RESUMEN
BACKGROUND: Hemostasis is a tightly regulated physiological process to rapidly induce hemostatic plugs at sites of vascular injury. Inappropriate activation of this process may lead to thrombosis, i.e. pathological blood clot formation in uninjured vessels or on atherosclerotic lesions. ATP release through Pannexin1 (Panx1) membrane channels contributes to collagen-induced platelet aggregation in vitro. OBJECTIVE: To investigate the effects of genetic and pharmacological inhibition of Panx1 on hemostasis and thrombosis in vivo. RESULTS: Bleeding time after tail clipping was increased by 2.5-fold in Panx1-/- mice compared to wild-type controls, suggesting that Panx1 deficiency impairs primary hemostasis. Wire myography on mesenteric arteries revealed diminished vasoconstriction in response to phenylephrine or U446619 in Panx1-/- mice. Mice with platelet-specific deletion of Panx1 (Panx1PDel) displayed 2-fold longer tail bleeding times than Panx1fl/fl controls. Moreover, venous thromboembolism (VTE) after injection of collagen/epinephrine in the jugular vein was reduced in Panx1-/- and Panx1PDel mice. Panx1PDel mice also showed reduced FeCl3-induced thrombosis in mesenteric arteries. BrilliantBlue-FCF, a Panx1 channel inhibitor, decreased collagen-induced platelet aggregation in vitro, increased tail bleeding time and reduced VTE in wild-type mice. Furthermore, we developed a specific Panx1 blocking antibody targeting a Panx1 extracellular loop, which reduced ATP release from platelets in vitro. Treating wild-type mice with this antibody increased tail bleeding time and decreased VTE compared to control antibody. CONCLUSIONS: Panx1 channel deletion or inhibition diminishes clot formation during hemostasis and thrombosis in vivo. Blocking Panx1 channels may be an attractive strategy for modulating platelet aggregation in thrombotic disease.
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Conexinas/antagonistas & inhibidores , Hemostasis/fisiología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Trombosis/terapia , Animales , Humanos , Masculino , RatonesRESUMEN
Ammonia detoxification and gluconeogenesis are major hepatic functions mutually connected through amino acid metabolism. The liver is rich in glutamate dehydrogenase (GDH) that catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate and ammonia, thus bridging amino acid-to-glucose pathways. Here we generated inducible liver-specific GDH-knockout mice (HepGlud1-/- ) to explore the role of hepatic GDH on metabolic homeostasis. Investigation of nitrogen metabolism revealed altered ammonia homeostasis in HepGlud1-/- mice characterized by increased circulating ammonia associated with reduced detoxification process into urea. The abrogation of hepatic GDH also modified energy homeostasis. In the fasting state, HepGlud1-/- mice could barely produce glucose in response to alanine due to impaired liver gluconeogenesis. Compared with control mice, lipid consumption in HepGlud1-/- mice was favored over carbohydrates as a compensatory energy fuel. The changes in energy partitioning induced by the lack of liver GDH modified the circadian rhythm of food intake. Overall, this study demonstrates the central role of hepatic GDH as a major regulator for the maintenance of ammonia and whole-body energy homeostasis.
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Amoníaco/metabolismo , Gluconeogénesis/fisiología , Glutamato Deshidrogenasa/metabolismo , Hígado/metabolismo , Animales , Femenino , Gluconeogénesis/genética , Homeostasis/genética , Homeostasis/fisiología , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Hígado/enzimología , Masculino , Ratones , Ratones Noqueados , Ratones Mutantes , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismoRESUMEN
Recruitment of circulating monocytes is critical for tumour angiogenesis. However, how human monocyte subpopulations extravasate to tumours is unclear. Here we show mechanisms of extravasation of human CD14dimCD16+ patrolling and CD14+CD16+ intermediate proangiogenic monocytes (HPMo), using human tumour xenograft models and live imaging of transmigration. IFNγ promotes an increase of the chemokine CX3CL1 on vessel lumen, imposing continuous crawling to HPMo and making these monocytes insensitive to chemokines required for their extravasation. Expression of the angiogenic factor VEGF and the inflammatory cytokine TNF by tumour cells enables HPMo extravasation by inducing GATA3-mediated repression of CX3CL1 expression. Recruited HPMo boosts angiogenesis by secreting MMP9 leading to release of matrix-bound VEGF-A, which amplifies the entry of more HPMo into tumours. Uncovering the extravasation cascade of HPMo sets the stage for future tumour therapies.
