Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular ...communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid 12(S)-HETE as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A₂ IIA (sPLA₂-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA₂-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA₂-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA₂-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.
Inflammatory bone loss in septic and inflammatory conditions is due to increased activity of osteoclasts that requires receptor activator of NF-kappa B-ligand (RANKL). Neutrophils are the predominant ...infiltrating cells in these conditions. Although disease severity is linked to neutrophils, their role in evolution of bony lesions is not clear. We show that lipopolysaccharide (LPS), a toll-like receptor 4 ligand, up-regulated the expression of membrane RANKL in human blood neutrophils and murine air pouch–derived neutrophils. LPS-activated human and murine neutrophils, cocultured with human monocyte-derived osteoclasts and RAW 264.7 cells, respectively, stimulated bone resorption. Transfection of PLB-985 neutrophil-like cells with RANKL antisense RNA reduced osteoclastogenesis. Synovial fluid neutrophils of patients with exacerbation of rheumatoid arthritis strongly expressed RANKL and activated osteoclastogenesis in coculture systems. Osteoprotegerin, the RANKL decoy receptor, suppressed osteoclast activation by neutrophils from these different sources. Moreover, direct cell-cell contact between neutrophils and osteoclasts was visualized by confocal laser microscopy. Activation of neutrophil membrane-bound RANKL was linked to tyrosine phosphorylation of Src-homology domain–containing cytosolic phosphatase 1 with concomitant down-regulation of cytokine production. The demonstration of these novel functions of neutrophils highlights their potential role in osteoimmunology and in therapeutics of inflammatory bone disease.
Monosodium urate (MSU) microcrystals present in bone tissues of chronic gout can be ingested by nonprofessional phagocytes like osteoblasts (OBs) that express NLRP3 (nucleotide-binding domain and ...leucine-rich repeat region containing family of receptor protein 3). MSU is known to activate NLRP3 inflammasomes in professional phagocytes. We have identified a new role for NLRP3 coupled to autophagy in MSU-stimulated human OBs.
Normal human OBs cultured in vitro were investigated for their capacity for phagocytosis of MSU microcrystals by using confocal microscopy. Subsequent mineralization and matrix metalloproteinase activity were evaluated, whereas regulatory events of phagocytosis were deciphered by using signaling inhibitors, phosphokinase arrays, and small interfering RNAs. Statistics were carried out by using paired or unpaired t tests, and the one-way ANOVA, followed by multiple comparison test.
Most of the OBs internalized MSU in vacuoles. This process depends on signaling via PI3K, protein kinase C (PKC), and spleen tyrosine kinase (Syk), but is independent of Src kinases. Simultaneously, MSU decreases phosphorylation of the protein kinases TOR (target of rapamycin) and p70S6K. MSU activates the cleavage of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, and MSU microcrystals are coated with GFP-tagged LC3. However, MSU-stimulated autophagy in OBs absolutely requires the phagocytosis process. We find that MSU upregulates NLRP3, which positively controls the formation of MSU-autophagosomes in OBs. MSU does not increase death and late apoptosis of OBs, but reduces their proliferation in parallel to decreasing their competence for mineralization and to increasing their matrix metalloproteinase activity.
MSU microcrystals, found locally encrusted in the bone matrix of chronic gout, activate phagocytosis and NLRP3-dependent autophagy in OBs, but remain intact in permanent autophagosomes while deregulating OB functions.
