P2X7 receptor induces mitochondrial failure in monocytes and compromises NLRP3 inflammasome activation during sepsis

International audienceSepsis is characterized by a systemic inflammatory response followed by immunosuppres-sion of the host. Metabolic defects and mitochondrial failure are common in immunocom-promised patients with sepsis. The NLRP3 inflammasome is important for establishing an inflammatory response after activation by the purinergic P2X7 receptor. Here, we study a cohort of individuals with intra-abdominal origin sepsis and show that patient monocytes have impaired NLRP3 activation by the P2X7 receptor. Furthermore, most sepsis-related deaths are among patients whose NLRP3 activation is profoundly altered. In monocytes from sepsis patients, the P2X7 receptor is associated with mitochondrial dysfunction. Furthermore, activation of the P2X7 receptor results in mitochondrial damage, which in turn inhibits NLRP3 activation by HIF-1α. We show that mortality increases in a mouse model of sepsis when the P2X7 receptor is activated in vivo. These data reveal a molecular mechanism initiated by the P2X7 receptor that contributes to NLRP3 impairment during infection

Longitudinal dynamics of SARS-CoV-2-specific cellular and humoral immunity after natural infection or BNT162b2 vaccination

The timing of the development of specific adaptive immunity after natural SARS-CoV-2 infection, and its relevance in clinical outcome, has not been characterized in depth. Description of the long-term maintenance of both cellular and humoral responses elicited by real-world anti-SARS-CoV-2 vaccination is still scarce. Here we aimed to understand the development of optimal protective responses after SARS-CoV-2 infection and vaccination. We performed an early, longitudinal study of S1-, M- and N-specific IFN-γ and IL-2 T cell immunity and anti-S total and neutralizing antibodies in 88 mild, moderate or severe acute COVID-19 patients. Moreover, SARS-CoV-2-specific adaptive immunity was also analysed in 234 COVID-19 recovered subjects, 28 uninfected BNT162b2-vaccinees and 30 uninfected healthy controls. Upon natural infection, cellular and humoral responses were early and coordinated in mild patients, while weak and inconsistent in severe patients. The S1-specific cellular response measured at hospital arrival was an independent predictive factor against severity. In COVID-19 recovered patients, four to seven months post-infection, cellular immunity was maintained but antibodies and neutralization capacity declined. Finally, a robust Th1-driven immune response was developed in uninfected BNT162b2-vaccinees. Three months post-vaccination, the cellular response was comparable, while the humoral response was consistently stronger, to that measured in COVID-19 recovered patients. Thus, measurement of both humoral and cellular responses provides information on prognosis and protection from infection, which may add value for individual and public health recommendations

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

Relación funcional y metabólica entre los metabolitos de la metionina, la esfingomielinasa ácida y la fosfatidilcolina: Impacto en esteatohepatitis

