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BACKGROUND: Venous thromboembolism is a major health problem. After thrombus formation, its resolution is essential to re-establish blood flow, which is crucially mediated by infiltrating neutrophils and monocytes in concert with activated platelets and endothelial cells. Thus, we aimed to modulate leukocyte function during thrombus resolution post-thrombus formation by blocking P-selectin/CD62P-mediated cell interactions. METHODS: Thrombosis was induced by inferior vena cava stenosis through ligation in mice. After 1 day, a P-selectin-blocking antibody or isotype control was administered and thrombus composition and resolution were analyzed. RESULTS: Localizing neutrophils and macrophages in thrombotic lesions of wild-type mice revealed that these cells enter the thrombus and vessel wall from the caudal end. Neutrophils were predominantly present 1 day and monocytes/macrophages 3 days after vessel ligation. Blocking P-selectin reduced circulating platelet-neutrophil and platelet-Ly6Chigh monocyte aggregates near the thrombus, and diminished neutrophils and Ly6Chigh macrophages in the cranial thrombus part compared with isotype-treated controls. Depletion of neutrophils 1 day after thrombus initiation did not phenocopy P-selectin inhibition but led to larger thrombi compared with untreated controls. In vitro, P-selectin enhanced human leukocyte function as P-selectin-coated beads increased reactive oxygen species production by neutrophils and tissue factor expression of classical monocytes. Accordingly, P-selectin inhibition reduced oxidative burst in the thrombus and tissue factor expression in the adjacent vessel wall. Moreover, blocking P-selectin reduced thrombus density determined by scanning electron microscopy and increased urokinase-type plasminogen activator levels in the thrombus, which accelerated caudal fibrin degradation from day 3 to day 14. This accelerated thrombus resolution as thrombus volume declined more rapidly after blocking P-selectin. CONCLUSIONS: Inhibition of P-selectin-dependent activation of monocytes and neutrophils accelerates venous thrombosis resolution due to reduced infiltration and activation of innate immune cells at the site of thrombus formation, which prevents early thrombus stabilization and facilitates fibrinolysis.
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Monocitos , Trombosis , Ratones , Humanos , Animales , Monocitos/patología , Selectina-P , Células Endoteliales , Tromboplastina , Infiltración Neutrófila , NeutrófilosRESUMEN
Changes in Ca2+ influx during proinflammatory stimulation modulates cellular responses, including the subsequent activation of inflammation. Whereas the involvement of Ca2+ has been widely acknowledged, little is known about the role of Na+. Ranolazine, a piperazine derivative and established antianginal drug, is known to reduce intracellular Na+ as well as Ca2+ levels. In stable coronary artery disease patients (n = 51) we observed reduced levels of high-sensitive C-reactive protein (CRP) 3 mo after the start of ranolazine treatment (n = 25) as compared to the control group. Furthermore, we found that in 3,808 acute coronary syndrome patients of the MERLIN-TIMI 36 trial, individuals treated with ranolazine (1,934 patients) showed reduced CRP values compared to placebo-treated patients. The antiinflammatory effects of sodium modulation were further confirmed in an atherosclerotic mouse model. LDL-/- mice on a high-fat diet were treated with ranolazine, resulting in a reduced atherosclerotic plaque burden, increased plaque stability, and reduced activation of the immune system. Pharmacological Na+ inhibition by ranolazine led to reduced express of adhesion molecules and proinflammatory cytokines and reduced adhesion of leukocytes to activated endothelium both in vitro and in vivo. We demonstrate that functional Na+ shuttling is required for a full cellular response to inflammation and that inhibition of Na+ influx results in an attenuated inflammatory reaction. In conclusion, we demonstrate that inhibition of Na+-Ca2+ exchange during inflammation reduces the inflammatory response in human endothelial cells in vitro, in a mouse atherosclerotic disease model, and in human patients.
