Assessment of pathophysiological mechanisms in obesity-related diseases through metabolomics, transcriptomics and mouse models of disease

L'exposició a quantitats excessives de nutrients i la manca d'activitat física promouen el desenvolupament de patologies metabòliques. En el cas de l'obesitat, l'excés de nutrients supera la capacitat d'emmagatzematge dels teixits metabòlics i activa les vies metabòliques implicades en respostes inflamatòries. Els macròfags mostren un ampli espectre d'estats de "polarització" i són els desencadenants de les respostes inflamatòries en les malalties metabòliques com l'obesitat. Els excessos de greix indueixen un canvi de polarització en els macròfags que adquireixen funcions pro-inflamatòries i potencien l'estat d'inflamació associat a l'obesitat. En el primer estudi es van abordar les conseqüències de la ingesta excessiva de nutrients en el metabolisme energètic. Vam desenvolupar un mètode basat en GC-EI-QTOF-MS per identificar i quantificar els metabòlits en mostres biològiques per predir alteracions mitocondrials, una característica comú en els trastorns metabòlics. El segon estudi es va centrar en la identificació d'estratègies terapèutiques per millorar les alteracions metabòliques induïdes per una dieta rica en greixos en ratolins deficients en el receptor de lipoproteïnes de baixa densitat. Els nostres resultats van indicar que la combinació de metformina i restricció calòrica proporciona una bona alternativa contra el dany metabòlic i podria ser una estratègia terapèutica per millorar la disfunció metabòlica. En el tercer estudi s'analitzen els efectes del palmitat (àcid gras saturat) sobre la polarització dels macròfags humans. L'exposició de macròfags humans derivats de monòcits dependents de M-CSF a palmitat disminueix l'expressió de factors de transcripció que promouen l'expressió del "conjunt de gens antiinflamatoris" i simultàniament promou l'adquisició d'un perfil pro-inflamatori mitjançant l'activació de JNK. A més, el palmitat condiciona els macròfags de manera que potenciïn les respostes inflamatòries cap a un estímul patogen com el LPS, del qual el palmitat difereix en efectes transcripcionals i funcionals.La exposición a cantidades excesivas de nutrientes y la falta de actividad física promueven el desarrollo de patologías metabólicas. En el caso de la obesidad, el exceso de nutrientes supera la capacidad de almacenamiento de los tejidos metabólicos y activa las vías metabólicas implicadas en respuestas inflamatorias. Los macrófagos muestran un amplio espectro de estados de "polarización" y son los desencadenantes de las respuestas inflamatorias en las enfermedades metabólicas como la obesidad. Los excesos de grasa inducen un cambio de polarización en los macrófagos que adquieren funciones pro-inflamatorias y potencian el estado de inflamación asociado a la obesidad. En el primer estudio se abordaron las consecuencias de la ingesta excesiva de nutrientes en el metabolismo energético. Desarrollamos un método basado en GC-EI-QTOF-MS para identificar y cuantificar los metabolitos en muestras biológicas para predecir alteraciones mitocondriales, una característica común en los trastornos metabólicos. El segundo estudio se centró en la identificación de estrategias terapéuticas para mejorar las alteraciones metabólicas inducidas por una dieta rica en grasas en ratones deficientes en el receptor de lipoproteínas de baja densidad. Nuestros resultados indicaron que la combinación de metformina y restricción calórica proporciona una buena alternativa contra el daño metabólico y podría ser una estrategia terapéutica para mejorar la disfunción metabólica. En el tercer estudio se analizan los efectos del palmitato (ácido graso saturado) sobre la polarización de los macrófagos humanos. La exposición de macrófagos humanos derivados de monocitos dependientes de M-CSF a palmitato disminuye la expresión de factores de transcripción que promueven la expresión del "conjunto de genes anti-inflamatorios" y simultáneamente promueve la adquisición de un perfil pro-inflamatorio mediante la activación de JNK. Además, el palmitato condiciona a los macrófagos de manera que potencien las respuestas inflamatorias hacia un estímulo patógeno como el LPS, del cual el palmitato difiere en efectos transcripcionales y funcionales.Current lifestyle patterns, like exposure to excessive amounts of nutrients and lack of physical activity, promote the development of metabolic pathologies. In the case of obesity, chronic over nutrition exceed the storage capacity of metabolic tissues and activate metabolic pathways involved in the development of inflammatory responses and insulin resistance. Macrophages are considered as triggers of the inflammatory responses underlying metabolic diseases like obesity. By virtue of their plasticity, macrophages can display a wide spectrum of functional “polarization” states. An excessive amount of fat induces a macrophage polarization switch that leads to the acquisition of pro-inflammatory effector functions that exacerbate the obesity-associated inflammation state. In the first study, we addressed the consequences of excessive nutrient intake in energy metabolism. Since mitochondrial dysfunction is a common feature in metabolic disorders, we develop a GC-EI-QTOF-MS method to identify and quantify metabolites in biological samples as a mean to predict mitochondrial alterations. The second study focused on the identification of therapeutic strategies to ameliorate the metabolic disturbances induced by high-fat diet in low density lipoprotein receptor-deficient mice. Our findings reveal that the combination of metformin and caloric restraint provides a better alternative against the metabolic damage and might be a therapeutic strategy to ameliorate the metabolic dysfunction induced by excessive nutrient intake. The effects of palmitate (saturated fatty acid) on the human macrophage polarization are reported in the third study. We observed that exposure of M-CSF-dependent monocyte-derived human macrophages to palmitate lowers the expression of transcription factors that drive “anti-inflammatory gene set” expression and simultaneously promote the acquisition of a pro-inflammatory transcriptional and functional profile via JNK activation. Besides, palmitate was found to prime macrophages for exacerbated inflammatory responses towards a pathogenic stimulus like LPS. Importantly, we also report that the transcriptional and functional effects of palmitate differ from those triggered by LPS

