Regulación de la activación del inflasoma NLRP3 por LIPINA-2 en macrófagos

The innate immune system constitutes the first line of defense against invading microbial pathogens, as it can discriminate self from non-self. It can also sense endogenous danger signals that are released from disrupted host tissue or stressed cells. Macrophages, cells of the innate immune system, are able to recognize PAMPs and DAMPs by different receptors that modulate inflammation through transcriptional (TLRs) and post-transcriptional (NLRs) mechanisms. Key players in those pathways are large intracellular multiprotein complexes called inflammasomes. After exposure to pathogens and danger signals, inflammasomes orchestrate innate immune responses through activation of caspase-1 leading to the maturation of pro-inflammatory cytokines pro-IL-1β and pro-IL-18 and an inflammatory form of death known as pyroptosis. NLRP3 inflammasome is the best characterized to date. It is composed of an NLR receptor, the adaptor protein ASC and pro-caspase-1. In macrophages, its activation and assembly need two signals. The first signal, also known as priming, leads to transcription of genes encoding pro-IL-1β and NLRP3 and it is mediated by activation of the transcription factor NF-κB through various receptors, such as TLRs, IL1R or TNFR. The second signal, provided by DAMPs, activates the inflammasome assembly, resulting in the catalytic processing of pro-caspase-1 into its enzymatically active form. NLRP3 inflammasome is activated by a wide range of stimuli, such as bacterial and viral pathogens, pore-forming toxins, lipids, crystals, vaccine adjuvants and stress cellular signals such as ATP. It is broadly agreed that detection of such a diverse variety of agents cannot bind to NLRP3 directly. Instead, it is thought that NLRP3 monitors a common host-derived factor triggered by all these agents. Several hypotheses on molecular mechanisms leading to NLRP3 activation have been formulated. Three models that may not be exclusive are the most widely accepted. The first model suggests that a decrease in intracellular potassium concentration causes inflammasome activation, the second model points at mitochondrial damage and ROS production, and the third model claims that lysosomal destabilization is the mechanism triggering caspase-1 activation.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

PHLPP1 counter-regulates STAT1-mediated inflammatory signaling.

Inflammation is an essential aspect of innate immunity but also contributes to diverse human diseases. Although much is known about the kinases that control inflammatory signaling, less is known about the opposing phosphatases. Here we report that deletion of the gene encoding PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) protects mice from lethal lipopolysaccharide (LPS) challenge and live Escherichia coli infection. Investigation of PHLPP1 function in macrophages reveals that it controls the magnitude and duration of inflammatory signaling by dephosphorylating the transcription factor STAT1 on Ser727 to inhibit its activity, reduce its promoter residency, and reduce the expression of target genes involved in innate immunity and cytokine signaling. This previously undescribed function of PHLPP1 depends on a bipartite nuclear localization signal in its unique N-terminal extension. Our data support a model in which nuclear PHLPP1 dephosphorylates STAT1 to control the magnitude and duration of inflammatory signaling in macrophages

Lipin-1 integrates lipid synthesis with proinflammatory responses during TLR activation in macrophages