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Síndrome Coronario Agudo , Proteína C-Reactiva , Fármacos Cardiovasculares , Enfermedad de la Arteria Coronaria , Ranolazina , Bloqueadores de los Canales de Sodio , Sodio , Síndrome Coronario Agudo/tratamiento farmacológico , Animales , Proteína C-Reactiva/análisis , Proteína C-Reactiva/metabolismo , Fármacos Cardiovasculares/farmacología , Fármacos Cardiovasculares/uso terapéutico , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Células Endoteliales/metabolismo , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Ratones , Ranolazina/farmacología , Ranolazina/uso terapéutico , Sodio/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Bloqueadores de los Canales de Sodio/uso terapéuticoRESUMEN
Platelets are unique anucleated blood cells that constantly patrol the vasculature to seal and prevent injuries in a process termed haemostasis. Thereby they rapidly adhere to the subendothelial matrix and recruit further platelets, resulting in platelet aggregates. Apart from their central role in haemostasis, they also kept some of their features inherited by their evolutionary ancestor-the haemocyte, which was also involved in immune defences. Together with leukocytes, platelets fight pathogenic invaders and guide many immune processes. In addition, they rely on several signalling pathways which are also relevant to immune cells. Among these, one of the central signalling hubs is the PI3K pathway. Signalling processes in platelets are unique as they lack a nucleus and therefore transcriptional regulation is absent. As a result, PI3K subclasses fulfil distinct roles in platelets compared to other cells. In contrast to leukocytes, the central PI3K subclass in platelet signalling is PI3K class Iß, which underlines the uniqueness of this cell type and opens new ways for potential platelet-specific pharmacologic inhibition. An overview of platelet function and signalling with emphasis on PI3K subclasses and their respective inhibitors is given in this chapter.
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Plaquetas , Trombosis , Plaquetas/metabolismo , Plaquetas/patología , Hemostasis/fisiología , Humanos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Isoformas de Proteínas/metabolismo , Trombosis/metabolismo , Trombosis/patologíaRESUMEN
Interleukin-4 (IL-4) and its receptors (IL-4R) promote the proliferation and polarization of macrophages. However, it is unknown if IL-4R also influences monocyte homeostasis and if steady state IL-4 levels are sufficient to affect monocytes. Employing full IL-4 receptor alpha knockout mice (IL-4Rα-/- ) and mice with a myeloid-specific deletion of IL-4Rα (IL-4Rαf/f LysMcre ), we show that IL-4 acts as a homeostatic factor regulating circulating monocyte numbers. In the absence of IL-4Rα, murine monocytes in blood were reduced by 50% without altering monocytopoiesis in the bone marrow. This reduction was accompanied by a decrease in monocyte-derived inflammatory cytokines in the plasma. RNA sequencing analysis and immunohistochemical staining of splenic monocytes revealed changes in mRNA and protein levels of anti-apoptotic factors including BIRC6 in IL-4Rα-/- knockout animals. Furthermore, assessment of monocyte lifespan in vivo measuring BrdU+ cells revealed that the lifespan of circulating monocytes was reduced by 55% in IL-4Rα-/- mice, whereas subcutaneously applied IL-4 prolonged it by 75%. Treatment of human monocytes with IL-4 reduced the amount of dying monocytes in vitro. Furthermore, IL-4 stimulation reduced the phosphorylation of proteins involved in the apoptosis pathway, including the phosphorylation of the NFκBp65 protein. In a cohort of human patients, serum IL-4 levels were significantly associated with monocyte counts. In a sterile peritonitis model, reduced monocyte counts resulted in an attenuated recruitment of monocytes upon inflammatory stimulation in IL-4Rαf/f LysMcre mice without changes in overall migratory function. Thus, we identified a homeostatic role of IL-4Rα in regulating the lifespan of monocytes in vivo.
