes in terms of the economic exploitation of ...rural areas as well as environmental, cultural, and social factors. The agri-food system proposed by the Slow Food (SF) Presidia Project, which focuses on farm-to-market systems for local, high-quality, sustainable products, can respond to the new and emerging needs of both rural and urban populaces via several approaches in addition to food production itself. However, evaluating these parameters is challenging. The aim of this study was to develop an indicator-based tool to monitor the sustainability in agri-food systems that considers quality as well as economic, ecological, social, and cultural aspects. We: (i) translated the major SF principles of “good,” “clean,” and “fair” into five major criteria to evaluate sustainability; (ii) designed multiple indicators to monitor progress toward sustainability for each of those criteria; and (iii) applied the monitoring tools to three case studies as a first attempt at end-use validation. Indicators and criteria were weighted either equally or based on their importance to surveyed stakeholders, i.e., consumers, producers, and scientists/experts. The proposed approach performed well as a tool for a broad sustainability evaluation by effectively combining the indicators with the same feedback. With this approach, we demonstrated that the SF Presidia project increases all dimensions of sustainability and in particular socioeconomic and cultural capital by preserving the environmental and quality aspects of the food products.
Dysregulated production of cytokines has a critical role in systemic lupus. The aim of this work is to identify, by a comprehensive analysis of IL-1 family cytokines and receptors in serum, ...correlation between cytokines/receptors' levels and the clinical and serological features of the disease.
A full clinical evaluation was performed in 74 patients with systemic lupus erythematosus (SLE). C3, C4, anti-dsDNA and anti-C1q antibodies were measured. Cytokines of the IL-1 family (IL-1α, IL-1β, IL-33, IL-18), soluble receptors (sIL-1R1, sIL-1R2, sIL-1R3, ST2/sIL-1R4) and antagonists (IL-1Ra, IL-18 binding protein (IL-18BP)) were measured in serum by multiarray ELISA. Free IL-18 was calculated as the amount of IL-18 not inhibited by IL-18BP. Data were analysed by non-parametric tests and by multivariate analysis, using partial least squares (PLS) models.
Total IL-18, IL-18BP, sIL-1R4 and IL-1Ra levels were higher in SLE vs.
Total and free IL-18 and sIL-1R4 were higher in patients with active vs. inactive disease and correlated with ECLAM, anti-C1q and anti-dsDNA antibodies. sIL-1R2 was higher in patients with inactive disease, was negatively correlated with ECLAM and anti-C1q antibodies and was positively correlated with C3 levels. PLS identified sIL-1R4, sIL-1R2 and anti-dsDNA as variables distinguishing patients with active from those with inactive disease; sIL-1R4, IL-18BP and anti-dsDNA identified patients with active nephritis; sIL-1R4, C3, IL-18 and free IL-18 identified patients with haematological involvement.
The data support the use of IL-18, sIL-1R2 and sIL-1R4 as biomarkers of disease activity and organ involvement, and suggest that failure in the inhibition of IL-1 activation may be a critical event in the active stages of SLE.
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease leading to the death of affected individuals within years. The involvement of inflammation in the pathogenesis of ...neurodegenerative diseases, including ALS, is increasingly recognized but still not well understood. The aim of this study is to evaluate the levels of inflammation-related IL-1 family cytokines (IL-1β, IL-18, IL-33, IL-37) and their endogenous inhibitors (IL-1Ra, sIL-1R2, IL-18BP, sIL-1R4) in patients with sporadic ALS (sALS), METHODS: Sera were collected from 144 patients (125 patients were characterized by disease form, duration, and disability, using the revised ALS functional rating scale (ALSFRS-R) and from 40 matched controls. Cerebrospinal fluid (CSF) was collected from 54 patients with sALS and 65 patients with other non-infectious non-oncogenic diseases as controls. Cytokines and inhibitors were measured by commercial ELISA.
Among the IL-1 family cytokines tested total IL-18, its endogenous inhibitor IL-18BP, and the active form of the cytokine (free IL-18) were significantly higher in the sALS sera than in controls. No correlation between these soluble mediators and different clinical forms of sALS or the clinical setting of the disease was found. IL-18BP was the only mediator detectable in the CSF of patients.
Among the IL-1 family cytokines, only IL-18 correlates with this disease and may therefore have a pathological role in sALS. The increase of total IL-18 suggests the activation of IL-18-cleaving inflammasome. Whether IL-18 upregulation in circulation of sALS patients is a consequence of inflammation or one of the causes of the pathology still needs to be addressed.
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