RESUMEN
ABSTRACT: Background : Mesenchymal stem cells (MSCs) can be activated by different bacterial toxins. Lipopolysaccharides and Shiga Toxin (Stx) are the main toxins necessary for hemolytic uremic syndrome development. The main etiological event in this disease is endothelial damage that causes glomerular destruction. Considering the repairing properties of MSC, we aimed to study the response of MSC derived from induced pluripotent stem cells (iPSC-MSC) to LPS and/or Stx and its effect on the restoration of injured endothelial cells. Methods : iPSC-MSC were treated with LPS and or/Stx for 24 h and secretion of cytokines, adhesion, and migration were measured in response to these toxins. In addition, conditioned media from treated iPSC-MSC were collected and used for proteomics analysis and evaluation of endothelial cell healing and tubulogenesis using human microvascular endothelial cells 1 as a source of endothelial cells. Results : The results obtained showed that LPS induced a proinflammatory profile on iPSC-MSC, whereas Stx effects were less evident, even though cells expressed the Gb 3 receptor. Moreover, LPS induced on iPSC-MSC an increment in migration and adhesion to a gelatin substrate. Addition of conditioned media of iPSC-MSC treated with LPS + Stx, decreased the capacity of human microvascular endothelial cells 1 to close a wound, and did not favor tubulogenesis. Proteomic analysis of iPSC-MSC treated with LPS and/or Stx revealed specific protein secretion patterns that support the functional results described. Conclusions : iPSC-MSC activated by LPS acquired a proinflammatory profile that induces migration and adhesion to extracellular matrix proteins but the addition of Stx did not activate any repair program to ameliorate endothelial damage, indicating that the use of iPSC-MSC to regenerate endothelial injury caused by LPS and/or Stx in hemolytic uremic syndrome could not be the best option to consider to regenerate a tissue injury.
Asunto(s)
Síndrome Hemolítico-Urémico , Células Madre Pluripotentes Inducidas , Humanos , Toxina Shiga , Lipopolisacáridos/farmacología , Células Endoteliales/metabolismo , Medios de Cultivo Condicionados , ProteómicaRESUMEN
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.
Asunto(s)
Trampas Extracelulares , Síndrome Hemolítico-Urémico , Trombosis , Animales , Niño , Células Endoteliales/metabolismo , Trampas Extracelulares/metabolismo , Humanos , Lipopolisacáridos/toxicidad , Ratones , Toxina Shiga , Trombosis/complicacionesRESUMEN
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.
Asunto(s)
Plaquetas/metabolismo , Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Activación Plaquetaria , Transducción de Señal , Células Endoteliales/metabolismo , Humanos , Lipopéptidos/inmunología , Lipopolisacáridos/inmunología , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/inmunología , Receptores de Superficie Celular/metabolismoRESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Espacio Extracelular/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Acidosis/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Apoptosis/efectos de los fármacos , Aspirina/farmacología , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/prevención & control , Western Blotting , Permeabilidad Capilar/efectos de los fármacos , ADN/biosíntesis , ADN/genética , Dexametasona/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Lavado Peritoneal , Sulfonas/farmacologíaRESUMEN
OBJECTIVE: Megakaryo/thrombopoiesis is a complex process regulated by multiple signals provided by the bone marrow microenvironment. Because macrophages are relevant components of the bone marrow stroma and their activation induces an upregulation of molecules that can regulate hematopoiesis, we analyzed the impact of these cells on the control of megakaryocyte development and platelet biogenesis. MATERIALS AND METHODS: The different stages of megakaryo/thrombopoiesis were analyzed by flow cytometry using an in vitro model of human cord blood CD34(+) cells stimulated with thrombopoietin in either a transwell system or conditioned media from monocyte-derived macrophages isolated from peripheral blood. Cytokines secreted from macrophages were characterized by protein array and enzyme-linked immunosorbent assay. RESULTS: Resting macrophages released soluble factors that promoted megakaryocyte growth, cell ploidy, a size increase, proplatelet production, and platelet release. Lipopolysaccharide stimulation triggered the secretion of cytokines that exerted opposite effects together with a dramatic switch of CD34(+) commitment to the megakaryocytic lineage toward the myeloid lineage. Neutralization of interleukin-8 released by stimulated macrophages partially reversed the inhibition of megakaryocyte growth. Activation of nuclear factor κB had a major role in the synthesis of molecules involved in the megakaryocyte inhibition mediated by lipopolysaccharide-stimulated macrophages. CONCLUSIONS: Our study extends our understanding about the role of the bone marrow microenvironment in the regulation of megakaryo/thrombopoiesis by showing that soluble factors derived from macrophages positively or negatively control megakaryocyte growth, differentiation, maturation, and their ability to produce platelets.