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Barcelona.--2017-09-28.--ExcelenteLa esteatohepatltis es una enfermedad hepática crónica que se caracteriza por la presencia de esteatosis, inflamación, daño hepatocelular y fibrosís que puede progresar hasta el carclnoma hepatocelular. Ésta, constituye un grave problema de salud a nivel global para et que es necesario el hallazgo de nuevos tratamientos efectivos. Existen evidencias claras que otorgan a la alteración en el metabolismo de ta metionina (bajos niveles de S-adenosilmetlonlna, SAMe, debido a la deficiencia de la encima metionina adenosiltransferasa lA, MATlA); al incremento en la actividad de la esfingomiellnasa ácida (ASMase) y al descenso en los niveles de fosfatidilcolina (PC) un papel clave en el proceso, tanto en modelos experimentales como en biopsias de pacientes con enfermedad hepática alcohólica (EHA) y no alcohóllca (EHNA). Por lo tanto, los objetivos fueron: 1. Determinar el mecanismo de activación de ASMase en modelos nutricionates de EHNA y EHA en ratones caracterizados por poseer un metabolismo alterado de la metionina. 2. Caracterización neuronal del del ratón generado deficiente en los genes MATlA y ASMase (Mat-/-ASM-/-).l. Se generó y caracterizó hepáticamente el ratón Mat-/-ASM-/- y se llevaron a cabo diversos modelos nutrlcionales de EHNA (con una dieta deficiente en colina) in vivo con diferentes backgrounds genéticos (Matla -/- y Mat-/-ASM-/-). Los ratones Mat-/­ASM-/- presentan daño hepático per se, con altos niveles de AST y ALT, así como una alteración de la síntesis de los metabolltos implicados en la ruta de la metionina. Dado que nuestra hipótesis se basa en que la deficiencia de ASMase puede influenciar en la progresión de la esteatohepatitis que presenta de forma espontánea el ratón Matla-/-, alimentamos a los ratones Matla-/- y Mat-/-ASM-/- con una dieta deficiente en colina (CD). Los ratones Matla-/- presentaron una sobreexpresión de ASMase de forma basal que aumentó de forma significativa tras recibir una dieta CD. Este aumento se acompañó con un aumento de ALT y AST, acumulación de triglicéridos, aumento de marcadores de inflamación y del estrés del retículo endoplasmático (ER estrés). LQs ratones Mat-/-ASM-/- alimentados con una dieta CD, presentaron menor acumulación de trigliééridos, una disminución de los marcadores de inflamación y del estrés del ER. Por lo tanto, la deficiencia de ASMase frena la progresión de la steatohepatitis inducida por una dieta CD. Debido a la baja viabilidad de los ratones Mat-/-ASM-/-, decidimos utilizar una aproximación al modelo genético Inhibiendo ASMase a partir de un vector adenoviral que expresaba un shRNA para ASMase (ASMSH) con los ratones Matla-/-. Se alimentaron los ratones con una dieta CD y se les inyectó el ASMSH para observar el efecto de la inhibición de ASMase a desarrollar EHNA. Los ratones alimentados con una dieta CD y tratados con ASMSH, presentaron una disminución de las transaminasas, de la expresión de marcadores proinflamatorios, genes lipogénicos y del ER estrés. Además, empleamos una alternativa farmacológica para inhibir la ASMase en este modelo nutricional y proponer así una opción terapéutica, utilizando ta Amitriptilina. Se alimentó a los ratones Matla-/- con una dieta CD y se les inyectó el fármaco. Los ratones Matla-/- que reclbieron una dieta CD y fueron tratados con el inhlbidor farmacológico Amitriptilina, presentaron una disminución de los niveles de ALT, de la expresión de marcadores proinftamatorios tales como IL-lb y TNF y mostraron una clara mejora a nivel histológico y una disminución de la acumulación de tri licéridos· todo esto acompañado con una clara disminución de la actividadPeer reviewe

Purinergic receptors and the inflammatory response mediated by lipids.

The inflammatory response is regulated by the production of different extracellular mediators, including lipids and extracellular nucleotides. In the extracellular environment, intermediate lipids activate specific G-protein-coupled receptors (GPCRs) in target cells and promote cell recruitment and activation. Extracellular nucleotides activate two types of receptors, the ionotropic purinergic P2X and the metabotropic purinergic P2Y receptors, inducing the release of cytokines and promoting cell recruitment. Several P2X receptors are associated with an increase in the production of immunoactive lipids mediators, which in turn are able to interfere with the activation of different P2Y receptors, establishing a tight signalling link between purinergic receptors and lipid mediators. In this review, we summarise recent studies indicating signalling crosstalk between purinergic P2X and P2Y receptor activation and lipid mediators with a focus on inflammatory diseases. Novel concepts arising from this crosstalk would result in the development of combinatorial therapies targeting lipid synthesis together with individual P2 receptors for the management of inflammatory diseases.info:eu-repo/semantics/publishe

Key role of ASMase in the susceptibility of MAT1A deficient mice to choline deficient diet-induced steatohepatitis

Trabajo presentado en la The Liver Meeting AASLD, celebrada en San Francisco, California, del 13 al 17 de noviembre de 2015Peer Reviewe

Myristic acid potentiates palmitic acid-induced lipotoxicity and steatohepatitis associated with lipodystrophy by sustaning de novo ceramide synthesis

