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1.
Mol Ther ; 29(1): 338-346, 2021 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-32966769

RESUMEN

Complement factor C5a was originally identified as a powerful promoter of inflammation through activation of the C5a receptor 1 (C5ar1). Recent evidence suggests involvement of C5a not only in pro- but also in anti-inflammatory signaling. The present study aims to unveil the role of C5ar1 as potential therapeutic target in a murine sepsis model. Our study discloses a significantly increased survival in models of mild to moderate but not severe sepsis of C5ar1-deficient mice. The decreased mortality of C5ar1-deficient mice is accompanied by improved pathogen clearance and largely preserved liver function. C5ar1-deficient mice exhibited a significantly increased production of the pro-inflammatory mediator interferon-γ (IFN-γ) and a decreased production of the anti-inflammatory cytokine interleukin-10 (IL-10). Together, these data uncover C5a signaling as a mediator of immunosuppressive processes during sepsis and describe the C5ar1 and related changes of the IFN-γ to IL-10 ratio as markers for the immunological (dys)function accompanying sepsis.


Asunto(s)
Biomarcadores , Susceptibilidad a Enfermedades/inmunología , Inmunomodulación , Receptor de Anafilatoxina C5a/metabolismo , Sepsis/metabolismo , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inmunidad Innata , Inmunomodulación/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Ratones , Ratones Noqueados , Terapia Molecular Dirigida , Fenotipo , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/genética , Sepsis/diagnóstico , Sepsis/tratamiento farmacológico , Sepsis/etiología
2.
Trends Immunol ; 39(6): 438-445, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29716792

RESUMEN

Recent investigations reveal memory-like adaptive responses of the innate immune system to sequential pathogen challenge. Of note, opposing effects that include both sensitization ('training') and desensitization ('tolerance') have been reported. While hitherto the nature of the pathogen was thought to be of prime importance, we propose that pathogen dose plays a key role in determining these opposing effects. Within this concept, training and tolerance of innate immune cells emerge as adaptive responses to increasing pathogen load. Furthermore, environmental stressors significantly impact the pathogen-induced responses of these innate immune cells. Therefore, we hypothesize that pathogens, like other stressors, provoke hormetic responses of the affected cells. This concept could explain the tight interplay of dose-related effects of pathogens and other stressors in infectious diseases.


Asunto(s)
Inmunidad Adaptativa/inmunología , Enfermedades Transmisibles/inmunología , Tolerancia Inmunológica/inmunología , Inmunidad Innata/inmunología , Animales , Humanos , Lipopolisacáridos/inmunología , Modelos Inmunológicos , Monocitos/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología
3.
Crit Care ; 25(1): 368, 2021 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-34674733

RESUMEN

BACKGROUND: Fever and hypothermia have been observed in septic patients. Their influence on prognosis is subject to ongoing debates. METHODS: We did a secondary analysis of a large clinical dataset from a quality improvement trial. A binary logistic regression model was calculated to assess the association of the thermal response with outcome and a multinomial regression model to assess factors associated with fever or hypothermia. RESULTS: With 6542 analyzable cases we observed a bimodal temperature response characterized by fever or hypothermia, normothermia was rare. Hypothermia and high fever were both associated with higher lactate values. Hypothermia was associated with higher mortality, but this association was reduced after adjustment for other risk factors. Age, community-acquired sepsis, lower BMI and lower outside temperatures were associated with hypothermia while bacteremia and higher procalcitonin values were associated with high fever. CONCLUSIONS: Septic patients show either a hypothermic or a fever response. Whether hypothermia is a maladaptive response, as indicated by the higher mortality in hypothermic patients, or an adaptive response in patients with limited metabolic reserves under colder environmental conditions, remains an open question. Trial registration The original trial whose dataset was analyzed was registered at ClinicalTrials.gov (NCT01187134) on August 23, 2010, the first patient was included on July 1, 2011.


Asunto(s)
Fiebre , Hipotermia , Sepsis , Fiebre/complicaciones , Humanos , Hipotermia/complicaciones , Pronóstico , Sepsis/terapia , Temperatura
4.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-33806610