Lipin-1 is a Mg2+-dependent phosphatidic acid phosphatase involved in the de novo synthesis of phospholipids and triglycerides. Using macrophages from lipin-1-deficient animals and human macrophages deficient in the enzyme, we show in this work that this phosphatase acts as a proinflammatory mediator during TLR signaling and during the development of in vivo inflammatory processes. After TLR4 stimulation lipin-1-deficient macrophages showed a decreased production of diacylglycerol and activation of MAPKs and AP-1. Consequently, the generation of proinflammatory cytokines like IL-6, IL-12, IL-23, or enzymes like inducible NO synthase and cyclooxygenase 2, was reduced. In addition, animals lacking lipin-1 had a faster recovery from endotoxin administration concomitant with a reduced production of harmful molecules in spleen and liver. These findings demonstrate an unanticipated role for lipin-1 as a mediator of macrophage proinflammatory activation and support a critical link between lipid biosynthesis and systemic inflammatory responses.This work was supported by the Spanish Ministry of Science and Innovation (Grants SAF2007-60055, SAF2010-18831, and BFU2010-18826) and the Regional Government of Castile and Leon (Grants BIO39/VA04/10 and CSI168A12-1). L.P. and G.L. were supported by predoctoral fellowships from the Spanish Ministry of Science and Innovation (Plan de Formación de Personal Investigador and Plan de Formación de Profesorado Universitario programs). M.V. was supported by a predoctoral fellowship from the Regional Government of Castile and Leon. E.E. was supported by a predoctoral fellowship from the Spanish National Research Council (Junta de Ampliación de Estudios Program). C.G. was supported by a predoctoral fellowship from the University of Valladolid.Peer Reviewe

Regulación de la activación del inflamasoma NLRP3 por lipina-2 en macrófagos

Programa de Doctorado en Investigación Biomédica por la Universidad de Valladolid.The innate immune system constitutes the first line of defense against invading microbial pathogens, as it can discriminate self from non-self. It can also sense endogenous danger signals that are released from disrupted host tissue or stressed cells. Macrophages, cells of the innate immune system, are able to recognize PAMPs and DAMPs by different receptors that modulate inflammation through transcriptional (TLRs) and post-transcriptional (NLRs) mechanisms. Key players in those pathways are large intracellular multiprotein complexes called inflammasomes. After exposure to pathogens and danger signals, inflammasomes orchestrate innate immune responses through activation of caspase-1 leading to the maturation of pro-inflammatory cytokines pro-IL-1β and pro-IL-18 and an inflammatory form of death known as pyroptosis. NLRP3 inflammasome is the best characterized to date. It is composed of an NLR receptor, the adaptor protein ASC and pro-caspase-1. In macrophages, its activation and assembly need two signals. The first signal, also known as priming, leads to transcription of genes encoding pro-IL-1β and NLRP3 and it is mediated by activation of the transcription factor NF-κB through various receptors, such as TLRs, IL1R or TNFR. The second signal, provided by DAMPs, activates the inflammasome assembly, resulting in the catalytic processing of pro-caspase-1 into its enzymatically active form. NLRP3 inflammasome is activated by a wide range of stimuli, such as bacterial and viral pathogens, pore-forming toxins, lipids, crystals, vaccine adjuvants and stress cellular signals such as ATP. It is broadly agreed that detection of such a diverse variety of agents cannot bind to NLRP3 directly. Instead, it is thought that NLRP3 monitors a common host-derived factor triggered by all these agents. Several hypotheses on molecular mechanisms leading to NLRP3 activation have been formulated. Three models that may not be exclusive are the most widely accepted. The first model suggests that a decrease in intracellular potassium concentration causes inflammasome activation, the second model points at mitochondrial damage and ROS production, and the third model claims that lysosomal destabilization is the mechanism triggering caspase-1 activation.Peer Reviewe

Macrophages lacking lipin-2 enhance IL-1β production through the overactivation of NLRP3 inflammasome

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.Lipin-2 is a member of the lipin family of proteins, which catalyze the enzymatic conversion of phosphatidic acid to diacylglycerol. Mutations found in the human LPIN2 gene are known to cause Majeed Syndrome, an autoinflammatory disorder in which IL-1βis involved. Since NLRP3 inflammasome orchestrate innate immune responses through activation of caspase-1 leading to the maturation of pro-inflammatory cytokines pro-IL-1β and pro-IL-18, it was hypothesized that lipin-2 could modulate its production and thereby be involved in the regulation of inflammasome activity. We found out that both, classic (LPS and ATP) and metabolic (LPS and palmitic acid) activation of NLRP3 inflammasome, promote an increased production of the proinflammatory cytokine IL-1β in macrophages lacking lipin-2, which depends on the overactivation of NF-κB by LPS. Besides, depletion of lipin-2 in macrophages alter cellular responses to ATP, what leads to overstimulation of the NLRP3 pathway triggering the IL-1β and IL-18 production in an ASC and caspase-1 dependent manner. These effects may be due to the fact that absence of lipin-2 modifies lipid membrane levels, leading to an increase of P2X7 receptor activity, triggering potassium efflux from the cell, and increasing the assembly of the inflammasome and its activity. Collectively, these studies confirm a protective role for lipin-2 in the pathology of inflammatory diseases mediated by activation of inflamasome NLRP3.Peer reviewe

