Overexpression of cytosolic group IVA phospholipase A2 protects cells from Ca2+ -dependent death

El pdf del artículo es la versión post-print.-- et al.The calcium ionophore ionomycin induces apoptosis-like events in the human embryonic kidney cell line at early times. Plasma membrane blebbing, mitochondrial depolarization, externalization of phosphatidylserine, and nuclear permeability changes can all be observed within 15 min of treatment. However, there is no activation of caspases or chromatin condensation. Expression of a fusion protein containing the enhanced green fluorescent protein (EGFP) and human cytosolic Group IVA phospholipase A2α (EGFP-cPLA 2α) in these cells prevents ionomycin-induced phosphatidylserine externalization and death. Cells expressing the cPLA 2α mutant D43N, which does not bind calcium, retain their susceptibility to ionomycin-induced cell death. Both nonexpressing and EGFP-D43N-cPLA2α-expressing human embryonic kidney cells can be spared from ionomycin-induced cell death by pretreating them with exogenous arachidonic acid. Moreover, during calcium overload, mitochondrial depolarization is significantly lower in the EGFP-cPLA2α- expressing cells than in cells expressing normal amounts of cPLA 2α. These results suggest that early cell death events promoted by an overload of calcium can be prevented by the presence of high levels of arachidonic acid. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported in part by Grant BMC2001- 2244 from the Spanish Ministry of Science and Technology, Grants BFU2004-01886/BMC, SAF2004-04676, and SAF2004-01232 from the Spanish Ministry of Education and Science, and Red Brucella, Red Respira, and Red Temática de Investigación Cardiovascular, from the Instituto de Salud Carlos III.Peer Reviewe

Secretory phospholipase A2 elicits proinflammatory changes and upregulates the surface expression of fas ligand in monocytic cells: potential relevance for atherogenesis

Producción CientíficaType IIA secretory phospholipase A2 (sPLA2) is an acute-phase reactant that plays a role in atherogenesis and is expressed in atherosclerotic arterial walls displaying inflammatory features. This generates a relevant question addressing the biological effects of this enzyme on monocytic cells, in view of the role of these cells in the inflammatory process associated with atherosclerosis. sPLA2 produced a mild activation of the p42 mitogen-activated protein module of the mitogen-activated protein kinase (MAPK) cascade and a prominent activation of c-Jun N-terminal kinase in THP-1 monocytes. This activation showed both an early and a late peak, different from that elicited by tumor necrosis factor-α (TNF-α), which only showed the first peak. This was accompanied by activation of arachidonate metabolism, as judged from both the activation of the cytosolic phospholipase A2 (cPLA2) and the induction of cyclooxygenase-2 (COX-2) expression. sPLA2 also elicited the production of monocyte chemoattractant protein-1 (MCP-1) and showed a synergistic effect with TNF-α on both COX-2 induction and MCP-1 production. sPLA2 upregulated the expression of Fas ligand at the cell surface, but it did not influence Fas expression nor cell survival of monocytes. In summary, these data indicate that some of the atherogenic effects of sPLA2 can be exerted by engagement of an sPLA2-binding structure on monocytic cells, most probably the M-type receptor for sPLA2, which produces the activation of the MAPK cascade, induces a proinflammatory phenotype, and upregulates the cell surface expression of Fas ligand.Plan Nacional de Salud y Farmacia (grant SAF98/0176)Comisión Interministerial de Ciencia y Tecnología - Comisión Europea (grant 1FD97-0590)Fondo de Investigación Sanitaria (grant FIS00/0393

Valor nutritivo y efectos metabólicos de la reutilización de aceites comestibles calentados y oxidados

Las grasas y los aceites procesados, oxidados y calentados pueden tener propiedades nutricionales diferentes de la materia prima de donde provienen; existen cambios físico-químicos inducidos o provocados en las diferentes etapas a las que se someten durante la obtención comercial, lo que trae como consecuencia una modificación desde el punto de vista de la nutrición. Estos cambios van desde la modificación isomérica de los dobles enlaces naturales de las grasas poli insaturadas que se someten a procesos de hidrogenación, con la consiguiente formación de isómeros trans, con los efectos biológicos que éstos ocasionan al organismo humano. Por otra parte, los fenómenos de auto oxidación de las grasas en general, así como a los cambios suscitados durante el proceso térmico como consecuencia del freído profundo, contribuyen a cambios radicales en la estructura molecular de las grasas produciéndose no sólo radicales libres de oxígeno, sustancias altamente reactivas y dañinas para las células, sino también un sin número de sustancias, algunas de ellas verdaderos carcinógenos. Estudios como el nuestro, efectuados tanto de grasas calentadas como de ácidos grasos isoméricos, se han ideado experimentos para identificar isómeros o compuestos con efectos biológicos y/o nutricionales potencialmente indeseables en condiciones extremas de dieta o en circunstancias específicas. En general, los mismos síntomas que sugieren toxicidad en casos específicos no han sido reproducidos cuando se ingieren dietas con el nivel de grasas calentadas o hidrogenadas usualmente en Estados Unidos. Sin embargo, aún quedan muchas preguntas sin responder acerca de los efectos en la nutrición y fundamentalmente sobre el metabolismo lipoproteico de los aceites hidrogenados y calentados. Se tiene relativamente poca información acerca del umbral biológico de tolerancia de grasas parcialmente hidrogenadas o calentadas sin afectar el bienestar