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Adenocarcinoma/inmunología , Neoplasias de la Mama/inmunología , Movimiento Celular/inmunología , Neoplasias Colorrectales/inmunología , Inflamación/inmunología , Monocitos/inmunología , Neovascularización Patológica/inmunología , Animales , Línea Celular Tumoral , Quimiocina CX3CL1/inmunología , Factor de Transcripción GATA3/inmunología , Humanos , Interferón gamma/inmunología , Metaloproteinasa 9 de la Matriz/inmunología , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Factor de Necrosis Tumoral alfa/inmunología , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Leukocyte recruitment is an important process in combating pathogens. The largest class of circulating leukocytes are neutrophils, which rapidly invade inflamed tissue, followed by inflammatory Ly6C+ monocytes. Ly6Clow monocytes patrol the endothelial wall routinely in the steady state. We recently reported early luminal recruitment of Ly6Clow monocytes, which preceded and orchestrated neutrophil arrival and extravasation in response to TLR7/8-mediated vascular inflammation. Here we dissected the kinetics of recruitment of monocytes and neutrophils and examined the dynamics of Ly6Clow monocytes in response to several other Toll-like receptor (TLR) agonists, using intravital confocal microscopy. We observed two types of kinetics in mesenteric veins. TLR2, TLR5 and TLR9 agonists caused early monocyte and neutrophil influx whereas TLR3 and TLR4 agonists rapidly recruited neutrophils and caused Ly6Clow monocytes to arrive at low levels later on. All TLR agonists, except TLR9, led Ly6Clow monocytes to meticulously patrol the vascular wall. Finally, these monocytes released pro-inflammatory cytokines and chemokines implicated in neutrophil recruitment in response to TLR2, TLR4, and TLR9 stimulation but not to TLR3 and TLR5 agonists. These results refine our understanding of the early events in the leukocyte recruitment cascade, including the patrolling behavior of Ly6Clow monocytes, in TLR-mediated acute vascular inflammation.
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Endotelio/inmunología , Inflamación/inmunología , Monocitos/inmunología , Infiltración Neutrófila , Receptores Toll-Like/inmunología , Animales , Antígenos Ly/inmunología , Antígenos Ly/metabolismo , Quimiocinas/biosíntesis , Quimiocinas/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Cinética , Ratones , Monocitos/metabolismo , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/inmunología , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismoRESUMEN
Inflammation is characterized by the recruitment of leukocytes from the bloodstream. The rapid arrival of neutrophils is followed by a wave of inflammatory lymphocyte antigen 6 complex (Ly6C)-positive monocytes. In contrast Ly6C(low) monocytes survey the endothelium in the steady state, but their role in inflammation is still unclear. Here, using confocal intravital microscopy, we show that upon Toll-like receptor 7/8 (TLR7/8)-mediated inflammation of mesenteric veins, platelet activation drives the rapid mobilization of Ly6C(low) monocytes to the luminal side of the endothelium. After repeatedly interacting with platelets, Ly6C(low) monocytes commit to a meticulous patrolling of the endothelial wall and orchestrate the subsequent arrival and extravasation of neutrophils through the production of proinflammatory cytokines and chemokines. At a molecular level, we show that cysteine-rich protein 61 (CYR61)/CYR61 connective tissue growth factor nephroblastoma overexpressed 1 (CCN1) protein is released by activated platelets and enables the recruitment of Ly6C(low) monocytes upon vascular inflammation. In addition endothelium-bound CCN1 sustains the adequate patrolling of Ly6C(low) monocytes both in the steady state and under inflammatory conditions. Blocking CCN1 or platelets with specific antibodies impaired the early arrival of Ly6C(low) monocytes and abolished the recruitment of neutrophils. These results refine the leukocyte recruitment cascade model by introducing endothelium-bound CCN1 as an inflammation mediator and by demonstrating a role for platelets and patrolling Ly6C(low) monocytes in acute vascular inflammation.
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Antígenos Ly/análisis , Proteína 61 Rica en Cisteína/fisiología , Monocitos/fisiología , Vasculitis/etiología , Animales , Plaquetas/fisiología , Movimiento Celular , Ratones , Ratones Endogámicos C57BL , Neutrófilos/fisiología , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 8/fisiologíaRESUMEN
Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest. Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes. The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.
RESUMEN
Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite's ability to modulate host signalling pathways and immune responses.
