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 TLR-4 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.

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.

Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets.

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.

Myelin Basic Protein and a Multiple Sclerosis-related MBP-peptide Bind to Oligonucleotides.

Aptamer ligands for myelin basic protein (MBP) were obtained using the systematic evolution of ligand by exponential enrichment (SELEX) method. Two clones were isolated from a pool of oligonucleotides and tested for MBP targeting. Using purified MBP, we demonstrated the binding activity of the aptamers and we also showed the affinity of MBP for oligonucleotides of specific length. Moreover, one selected aptamer competitively inhibited the binding of an MBP-specific antibody to MBP and the aptamer was found more sensitive than a commercial antibody. In addition, we showed the ability of the aptamer to detect myelin-rich regions in paraffin-embedded mouse brain tissue. Therefore, the MBP-binding activity of the selected oligonucleotide may prove useful as a tool for life science and medical research for myelin detection and might be a good lead for testing it in autoimmune diseases such as multiple sclerosis.

Rol de las plaquetas en la formación de trampas de ADN derivadas de neutrófilos / Role of platelets in the formation of neutrophil extracellular traps / 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 patogênico 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.