Eicosanoids in the Innate Immune Response: TLR and Non-TLR Routes

The variable array of pattern receptor expression in different cells of the innate immune system explains the induction of distinct patterns of arachidonic acid (AA) metabolism. Peptidoglycan and mannan were strong stimuli in neutrophils, whereas the fungal extract zymosan was the most potent stimulus in monocyte-derived dendritic cells since it induced the production of PGE2, PGD2, and several cytokines including a robust IL-10 response. Zymosan activated κB-binding activity, but inhibition of NF-κB was associated with enhanced IL-10 production. In contrast, treatments acting on CREB (CRE binding protein), including PGE2, showed a direct correlation between CREB activation and IL-10 production. Therefore, in dendritic cells zymosan induces il10 transcription by a CRE-dependent mechanism that involves autocrine secretion of PGE2, thus unraveling a functional cooperation between eicosanoid production and cytokine production

The Unfolded Protein Response and the Phosphorylations of Activating Transcription Factor 2 in the trans-Activation of il23a Promoter Produced by β-Glucans

Producción CientíficaCurrent views on the control of IL-23 production focus on theregulation ofil23a, the gene encoding IL-23 p19, by NF- Bincombination with other transcription factors. C/EBP homolo-gous protein (CHOP), X2-Box-binding protein 1 (XBP1), acti-vator protein 1 (AP1), SMAD, CCAAT/enhancer-binding pro-tein (C/EBP ), and cAMP-response element-binding protein(CREB) have been involved in response to LPS, but no data areavailable regarding the mechanism triggered by the fungalmimic and -glucan-containing stimulus zymosan, which pro-duces IL-23 and to a low extent the related cytokine IL-12 p70.Zymosan induced the mobilization of CHOP from the nuclearfractions to phagocytic vesicles. Hypha-formingCandidaalsoinduced the nuclear disappearance of CHOP. Assay of tran-scription factor binding to theil23apromoter showed anincrease of Thr(P)-71–Thr(P)-69-activating transcription fac-tor 2 (ATF2) binding in response to zymosan. PKC and PKA/mitogen- and stress-activated kinase inhibitors down-regulatedThr(P)-71–ATF2 binding to theil23apromoter andil23amRNA expression. Consistent with the current concept ofcomplementary phosphorylations on N-terminal Thr-71 andThr-69 of ATF2 by ERK and p38 MAPK, MEK, and p38 MAPKinhibitors blunted Thr(P)-69–ATF2 binding. Knockdown ofatf2mRNA with siRNA correlated with inhibition ofil23amRNA, but it did not affect the expression ofil12/23bandil10mRNA. These data indicate the following: (i) zymosan decreasesnuclear proapoptotic CHOP, most likely by promoting its accu-mulation in phagocytic vesicles; (ii) zymosan-inducedil23amRNA expression is best explained through coordinated B-and ATF2-dependent transcription; and (iii)il23aexpressionrelies on complementary phosphorylation of ATF2 on Thr-69and Thr-71 dependent on PKC and MAPK activities

Polymorphisms in receptors involved in opsonic and nonopsonic phagocytosis, and correlation with risk of infection in oncohematology patients

Producción CientíficaHigh-risk hematological malignancies are a privileged setting for infection by opportunistic microbes, with invasive mycosis being one of the most serious complications. Recently, genetic background has emerged as an unanticipated risk factor. For this reason, polymorphisms for genes encoding archetypal receptors involved in the opsonic and nonopsonic clearance of microbes, pentraxin-3 (PTX3) and Dectin-1, respectively, were studied and correlated with the risk of infection. Fungal, bacterial, and viral infections were registered for a group of 198 patients with highrisk hematological malignancies. Polymorphisms for the pentraxin-3 gene (PTX3) showed a significant association with the risk of fungal infection by Candida spp. and, especially, by Aspergillus spp. This link remained even for patients undergoing antifungal prophylaxis, thus demonstrating the clinical relevance of PTX3 in the defense against fungi. CLEC7A polymorphisms did not show any definite correlation with the risk of invasive mycosis, nor did they influence the expression of Dectin-1 isoforms generated by alternative splicing. The PTX3 mRNA expression level was significantly lower in samples from healthy volunteers who showed these polymorphisms, although no differences were observed in the extents of induction elicited by bacterial lipopolysaccharide and heat-killed Candida albicans, thus suggesting that the expression of PTX3 at the start of infection may influence the clinical outcome. PTX3 mRNA expression can be a good biomarker to establish proper antifungal prophylaxis in immunodepressed patients