Transcriptomic microRNA profiling of dendritic cells in response to gut microbiota-secreted vesicles

The interconnection between nutrients, metabolites and microbes is a key factor governing the healthy/pathological status of an individual. Thus, microbiota-based research is essential in order to better understand human health and nutrition. Gut bacteria release membrane vesicles (MVs) as an intercellular communication mechanism that allows the direct delivery of factors that prime the host's innate immune system. We have previously shown that MVs from intestinal E. coli activate dendritic cells (DCs) in a strain-specific manner. To gain insights into the regulatory mechanisms involved, here, we have used an RNA deep sequencing approach to identify differentially expressed miRNAs (microRNAs) in DCs which are challenged by the MVs of the probiotic Nissle 1917 (EcN) or the commensal ECOR12. MicroRNAs are post-transcriptional regulatory mediators that permit the fine tuning of signaling pathways. This approach allowed the identification of a common set of miRNAs which are modulated by MVs from both strains and miRNAs which are differentially expressed in response to EcN or ECOR12 MVs. Based on the differential expression of the target genes and subsequent validation experiments, we correlated some of the selected miRNAs with the reported cytokine profile and specific T cell responses. As far as we know, this is the first study to analyze the regulation of miRNAs in DCs by MVs released by gut microbiota

Metformin Potentiates the Benefits of Dietary Restraint: A Metabolomic Study

Prevention of the metabolic consequences of a chronic energy-dense/high-fat diet (HFD) represents a public health priority. Metformin is a strong candidate to be incorporated in alternative therapeutic approaches. We used a targeted metabolomic approach to assess changes related to the multi-faceted metabolic disturbances provoked by HFD. We evaluated the protective effects of metformin and explored how pro-inflammatory and metabolic changes respond when mice rendered obese, glucose-intolerant and hyperlipidemic were switched to diet reversal with or without metformin. Mice treated with metformin and diet-reversal showed a dramatically improved protection against HFD-induced hepatic steatosis, a beneficial effect that was accompanied by a lowering of liver-infiltrating pro-inflammatory macrophages and lower release of pro-inflammatory cytokines. Metformin combined with diet reversal promoted effective weight loss along with better glucose control, lowered levels of circulating cholesterol and triglycerides, and reduced adipose tissue content. Our findings underscored the ability of metformin to target the contribution of branched chain amino acids to adipose tissue metabolism while suppressing mitochondrial-dependent biosynthesis in hepatic tissue. The relationship between adipose tissue and liver might provide clinical potential for combining metformin and dietary modifications to protect against the metabolic damage occurring upon excessive dietary fat intake