Asunto(s)
Citocinas/farmacología , Macrófagos/metabolismo , Comunicación Paracrina , Trombopoyesis/efectos de los fármacos , Antígenos CD34/metabolismo , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Células Cultivadas , Quimiocinas/farmacología , Técnicas de Cocultivo , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Sangre Fetal/citología , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Lipopolisacáridos/farmacología , Macrófagos/citología , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , FN-kappa B/metabolismo , Trombopoyetina/farmacologíaRESUMEN
El objetivo de este proyecto fue estudiar los efectos del tratamiento con LIP-CLOD en el compartimiento megacariocítico y así determinar los posibles efectos secundarios del tratamiento con liposomas para determinar su uso potencial en tratamientos de PTI. Para cumplir con este objetivo durante el transcurso de la beca hemos desarrollado los siguientes puntos:. Evaluar la expresión RNA de citoquinas implicadas en la regulación de la megacariopoyesis. Evaluación de la expresión de c-Mpl receptor de trombopoietina. Determinar el papel regulatorio de las células adherentes de medula ósea y bazo, entre los que se encuentran los macrófagos, sobre el compartimiento gacariocítico. Evaluar el efecto del oxido nítrico sobre el compartimiento megacariocítico. Evaluar el efecto del LIP-CLOD en ratones con trombocitopenia severa inducida por citostáticos como el Carboplatino.
Asunto(s)
Ratones , Liposomas , Trombocitopenia , Ácido Clodrónico , BecasRESUMEN
El objetivo de este proyecto fue estudiar los efectos del tratamiento con LIP-CLOD en el compartimiento megacariocítico y así determinar los posibles efectos secundarios del tratamiento con liposomas para determinar su uso potencial en tratamientos de PTI. Para cumplir con este objetivo durante el transcurso de la beca hemos desarrollado los siguientes puntos:. Evaluar la expresión RNA de citoquinas implicadas en la regulación de la megacariopoyesis. Evaluación de la expresión de c-Mpl receptor de trombopoietina. Determinar el papel regulatorio de las células adherentes de medula ósea y bazo, entre los que se encuentran los macrófagos, sobre el compartimiento gacariocítico. Evaluar el efecto del oxido nítrico sobre el compartimiento megacariocítico. Evaluar el efecto del LIP-CLOD en ratones con trombocitopenia severa inducida por citostáticos como el Carboplatino.
Asunto(s)
Ratones , Trombocitopenia , Ácido Clodrónico , Liposomas , BecasRESUMEN
El objetivo de este proyecto fue estudiar los efectos del tratamiento con LIP-CLOD en el compartimiento megacariocítico y así determinar los posibles efectos secundarios del tratamiento con liposomas para determinar su uso potencial en tratamientos de PTI. Para cumplir con este objetivo durante el transcurso de la beca hemos desarrollado los siguientes puntos:. Evaluar la expresión RNA de citoquinas implicadas en la regulación de la megacariopoyesis. Evaluación de la expresión de c-Mpl receptor de trombopoietina. Determinar el papel regulatorio de las células adherentes de medula ósea y bazo, entre los que se encuentran los macrófagos, sobre el compartimiento gacariocítico. Evaluar el efecto del oxido nítrico sobre el compartimiento megacariocítico. Evaluar el efecto del LIP-CLOD en ratones con trombocitopenia severa inducida por citostáticos como el Carboplatino.
