Platelets: New bricks in the building of neutrophil extracellular traps

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.Fil: Carestia, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Kaufman, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Mecanismos moleculares involucrados en las respuestas inflamatorias mediadas por plaquetas en acidosis

La acidosis es una característica del microambiente inflamatorio presente en situaciones de injuria vascular, tumor, aterosclerosis y enfermedades inflamatorias pulmonares. Las plaquetas además de ser elementos claves en la hemostasia y la trombosis, también juegan un rol importante en estas patologías. En estudios previos mostramos que el descenso del pH apaga las funciones hemostáticas de las plaquetas y promueve respuestas inflamatorias mediadas por la expresión de P-selectina en la superficie plaquetaria. En este trabajo estudiamos los mecanismos moleculares involucrados en estos procesos. Mientras la fosforilación de algunas moléculas involucradas en la activación plaquetaria (Src, Akt y ERK) fue significativamente inhibida en acidosis, la activación de p38 o NFκB no fue modificada o aumentó respectivamente, con el descenso del pH. El incremento en la expresión de P-selectina observado en acidosis se mantuvo en presencia de inhibidores de Src (PP1), PI3K/Akt (Ly294002) y ERK (U0126). Este fenómeno tampoco fue modificado por la incubación individual de las plaquetas con bloqueantes de p38 (SB203580) o NFκB (Ro1069920), pero sí disminuyó significativamente ante la presencia simultánea de ambos inhibidores. En concordancia con el aumento en la expresión de P-selectin, la formación de agregados plaquetas-leucocitos, así como la migración de leucocitos en presencia de plaquetas, aumentó en acidosis y ambos fenómenos fueron revertidos por la presencia conjunta de SB203580 y Ro1069920. Estos resultados indican que las respuestas inflamatorias mediadas por plaquetas en condiciones de acidosis requieren la activación simultánea de p38 y NFκB y plantean la posibilidad de nuevos blancos terapéuticos para el tratamiento de la inflamación.Fil: Etulain, Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Carestia, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Rivadeneyra, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fondevila Sancet, Juan Carlos. Clínica Bazterrica. Servicio de Hematología; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Nucleosomes and neutrophil extracellular traps in septic and burn patients

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 4days post-admission (dpa). Nucleosomes were elevated in patients. In septics, they correlated with Human Neutrophil Elastase (HNE)-DNA complexes and SOFA score at 1dpa, 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 TLR4 expression, VWF levels and IL-6 were found in septics at 1dpa. 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.Fil: Kaufman, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Magosevich, Débora. Clínica Sagrado Corazón; ArgentinaFil: Moreno, María Carolina. Clínica Bazterrica; ArgentinaFil: Guzman, María Alejandra. Gobiernos de la Ciudad de Buenos Aires. Hospital de Quemados Dr. Arturo Umberto Illia; ArgentinaFil: D'Atri, Lina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Carestia, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fandiño, María Eugenia. Clínica Sagrado Corazón; ArgentinaFil: Fondevila, Carlos. Clínica Bazterrica; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Platelets promote macrophage polarization toward pro-inflammatory phenotype and increase survival of septic mice

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.Fil: Carestia, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. University of Calgary; CanadáFil: Mena, Hebe Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Olexen, Cinthia Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Ortiz Wilczyñski, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Negrotto, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Errasti, Andrea Emilse. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaFil: Gomez, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Jenne, Craig N.. University of Calgary; CanadáFil: Carrera Silva, Eugenio Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Neutrophil extracellular traps are involved in the innate immune response to infection with <i>Leptospira</i>

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.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias ExactasFacultad de Ciencias Médica

Neutrophil extracellular traps are involved in the innate immune response to infection with <i>Leptospira</i>

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.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias ExactasFacultad de Ciencias Médica