Palmitic acid (PA) induces hepatocyte apoptosis and fuels de novo ceramide synthesis in the endoplasmic reticulum (ER). Myristic acid (MA), a free fatty acid highly abundant in copra/palmist oils, is a predictor of nonalcoholic steatohepatitis (NASH) and stimulates ceramide synthesis. Here we investigated the synergism between MA and PA in ceramide synthesis, ER stress, lipotoxicity and NASH. Unlike PA, MA is not lipotoxic but potentiated PA-mediated lipoapoptosis, ER stress, caspase-3 activation and cytochrome c release in primary mouse hepatocytes (PMH). Moreover, MA kinetically sustained PA-induced total ceramide content by stimulating dehydroceramide desaturase and switched the ceramide profile from decreased to increased ceramide 14:0/ceramide16:0, without changing medium and long-chain ceramide species. PMH were more sensitive to equimolar ceramide14:0/ceramide16:0 exposure, which mimics the outcome of PA plus MA treatment on ceramide homeostasis, than to either ceramide alone. Treatment with myriocin to inhibit ceramide synthesis and tauroursodeoxycholic acid to prevent ER stress ameliorated PA plus MA induced apoptosis, similar to the protection afforded by the antioxidant BHA, the pan-caspase inhibitor z-VAD-Fmk and JNK inhibition. Moreover, ruthenium red protected PMH against PA and MA-induced cell death. Recapitulating in vitro findings, mice fed a diet enriched in PA plus MA exhibited lipodystrophy, hepatosplenomegaly, increased liver ceramide content and cholesterol levels, ER stress, liver damage, inflammation and fibrosis compared to mice fed diets enriched in PA or MA alone. The deleterious effects of PA plus MA-enriched diet were largely prevented by in vivo myriocin treatment. These findings indicate a causal link between ceramide synthesis and ER stress in lipotoxicity, and imply that the consumption of diets enriched in MA and PA can cause NASH associated with lipodystrophy.This work was supported by grants SAF-2011- 23031, SAF-2012-34831 from Plan Nacional de I+D, Spain, Fundació Marató de TV3, La Mutua Madrileña, PI11/0325 (META) grant from the Instituto Salud Carlos III, and by the support of CIBEREHD; the center grant P50-AA-11999 Research Center for Liver and Pancretic Diseases funded by NIAAA/NIH. Laura Martinez acknowledges support from Formación de Personal Investigador (FPI) fellowship BES-2009-027637, Boehringer Ingelheim and Journal of Cell Science Travel Fellowships.Peer Reviewe

Chronic alcohol feeding increases mitochondrial respiration in frg mice with humanized liver

Trabajo presentado en el Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases (AASLD) / Liver Meeting, celebrado en San Francisco del 9 al 13 de noviembre de 2018[Background] Alcoholic liver disease (ALD) is a spectrum of disorders that begin with hepatic steatosis, which can progress to alcoholic steatohepatitis (ASH) and cirrhosis. The incomplete understanding of mechanisms contributing to ALD progression has limited the availability of effective therapy. Mitochondrial dysfunction is a hallmark of ALD. Altered mitochondrial morphology in human ALD and indirect metabolic determinations suggested impaired mitochondrial function in ALD patients. In experimental models, impaired mitochondrial respiration seems to be species-dependent (rats vs. mice), with evidence in mice showing a correlation between increased mitochondrial respiration and ALD severity. Therefore, we examined the mitochondrial function from alcohol-fed FRG mice xenotransplanted with human adult hepatocytes (HH).[Methods] FRG mice were injected with HH through the spleen followed by controlled cycles of NTBC withdrawal. Humanized FRG mice were fed an alcohol (5%, ethanol; 36% calories) or control liquid diets for 10 days after 5 days of acclimation. HH were isolated and oxygen consumption rates (OCR) determined by a flux analyzer to examine respiratory parameters. In parallel, hepatocyte mitochondrial respiration was determined in wild type mice fed alcohol diet for comparison. In some cases, perivenous (PV) and periportal (PP) hepatocytes were isolated from wild type mice fed alcohol.[Results] FRG mice were repopulated with HH by 80-85%, as estimated by the determination of serum human albumin levels and expression of fumaroylacetoacetate by immunohistochemistry. OCR significantly increased in HH from FRG mice fed alcohol compared to FRG mice fed control diet. This outcome translated in a significant increase (2-fold) in basal OCR, ATP production and maximal respiration, without changes in proton leak, coupling efficiency or mitochondrial mass (cytochrome c immunostaining). Interestingly, these findings in OCR and respiratory parameters were similar to those found in mitochondrial respiration from wild type mice fed alcohol for the same period of time. Moreover, the mitochondrial respiratory alterations reflected predominant changes in PV rather than PP hepatocytes from wild type mice fed alcohol. Conclusion: Using this subrogate model of humanized ALD, our findings show that alcohol feeding increases mitochondrial respiration in human hepatocytes metabolizing alcohol, suggesting that increased mitochondrial respiration may contribute to human ALD.Peer reviewe

Sensing low intracellular potassium by NLRP3 results in a stable open structure that promotes inflammasome activation

The NLRP3 inflammasome is activated by a wide range of stimuli and drives diverse inflammatory diseases. The decrease of intracellular K+ concentration is a minimal upstream signal to most of the NLRP3 activation models. Here, we found that cellular K+ efflux induces a stable structural change in the inactive NLRP3, promoting an open conformation as a step preceding activation. This conformational change is facilitated by the specific NLRP3 FISNA domain and a unique flexible linker sequence between the PYD and FISNA domains. This linker also facilitates the ensemble of NLRP3PYD into a seed structure for ASC oligomerization. The introduction of the NLRP3 PYD-linker-FISNA sequence into NLRP6 resulted in a chimeric receptor able to be activated by K+ efflux–specific NLRP3 activators and promoted an in vivo inflammatory response to uric acid crystals. Our results establish that the amino-terminal sequence between PYD and NACHT domain of NLRP3 is key for inflammasome activation.I.H.-B. would like to acknowledge the funding by the Slovenian Research Agency (project grant J3-1746 and core funding P4-0176), and B.O. would like to acknowledge the funding by the Ministerio de Economía, Industria y Competitividad and ERDF (BIO2017-85329-R). This work was supported by grants to A.T.-A. from the internal support program of the Medical Faculty, University of Tübingen, Fortüne-Antrag Nr. 2615-0-0 and to P.P. from FEDER/Ministerio de Ciencia, Innovación y Universidades—Agencia Estatal de Investigación (grant SAF2017-88276-R), Fundación Séneca (grants 20859/PI/18, 21081/PDC/19, and 0003/COVI/20), and the European Research Council (ERC-2013-CoG grant 614578 and ERC-2019-PoC grant 899636)

Dietary and genetic disruption of hepatic methionine metabolism induce acid sphingomyelinase to promote steatohepatitis

Alcoholic (ASH) and nonalcoholic. (NASH).steatohepatitis are advanced.stages.of.fatty.liver.disease.Methionine adenosyltransferase 1A (MAT1A) plays a key role in hepatic methionine metabolism and germline Mat1a deletion in mice promotes NASH. Acid sphingomyelinase (ASMase) triggers hepatocellular apoptosis and liver fibrosis and has been shown to downregulate MAT1A expression in the context of fulminant liver failure. Given the role of ASMase in steatohepatitis development, we investigated the status of ASMase in Mat1a−/− mice and the regulation of ASMase by SAM/SAH. Consistent with its role in NASH, Mat1a−/− mice fed a choline-deficient (CD) diet exhibited macrosteatosis, inflammation, fibrosis and liver injury as well as reduced total and mitochondrial GSH levels. Our data uncovered an increased basal expression and activity of ASMase but not neutral SMase in Mat1a−/− mice, which further increased upon CD feeding. Interestingly, adenovirus-mediated shRNA expression targeting ASMase reduced ASMase activity and protected Mat1a−/− mice against CD diet-induced NASH. Similar results were observed in CD fed Mat1a−/− mice by pharmacological inhibition of ASMase with amitriptyline. Moreover, Mat1a/ASMase double knockout mice were resistant to CD-induced NASH. ASMase knockdown protected wild type mice against NASH induced by feeding a diet deficient in methionine and choline. Furthermore, Mat1a−/− mice developed acute-on-chronic ASH and this outcome was ameliorated by amitriptyline treatment. In vitro data in primary mouse hepatocytes revealed that decreased SAM/SAH ratio increased ASMase mRNA level and activity. MAT1A and ASMase mRNA levels exhibited an inverse correlation in liver samples from patients with ASH and NASH. Thus, disruption of methionine metabolism sensitizes to steatohepatitis by ASMase activation via decreased SAM/SAH. These findings imply that MAT1A deletion and ASMase activation engage in a self-sustained loop of relevance for steatohepatitis