Neutrophil extracellular traps (NETs) can be released in the vasculature. In addition to trapping microbes, they promote inflammatory and thrombotic diseases. Considering that P-selectin induces ...prothrombotic and proinflammatory signaling, we studied the role of this selectin in NET formation. NET formation (NETosis) was induced by thrombin-activated platelets rosetting with neutrophils and was inhibited by anti-P-selectin aptamer or anti-P-selectin glycoprotein ligand-1 (PSGL-1) inhibitory antibody but was not induced by platelets from P-selectin−/− mice. Moreover, NETosis was also promoted by P-selectin–immunoglobulin fusion protein but not by control immunoglobulin. We isolated neutrophils from mice engineered to overproduce soluble P-selectin (P-selectinΔCT/ΔCT mice). Although the levels of circulating DNA and nucleosomes (indicative of spontaneous NETosis) were normal in these mice, basal neutrophil histone citrullination and presence of P-selectin on circulating neutrophils were elevated. NET formation after stimulation with platelet activating factor, ionomycin, or phorbol 12-myristate 13-acetate was significantly enhanced, indicating that the P-selectinΔCT/ΔCT neutrophils were primed for NETosis. In summary, P-selectin, cellular or soluble, through binding to PSGL-1, promotes NETosis, suggesting that this pathway is a potential therapeutic target for NET-related diseases.
•NET formation is stimulated by platelet or soluble P-selectin.
In addition to being key elements in hemostasis and thrombosis, platelets have an important role in the inflammatory and innate immune response. This activity is associated with their capability to ...recognize pathogens through the expression of toll-like receptors, the secretion of various cytokines, chemokines, and growth factors stored within their granules, and the expression of cell adhesion molecules that allows interaction with other immune cells, mainly neutrophils and monocytes. As part of the first line of defense, neutrophils control invading pathogens by phagocytosis, the release of antimicrobial proteins during degranulation, or through the formation of web-like structures named neutrophil extracellular traps (NETs). NETs are formed by chromatin, proteases, and antimicrobial proteins, and their main function is to trap and kill bacteria, virus, and fungi, avoiding their dissemination. Besides microorganisms, NET formation is also triggered by proinflammatory molecules and platelets. The uncontrolled formation of NETs might exert tissue damage and has been involved in a pathogenic mechanism of autoimmune and prothrombotic clinical conditions. In this review, we discuss the role of platelets in NET generation highlighting the mediators, stimuli, and molecular mechanisms involved in this phenomenon, both in human and murine models.
Platelet TLR‐4 activation by pathogen‐ or damage‐associated molecular pattern molecules triggers pro‐thrombotic, proinflammatory, and pro‐coagulant effector responses. Moreover, platelet TLR4 has a ...prominent role as a sensor of high lipopolysaccharide circulating levels during sepsis and in the clearance of pathogens mediated by neutrophils. This review presents evidence pointing to TLR4 as a bridge connecting thrombosis and innate immunity.
Review of how platelet TLR4 activation links hemostasis and immunity.
Platelet GPIb and neutrophil CD18 together with TXA2, PF4, and vWF release mediate platelet‐triggered NET formation, while prostacyclin inhibits NETosis.
In addition to being key elements in ...hemostasis and thrombosis, platelets amplify neutrophil function. We aimed to gain further insight into the stimuli, mediators, molecular pathways, and regulation of neutrophil extracellular trap formation mediated by human platelets. Platelets stimulated by lipopolysaccharide, a wall component of gram‐negative bacteria, Pam3‐cysteine‐serine‐lysine 4, a mimetic of lipopeptide from gram‐positive bacteria, Escherichia coli, Staphylococcus aureus, or physiologic platelet agonists promoting neutrophil extracellular trap formation and myeloperoxidase‐associated DNA activity under static and flow conditions. Although P‐selectin or glycoprotein IIb/IIIa were not involved, platelet glycoprotein Ib, neutrophil cluster of differentiation 18, and the release of von Willebrand factor and platelet factor 4 seemed to be critical for the formation of neutrophil extracellular traps. The secretion of these molecules depended on thromboxane A2 production triggered by lipopolysaccharide or Pam3‐cysteine‐serine‐lysine 4 but not on high concentrations of thrombin. Accordingly, aspirin selectively inhibited platelet‐mediated neutrophil extracellular trap generation. Signaling through extracellular signal‐regulated kinase, phosphatidylinositol 3‐kinase, and Src kinases, but not p38 or reduced nicotinamide adenine dinucleotide phosphate oxidase, was involved in platelet‐triggered neutrophil extracellular trap release. Platelet‐mediated neutrophil extracellular trap formation was inhibited by prostacyclin. Our results support a role for stimulated platelets in promoting neutrophil extracellular trap formation, reveal that an endothelium‐derived molecule contributes to limiting neutrophil extracellular trap formation, and highlight platelet inhibition as a potential target for controlling neutrophil extracellular trap cell death.