Lipin-2 regulates NLRP3 inflammasome activation in macrophages

Resumen del póster presentado presentado al CIBERDEM Annual Meeting, celebrado en Cerdanyola del Vallès, Barcelona (España) del 11 al 13 de mayo de 2016.Cardiovascular disease is one of the most frequent inflammatory diseases. Atherosclerosis is the primary cause for cardiovascular disease, and diabetes increases the risk several-fold by enhancing the formation and/or progression of atherosclerotic lesions, a process in which abnormally-activated monocytes and macrophages appear to play a major role. Macrophages and other cell types are recruited to the subendothelial space of the arterial wall, where they engulf modified lipoproteins and become foam cells that constitute the core of atherosclerotic plaques. In a wide range of metabolic conditions, including atherosclerosis and Type 2 diabetes, the NLRP3 inflammasome is activated and contributes to disease by producing the proinflammatory cytokines IL-1β and IL-18. In the present work we have investigated the role of Lipin-2, a key enzyme in lipid metabolism, in the activation of the NLRP3 inflammasome. We found out that depletion of lipin-2 in macrophages promotes an increased expression of the proinflammatory cytokine IL-1β, which depends on the overactivation of NF-kB and MAPK by LPS. Cells lacking lipin-2 alter lipid cellular levels increasing ATP receptor activity, leading to overstimulation of the NLRP3 pathway, thereby triggering IL-1β and IL-18 production in an ASC and caspase-1 dependent manner. The absence of lipin-2 may modify lipid membrane lipids, leading to an increase of ATP receptor activity, potassium efflux from the cell, and assembly of the inflammasome and its activity. Our studies confirm a protective role for lipin-2 in the pathology of inflammatory diseases mediated by classical activation of inflamasoma NLRP3, and may open new avenues for controlling metabolic diseases in which macrophages may contribute to the development of the disease.Peer reviewe

Lipin-1 links lipid signaling with Iinflammation

Resumen del trabajo presentado al American Society for Biochemistry and Molecular Biology Annual Meeting, celebrado en Boston (US) del 2 al 6 de abril de 2015.Lipins are Mg2+-dependent phosphatidic acid phosphatases involved in the de novo synthesis of phospholipids and triglycerides. Although they regulate cellular levels of important signaling lipids, little is known about the role of lipins in the immune response. Using lipin-1-deficient animals, we show in this work that this phosphatase acts as a proinflammatory modulator during TLR signaling and also in the development of in vivo inflammatory processes. After stimulation with lipopolysaccharide (LPS), lipin-1 deficient macrophages had a decreased production of DAG, activation of MAPKs and phosphorylation of the AP-1 transcription factor c-jun. Consequently, the generation of cytokines like IL-6, IL12, IL23, or proteins like iNOS and COX-2 was reduced. In the same line, responses to Salmonella were diminished, although no differences in phagocytosis rates were found. During the development of chemically-induced colitis these animals exhibited reduced inflammatory responses and were protected against tissue damage and disease severity. These effects correlated with a lower generation of gut pathogenic factors like IL-17 and IFNg and increased production of the gut protective factor TGFa. These findings demonstrate an unanticipated role for lipin-1 as a mediator of inflammatory-based pathological states and support a critical link between lipid signaling and inflammatory responses.Peer Reviewe