Our knowledge of the role of cytokines in pathologic conditions has increased considerably with the emergence of molecular and genetic studies, particularly in the case of autoinflammatory monogenic ...diseases. Many rare disorders, considered orphan until recently, are directly related to abnormal gene regulation, and the treatment with biologic agents (biologics) targeting cytokine receptors, intracellular signaling or specific cytokines improve the symptoms of an increasing number of chronic inflammatory diseases. As it is currently impossible to systematically conduct genetic studies for all patients with autoinflammatory and autoimmune diseases, the evaluation of cytokines can be seen as a simple, less time consuming, and less expensive alternative. This approach could be especially useful when the diagnosis of syndromes of diseases of unknown etiology remains problematic. The evaluation of cytokines could also help avoid the current trial-and-error approach, which has the disadvantages of exposing patients to ineffective drugs with possible unnecessary side effects and permanent organ damages. In this review, we discuss the various possibilities, as well as the limitations of evaluating the cytokine profiles of patients suffering from autoinflammatory and autoimmune diseases, with methods such as direct detection of cytokines in the plasma/serum or following ex vivo stimulation of PBMCs leading to the production of their cytokine secretome. The patients' secretome, combined with biomarkers ranging from genetic and epigenetic analyses to immunologic biomarkers, may help not only the diagnosis but also guide the choice of biologics for more efficient and rapid treatments.
Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on ...phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.
IL-32γ is a multifunctional cytokine involved in various inflammatory and auto-immune diseases in which neutrophils can affect the evolution of these diseases. To persist at inflammatory sites, ...neutrophils require inhibition of their rapid and constitutive apoptosis, an inhibitory effect that phlogogenic cytokines support. To date, the effects of IL-32γ on neutrophils remain unknown. We demonstrate that IL-32γ delays, in a dose-dependent manner, the spontaneous apoptosis of human blood neutrophils by activating mainly p38 MAPK through rapid p38 phosphorylation. PI3-K and ERK1/2 MAPK are also involved, but to a lesser extent. Most of cytokines that induce retardation of neutrophil apoptosis activate the expression of MCL-1 at both mRNA and protein levels. IL-32γ added to human blood neutrophils in vitro is associated with sustained levels of MCL-1 protein. This effect in neutrophils corresponds to a decrease of MCL-1 protein degradation without any effect on MCL-1 mRNA levels. The sustained levels of MCL-1 induced by IL-32γ are only abrogated by the p38β MAPK inhibitor SB202190. Additionally, IL-32γ induces a reduction in caspase 3 activity in neutrophils. In conclusion, IL-32γ affects human blood neutrophils in vitro by increasing their survival, suggesting that this cytokine could have profound effects on the deleterious functions of neutrophils in several diseases.
Apoptosis of neutrophils is an essential checkpoint for the resolution of inflammation by shutting down the deleterious functions of these immune cells. This study investigated the role of ...anhydroglucitol-core gallotannins (ACGs) in apoptosis increase of human blood neutrophils treated by the hot water extract from red maple buds (RMB). Fractions obtained by liquid–liquid partitioning (ethyl acetate, butanol and water-remaining fractions) of the hot water extract from RMB were assessed for their effects on neutrophil viability by using flow cytometry. These fractions were then phytochemically analyzed to investigate the ability of major compounds to induce neutrophil apoptosis individually. Ethyl acetate and butanol fractions that contained the major ACGs ginnalin A, ginnalin 3,6 and ginnalin C stimulated the apoptosis of neutrophils. The three ACGs at 100 μM significantly increased the rate of the late apoptotic cells. When differentially combined, these ACGs have additive or antagonist effects. These effects are related to the concentrations of the constituents in the mixtures studied, especially so for ginnalin C. GinA increased FADD, phospho-Rad17, SMAC/Diablo and cytochrome C, while decreasing the anti-apoptotic protein catalase. These compounds could be useful for the development of novel therapeutic approaches that facilitate resolution of neutrophil-mediated inflammatory diseases.
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•Ethyl acetate and butanol fractions of red maple buds' hot water extract increase the rate of neutrophil apoptosis.•Both fractions are rich in anhydroglucitol-core gallotannins (ACGs), particularly in GinA, Gin3,6 and GinC.•Human blood neutrophils respond to these three ACGs at 100 μM by increasing their spontaneous apoptosis.•GinA and Gin3,6 have additive effects on neutrophil apoptosis. GinC is antagonistic to apoptosis induced by GinA and Gin3,6.•FADD, phospho-Rad17, SMAC/Diablo and cytochrome c are the apoptotic proteins specifically targeted by the three ACGs.