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Proteasas de Ácido Aspártico/metabolismo , Aparato de Golgi/enzimología , Interacciones Huésped-Parásitos/fisiología , Toxoplasma/patogenicidad , Toxoplasmosis/enzimología , Animales , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Técnicas de Inactivación de Genes , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Transporte de Proteínas , Reacción en Cadena en Tiempo Real de la Polimerasa , Toxoplasma/enzimología , TransfecciónRESUMEN
The recruitment of dendritic cells to sites of infections and their migration to lymph nodes is fundamental for antigen processing and presentation to T cells. In the present study, we showed that antibody blockade of junctional adhesion molecule C (JAM-C) on endothelial cells removed JAM-C away from junctions and increased vascular permeability after L. major infection. This has multiple consequences on the output of the immune response. In resistant C57BL/6 and susceptible BALB/c mice, we found higher numbers of innate immune cells migrating from blood to the site of infection. The subsequent migration of dendritic cells (DCs) from the skin to the draining lymph node was also improved, thereby boosting the induction of the adaptive immune response. In C57BL/6 mice, JAM-C blockade after L. major injection led to an enhanced IFN-γ dominated T helper 1 (Th1) response with reduced skin lesions and parasite burden. Conversely, anti JAM-C treatment increased the IL-4-driven T helper 2 (Th2) response in BALB/c mice with disease exacerbation. Overall, our results show that JAM-C blockade can finely-tune the innate cell migration and accelerate the consequent immune response to L. major without changing the type of the T helper cell response.
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Moléculas de Adhesión Celular/inmunología , Células Dendríticas/inmunología , Inmunidad Celular , Inmunidad Innata , Inmunoglobulinas/inmunología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Animales , Células Dendríticas/patología , Femenino , Leishmaniasis Cutánea/patología , Ratones , Ratones Endogámicos BALB C , Piel/inmunología , Piel/parasitología , Piel/patología , Células TH1/inmunología , Células TH1/patología , Células Th2/inmunología , Células Th2/patologíaRESUMEN
Cystein-rich protein 61 (CYR61/CCN1) is a component of the extracellular matrix, which is produced and secreted by several cell types including endothelial cells, fibroblasts and smooth muscle cells. CCN1 has been implicated in leukocyte migration and the inflammatory process, but it is also involved in cardiovascular development and carcinogenesis. It exerts its functions through binding to multiple integrins present in many different cell types. This multiplicity in function is now known to contribute to the diverse array of cellular processes it can regulate. The expression of CCN1 is tightly regulated by cytokines and growth factors. However, CCN1 can directly modulate cell adhesion and migratory processes whilst simultaneously regulating the production of other cytokines and chemokines through paracrine and autocrine feedback loops. This complex functionality of CCN1 has highlighted the pivotal role this molecule can play in regulating the immunosurveillance process. Furthermore, CCN1 has now emerged as an important partner when targeting components of the infectious or chronic inflammatory disease processes such as atherosclerosis or rheumatoid arthritis. This review will focus on CYR61/CCN1 and its ability to control the migration of leukocytes, the production of cytokines and cell proliferation or senescence at the site of inflammation.
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Movimiento Celular , Proteína 61 Rica en Cisteína/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Animales , Adhesión Celular , Muerte Celular , Proliferación Celular , Supervivencia Celular , Senescencia Celular , Proteína 61 Rica en Cisteína/genética , Citocinas/biosíntesis , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Homeostasis , Humanos , Integrinas/metabolismo , Unión ProteicaRESUMEN
Thymic epithelial cells (TEC) are heterogeneous stromal cells that generate microenvironments required for the formation of T cells within the thymus. Defects in TEC lead to immunodeficiency or autoimmunity. Here we identify TEC as the major source of cysteine-rich protein 61 (CYR61), a matricellular protein implicated in cell proliferation and migration. Binding of CYR61 to LFA-1, ICAM-1 and integrin α6 supports the adhesion of TEC and thymocytes as well as their interaction. Treatment of thymic lobes with recombinant CYR61 expands the stromal compartment by inducing the proliferation of TEC and activates Akt signalling. Engraftment of CYR61-overexpressing thymic lobes into athymic nude mice drastically boosts the yield of thymic output via expansion of TEC. This increases the space for the recruitment of circulating hematopoietic progenitors and the development of T cells. Our discovery paves the way for therapeutic interventions designed to restore thymus stroma and T-cell generation.