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Interleucina-4/metabolismo , Monocitos , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Animales , Homeostasis , Humanos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Monocitos/metabolismoRESUMEN
PURPOSE OF THE REVIEW: In this review, we summarize current insights into the versatile roles of endothelial cells in atherogenesis. RECENT FINDINGS: The vascular endothelium represents the first barrier that prevents the entry of lipoproteins and leukocytes into the vessel wall, thereby controlling two key events in the pathogenesis of atherosclerosis. Disturbance of endothelial homeostasis increases vascular permeability, inflammation, and cellular trans-differentiation, which not only promotes the build-up of atherosclerotic plaques but is also involved in life-threatening thromboembolic complications such as plaque rupture and erosion. In this review, we focus on recent findings on endothelial lipoprotein transport, inflammation, cellular transitions, and barrier function. By using cutting-edge technologies such as single-cell sequencing, epigenetics, and cell fate mapping, novel regulatory mechanisms and endothelial cell phenotypes have been discovered, which have not only challenged established concepts of endothelial activation, but have also led to a different view of the disease.
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Aterosclerosis , Placa Aterosclerótica , Aterosclerosis/etiología , Células Endoteliales , Endotelio Vascular , Humanos , Inflamación/complicaciones , Placa Aterosclerótica/complicacionesRESUMEN
Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze-thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task.
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Técnicas de Cultivo de Célula/métodos , Criopreservación/métodos , Leucocitos Mononucleares/citología , Supervivencia Celular , Células Cultivadas , Femenino , Citometría de Flujo , Humanos , Recuento de Leucocitos , Leucocitos Mononucleares/metabolismo , MasculinoRESUMEN
BACKGROUND: The IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKß, is the main activator of the inflammatory transcription factor NF-κB, which is constitutively active in many cancers. While several connections between NF-κB signaling and the oncogene c-Myc have been shown, functional links between the signaling molecules are still poorly studied. METHODS: Molecular interactions were shown by co-immunoprecipitation and FRET microscopy. Phosphorylation of c-Myc was shown by kinases assays and its activity by improved reporter gene systems. CRISPR/Cas9-mediated gene knockout and chemical inhibition were used to block IKK activity. The turnover of c-Myc variants was determined by degradation in presence of cycloheximide and by optical pulse-chase experiments.. Immunofluorescence of mouse prostate tissue and bioinformatics of human datasets were applied to correlate IKKα- and c-Myc levels. Cell proliferation was assessed by EdU incorporation and apoptosis by flow cytometry. RESULTS: We show that IKKα and IKKß bind to c-Myc and phosphorylate it at serines 67/71 within a sequence that is highly conserved. Knockout of IKKα decreased c-Myc-activity and increased its T58-phosphorylation, the target site for GSK3ß, triggering polyubiquitination and degradation. c-Myc-mutants mimicking IKK-mediated S67/S71-phosphorylation exhibited slower turnover, higher cell proliferation and lower apoptosis, while the opposite was observed for non-phosphorylatable A67/A71-mutants. A significant positive correlation of c-Myc and IKKα levels was noticed in the prostate epithelium of mice and in a variety of human cancers. CONCLUSIONS: Our data imply that IKKα phosphorylates c-Myc on serines-67/71, thereby stabilizing it, leading to increased transcriptional activity, higher proliferation and decreased apoptosis.