Papel das plaquetas na formação de armadilhas de DNA derivadas de neutrófilos

Además de ser elementos clave en la hemostasia y trombosis, las plaquetas tienen un rol preponderante en la respuesta inflamatoria e inmune asociada con su capacidad para reconocer patógenos a través de la expresión de los receptores tipo Toll, la secreción de una amplia variedad de citoquinas, quemoquinas y factores de crecimiento almacenados en sus gránulos y por la expresión de moléculas de adhesión que permiten la interacción con otras células vasculares. Como parte de la primera línea de defensa, los neutrófilos controlan la infección por fagocitosis, liberación de proteínas antimicrobianas durante la degranulación o a través de la formación de estructuras tipo red, conocidas como trampas extracelulares de neutrófilos (NETs). Estas están formadas por cromatina, proteasas y proteínas antimicrobianas cuya función principal es atrapar y eliminar bacterias, virus y hongos, impidiendo su diseminación. Además de microorganismos, la formación de NETs es gatillada por moléculas proinflamatorias y plaquetas. Su formación descontrolada puede ocasionar daño tisular y es considerada un mecanismo patogénico de eventos protrombóticos, inflamatorios y autoinmunes. En esta revisión se discute el rol de las plaquetas en la formación de NETs y se destacan los mediadores, estímulos y mecanismos moleculares participantes de este fenómeno, en humanos y modelos murinos.In addition to being key elements in hemostasis and thrombosis, platelets have an important role in inflammatory and innate immune responses. This activity is associated with their capability to recognize pathogens through the expression of TLRs, the secretion of a wide variety of cytokines, chemokines and growth factors stored within their granules and cell adhesion molecule expresssion that enable interaction with other vascular cells. 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 known as 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, thus avoiding their dissemination. Besides microorganisms, NETs formation is also triggered by proinflammatory molecules, and platelets. The uncontrolled formation of NETs might exert tissue damage and has been involved as a pathogenic mechanism of autoimmune and prothrombotic events. In this review, the role of platelets in NET generation is discussed, highlighting the mediators, stimuli and molecular mechanisms involved in this phenomenon, both in human and murine models.Além de serem elementos chave na hemostasia e trombose, as plaquetas têm um papel preponderante na resposta inflamatória e imune associada a sua capacidade para reconhecer patógenos através da expressão dos receptores tipo Toll, a secreção de uma ampla variedade de citocinas, quemocinas e fatores de crescimento armazenados em seus grânulos e pela expressão de moléculas de adesão que permitem a interação com outras células vasculares. Como parte da primeira linha de defesa, os neutrófilos controlam a infecção por fagocitose, liberação de proteínas antimicrobianas durante a degranulação ou através da formação de estruturas tipo rede, conhecidas como armadilhas extracelulares de neutrófilos (NETs). Elas estão formadas por cromatina, proteases e proteínas antimicrobianas cuja função principal é prender e eliminar bactérias, vírus e fungos, impedindo sua disseminação. Além de microorganismos, a formação de NETs é disparada por moléculas pró-inflamatórias e plaquetas. Sua formação descontrolada pode provocar dano tissular e é considerada um mecanismo patogê- nico de eventos pró-trombóticos, inflamatórios e autoimunes. Nesta revisão é discutido o papel das plaquetas na formação de NETs, destacando os mediadores, estímulos e mecanismos moleculares participantes deste fenômeno, em humanos e modelos murídeos.Fil: Carestia, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Kaufman, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

Inhibition of immunothrombosis does not affect pathogen capture and does not promote bacterial dissemination in a mouse model of sepsis

After infection, neutrophils release neutrophil extracellular traps (NETs), decondensed DNA fibers decorated with both nuclear proteins and proteins derived from intracellular granules. These structures have a fundamental role in the development of immunothrombosis; a physiological process mediated by immune cells and molecules from the coagulation system that facilitates the recognition, containment, and destruction of pathogens. Although NETs and immunothrombi are widely hypothesized to be key host defense responses responsible for limiting bacterial dissemination, their actual role in this process has not been formally assessed within the context of a bloodstream infection. Mice were first treated with LPS to generate inflammation (NETs and immunothrombi) and then bacteria dissemination was analyzed by intravital microscopy and colony-forming units (CFU) assay. Blocking NETs or coagulation by the administration of DNase or Argatroban (thrombin inhibitor), respectively, did not modify the percentage of bacteria capture by Kupffer cells, neutrophils or platelets. Moreover, both inhibitors reduced the number of bacteria in the spleen, without modifying CFUs in the liver or lung. In conclusion, we demonstrate that immunothrombi are not necessary to limit the dissemination of bloodstream bacterial infections

Functional responses and molecular mechanisms involved in histone-mediated platelet activation

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.Fil: Carestia, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Rivadeneyra, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Romaniuk, María Albertina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fondevila, Carlos. Clínica Bazterrica;Fil: Negrotto, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin