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Leptospirosis is a global zoonosis caused by pathogenic Leptospira. Neutrophils are key cells against bacterial pathogens but can also contribute to tissue damage. Because the information regarding the role of human neutrophils in leptospirosis is scant, we comparatively analysed the human neutrophil's response to saprophytic Leptospira biflexa serovar Patoc (Patoc) and the pathogenic Leptospira interrogans serovar Copenhageni (LIC). Both species triggered neutrophil responses involved in migration, including the upregulation of CD11b expression, adhesion to collagen, and the release of IL-8. In addition, both species increased levels of pro-inflammatory IL-1β and IL-6 associated with the inflammasome and NFκB pathway activation and delayed neutrophil apoptosis. LIC was observed on the neutrophil surface and not phagocytized. In contrast, Patoc generated intracellular ROS associated with its uptake. Neutrophils express the TYRO3, AXL, and MER receptor protein tyrosine kinases (TAM), but only LIC selectively increased the level of AXL. TLR2 but not TLR4-blocking antibodies abrogated the IL-8 secretion triggered by both Leptospira species. In summary, we demonstrate that Leptospira species trigger a robust neutrophil activation and pro-inflammatory response. These findings may be useful to find new diagnostic markers and therapeutic strategies against leptospirosis., (© 2018 John Wiley & Sons Ltd.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

M strain, the most prevalent multidrug-resistant strain of Mycobacterium tuberculosis ( Mtb ) in Argentina, has mounted mechanisms to evade innate immune response. The role of human bronchial epithelium in Mtb infection remains unknown as well as its crosstalk with neutrophils (PMN). In this work, we evaluate whether M and H37Rv strains invade and replicate within bronchial epithelial cell line Calu-6 and how conditioned media (CM) derived from infected cells alter PMN responses. We demonstrated that M infects and survives within Calu-6 without promoting death. CM from M-infected Calu-6 (M-CM) did not attract PMN in correlation with its low IL-8 content compared to H37Rv-CM. Also, PMN activation and ROS production in response to irradiated H37Rv were impaired after treatment with M-CM due to the lack of TNF- α . Interestingly, M-CM increased H37Rv replication in PMN which would allow the spreading of mycobacteria upon PMN death and sustain IL-8 release. Thus, our results indicate that even at low invasion/replication rate within Calu-6, M induces the secretion of factors altering the crosstalk between these nonphagocytic cells and PMN, representing an evasion mechanism developed by M strain to persist in the host. These data provide new insights on the role of bronchial epithelium upon M infection.

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 A(2) 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., (© Society for Leukocyte Biology.)

NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden.

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)tetrazolo[1,5-b]pyridazine], 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.

We report the study of a predicted outer-membrane leptospiral protein encoded by the gene lic11207. This protein is conserved in several pathogenic leptospiral strains but is absent in the saprophyte Leptospira biflexa. This putative outer-membrane protein has a domain of unknown function (DUF) 1565 found in several phylogenetically diverse bacteria and in the archaeon Methanosarcina acetivorans. The gene was cloned and expressed in Escherichia coli BL21 (SI) strain using the expression vector pDEST17. The 34 kDa recombinant protein was tagged with N-terminal hexahistidine and purified by metal-charged chromatography. The purified protein was used to assess: reactivity with human convalescent sera; in vivo expression; ability to activate endothelial cells (EC); and ability to modulate the apoptosis of polymorphonuclear cells (PMNs). The LIC11207 coding sequence was identified in vivo in the hamster renal tubules during experimental infection with Leptospira interrogans. The rLIC11207 showed significant antigenicity against human convalescent sera when compared with sera from healthy donors. The recombinant protein did not alter the surface expression of E-selectin or intercellular adhesion molecule 1 (ICAM-1) in EC and failed to induce the release of von Willebrand factor (vWF). Interestingly, rLIC11207 delayed apoptosis of PMNs suggesting a possible role of this protein during the infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Acidosis is one of the hallmarks of tissue injury such as trauma, infection, inflammation, and tumour growth. Although platelets participate in the pathophysiology of all these processes, the impact of acidosis on platelet biology has not been studied outside of the quality control of laboratory aggregation assays or platelet transfusion optimization. Herein, we evaluate the effect of physiologically relevant changes in extracellular acidosis on the biological function of platelets, placing particular emphasis on haemostatic and secretory functions. Platelet haemostatic responses such as adhesion, spreading, activation of αIIbβ3 integrin, ATP release, aggregation, thromboxane B2 generation, clot retraction and procoagulant activity including phosphatidilserine exposure and microparticle formation, showed a statistically significant inhibition of thrombin-induced changes at pH of 7.0 and 6.5 compared to the physiological pH (7.4). The release of alpha granule content was differentially regulated by acidosis. At low pH, thrombin or collagen-induced secretion of vascular endothelial growth factor and endostatin were dramatically reduced. The release of von Willebrand factor and stromal derived factor-1α followed a similar, albeit less dramatic pattern. In contrast, the induction of CD40L was not changed by low pH, and P-selectin exposure was significantly increased. While the generation of mixed platelet-leukocyte aggregates and the increased chemotaxis of neutrophils mediated by platelets were further augmented under acidic conditions in a P-selectin dependent manner, the increased neutrophil survival was independent of P-selectin expression. In conclusion, our results indicate that extracellular acidosis downregulates most of the haemostatic platelet functions, and promotes those involved in amplifying the neutrophil-mediated inflammatory response.

During inflammation, polymorphonuclear leukocyte (PMN) apoptosis can be delayed by different proinflammatory mediators. Classically, it has been accepted that the widely used anti-inflammatory drug acetyl salicylic acid (ASA) exerts its action through inhibition of cyclooxygenases and subsequent prostaglandin synthesis. We hypothesized that another anti-inflammatory action of ASA could be the shortening of PMN survival. We found that at therapeutic concentrations (1-3 mM), ASA and its metabolite salicylate (NaSal), but not indomethacin or ibuprofen, counteracted the prolonged PMN survival mediated by lipopolysaccharide (LPS) through inhibition of nuclear factor-kappaB (NF-kappaB) activation. Both salicylates also inhibited interleukin (IL)-1alpha or acidic conditions antiapoptotic activity. Higher concentrations of both drugs had a direct apoptotic effect. Salicylates were not effective when PMN apoptosis delay was induced by granulocyte macrophage-colony-stimulating factor (GM-CSF), a NF-kappaB-independent cytokine. Promotion of PMN survival by the combination of IL-1alpha and LPS was also reversed by salicylates, but higher concentrations were required. ASA concentrations that did not trigger PMN death increase the zymosan- or tumor necrosis factor-alpha-mediated proapoptotic effect. The LPS- and IL-1alpha- but not GM-CSF-mediated antiapoptotic effect was markedly reduced in PMNs from donors who had ingested ASA. Using a thioglycolate-induced peritonitis model, we showed that in ASA- or NaSal-treated mice there was not only a decrease in the number of cells recruited but also an increase in the percentage of apoptotic PMNs as well as an enhancement of phagocytosis compared with controls. Our findings demonstrate that acceleration of PMN apoptosis by turning off the NF-kappaB-mediated survival signals elicited by proinflammatory stimuli is another anti-inflammatory action of ASA and NaSal.

Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in the pediatric population. The etiology of HUS is linked to Gram-negative, Shiga toxin (Stx)-producing enterohemorrhagic bacterial infections. While the effect of Stx is focused on endothelial damage of renal glomerulus, cytokines induced by Stx or bacterial lipopolysaccharide (LPS) and polymorphonuclear cells (PMNs) are involved in the development of the disease. PMN release neutrophil extracellular traps (NETs) to eliminate pathogens, although NETs favor platelets (Plts) adhesion/thrombus formation and can cause tissue damage within blood vessels. Since thrombus formation and occlusion of vessels are characteristic of HUS, PMN–Plts interaction in the context of Stx may promote netosis and contribute to the endothelial damage observed in HUS. The aim of this study was to determine the relevance of netosis induced by Stx in the context of LPS-sensitized Plts on endothelial damage. We observed that Stx2 induced a marked enhancement of netosis promoted by Plts after LPS stimulation. Several factors seemed to promote this phenomenon. Stx2 itself increased the expression of its receptor on Plts, increasing toxin binding. Stx2 also increased LPS binding to Plts. Moreover, Stx2 amplified LPS induced P-selectin expression on Plts and mixed PMN–Plts aggregates formation, which led to activation of PMN enhancing dramatically NETs formation. Finally, experiments revealed that endothelial cell damage mediated by PMN in the context of Plts treated with LPS and Stx2 was decreased when NETs were disrupted or when mixed aggregate formation was impeded using an anti-P-selectin antibody. Using a murine model of HUS, systemic endothelial damage/dysfunction was decreased when NETs were disrupted, or when Plts were depleted, indicating that the promotion of netosis by Plts in the context of LPS and Stx2 plays a fundamental role in endothelial toxicity. These results provide insights for the first time into the pivotal role of Plts as enhancers of endothelial damage through NETs promotion in the context of Stx and LPS. Consequently, therapies designed to reduce either the formation of PMN–Plts aggregates or NETs formation could lessen the consequences of endothelial damage in HUS. [ABSTRACT FROM AUTHOR]

Platelets have a well-recognized role in hemostasis and thrombosis, and they are important amplifiers of inflammation and innate immune responses. The formation of DNA extracellular traps (ETs) is a complex cellular mechanism, which occurs in response to microbial infections and sterile inflammation, and results in the release of DNA complexed with histones and various granular proteins. ETs were first discovered in neutrophils (NETs); however, it is now accepted that other leukocytes, including eosinophils (EETs) and monocytes/macrophages (MoETs/METs), can also generate them. Moreover, several types of ETs have been described. Increasing evidence has demonstrated that platelets modulate the formation of ETs. This review summarizes recent findings about the physiopathological role of platelets in the formation of ETs during infection and future perspectives in the field. [ABSTRACT FROM AUTHOR]

The article discusses how bears hibernate for months without experiencing deep vein thrombosis (DVT), which occurs commonly in humans during short-term immobility, and the potential therapeutic target this presents. The authors explain that hibernating bears have antithrombotic platelets with reduced biomarkers of thromboinflammation and that this characteristic is found in other animal species, including humans, suggesting it could be a potential treatment for immobilized patients.

Keywords: platelets; thromboinflammation; neutrophils; endothelial cells; inflammation; sepsis; cancer EN platelets thromboinflammation neutrophils endothelial cells inflammation sepsis cancer 1 3 3 06/02/20 20200528 NES 200528 The word thromboinflammation appeared in 2004 to describe the interactions and cooperation between platelets and neutrophils in the context of arterial in-stent restenosis ([1]). Cognasse et al. broaden our perspective of the immunomodulatory properties of platelets by presenting how platelets can themselves release non-infectious danger signals during storage of platelet concentrates, thus contributing to adverse immune reactions to platelet transfusion. Platelets, thromboinflammation, neutrophils, endothelial cells, inflammation, sepsis, cancer. [Extracted from the article]

Introduction and Objective: 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. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

NETosis is a host defense mechanism associated with inflammation and tissue damage. Experimental models show that platelets and von Willebrand factor (VWF) are key elements for intravascular NETosis. We determined NETosis in septic and burn patients at 1 and 4 days post-admission (dpa). Nucleosomes were elevated in patients. In septics, they correlated with Human Neutrophil Elastase (HNE)-DNA complexes and SOFA score at 1 dpa, and were associated with mortality. Patient's neutrophils had spontaneous NETosis and were unresponsive to stimulation. Although platelet P-selectin and TNF-α were increased in both groups, higher platelet TLR-4 expression, VWF levels and IL-6 were found in septics at 1 dpa. Neither platelet activation markers nor cytokines correlated with nucleosomes or HNE-DNA. Nucleosomes could be indicators of organ damage and predictors of mortality in septic but not in burn patients. Platelet activation, VWF and cytokines do not appear to be key mediators of NETosis in these patient groups. [ABSTRACT FROM AUTHOR]

Neutrophil extracellular traps (NETs) are released in the vasculature. Besides 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 release (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 not induced by platelets from P-selectin-/- mice. Moreover, NETosis was also promoted by P-selectin-Ig fusion protein but not by control Ig. We isolated neutrophils from mice engineered to overproduce soluble P-selectin, the P-selectinΔCT/ΔCT mice. While 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 PAF, ionomycin, or PMA 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. [ABSTRACT FROM AUTHOR]