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Quinasa I-kappa B/metabolismo , Inflamación/enzimología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Células HEK293 , Humanos , Quinasa I-kappa B/química , Inflamación/patología , Masculino , Ratones , Modelos Biológicos , Mutación/genética , Fosforilación , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Próstata/metabolismo , Unión Proteica , Estabilidad Proteica , Transcripción GenéticaRESUMEN
Genetically modified mice have advanced our knowledge on platelets in hemostasis and beyond tremendously. However, mouse models harbor certain limitations, including availability of platelet specific transgenic strains, and off-target effects on other cell types. Transfusion of genetically modified platelets into thrombocytopenic mice circumvents these problems. Additionally, ex vivo treatment of platelets prior to transfusion eliminates putative side effects on other cell types. Thrombocytopenia is commonly induced by administration of anti-platelet antibodies, which opsonize platelets to cause rapid clearance. However, antibodies do not differentiate between endogenous or exogenous platelets, impeding transfusion efficacy. In contrast, genetic depletion with the inducible diphtheria toxin receptor (iDTR) system induces thrombocytopenia via megakaryocyte ablation without direct effects on circulating platelets. We compared the iDTR system with antibody-based depletion methods regarding their utility in platelet transfusion experiments, outlining advantages and disadvantages of both approaches. Antibodies led to thrombocytopenia within two hours and allowed the dose-dependent adjustment of the platelet count. The iDTR model caused complete thrombocytopenia within four days, which could be sustained for up to 11 days. Neither platelet depletion approach caused platelet activation. Only the iDTR model allowed efficient platelet transfusion by keeping endogenous platelet levels low and maintaining exogenous platelet levels over longer time periods, thus providing clear advantages over antibody-based methods. Transfused platelets were fully functional in vivo, and our model allowed examination of transgenic platelets. Using donor platelets from already available genetically modified mice or ex vivo treated platelets, may decrease the necessity of platelet-specific mouse strains, diminishing off-target effects and thereby reducing animal numbers.
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Recuento de Plaquetas , Transfusión de Plaquetas , Trombocitopenia , Animales , Plaquetas , Hemostasis , Ratones , Trombocitopenia/genética , Trombocitopenia/terapiaRESUMEN
AIM: Periodontitis results in platelet activation and enhanced risk for cardiovascular disease. As it is currently unknown whether periodontal treatment reverses platelet hyper-reactivity, we aimed to investigate the role of periodontal treatment on platelet activation. MATERIALS AND METHODS: In a prospective controlled therapeutic trial, 52 patients were enrolled and randomly selected for periodontal treatment or monitored without treatment for 3 months. Patient blood was analysed by flow cytometry for platelet activation markers and by light transmission aggregometry for platelet aggregation in response to pro-thrombotic stimuli. RESULTS: In this study, platelet activation in the control group aggravated over the observation period of 3 months, whereas patients that underwent periodontal treatment showed unchanged levels of platelet activation, measured by surface expression of CD62P, CD40L, generation of reactive oxygen production, activation of GPIIb/IIIa and fibrinogen binding. Moreover, platelet turnover, measured by platelet RNA content and platelet aggregation in response to collagen, differed significantly between patients that were treated and those who were untreated. CONCLUSIONS: Subgingival debridement reduces the risk of aggravated platelet activation and therefore might potentially diminish subsequent diseases such as cardiovascular disease in periodontal patients.
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Periodontitis , Activación Plaquetaria , Humanos , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Estudios ProspectivosRESUMEN
Platelets, besides their specialised role in haemostasis and atherothrombosis, actively modulate innate and adaptive immune responses with crucial roles in immune surveillance, inflammation and host defence during infection. An important prerequisite for platelet-mediated changes of immune functions involves direct engagement with different types of leukocytes. Indeed, increased platelet-leukocyte aggregates (PLAs) within the circulation and/or locally at the site of inflammation represent markers of many thrombo-inflammatory diseases, such as cardiovascular diseases, acute lung injury, renal and cerebral inflammation. Therefore, measurement of PLAs could provide an attractive and easily accessible prognostic and/or diagnostic tool for many diseases. To measure PLAs in different (patho-)physiological settings in human and animal models flow cytometric and microscopic approaches have been applied. These techniques represent complementary tools to study different aspects relating to the involvement of leukocyte subtypes and molecules, as well as location of PLAs within tissues, dynamics of their interactions and/or dynamic changes in leukocyte and platelet behaviour. This review summarises various approaches to measure and interpret PLAs and discusses potential experimental factors influencing platelet binding to leukocytes. Furthermore, we summarise insights gained from studies regarding the underlying mechanism of platelet-leukocyte interactions and discuss implications of these interactions in health and disease.
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Plaquetas/fisiología , Leucocitos/fisiología , Agregación Plaquetaria , Pruebas de Función Plaquetaria , Animales , Biomarcadores , Comunicación Celular , Susceptibilidad a Enfermedades , Humanos , Microscopía , Modelos Animales , Imagen Molecular , Activación PlaquetariaRESUMEN
Infection induces platelet activation and consumption, which leads to thrombocytopenia, enhances microvascular thrombosis, impairs microcirculation and eventually triggers disseminated intravascular coagulation (DIC). It is well characterized that endotoxemia results in a pro-inflammatory and pro-coagulatory state, which favors platelet activation. However the early, direct effects of endotoxemia on platelets have not been investigated so far. Therefore we aimed to determine the early effects of the endotoxin lipopolysaccharide (LPS) on platelet function in vivo. In a human endotoxemia model, 15 healthy volunteers were stimulated with LPS (2 ng/kg). Blood was drawn before, 10, 30 and 60 min after LPS challenge and platelet activation analyzed by flow cytometry (GPIIb/IIIa activation, surface CD62P and CD40L, intraplatelet reactive oxygen formation and platelet-leukocyte aggregates) and ELISA (sCD40L, sCD62P and CXCL4). In parallel, blood samples and platelets were spiked with LPS (50 pg/ml) in vitro and monitored over 60 min for the same platelet activation markers. In vitro platelet stimulation with LPS activated platelets independent of the presence of leukocytes and enhanced their adhesion to endothelial cells. In contrast, in vivo no increase in GPIIb/IIIa activation or surface expression of CD62P was observed. However, endotoxemia resulted in a significant drop in platelet count and elevated the plasma CXCL4 levels already 10 min after the LPS challenge. These data indicate that LPS rapidly activates platelets, leading to α-granule release and endothelial adhesion. This might explain the drop in platelet count observed at the onset of endotoxemia.
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Plaquetas/metabolismo , Endotoxemia/sangre , Endotoxemia/diagnóstico , Activación Plaquetaria , Biomarcadores , Ligando de CD40/sangre , Células Endoteliales/metabolismo , Endotoxemia/etiología , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lipopolisacáridos/efectos adversos , Masculino , Selectina-P/sangre , Adhesividad Plaquetaria , Recuento de Plaquetas , Factor Plaquetario 4/sangre , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Beyond their traditional role in haemostasis and thrombosis, platelets are increasingly recognised as immune modulatory cells. Activated platelets and platelet-derived microparticles can bind to leukocytes, which stimulates mutual activation and results in rapid, local release of platelet-derived cytokines. Thereby platelets modulate leukocyte effector functions and contribute to inflammatory and immune responses to injury or infection. Platelets enhance leukocyte extravasation, differentiation and cytokine release. Platelet-neutrophil interactions boost oxidative burst, neutrophil extracellular trap formation and phagocytosis and play an important role in host defence. Platelet interactions with monocytes propagate their differentiation into macrophages, modulate cytokine release and attenuate macrophage functions. Depending on the underlying pathology, platelets can enhance or diminish leukocyte cytokine production, indicating that platelet-leukocyte interactions represent a fine balanced system to restrict excessive inflammation during infection. In atherosclerosis, platelet interaction with neutrophils, monocytes and dendritic cells accelerates key steps of atherogenesis by promoting leukocyte extravasation and foam cell formation. Platelet-leukocyte interactions at sites of atherosclerotic lesions destabilise atherosclerotic plaques and promote plaque rupture. Leukocytes in turn also modulate platelet function and production, which either results in enhanced platelet destruction or increased platelet production. This review aims to summarise the key effects of platelet-leukocyte interactions in inflammation, infection and atherosclerosis.
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BACKGROUND: During infection, neutrophil extracellular traps (NETs) are associated with severity of pulmonary diseases such as acute respiratory disease syndrome. NETs induce subsequent immune responses, are directly cytotoxic to pulmonary cells, and are highly procoagulant. Anticoagulation treatment was shown to reduce in-hospital mortality, indicating thromboinflammatory complications. However, data are sparsely available on the involvement of NETs in secondary events after virus clearance, which can lead to persistent lung damage and postacute sequelae with chronic fatigue and dyspnea. OBJECTIVES: This study focuses on late-phase events using a murine model of viral lung infection with postacute sequelae after virus resolution. METHODS: C57BL/6JRj mice were infected intranasally with the betacoronavirus murine coronavirus (MCoV, strain MHV-A95), and tissue samples were collected after 2, 4, and 10 days. For NET modulation, mice were pretreated with OM-85 or GSK484 and DNase I were administered intraperitoneally between days 2 to 5 and days 4 to 7, respectively. RESULTS: Rapid, platelet-attributed thrombus formation was followed by a second, late phase of thromboinflammation. This phase was characterized by negligible virus titers but pronounced tissue damage, apoptosis, oxidative DNA damage, and presence of NETs. Inhibition of NETs during the acute phase did not impact virus burden but decreased lung cell apoptosis by 67% and oxidative stress by 94%. Prevention of neutrophil activation by immune training before virus infection reduced damage by 75%, NETs by 31%, and pulmonary thrombi by 93%. CONCLUSION: NETs are detrimental inducers of tissue damage during respiratory virus infection but do not contribute to virus clearance.
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Infecciones por Coronavirus , Coronavirus , Trampas Extracelulares , Trombosis , Animales , Ratones , Neutrófilos , Tromboinflamación , Modelos Animales de Enfermedad , Inflamación/complicaciones , Trombosis/complicaciones , Ratones Endogámicos C57BL , Pulmón , Infecciones por Coronavirus/complicacionesRESUMEN
BACKGROUND: Posthepatectomy liver failure (PHLF) represents a life-threatening complication with limited therapeutic options. Neutrophils play a critical and dynamic role during regeneratory processes, but their role in human liver regeneration is incompletely understood, especially as underlying liver disease, detectable in the majority of patients, critically affects hepatic regeneration. Here we explored intrahepatic neutrophil accumulation and neutrophil extracellular traps (NETs) in patients with PHLF and validated the functional relevance of NETs in a murine partial hepatectomy (PHx) model. METHODS: We investigated the influx of neutrophils, macrophages, eosinophils, and mast cells and the presence of their respective extracellular traps in liver biopsies of 35 patients undergoing hepatectomy (10 patients with PHLF) before and after the initiation of liver regeneration by fluorescence microscopy. In addition, NET formation and neutrophil activation were confirmed by plasma analysis of 99 patients (24 patients with PHLF) before and up to 5 days after surgery. Furthermore, we inhibited NETs via DNase I in a murine PHx model of mice with metabolically induced liver disease. RESULTS: We detected rapid intrahepatic neutrophil accumulation, elevated levels of myeloperoxidase release, and NET formation in regenerating human livers, with a significantly higher increase of infiltrating neutrophils and NETs in patients with PHLF. Circulating markers of neutrophil activation, including elastase, myeloperoxidase, and citrullinated histone H3, correlated with markers of liver injury. In a murine PHx model, we showed that the inhibition of NET accelerated hepatocyte proliferation and liver regeneration. CONCLUSIONS: Patients with PHLF showed accelerated intrahepatic neutrophil infiltration and NET formation, which were associated with liver damage. Further, we identified postsurgical myeloperoxidase levels as predictive markers for adverse outcomes and observed that blocking NETs in a murine PHx model accelerated tissue regeneration.
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Trampas Extracelulares , Hiperplasia Nodular Focal , Fallo Hepático , Humanos , Animales , Ratones , Neutrófilos , Fallo Hepático/etiología , PeroxidasaRESUMEN
The main function of the epidermis is to protect us against a multitude of hostile attacks from the environment. Its main cell type, the keratinocytes have a sophisticated system of different proteins and lipids available to form the cornified envelope, which is responsible for the barrier function of the skin. During ageing, dramatic changes are taking place. Some proteins of the SPRR-, S100- and LCE3-family are massively up-regulated, whereas others like loricrin, filaggrin and the LCE1&2 protein families are significantly down-regulated. The latter ones are known to be under control of calcium and/or 'calcium response elements'. We were able to show that the calcium peak specific for the stratum granulosum, which is the site where loricrin and the LCE1&2 families are synthesized, is reduced during ageing. The resulting cornified envelope in old skin has an extensively changed composition on the molecular level compared to young skin. This knowledge is of critical importance to understand chronic wound formation and ulcers in old age.
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Proteínas Ricas en Prolina del Estrato Córneo/genética , Epidermis/fisiología , Queratinocitos/fisiología , Envejecimiento de la Piel/genética , Transcriptoma , Adolescente , Adulto , Anciano , Calcio/metabolismo , Calgranulina B/genética , Niño , Preescolar , Células Epidérmicas , Femenino , Proteínas Filagrina , Prepucio/citología , Prepucio/fisiología , Humanos , Lactante , Recién Nacido , Proteínas de Filamentos Intermediarios/genética , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Proteínas/genética , Úlcera Cutánea/genética , Adulto JovenRESUMEN
Atherosclerosis is a chronic, inflammatory disease of the vessel wall where triggered immune cells bind to inflamed endothelium, extravasate and sustain local inflammation. Leukocyte adhesion and extravasation are mediated by adhesion molecules expressed by activated endothelial cells, like intercellular adhesion molecule 1 (ICAM-1). Extracellular adherence protein (Eap) from Staphylococcus aureus binds to a plethora of extracellular matrix proteins, including ICAM-1 and its ligands macrophage-1 antigen (Mac-1, αMß2) and lymphocyte function-associated antigen 1 (LFA-1, αLß2), thereby disrupting the interaction between leukocytes and endothelial cells. We aimed to use Eap to inhibit the interaction of leukocytes with activated endothelial cells in settings of developing and established atherosclerosis in apolipoprotein E (ApoE) deficient mice on high-fat diet. In developing atherosclerosis, Eap treatment reduced circulating platelet-neutrophil aggregates as well as infiltration of T cells and neutrophils into the growing plaque, accompanied by reduced formation of neutrophil extracellular traps (NETs). However, plaque size did not change. Intervention treatment with Eap of already established plaques did not result in cellular or morphological plaque changes, whereas T cell infiltration was increased and thereby again modulated by Eap. We conclude that although Eap leads to cellular changes in developing plaques, clinical implications might be limited as patients are usually treated at a more advanced stage of disease progression. Hence, usage of Eap might be an interesting mechanistic tool for cellular infiltration during plaque development in basic research but not a clinical target.
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Aterosclerosis , Placa Aterosclerótica , Ratones , Animales , Molécula 1 de Adhesión Intercelular/genética , Staphylococcus aureus/metabolismo , Células Endoteliales/metabolismo , Antígeno-1 Asociado a Función de Linfocito/genética , FenotipoRESUMEN
AIMS: Ischaemia-reperfusion injury (IRI) following myocardial infarction remains a challenging topic in acute cardiac care and consecutively arising heart failure represents a severe long-term consequence. The extent of neutrophil infiltration and neutrophil-mediated cellular damage are thought to be aggravating factors enhancing primary tissue injury. Toll-like receptor 9 was found to be involved in neutrophil activation as well as chemotaxis and may represent a target in modulating IRI, aspects we aimed to illuminate by pharmacological inhibition of the receptor. METHODS AND RESULTS: Forty-nine male adult Sprague-Dawley rats were used. IRI was induced by occlusion of the left coronary artery and subsequent snare removal after 30 min. Oligonucleotide (ODN) 2088, a toll-like receptor 9 (TLR9) antagonist, control-ODN, or DNase, were administered at the time of reperfusion and over 24 h via a mini-osmotic pump. The hearts were harvested 24 h or 4 weeks after left coronary artery occlusion and immunohistochemical staining was performed. Echocardiography was done after 1 and 4 weeks to determine ventricular function. Inhibition of TLR9 by ODN 2088 led to left ventricular wall thinning (P = 0.003) in association with drastically enhanced neutrophil infiltration (P = 0.005) and increased markers of tissue damage. Additionally, an up-regulation of the chemotactic receptor CXCR2 (P = 0.046) was found after TLR9 inhibition. No such effects were observed in control-ODN or DNase-treated animals. We did not observe changes in monocyte content or subset distribution, hinting towards neutrophils as the primary mediators of the exerted tissue injury. CONCLUSIONS: Our data indicate a TLR9-dependent, negative regulation of neutrophil infiltration. Blockage of TLR9 appears to prevent the down-regulation of CXCR2, followed by an uncontrolled migration of neutrophils towards the area of infarction and the exertion of disproportional tissue injury resulting in potential aneurysm formation. In comparison with previous studies conducted in TLR-/- mice, we deliberately chose a transient pharmacological inhibition of TLR9 to highlight effects occurring in the first 24 h following IRI.