Coxsackievirus B3 (CVB3) is a globally prevalent enterovirus of the Picornaviridae family that is frequently associated with viral myocarditis (VM). Neutrophils, as first responders, may be key cells ...in determining viral disease outcomes; however, neutrophils have been poorly studied with respect to viral infection. Although neutrophils have been ascribed a relevant role in early cardiac inflammation, their precise role in CVB3 infection has not yet been evaluated. In this study, we aimed to determine if the interaction between human neutrophils and CVB3 could lead to viral replication and/or modulation of neutrophil survival and biological functions, and whether neutrophil depletion in a murine model has a beneficial or harmful effect on CVB3 infection. Our results show that CVB3 interacted with but did not replicate in human neutrophils. Neutrophils recognized CVB3 mainly through endosomal TLR-8, and infection triggered NFκB activation. Virus internalization resulted in increased cell survival, up-regulation of CD11b, enhanced adhesion to fibrinogen and fibronectin, and the secretion of IL-6, IL-1β, TNF-α, and IL-8. Supernatants from infected neutrophils exerted chemotactic activity partly mediated by IL-8. The infected neutrophils released myeloperoxidase and triggered neutrophil extracellular trap formation in the presence of TNF-α. In mice infected with CVB3, viral RNA was detected in neutrophils as well as in mononuclear cells. After neutrophil depletion, mice showed reduced VM reflected by a reduction in viral titers, cell exudates, and CCL-2 mRNA levels, as well as the abrogation of reactive cardiomyocyte hypertrophy. Our results indicate that neutrophils have relevant direct and indirect roles in the pathogenesis of CVB3-induced VM.
•-Coxsackievirus B3 (CVB3) interacts with, but does not replicate in human neutrophils.•-CVB3 increases neutrophil survival, CD11b expression, cytokine and IL-8 release.•-CVB3 triggers neutrophil release of myeloperoxidase and NETs.•-Neutrophils sense CVB3 mainly through TLR-8 and the activation of NFκB.•-Neutrophil-depleted CVB3-infected mice exhibit reduced viral myocarditis.
We investigated the contribution of human platelets to macrophage effector properties in the presence of lipopolysaccharide (LPS), as well as the beneficial effects and time frame for platelet ...transfusion in septic animals. Our results show that platelets sequester both pro-(TNF-α/IL-6) and anti-(IL-10) inflammatory cytokines released by monocytes. Low LPS concentrations (0.01 ng/mL) induced M2 macrophage polarization by decreasing CD64 and augmenting CD206 and CD163 expression; yet, the presence of platelets skewed monocytes toward type 1 macrophage (M1) phenotype in a cell-contact-dependent manner by the glycoprotein Ib (GPIb)-CD11b axis. Accordingly, platelet-licensed macrophages showed increased TNF-α levels, bacterial phagocytic activity, and a reduced healing capability. Platelet transfusion increased inducible nitric oxide synthase (iNOS)+ macrophages, improving bacterial clearance and survival rates in septic mice up to 6 h post-infection, an effect that was abolished by CD11b and GPIb blockade. Our results demonstrate that platelets orchestrate macrophage effector responses, improving the clinical outcome of sepsis in a narrow but relevant time frame.
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•Platelets sequester pro- and anti-inflammatory cytokines released by monocytes•In the presence of LPS, platelets skew monocytes toward a pro-inflammatory phenotype•During sepsis, platelet transfusion increases iNOS+ macrophages and bacterial clearance•Platelet transfusion increases septic mice survival in a narrow time frame
Carestia et al. describe the beneficial role of platelet transfusion for animal survival during sepsis by reprogramming macrophages and fostering antimicrobial functions. The cross-talk between platelets and monocytes that promotes pro-inflammatory macrophages depends on the intimal cellular contact between platelet GPIb and CD11b in the monocyte surface.