Macrophage-specific MHCII expression is regulated by a remote Ciita enhancer controlled by NFAT5

MHCII in antigen-presenting cells (APCs) is a key regulator of adaptive immune responses. Expression of MHCII genes is controlled by the transcription coactivator CIITA, itself regulated through cell type-specific promoters. Here we show that the transcription factor NFAT5 is needed for expression of Ciita and MHCII in macrophages, but not in dendritic cells and other APCs. NFAT5-deficient macrophages showed defective activation of MHCII-dependent responses in CD4+ T lymphocytes and attenuated capacity to elicit graft rejection in vivo. Ultrasequencing analysis of NFAT5-immunoprecipitated chromatin uncovered an NFAT5-regulated region distally upstream of Ciita This region was required for CIITA and hence MHCII expression, exhibited NFAT5-dependent characteristics of active enhancers such as H3K27 acetylation marks, and required NFAT5 to interact with Ciita myeloid promoter I. Our results uncover an NFAT5-regulated mechanism that maintains CIITA and MHCII expression in macrophages and thus modulates their T lymphocyte priming capacity.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO), Agencia Estatal de Investigación, and European Regional Development Fund (SAF2012-36535, SAF2015-71363-R, and BFU2016-77961-P), and Fundació la Marató de TV3 (1225-30 and 201619-30). We also acknowledge funding support from Generalitat de Catalunya (2014SGR1153, 2017SGR888, and 2017SGR702) and MINECO through the “Unidad de Excelencia María de Maeztu” funded by MINECO (MDM-2014-0370). H. Huerga Encabo was supported by a predoctoral fellowship of the Spanish Ministerio de Educación, Cultura y Deporte (FPU13/01798). M. Tellechea was supported by fellowships from Fundació Catalunya-La Pedrera (2011) and Generalitat de Catalunya (FI-DGR program 2013). C. López-Rodríguez is a recipient of an ICREA Acadèmia award from Institució Catalana de Recerca i Estudis Avançats (ICREA, Generalitat de Catalunya)

Macrophage-specific MHCII expression is regulated by a remote Ciita enhancer controlled by NFAT5

MHCII in antigen-presenting cells (APCs) is a key regulator of adaptive immune responses. Expression of MHCII genes is controlled by the transcription coactivator CIITA, itself regulated through cell type-specific promoters. Here we show that the transcription factor NFAT5 is needed for expression of Ciita and MHCII in macrophages, but not in dendritic cells and other APCs. NFAT5-deficient macrophages showed defective activation of MHCII-dependent responses in CD4+ T lymphocytes and attenuated capacity to elicit graft rejection in vivo. Ultrasequencing analysis of NFAT5-immunoprecipitated chromatin uncovered an NFAT5-regulated region distally upstream of Ciita This region was required for CIITA and hence MHCII expression, exhibited NFAT5-dependent characteristics of active enhancers such as H3K27 acetylation marks, and required NFAT5 to interact with Ciita myeloid promoter I. Our results uncover an NFAT5-regulated mechanism that maintains CIITA and MHCII expression in macrophages and thus modulates their T lymphocyte priming capacity.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO), Agencia Estatal de Investigación, and European Regional Development Fund (SAF2012-36535, SAF2015-71363-R, and BFU2016-77961-P), and Fundació la Marató de TV3 (1225-30 and 201619-30). We also acknowledge funding support from Generalitat de Catalunya (2014SGR1153, 2017SGR888, and 2017SGR702) and MINECO through the “Unidad de Excelencia María de Maeztu” funded by MINECO (MDM-2014-0370). H. Huerga Encabo was supported by a predoctoral fellowship of the Spanish Ministerio de Educación, Cultura y Deporte (FPU13/01798). M. Tellechea was supported by fellowships from Fundació Catalunya-La Pedrera (2011) and Generalitat de Catalunya (FI-DGR program 2013). C. López-Rodríguez is a recipient of an ICREA Acadèmia award from Institució Catalana de Recerca i Estudis Avançats (ICREA, Generalitat de Catalunya)