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Proteína 61 Rica en Cisteína/metabolismo , Células Epiteliales/citología , Células Madre/citología , Linfocitos T/citología , Timo/citología , Animales , Adhesión Celular , Proliferación Celular , Células Cultivadas , Proteína 61 Rica en Cisteína/genética , Células Epiteliales/metabolismo , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Unión Proteica , Células Madre/metabolismo , Linfocitos T/metabolismo , Timo/metabolismoRESUMEN
Uncoupling protein 2 (UCP2) belongs to the family of mitochondrial carriers. Here, we highlight recent findings regarding UCP2 function in the immune system. UCP2 controls immune cell activation by modulating MAPK pathways and the production of mitochondrial reactive oxygen species. In several models of infection, inflammation and autoimmunity, a regulatory impact of UCP2 was demonstrated by its direct implication in the production of cytokines and nitric oxide and in cell migration. In addition, UCP2 is reported as a key protein for oxidation of fatty acids, glutamine and glucose. Therefore we present a model of how the regulation of nutrient oxidation by UCP2 promotes immune cell activation.
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Inmunidad , Canales Iónicos/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Genómica , Humanos , Canales Iónicos/deficiencia , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas Mitocondriales/deficiencia , Transducción de Señal , Proteína Desacopladora 2Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Células Dendríticas/fisiología , Homeostasis/fisiología , Tejido Linfoide/citología , Animales , Linfocitos T CD4-Positivos/inmunología , Humanos , Ganglios Linfáticos/fisiología , Sistema Linfático/fisiología , Linfocitos/fisiología , Tejido Linfoide/fisiologíaRESUMEN
Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane and is reported to uncouple respiration from ATP synthesis. Our observation that the amino acid glutamine specifically induces UCP2 protein expression prompted us to investigate metabolic consequences of a UCP2 knockdown (Ucp2-KO) when glutamine is offered as a substrate. We found that Ucp2-KO macrophages incubated in the presence of glutamine exhibit a lower ammonium release, a decreased respiratory rate, and an intracellular accumulation of aspartate. Therefore, we conclude that UCP2 expression is required for efficient oxidation of glutamine in macrophages. This role of UCP2 in glutamine metabolism appears independent from the uncoupling activity of UCP2.
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Glutamina/metabolismo , Canales Iónicos/fisiología , Macrófagos/metabolismo , Proteínas Mitocondriales/fisiología , Animales , Células Cultivadas , Canales Iónicos/genética , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Proteína Desacopladora 2RESUMEN
Infiltration of inflammatory cells into pancreatic islets of Langerhans and selective destruction of insulin-secreting beta-cells are characteristics of type 1 diabetes. Uncoupling protein 2 (UCP2) is a mitochondrial protein expressed in immune cells. UCP2 controls macrophage activation by modulating the production of mitochondrial reactive oxygen species (ROS) and MAPK signaling. We investigated the role of UCP2 on immune cell activity in type 1 diabetes in Ucp2-deficient mice. Using the model of multiple low-dose streptozotocin (STZ)-induced diabetes, we found that autoimmune diabetes was strongly accelerated in Ucp2-KO mice, compared with Ucp2-WT mice with increased intraislet lymphocytic infiltration. Macrophages from STZ-treated Ucp2-KO mice had increased IL-1beta and nitric oxide (NO) production, compared with WT macrophages. Moreover, more macrophages were recruited in islets of STZ-treated Ucp2-KO mice, compared with Ucp2-WT mice. This finding also was accompanied by increased NO/ROS-induced damage. Altogether, our data show that inflammation is stronger in Ucp2-KO mice and islets, leading to the exacerbated disease in these mice. Our results highlight the mitochondrial protein UCP2 as a new player in autoimmune diabetes.
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Diabetes Mellitus Experimental/inmunología , Canales Iónicos/fisiología , Islotes Pancreáticos/inmunología , Macrófagos Peritoneales/inmunología , Proteínas Mitocondriales/fisiología , Animales , Glucemia/análisis , Células Cultivadas/efectos de los fármacos , Citocinas/metabolismo , Diabetes Mellitus Experimental/patología , Progresión de la Enfermedad , Inflamación , Interferón gamma/farmacología , Canales Iónicos/deficiencia , Canales Iónicos/genética , Islotes Pancreáticos/patología , Lipopolisacáridos/farmacología , Linfocitos/inmunología , Linfocitos/patología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/fisiología , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Óxido Nítrico/biosíntesis , Óxido Nítrico/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Estreptozocina/administración & dosificación , Estreptozocina/toxicidad , Proteína Desacopladora 2RESUMEN
The understanding of mitochondrial functioning is of prime importance since it combines the production of energy as adenosine triphosphate (ATP) with an efficient chain of redox reactions, but also with the unavoidable production of reactive oxygen species (ROS) involved in aging. Mitochondrial respiration may be uncoupled from ATP synthesis by a proton leak induced by the thermogenic uncoupling protein 1 (UCP1). Mild uncoupling activity, as proposed for UCP2, UCP3, and avian UCP could theoretically control ROS production, but the nature of their transport activities is far from being definitively understood. The recent discovery of a UCP1 gene in fish has balanced the evolutionary view of uncoupling protein history. The thermogenic proton transport of mammalian UCP1 seems now to be a late evolutionary characteristic and the hypothesis that ancestral UCPs may carry other substrates is tempting. Using in silico genome analyses among taxa and a biochemical approach, we present a detailed phylogenetic analysis of UCPs and investigate whether avian UCP is a good candidate for pleiotropic mitochondrial activities, knowing that only one UCP has been characterized in the avian genome, unlike all other vertebrates. We show, here, that the avian class seems to be the only vertebrate lineage lacking two of the UCP1/2/3 homologues present in fish and mammals. We suggest, based on phylogenetic evidence and synteny of the UCP genes, that birds have lost UCP1 and UCP2. The phylogeny also supports the history of two rounds of duplication during vertebrate evolution. The avian uncoupling protein then represents a unique opportunity to explore how UCPs' activities are controlled, but also to understand why birds exhibit such a particular relationship between high metabolism and slow rate of aging.