The formation of neutrophil extracellular traps (NETs) is a newly described phenomenon that increases the bacteria-killing ability and the inflammatory response of neutrophils. Because NET generation ...occurs in an inflammatory microenvironment, we examined its regulation by anti-inflammatory drugs. Treatment of neutrophils with dexamethasone had no effect, but acetylsalicylic acid (ASA) treatment prevented NET formation. NETosis was also abrogated by the presence of BAY 11-7082 (E)-3-4-methylphenylsulfonyl-2-propenenitrile and Ro 106-9920 6-(phenylsulfinyl)tetrazolo1,5-bpyridazine, two structurally unrelated nuclear factor-κB (NF-κB) inhibitors. The decrease in NET formation mediated by ASA, BAY-11-7082, and Ro 106-9920 was correlated with a significant reduction in the phosphorylation of NF-κB p65 subunit, indicating that the activation of this transcription factor is a relevant signaling pathway involved in the generation of DNA traps. The inhibitory effect of these drugs was also observed when NET generation was induced under acidic or hyperthermic conditions, two stress signals of the inflammatory microenvironment. In a mouse peritonitis model, while pretreatment of animals with ASA or BAY 11-7082 resulted in a marked suppression of NET formation along with increased bacteremia, dexamethasone had no effect. Our results show that NETs have an important role in the local control of infection and that ASA and NF-κB blockade could be useful therapies to avoid undesired effect of persistent neutrophil activation.
Diabetes is characterized by chronic inflammation, endothelial dysfunction, increased risk of infections and early cardiovascular disease. By releasing neutrophil extracellular traps (NETs), ...neutrophils kill bacteria and exert pro-inflammatory and pro-thrombotic activities. Increased NETosis has been found in cross-sectional studies including treated type 2 diabetes mellitus (T2DM) patients. In this study, we determined whether the ability of neutrophils to form NETs differs in diabetic patients pre- and post-hyperglycemic control versus healthy donors (HD), and the relationship between NETosis with pro-thrombotic, pro-inflammatory biomarkers and thrombotic clinical events.
Diabetic patients recently diagnosed and after 6 and 12 months of treatment (N = 25) and HD (N = 25) were included. NET formation was studied by microscopy and fluorometry. Nucleosomes, HNE-DNA complexes, von Willebrand factor (vWF), IL6 and TNFα plasma levels were measured by ELISA and P-selectin on the platelet surface was assessed by cytometry.
Basal levels of NETs in recently diagnosed T2DM patients were higher compared to HD. While TNFα stimulation of control neutrophils resulted in DNA release, patient neutrophils were not responsive. Although glycemia decreased after 6 months of metformin treatment, basal and TNFα and PMA-stimulated NETs reached normal values after 12 months. Compared to controls, nucleosomes, HNE-DNA complexes, IL-6 and TNFα levels were increased in recently diagnosed patients and decreased after 12 months of treatment. P-selectin and vWF levels were similar in both populations.
Our data suggest that NETs could represent a biomarker for T2DM. Increased NETosis in T2DM patients does not appear to be the consequence of impaired glycemic control but rather due to pro-inflammatory cytokines and is not related to thrombotic events.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Histones are highly alkaline proteins found in cell nuclei and they can be released by either dying or inflammatory cells. The recent observations that histones are major components of neutrophil ...extracellular traps and promote platelet aggregation and platelet-dependent thrombin generation have shown that these proteins are potent prothrombotic molecules. Because the mechanism(s) of platelet activation by histones are not completely understood, we explored the ability of individual recombinant human histones H1, H2A, H2B, H3 and H4 to induce platelet activation as well as the possible molecular mechanisms involved. All histones were substrates for platelet adhesion and spreading and triggered fibrinogen binding, aggregation, von Willebrand factor release, P-selectin and phosphatidylserine (PS) exposure and the formation of platelet-leukocyte aggregates; however, H4 was the most potent. Histone-mediated fibrinogen binding, P-selectin and PS exposure and the formation of mixed aggregates were potentiated by thrombin. Histones induced the activation of ERK, Akt, p38 and NFκB. Accordingly, histone-induced platelet activation was significantly impaired by pretreatment of platelets with inhibitors of ERK (U 0126), PI3K/Akt (Ly 294002), p38 (SB 203580) and NFκB (BAY 11–7082 and Ro 106–9920). Preincubation of platelets with either aspirin or dexamethasone markedly decreased fibrinogen binding and the adhesion mediated by histones without affecting P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1, H2A and H2B, were partially mediated through interaction with Toll-like receptors –2 and –4. Our data identify histones as important triggers of haemostatic and proinflammatory platelet responses, and only haemostatic responses are partially inhibited by anti-inflammatory drugs.
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