Protein kinase Cα gain-of-function variant in Alzheimer’s disease displays enhanced catalysis by a mechanism that evades down-regulation

Conventional protein kinase C (PKC) family members are reversibly activated by binding to the second messengers Ca2+ and diacylglycerol, events that break autoinhibitory constraints to allow the enzyme to adopt an active, but degradation-sensitive, conformation. Perturbing these autoinhibitory constraints, resulting in protein destabilization, is one of many mechanisms by which PKC function is lost in cancer. Here, we address how a gain-of-function germline mutation in PKCα in Alzheimer's disease (AD) enhances signaling without increasing vulnerability to down-regulation. Biochemical analyses of purified protein demonstrate that this mutation results in an ∼30% increase in the catalytic rate of the activated enzyme, with no changes in the concentrations of Ca2+ or lipid required for half-maximal activation. Molecular dynamics simulations reveal that this mutation has both localized and allosteric effects, most notably decreasing the dynamics of the C-helix, a key determinant in the catalytic turnover of kinases. Consistent with this mutation not altering autoinhibitory constraints, live-cell imaging studies reveal that the basal signaling output of PKCα-M489V is unchanged. However, the mutant enzyme in cells displays increased sensitivity to an inhibitor that is ineffective toward scaffolded PKC, suggesting the altered dynamics of the kinase domain may influence protein interactions. Finally, we show that phosphorylation of a key PKC substrate, myristoylated alanine-rich C-kinase substrate, is increased in brains of CRISPR-Cas9 genome-edited mice containing the PKCα-M489V mutation. Our results unveil how an AD-associated mutation in PKCα permits enhanced agonist-dependent signaling via a mechanism that evades the cell's homeostatic down-regulation of constitutively active PKCα

Critical role for cytosolic group IVA phospholipase A2 in early adipocyte differentiation and obesity

Adipogenesis is the process of differentiation of immature mesenchymal stem cells into adipocytes. Elucidation of the mechanisms that regulate adipocyte differentiation is key for the development of novel therapies for the control of obesity and related comorbidities. Cytosolic group IVA phospholipase A (cPLAα) is the pivotal enzyme in receptor-mediated arachidonic acid (AA) mobilization and attendant eicosanoid production. Using primary multipotent cells and cell lines predetermined to become adipocytes, we show here that cPLAα displays a proadipogenic function that occurs very early in the adipogenic process. Interestingly, cPLAα levels decrease during adipogenesis, but cPLAα-deficient preadipocytes exhibit a reduced capacity to differentiate into adipocytes, which affects early and terminal adipogenic transcription factors. Additionally, the absence of the phospholipase alters proliferation and cell-cycle progression that takes place during adipogenesis. Preconditioning of preadipocytes with AA increases the adipogenic capacity of these cells. Moreover, animals deficient in cPLAα show resistance to obesity when fed a high fat diet that parallels changes in the expression of adipogenic transcription factors of the adipose tissue. Collectively, these results show that preadipocyte cPLAα activation is a hitherto unrecognized factor for adipogenesis in vitro and in vivo.This work was supported by the Spanish Ministry of Science and Innovation (Grants SAF2010-18831, BFU2010-18826 and SAF2013-48201-R), the Regional Government of Castile and Leon (Grants BIO39/VA04/10, CSI168A12-1 and BIO/VA03/14) and by grants-in aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and AMED-CREST from the Japan Agency for Medical Research and Development (MM and HS). L.P. and G.L. were supported by predoctoral fellowships from the Spanish Ministry of Science and Innovation (Plan de Formación de Personal Investigador and Plan de Formación de Profesorado Universitario programs, respectively). M.V. was supported by a predoctoral fellowship from the Regional Government of Castile and Leon. CIBERDEM is an initiative of Instituto de Salud Carlos III.Peer Reviewe