RESUMEN

Microglia, the innate immune cells of the CNS, exhibit long-term response changes indicative of innate immune memory (IIM). Our previous studies revealed IIM patterns of microglia with opposing immune phenotypes: trained immunity after a low dose and immune tolerance after a high dose challenge with pathogen-associated molecular patterns (PAMP). Compelling evidence shows that innate immune cells adopt features of IIM via immunometabolic control. However, immunometabolic reprogramming involved in the regulation of IIM in microglia has not been fully addressed. Here, we evaluated the impact of dose-dependent microglial priming with ultra-low (ULP, 1 fg/mL) and high (HP, 100 ng/mL) lipopolysaccharide (LPS) doses on immunometabolic rewiring. Furthermore, we addressed the role of PI3Kγ on immunometabolic control using naïve primary microglia derived from newborn wild-type mice, PI3Kγ-deficient mice and mice carrying a targeted mutation causing loss of lipid kinase activity. We found that ULP-induced IIM triggered an enhancement of oxygen consumption and ATP production. In contrast, HP was followed by suppressed oxygen consumption and glycolytic activity indicative of immune tolerance. PI3Kγ inhibited glycolysis due to modulation of cAMP-dependent pathways. However, no impact of specific PI3Kγ signaling on immunometabolic rewiring due to dose-dependent LPS priming was detected. In conclusion, immunometabolic reprogramming of microglia is involved in IIM in a dose-dependent manner via the glycolytic pathway, oxygen consumption and ATP production: ULP (ultra-low-dose priming) increases it, while HP reduces it.


Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/inmunología , Inmunidad Innata/inmunología , Memoria Inmunológica/inmunología , Adenosina Trifosfato/inmunología , Animales , Glucólisis/inmunología , Tolerancia Inmunológica/inmunología , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/inmunología , Consumo de Oxígeno/inmunología , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Transducción de Señal/inmunología
5.
J Neuroinflammation ; 17(1): 292, 2020 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-33028343

RESUMEN

BACKGROUND: Sepsis-associated encephalopathy (SAE) is an early and frequent event of infection-induced systemic inflammatory response syndrome. Phosphoinositide 3-kinase γ (PI3Kγ) is linked to neuroinflammation and inflammation-related microglial activity. In homeotherms, variations in ambient temperature (Ta) outside the thermoneutral zone lead to thermoregulatory responses, mainly driven by a gradually increasing sympathetic activity, and may affect disease severity. We hypothesized that thermoregulatory response to hypothermia (reduced Ta) aggravates SAE in PI3Kγ-dependent manner. METHODS: Experiments were performed in wild-type, PI3Kγ knockout, and PI3Kγ kinase-dead mice, which were kept at neutral (30 ± 0.5 °C) or moderately lowered (26 ± 0.5 °C) Ta. Mice were exposed to lipopolysaccharide (LPS, 10 µg/g, from Escherichia coli serotype 055:B5, single intraperitoneal injection)-evoked systemic inflammatory response (SIR) and monitored 24 h for thermoregulatory response and blood-brain barrier integrity. Primary microglial cells and brain tissue derived from treated mice were analyzed for inflammatory responses and related cell functions. Comparisons between groups were made with one-way or two-way analysis of variance, as appropriate. Post hoc comparisons were made with the Holm-Sidak test or t tests with Bonferroni's correction for adjustments of multiple comparisons. Data not following normal distribution was tested with Kruskal-Wallis test followed by Dunn's multiple comparisons test. RESULTS: We show that a moderate reduction of ambient temperature triggers enhanced hypothermia of mice undergoing LPS-induced systemic inflammation by aggravated SAE. PI3Kγ deficiency enhances blood-brain barrier injury and upregulation of matrix metalloproteinases (MMPs) as well as an impaired microglial phagocytic activity. CONCLUSIONS: Thermoregulatory adaptation in response to ambient temperatures below the thermoneutral range exacerbates LPS-induced blood-brain barrier injury and neuroinflammation. PI3Kγ serves a protective role in suppressing release of MMPs, maintaining microglial motility and reinforcing phagocytosis leading to improved brain tissue integrity. Thus, preclinical research targeting severe brain inflammation responses is seriously biased when basic physiological prerequisites of mammal species such as preferred ambient temperature are ignored.


Asunto(s)
Regulación de la Temperatura Corporal/fisiología , Fosfatidilinositol 3-Quinasa Clase Ib/deficiencia , Lipopolisacáridos/toxicidad , Encefalopatía Asociada a la Sepsis/enzimología , Encefalopatía Asociada a la Sepsis/fisiopatología , Animales , Animales Recién Nacidos , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/enzimología , Barrera Hematoencefálica/fisiopatología , Temperatura Corporal/efectos de los fármacos , Temperatura Corporal/fisiología , Regulación de la Temperatura Corporal/efectos de los fármacos , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Encefalopatía Asociada a la Sepsis/inducido químicamente
6.
Int J Mol Sci ; 21(6)2020 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-32197550

RESUMEN

Atherosclerosis is a multifactorial chronic disease that affects large arteries and may lead to fatal consequences. According to current understanding, inflammation and lipid accumulation are the two key mechanisms of atherosclerosis development. Animal models based on genetically modified mice have been developed to investigate these aspects. One such model is low-density lipoprotein (LDL) receptor knockout (KO) mice (ldlr-/-), which are characterized by a moderate increase of plasma LDL cholesterol levels. Another widely used genetically modified mouse strain is apolipoprotein-E KO mice (apoE-/-) that lacks the primary lipoprotein required for the uptake of lipoproteins through the hepatic receptors, leading to even greater plasma cholesterol increase than in ldlr-/- mice. These and other animal models allowed for conducting genetic studies, such as genome-wide association studies, microarrays, and genotyping methods, which helped identifying more than 100 mutations that contribute to atherosclerosis development. However, translation of the results obtained in animal models for human situations was slow and challenging. At the same time, genetic studies conducted in humans were limited by low sample sizes and high heterogeneity in predictive subclinical phenotypes. In this review, we summarize the current knowledge on the use of KO mice for identification of genes implicated in atherosclerosis and provide a list of genes involved in atherosclerosis-associated inflammatory pathways and their brief characteristics. Moreover, we discuss the approaches for candidate gene search in animals and humans and discuss the progress made in the field of epigenetic studies that appear to be promising for identification of novel biomarkers and therapeutic targets.


Asunto(s)
Aterosclerosis , Dislipidemias , Regulación de la Expresión Génica , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Dislipidemias/genética , Dislipidemias/metabolismo , Humanos , Ratones , Ratones Noqueados
7.
Int J Mol Sci ; 21(3)2020 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-32012706

RESUMEN

Excessive accumulation of lipid inclusions in the arterial wall cells (foam cell formation) caused by modified low-density lipoprotein (LDL) is the earliest and most noticeable manifestation of atherosclerosis. The mechanisms of foam cell formation are not fully understood and can involve altered lipid uptake, impaired lipid metabolism, or both. Recently, we have identified the top 10 master regulators that were involved in the accumulation of cholesterol in cultured macrophages induced by the incubation with modified LDL. It was found that most of the identified master regulators were related to the regulation of the inflammatory immune response, but not to lipid metabolism. A possible explanation for this unexpected result is a stimulation of the phagocytic activity of macrophages by modified LDL particle associates that have a relatively large size. In the current study, we investigated gene regulation in macrophages using transcriptome analysis to test the hypothesis that the primary event occurring upon the interaction of modified LDL and macrophages is the stimulation of phagocytosis, which subsequently triggers the pro-inflammatory immune response. We identified genes that were up- or downregulated following the exposure of cultured cells to modified LDL or latex beads (inert phagocytosis stimulators). Most of the identified master regulators were involved in the innate immune response, and some of them were encoding major pro-inflammatory proteins. The obtained results indicated that pro-inflammatory response to phagocytosis stimulation precedes the accumulation of intracellular lipids and possibly contributes to the formation of foam cells. In this way, the currently recognized hypothesis that the accumulation of lipids triggers the pro-inflammatory response was not confirmed. Comparative analysis of master regulators revealed similarities in the genetic regulation of the interaction of macrophages with naturally occurring LDL and desialylated LDL. Oxidized and desialylated LDL affected a different spectrum of genes than naturally occurring LDL. These observations suggest that desialylation is the most important modification of LDL occurring in vivo. Thus, modified LDL caused the gene regulation characteristic of the stimulation of phagocytosis. Additionally, the knock-down effect of five master regulators, such as IL15, EIF2AK3, F2RL1, TSPYL2, and ANXA1, on intracellular lipid accumulation was tested. We knocked down these genes in primary macrophages derived from human monocytes. The addition of atherogenic naturally occurring LDL caused a significant accumulation of cholesterol in the control cells. The knock-down of the EIF2AK3 and IL15 genes completely prevented cholesterol accumulation in cultured macrophages. The knock-down of the ANXA1 gene caused a further decrease in cholesterol content in cultured macrophages. At the same time, knock-down of F2RL1 and TSPYL2 did not cause an effect. The results obtained allowed us to explain in which way the inflammatory response and the accumulation of cholesterol are related confirming our hypothesis of atherogenesis development based on the following viewpoints: LDL particles undergo atherogenic modifications that, in turn, accompanied by the formation of self-associates; large LDL associates stimulate phagocytosis; as a result of phagocytosis stimulation, pro-inflammatory molecules are secreted; these molecules cause or at least contribute to the accumulation of intracellular cholesterol. Therefore, it became obvious that the primary event in this sequence is not the accumulation of cholesterol but an inflammatory response.


Asunto(s)
Células Espumosas/metabolismo , Células Espumosas/patología , Lipoproteínas LDL/metabolismo , Fagocitosis , Biomarcadores , Células Espumosas/inmunología , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunidad Innata , Metabolismo de los Lípidos , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Oxidación-Reducción , Fagocitosis/genética , Fagocitosis/inmunología , Transducción de Señal , Transcriptoma
8.
Basic Res Cardiol ; 114(3): 26, 2019 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-31016449

RESUMEN

Sepsis-induced myocardial depression (SIMD) is an early and frequent consequence of the infection-induced systemic inflammatory response syndrome. In homiotherms, variations in ambient temperature (Ta) outside the thermoneutral zone induce thermoregulatory responses mainly driven by a gradually increased sympathetic activity, which may affect disease severity. We hypothesized that thermoregulatory responses upon reduced Ta exposition aggravate SIMD in mice. Mice were kept at neutral Ta (30 ± 0.5 °C), moderately lowered Ta (26 ± 0.5 °C) or markedly lowered Ta (22 ± 0.5 °C), exposed to lipopolysaccharide- (LPS, 10 µg/g, from Escherichia coli serotype 055:B5, single intraperitoneal injection) evoked shock and monitored for survival, cardiac autonomic nervous system function and left ventricular performance. Primary adult cardiomyocytes and heart tissue derived from treated mice were analyzed for inflammatory responses and signaling pathways of myocardial contractility. We show that a moderate reduction of Ta to 26 °C led to a 40% increased mortality of LPS-treated mice when compared to control mice and that a marked reduction of Ta to 22 °C resulted in an early mortality of all mice. Mice kept at 26 °C exhibited increased heart rate and altered indices of heart rate variability (HRV), indicating sympathovagal imbalance along with aggravated LPS-induced SIMD. This SIMD was associated with reduced myocardial ß-adrenergic receptor expression and suppressed adrenergic signaling, as well as with increased myocardial iNOS expression, nitrotyrosine formation and leukocyte invasion as well as enhanced apoptosis and appearance of contraction band necrosis in heart tissue. While ineffective separately, combined treatment with the ß2-adrenergic receptor (AR) antagonist ICI 118551 (10 ng/gbw) and the inducible nitric oxide synthase (iNOS) inhibitor 1400 W (5 µg/gbw) reversed the increase in LPS-induced mortality and aggravation of SIMD at reduced Ta. Thus, consequences of thermoregulatory adaptation in response to ambient temperatures below the thermoneutral range increase the mortality from LPS-evoked shock and markedly prolong impaired myocardial function. These changes are mitigated by combined ß2-AR and iNOS inhibition.


Asunto(s)
Sistema Nervioso Autónomo/fisiopatología , Regulación de la Temperatura Corporal , Cardiopatías/inducido químicamente , Corazón/inervación , Vivienda para Animales , Contracción Miocárdica , Síndrome de Respuesta Inflamatoria Sistémica/inducido químicamente , Temperatura , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Cardiopatías/metabolismo , Cardiopatías/fisiopatología , Hemodinámica , Mediadores de Inflamación/metabolismo , Lipopolisacáridos , Masculino , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/fisiopatología
9.
Biogerontology ; 20(4): 571-581, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30895414

RESUMEN

Mild environmental stress might have beneficial effects in aging by activating maintenance and repair processes in cells and organs. These beneficial stress effects fit to the concept of hormesis. Prominent stressors acting in a hormetic way are physical exercises, fasting, cold and heat. This review will introduce some toxins, which have been found to induce hormetic responses in animal models of aging research. To highlight the molecular signature of these hormetic effects we will depict signaling pathways affected by low doses of toxins on cellular and organismic level. As prominent examples for signaling pathways involved in both aging processes as well as toxin responses, PI3K/Akt/mTOR- and AMPK-signal transduction will be described in more detail. Due to the striking overlap of signaling pathways mediating toxin induced responses and aging processes we propose considering the ability of low doses of toxins to slow down the rate of aging.


Asunto(s)
Envejecimiento/fisiología , Arsénico/toxicidad , Cadmio/toxicidad , Senescencia Celular/fisiología , Hormesis/fisiología , Envejecimiento/efectos de los fármacos , Animales , Modelos Animales , Transducción de Señal , Estrés Fisiológico
10.
Mol Cell ; 42(1): 84-95, 2011 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-21474070

RESUMEN

Adrenergic stimulation of the heart engages cAMP and phosphoinositide second messenger signaling cascades. Cardiac phosphoinositide 3-kinase p110γ participates in these processes by sustaining ß-adrenergic receptor internalization through its catalytic function and by controlling phosphodiesterase 3B (PDE3B) activity via an unknown kinase-independent mechanism. We have discovered that p110γ anchors protein kinase A (PKA) through a site in its N-terminal region. Anchored PKA activates PDE3B to enhance cAMP degradation and phosphorylates p110γ to inhibit PIP(3) production. This provides local feedback control of PIP(3) and cAMP signaling events. In congestive heart failure, p110γ is upregulated and escapes PKA-mediated inhibition, contributing to a reduction in ß-adrenergic receptor density. Pharmacological inhibition of p110γ normalizes ß-adrenergic receptor density and improves contractility in failing hearts.


Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Miocitos Cardíacos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Fosfatidilinositol 3-Quinasa Clase Ib/química , Fosfatidilinositol 3-Quinasa Clase Ib/deficiencia , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , ADN/genética , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Mapeo de Interacción de Proteínas , Quinoxalinas/farmacología , Receptores Adrenérgicos beta/metabolismo , Sistemas de Mensajero Secundario , Homología de Secuencia de Aminoácido , Tiazolidinedionas/farmacología
11.
Mol Cell Neurosci ; 78: 1-8, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27825984

RESUMEN

Microglial motility is tightly controlled by multitude of agonistic and antagonistic factors. Chemoattractants, released after infection or damage of the brain, provoke directed migration of microglia to the pathogenic incident. In contrast, noradrenaline and other stress hormones have been shown to suppress microglial movement. Here we asked for the signaling reactions involved in the positive and negative control of microglial motility. Using pharmacological and genetic approaches we identified the lipid kinase activity of phosphoinositide 3-kinase species γ (PI3Kγ) as an essential mediator of microglial migration provoked by the complement component C5a and other chemoattractants. Inhibition of PI3Kγ lipid kinase activity by protein kinase A was disclosed as mechanism causing suppression of microglial migration by noradrenaline. Together these data characterize PI3Kγ as a nodal point in the control of microglial motility.


Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Factores Quimiotácticos/farmacología , Quimiotaxis , Microglía/metabolismo , Norepinefrina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Línea Celular , Células Cultivadas , Complemento C5a/farmacología , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/fisiología , Fosfatidilinositol 3-Quinasas/genética
12.
Glia ; 65(2): 416-428, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27859601

RESUMEN

Microglia cells are brain macrophages whose proper functioning is essential for maintenance and repair processes of the central nervous system (CNS). Migration and phagocytosis are critical aspects of microglial activity. By using genetically modified cell lines and knockout mice we demonstrate here that the receptor protein-tyrosine phosphatase (PTP) DEP-1 (also known as PTPRJ or CD148) acts as a positive regulator of both processes in vitro and in vivo. Notably, reduced microglial migration was detectable in brains of Ptprj-/- mice using a wounding assay. Mechanistically, density-enhanced phosphatase-1 (DEP-1) may in part function by inhibiting the activity of the Src family kinase Fyn. In the microglial cell line BV2 DEP-1 depletion by shRNA-mediated knockdown resulted in enhanced phosphorylation of the Fyn activating tyrosine (Tyr420 ) and elevated specific Fyn-kinase activity in immunoprecipitates. Moreover, Fyn mRNA and protein levels were reduced in DEP-1 deficient microglia cells. Consistent with a negative regulatory role of Fyn for microglial functions, which is inhibited by DEP-1, microglial cells from Fyn-/- mice exhibited elevated migration and phagocytosis. Enhanced microglia migration to a site of injury was also observed in Fyn-/- mice in vivo. Taken together our data revealed a previously unrecognized role of DEP-1 and suggest the existence of a potential DEP-1-Fyn axis in the regulation of microglial functions. GLIA 2017;65:416-428.


Asunto(s)
Movimiento Celular/fisiología , Regulación de la Expresión Génica/genética , Microglía/fisiología , Fagocitosis/genética , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Animales , Animales Recién Nacidos , Línea Celular Transformada , Movimiento Celular/genética , Células Cultivadas , Corteza Cerebral/citología , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/fisiología , Proteínas Proto-Oncogénicas c-fyn/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo
13.
Biochem J ; 469(1): 59-69, 2015 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-26173259

RESUMEN

Class IB phosphoinositide 3-kinases γ (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody [mAb(A)p110γ] to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gßγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gßγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ, Gßγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gßγ-stimulated lipid kinase activity of p87-p110γ 30-fold more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gßγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.


Asunto(s)
Anticuerpos Monoclonales de Origen Murino/química , Fosfatidilinositol 3-Quinasa Clase Ib , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Animales , Fosfatidilinositol 3-Quinasa Clase Ib/química , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Medición de Intercambio de Deuterio , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Células HEK293 , Humanos , Células Sf9 , Spodoptera
14.
Br J Haematol ; 166(4): 529-39, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24766330

RESUMEN

Constitutive phosphatidylinositide 3-kinase (PI3K) signalling has been implicated in multiple myeloma (MM) pathophysiology and is regarded as an actionable target for pharmacological intervention. Isoform-specific PI3K inhibition may offer the most focused treatment approach and could result in greater clinical efficacy and reduced side effects. We therefore performed isoform-specific knockdown of PIK3CA, PIK3CB, PIK3CD, and PIK3CG to analyse their individual contributions to MM cell survival and downstream signalling. In addition, we tested the effectivity of the novel PI3K isoform-specific inhibitors BYL-719 (PIK3CA), TGX-221 (PIK3CB), CAL-101 (PIK3CD), and CAY10505 (PIK3CG). We found the PIK3CA isoform to be of paramount importance for constitutive Akt activity in MM cells, and - in contrast to inhibition of other class I isoforms - only the blockade of PIK3CA was sufficient to induce cell death in a sizeable subgroup of MM samples. Furthermore, pharmacological PIK3CA inhibition in combination treatments of BYL-719 and established anti-myeloma agents resulted in strongly enhanced MM cell death. Our data thus clearly indicate therapeutic potential of PIK3CA inhibitors and support their clinical evaluation in multiple myeloma.


Asunto(s)
Mieloma Múltiple/patología , Fosfatidilinositol 3-Quinasas/fisiología , Antineoplásicos/farmacología , Supervivencia Celular/fisiología , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I , Combinación de Medicamentos , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Mieloma Múltiple/metabolismo , Fosfatidilinositol 3-Quinasas/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiología , ARN Interferente Pequeño/farmacología , Transducción de Señal
15.
Trends Mol Med ; 2024 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-38744580

RESUMEN

Hormesis is a phenomenon whereby low-level stress can improve cellular, organ, or organismal fitness in response to a subsequent similar or other stress insult. Whereas hormesis is thought to contribute to the fitness benefits arising from symbiotic host-microbe interactions, the putative benefits of hormesis in host-pathogen interactions have yet to be explored. Hormetic responses have nonetheless been reported in experimental models of infection, a common feature of which is regulation of host mitochondrial function. We propose that these mitohormetic responses could be harnessed therapeutically to limit the severity of infectious diseases.

16.
J Cell Sci ; 124(Pt 11): 1819-30, 2011 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-21540297

RESUMEN

VEGF-, phosphoinositide 3-kinase (PI3K)- and protein kinase C (PKC)-regulated signaling in cardiac and vascular differentiation was investigated in mouse ES cells and in ES cell-derived Flk-1⁺ cardiovascular progenitor cells. Inhibition of PI3K by wortmannin and LY294002, disruption of PI3K catalytic subunits p110α and p110δ using short hairpin RNA (shRNA), or inhibition of p110α with compound 15e and of p110δ with IC-87114 impaired cardiac and vascular differentiation. By contrast, TGX-221, an inhibitor of p110ß, and shRNA knockdown of p110ß were without significant effects. Antagonists of the PKC family, i.e. bisindolylmaleimide-1 (BIM-1), GÖ 6976 (targeting PKCα/ßII) and rottlerin (targeting PKCδ) abolished vasculogenesis, but not cardiomyogenesis. Inhibition of Akt blunted cardiac as well as vascular differentiation. VEGF induced phosphorylation of PKCα/ßII and PKCδ but not PKCζ. This was abolished by PI3K inhibitors and the VEGFR-2 antagonist SU5614. Furthermore, phosphorylation of Akt and phosphoinositide-dependent kinase-1 (PDK1) was blunted upon inhibition of PI3K, but not upon inhibition of PKC by BIM-1, suggesting that activation of Akt and PDK1 by VEGF required PI3K but not PKC. In summary, we demonstrate that PI3K catalytic subunits p110α and p110δ are central to cardiovasculogenesis of ES cells. Akt downstream of PI3K is involved in both cardiomyogenesis and vasculogenesis, whereas PKC is involved only in vasculogenesis.


Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Cuerpos Embrioides/citología , Desarrollo de Músculos , Miocardio/citología , Neovascularización Fisiológica , Proteína Quinasa C/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Dominio Catalítico , Diferenciación Celular , Línea Celular , Cromonas/farmacología , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Cuerpos Embrioides/efectos de los fármacos , Activación Enzimática , Ratones , Morfolinas/farmacología , Miocardio/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Transducción de Señal
17.
Biochem J ; 441(3): 851-8, 2012 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-22054284

RESUMEN

G-protein-regulated PI3Kγ (phosphoinositide 3-kinase γ) plays a crucial role in inflammatory and allergic processes. PI3Kγ, a dimeric protein formed by the non-catalytic p101 and catalytic p110γ subunits, is stimulated by receptor-released Gßγ complexes. We have demonstrated previously that Gßγ stimulates both monomeric p110γ and dimeric p110γ/p101 lipid kinase activity in vitro. In order to identify the Gß residues responsible for the Gßγ-PI3Kγ interaction, we examined Gß1 mutants for their ability to stimulate lipid and protein kinase activities and to recruit PI3Kγ to lipid vesicles. Our findings revealed different interaction profiles of Gß residues interacting with p110γ or p110γ/p101. Moreover, p101 was able to rescue the stimulatory activity of Gß1 mutants incapable of modulating monomeric p110γ. In addition to the known adaptor function of p101, in the present paper we show a novel regulatory role of p101 in the activation of PI3Kγ.


Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/fisiología , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Animales , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase Ib/química , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Resistencia a Medicamentos/genética , Resistencia a Medicamentos/fisiología , Activación Enzimática/genética , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteolisis/efectos de los fármacos , Spodoptera , Transfección , Tripsina/metabolismo , Tripsina/farmacología
18.
Br J Pharmacol ; 180(4): 441-458, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36245399

RESUMEN

BACKGROUND AND PURPOSE: Prostaglandin E2 is considered a major mediator of inflammatory pain, by acting on neuronal Gs protein-coupled EP2 and EP4 receptors. However, the neuronal EP3 receptor, colocalized with EP2 and EP4 receptor, is Gi protein-coupled and antagonizes the pronociceptive prostaglandin E2 effect. Here, we investigated the cellular signalling mechanisms by which the EP3 receptor reduces EP2 and EP4 receptor-evoked pronociceptive effects in sensory neurons. EXPERIMENTAL APPROACH: Experiments were performed on isolated and cultured dorsal root ganglion (DRG) neurons from wild type, phosphoinositide 3-kinase γ (PI3Kγ)-/- , and PI3Kγkinase dead (KD)/KD mice. For subtype-specific stimulations, we used specific EP2, EP3, and EP4 receptor agonists from ONO Pharmaceuticals. As a functional readout, we recorded TTX-resistant sodium currents in patch-clamp experiments. Western blots were used to investigate the activation of intracellular signalling pathways. EP4 receptor internalization was measured using immunocytochemistry. KEY RESULTS: Different pathways mediate the inhibition of EP2 and EP4 receptor-dependent pronociceptive effects by EP3 receptor stimulation. Inhibition of EP2 receptor-evoked pronociceptive effect critically depends on the kinase-independent function of the signalling protein PI3Kγ, and adenosine monophosphate activated protein kinase (AMPK) is involved. By contrast, inhibition of EP4 receptor-evoked pronociceptive effect is independent on PI3Kγ and mediated through activation of G protein-coupled receptor kinase 2 (GRK2), which enhances the internalization of the EP4 receptor after ligand binding. CONCLUSION AND IMPLICATIONS: Activation of neuronal PI3Kγ, AMPK, and GRK2 by EP3 receptor activation limits cAMP-dependent pain generation by prostaglandin E2 . These new insights hold the potential for a novel approach in pain therapy.


Asunto(s)
Proteínas Quinasas Activadas por AMP , Prostaglandinas , Animales , Ratones , Fosfatidilinositol 3-Quinasa , Fosfatidilinositol 3-Quinasas , Dinoprostona/farmacología , Dinoprostona/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E , Subtipo EP2 de Receptores de Prostaglandina E , Células Receptoras Sensoriales/metabolismo , Dolor , Analgésicos , Subtipo EP3 de Receptores de Prostaglandina E/metabolismo
19.
Free Radic Biol Med ; 204: 151-160, 2023 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-37105418

RESUMEN

Ischemia-reperfusion injury is a critical liver condition during hepatic transplantation, trauma, or shock. An ischemic deprivation of antioxidants and energy characterizes liver injury in such cases. In the face of increased reactive oxygen production, hepatocytes are vulnerable to the reperfusion driving ROS generation and multiple cell-death mechanisms. In this study, we investigate the importance of hydrogen sulfide as part of the liver's antioxidant pool and the therapeutic potency of the hydrogen sulfide donors sodium sulfide (Na2S, fast releasing) and sodium thiosulfate (STS, Na2S2O3, slow releasing). The mitoprotection and toxicity of STS and Na2S were investigated on isolated mitochondria and a liver perfusion oxidative stress model by adding text-butyl hydroperoxide and hydrogen sulfide donors. The respiratory capacity of mitochondria, hepatocellular released LDH, glutathione, and lipid-peroxide levels were quantified. In addition, wild-type and cystathionine-γ-lyase knockout mice were subjected to warm selective ischemia-reperfusion injury by clamping the main inflow for 1 h followed by reperfusion of 1 or 24 h. A subset of animals was treated with STS shortly before reperfusion. Glutathione, plasma ALT, and lipid-peroxide levels were investigated alongside mitochondrial changes in structure (electron microscopy) and function (intravital microscopy). Liver tissue necrosis quantified 24 h after reperfusion indicates the net effects of the treatment on the organ. STS refuels and protects the endogenous antioxidant pool during liver ischemia-reperfusion injury. In addition, STS-mediated ROS scavenging significantly reduced lipid peroxidation and mitochondrial damage, resulting in better molecular and histopathological preservation of the liver tissue architecture. STS prevents tissue damage in liver ischemia-reperfusion injury by increasing the liver's antioxidant pool, thereby protecting mitochondrial integrity.


Asunto(s)
Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Sulfuro de Hidrógeno , Daño por Reperfusión , Ratones , Animales , Antioxidantes/farmacología , Especies Reactivas de Oxígeno , Hígado/patología , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/patología , Isquemia/patología , Glutatión , Peróxidos , Reperfusión , Lípidos
20.
PLoS Med ; 9(11): e1001338, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23152722

RESUMEN

BACKGROUND: Hepatic dysfunction and jaundice are traditionally viewed as late features of sepsis and portend poor outcomes. We hypothesized that changes in liver function occur early in the onset of sepsis, yet pass undetected by standard laboratory tests. METHODS AND FINDINGS: In a long-term rat model of faecal peritonitis, biotransformation and hepatobiliary transport were impaired, depending on subsequent disease severity, as early as 6 h after peritoneal contamination. Phosphatidylinositol-3-kinase (PI3K) signalling was simultaneously induced at this time point. At 15 h there was hepatocellular accumulation of bilirubin, bile acids, and xenobiotics, with disturbed bile acid conjugation and drug metabolism. Cholestasis was preceded by disruption of the bile acid and organic anion transport machinery at the canalicular pole. Inhibitors of PI3K partially prevented cytokine-induced loss of villi in cultured HepG2 cells. Notably, mice lacking the PI3Kγ gene were protected against cholestasis and impaired bile acid conjugation. This was partially confirmed by an increase in plasma bile acids (e.g., chenodeoxycholic acid [CDCA] and taurodeoxycholic acid [TDCA]) observed in 48 patients on the day severe sepsis was diagnosed; unlike bilirubin (area under the receiver-operating curve: 0.59), these bile acids predicted 28-d mortality with high sensitivity and specificity (area under the receiver-operating curve: CDCA: 0.77; TDCA: 0.72; CDCA+TDCA: 0.87). CONCLUSIONS: Liver dysfunction is an early and commonplace event in the rat model of sepsis studied here; PI3K signalling seems to play a crucial role. All aspects of hepatic biotransformation are affected, with severity relating to subsequent prognosis. Detected changes significantly precede conventional markers and are reflected by early alterations in plasma bile acids. These observations carry important implications for the diagnosis of liver dysfunction and pharmacotherapy in the critically ill. Further clinical work is necessary to extend these concepts into clinical practice. Please see later in the article for the Editors' Summary.


Asunto(s)
Peritonitis/fisiopatología , Sepsis/fisiopatología , Animales , Ácidos y Sales Biliares/sangre , Biomarcadores/sangre , Western Blotting , Colestasis/microbiología , Colestasis/fisiopatología , Coinfección/microbiología , Coinfección/fisiopatología , Heces/química , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Hígado/fisiopatología , Hepatopatías/microbiología , Hepatopatías/fisiopatología , Pruebas de Función Hepática , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Peritonitis/microbiología , Fosfatidilinositol 3-Quinasa/genética , Fosfatidilinositol 3-Quinasa/metabolismo , Ratas , Ratas Wistar , Sepsis/microbiología , Transducción de Señal , Espectrometría Raman , Xenobióticos/metabolismo
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