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Infarto del Miocardio , Receptor Toll-Like 9 , Ratas , Ratones , Masculino , Animales , Receptor Toll-Like 9/uso terapéutico , Ratas Sprague-Dawley , Infarto del Miocardio/tratamiento farmacológico , Corazón , Vasos CoronariosRESUMEN
Training of the innate immune system with orally ingested bacterial extracts was demonstrated to have beneficial effects on infection clearance and disease outcome. The aim of our study was to identify cellular and molecular processes responsible for these immunological benefits. We used a murine coronavirus (MCoV) A59 mouse model treated with the immune activating bacterial extract Broncho-Vaxom (BV) OM-85. Tissue samples were analysed with qPCR, RNA sequencing, histology, and flow cytometry. After BV OM-85 treatment, interstitial macrophages accumulated in lung tissue leading to a faster response of type I interferon (IFN) signalling after MCoV infection resulting in overall lung tissue protection. Moreover, RNA sequencing showed that lung tissue from mice receiving BV OM-85 resembled an intermediate stage between healthy and viral infected lung tissue at day 4, indicating a faster return to normal tissue homoeostasis. The pharmacologic effect was mimicked by adoptively transferring naive lung macrophages into lungs from recipient mice before virus infection. The beneficial effect of BV OM-85 was abolished when inhibiting initial type I IFN signalling. Overall, our data suggest that BV OM-85 enhances lung macrophages allowing for a faster IFN response towards a viral challenge as part of the oral-induced innate immune system training.
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Adyuvantes Inmunológicos , Betacoronavirus , Animales , Bacterias , Inmunidad Innata , Pulmón , Macrófagos , RatonesRESUMEN
According to genome-wide RNA sequencing data from human and mouse platelets, adipose triglyceride lipase (ATGL), the main lipase catalyzing triglyceride (TG) hydrolysis in cytosolic lipid droplets (LD) at neutral pH, is expressed in platelets. Currently, it is elusive to whether common lipolytic enzymes are involved in the degradation of TG in platelets. Since the consequences of ATGL deficiency in platelets are unknown, we used whole-body and platelet-specific (plat)Atgl-deficient (-/-) mice to investigate the loss of ATGL on platelet function. Our results showed that platelets accumulate only a few LD due to lack of ATGL. Stimulation with platelet-activating agonists resulted in comparable platelet activation in Atgl-/-, platAtgl-/-, and wild-type mice. Measurement of mitochondrial respiration revealed a decreased oxygen consumption rate in platelets from Atgl-/- but not from platAtgl-/- mice. Of note, global loss of ATGL was associated with an anti-thrombogenic phenotype, which was evident by reduced thrombus formation in collagen-coated channels in vitro despite unchanged bleeding and occlusion times in vivo. We conclude that genetic deletion of ATGL affects collagen-induced thrombosis without pathological bleeding and platelet activation.