Sugar maple (
Acer saccharum
M.) and red maple (
Acer rubrum
L.) barks were treated with hot water to extract nutrients in order to explore, for the first time, its potential as safe dietary ...antioxidants. The organic and inorganic nutrients of these extracts, as well as their safety on human PLB-985 cells differentiated into neutrophils-like cells, were determined. Proximate analysis showed that both bark extracts were low in moisture and fat. Sugar maple bark extract (SM-BX) showed crude protein and ash content higher than those found in red maple bark extract (RM-BX). In addition, SM-BX had total sugars higher than those evaluated in RM-BX, while complex sugars (oligo- and/or poly-saccharides) were similarly abundant in both bark extracts. Furthermore, SM-BX demonstrated a wide array of vital minerals (K, Ca, Mg, P, Na, Fe and Cu) in quantity larger than that evaluated in RM-BX, whereas RM-BX have Zn and Mn levels higher than those found in SM-BX. Phytochemical analyses showed that RM-BX exhibited total phenolic and flavonoid contents higher than those measured in SM-BX. Consequently, RM-BX presented an antioxidant activity higher than that of SM-BX: 2.85-fold ABTS radical cation scavenging capacity and 1.9-fold oxygen radical absorbance capacity. Finally, RM-BX and SM-BX were greatly safe since, at concentration up to 100 μg/ml, they did not modify the viability of neutrophils as determined by flow-cytometry assay using Annexin V-FITC/Propidum Iodide as markers. In conclusion, our
in vitro
studies indicate that both red and sugar maple bark extracts have a real potential as food additives.
Recent studies about hot-water extracts from sugar maple (Acer saccharum Marsh.) bark and buds demonstrated that they contain high amounts of phenolic structures that may be used as antioxidant food ...additives. However, the detailed chemical composition of these maple-derived extracts has yet to be determined. By performing high-performance liquid chromatography–diode array detector–high-resolution mass spectrometry (HPLC–DAD–HRMS)-based dereplication, we were able to spike and classify almost 100 metabolites in each hot-water extract. The sugar maple bark hot-water extract is rich in simple phenolic compounds and phenylpropanoid derivatives, while bud extract contains predominantly flavonoids, benzoic acids, and their complex derivatives (condensed and hydrolyzable tannins). Among those chemical structures, we tentatively identified 69 phenolic compounds potentially reported for the first time in the genus Acer. Considering the growing commercial demand in natural products, the phenolic fingerprints of sugar maple bark and bud hot-water extracts will help in promoting these two maple-derived products as new sources of bioactive compounds in the food, nutraceutical, and cosmetic industries.
Abstract
Parathyroid hormone (PTH) can be C-terminally extended without significant affinity loss for the PTH
1
receptor (PTHR
1
). We developed fusion protein ligands with enzymatic activity to ...probe PTHR
1
s at the cell surface. Two fusion proteins were generated by linking PTH to the N-terminus of either horseradish peroxidase (PTH-HRP) or the genetically modified soybean peroxidase APEX2 (PTH-APEX2). Alternatively, myc-tagged PTH (PTH-myc) was combined with antibodies, some of which HRP-conjugated, in the extracellular fluid. The three PTH-fusion proteins were produced as conditioned mediums (CM) by transfected producer HEK 293a cells. Binding of receptor-bound enzymatic ligands was revealed using widely available substrate/co-substrate systems. The stimulation of recipient HEK 293a expressing PTHR
1
s with the PTH-myc/antibodies combination or with PTH-APEX2 supported the histochemical or luminescent detection of recombinant PTHR
1
s (TrueBlue
TM
or luminol-based reagent). The PTH-HRP construction was the most sensitive and supported all tested peroxidase co-substrates (TrueBlue
TM
, tetramethylbenzidine (TMB), luminol, biotin-phenol with streptavidin-Qdots); the 3 latter schemes identified endogenous PTHR
1
in the osteoblastic HOS cell line. The specificity of the fusion protein binding to PTHR
1
was determined by its competition with an excess of PTH
1–34
. Bifunctional ligands possessing enzymatic activity detect intact receptors with various possible applications, including the screening of drugs that compete for receptor binding.
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