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Evolución Molecular , Canales Iónicos/genética , Proteínas Mitocondriales/genética , Secuencia de Aminoácidos , Animales , Aves , Canales Iónicos/química , Proteínas Mitocondriales/química , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Proteína Desacopladora 1RESUMEN
This study focused on the stability of UCP2 (uncoupling protein 2), a mitochondrial carrier located in the inner membrane of mitochondrion. UCP2 is very unstable, with a half-life close to 30min, compared to 30h for its homologue UCP1, a difference that may highlight different physiological functions. Heat production by UCP1 in brown adipocytes is generally a long and adaptive phenomenon, whereas control of mitochondrial ROS by UCP2 needs more subtle regulation. We show that a mutation in UCP2 shown to modify its activity, actually decreases its stability.
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Canales Iónicos/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Secuencia de Bases , Células CHO , Línea Celular , Cricetinae , Cricetulus , ADN/genética , Estabilidad de Medicamentos , Semivida , Humanos , Canales Iónicos/genética , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína Desacopladora 1 , Proteína Desacopladora 2RESUMEN
The mitochondrion is a major organelle contributing to energy metabolism but also a main site of ROS (reactive oxygen species) production. LPS (lipopolysaccharide)-induced ROS signalling is a critical event in macrophage activation. In the present paper we report that part of LPS-mediated ROS signalling comes from mitochondria inside a signal amplification loop that enhances MAPK (mitogen-activated protein kinase) activation. More precisely, we have identified the inner mitochondrial membrane UCP2 (uncoupling protein 2) as a physiological brake on ROS signalling. Stimulation of murine bone marrow-derived macrophages by LPS quickly down-regulated UCP2 through the JNK (c-Jun N-terminal kinase) and p38 pathways. UCP2 down-regulation was shown to be necessary to increase mitochondrial ROS production in order to potentiate MAPK activation. Consistent with this, UCP2-deficient macrophages exhibit an enhanced inflammatory state characterized by increased nitric oxide production and elevated migration ability. Additionally, we found that the absence of UCP2 renders macrophages more resistant to nitric oxide-induced apoptosis.
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Canales Iónicos/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Apoptosis , Células Cultivadas , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Canales Iónicos/deficiencia , Canales Iónicos/genética , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Proteína Desacopladora 2RESUMEN
The uncoupling protein 2 (UCP2) is located in the inner mitochondrial membrane and downregulates the production of reactive oxygen species (ROS). Recent data suggested a role for UCP2 in the immune response. We analyzed further this hypothesis during acute Listeria monocytogenes infection in mice. Death of infected Ucp2(-/-) mice was delayed in comparison with Ucp2(+/+), suggesting a role of UCP2 in the early step of the immune response. In vitro, the higher resistance of Ucp2(-/-) mice was not associated with a better control of bacterial growth by macrophages. In vivo, a significant increase of recruited phagocytes was observed in the spleen of Ucp2(-/-) mice. This was associated with a higher level of ROS in the spleen. Upregulation of pro-inflammatory cytokines IFNgamma, IL6, and IL1beta and of the chemokine MCP1 was observed in Ucp2(-/-) mice 4 days after infection, preceded by a decrease of the anti-inflammatory cytokine IL10 production. Present data highlight that, in an acute model of infection, UCP2 modulates innate immunity, via the modulation of ROS production, cytokine and chemokine production and consequently phagocyte recruitment.