Endoplasmic reticulum stress sensor IRE1α enhances IL-23 expression by human dendritic cells

Producción CientíficaHuman monocyte-derived dendritic cells (DCs) exposed to pathogen-associated molecular patterns (PAMPs) undergo bioenergetic changes that influence the immune response. We found that stimulation with PAMPs enhanced glycolysis in DCs, whereas oxidative phosphorylation remained unaltered. Glucose starvation and the hexokinase inhibitor 2-deoxy-d-glucose (2-DG) modulated cytokine expression in stimulated DCs. Strikingly, IL23A was markedly induced upon 2-DG treatment, but not during glucose deprivation. Since 2-DG can also rapidly inhibit protein N-glycosylation, we postulated that this compound could induce IL-23 in DCs via activation of the endoplasmic reticulum (ER) stress response. Indeed, stimulation of DCs with PAMPs in the presence of 2-DG robustly activated inositol-requiring protein 1α (IRE1α) signaling and to a lesser extent the PERK arm of the unfolded protein response. Additional ER stressors such as tunicamycin and thapsigargin also promoted IL-23 expression by PAMP-stimulated DCs. Pharmacological, biochemical, and genetic analyses using conditional knockout mice revealed that IL-23 induction in ER stressed DCs stimulated with PAMPs was IRE1α/X-box binding protein 1-dependent upon zymosan stimulation. Interestingly, we further evidenced PERK-mediated and CAAT/enhancer-binding protein β-dependent trans-activation of IL23A upon lipopolysaccharide treatment. Our findings uncover that the ER stress response can potently modulate cytokine expression in PAMP-stimulated human DCs.Plan Nacional de Salud y Farmacia (Proyecto SAF2013-44521-R

Tricarboxylic acid cycle activity and remodeling of glycerophosphocholine lipids support cytokine induction in response to fungal patterns

Producción CientíficaIncreased glycolysis parallels immune cell activation, but the role of pyruvate remains largely unexplored. We found that stimulation of dendritic cells with the fungal surrogate zymosan causes decreases of pyruvate, citrate, itaconate, and a-ketoglutarate, while increasing oxaloacetate, succinate, lactate, oxygen consumption, and pyruvate dehydrogenase activity. Expression of IL10 and IL23A (the gene encoding the p19 chain of IL-23) depended on pyruvate dehydrogenase activity. Mechanistically, pyruvate reinforced histone H3 acetylation, and acetate rescued the effect of mitochondrial pyruvate carrier inhibition, most likely because it is a substrate of the acetyl-CoA producing enzyme ACSS2. Mice lacking the receptor of the lipid mediator platelet-activating factor (PAF; 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) showed reduced production of IL-10 and IL-23 that is explained by the requirement of acetyl-CoA for PAF biosynthesis and its ensuing autocrine function. Acetyl-CoA therefore intertwines fatty acid remodeling of glycerophospholipids and energetic metabolism during cytokine induction.Plan Nacional de Salud y Farmacia (Proyectos SAF2013-44521-R, SAF2017-83079-R, BFU2014-53469-P, and BFU201)4- 53469-PJunta de Castilla y León - Fondo Social Europeo (Proyecto CSI035P17

Endoplasmic Reticulum Stress Sensor IRE1α Enhances IL-23 Expression by Human Dendritic Cells

Human monocyte-derived dendritic cells (DCs) exposed to pathogen-associated molecular patterns (PAMPs) undergo bioenergetic changes that influence the immune response. We found that stimulation with PAMPs enhanced glycolysis in DCs, whereas oxidative phosphorylation remained unaltered. Glucose starvation and the hexokinase inhibitor 2-deoxy-d-glucose (2-DG) modulated cytokine expression in stimulated DCs. Strikingly, IL23A was markedly induced upon 2-DG treatment, but not during glucose deprivation. Since 2-DG can also rapidly inhibit protein N-glycosylation, we postulated that this compound could induce IL-23 in DCs via activation of the endoplasmic reticulum (ER) stress response. Indeed, stimulation of DCs with PAMPs in the presence of 2-DG robustly activated inositol-requiring protein 1α (IRE1α) signaling and to a lesser extent the PERK arm of the unfolded protein response. Additional ER stressors such as tunicamycin and thapsigargin also promoted IL-23 expression by PAMP-stimulated DCs. Pharmacological, biochemical, and genetic analyses using conditional knockout mice revealed that IL-23 induction in ER stressed DCs stimulated with PAMPs was IRE1α/X-box binding protein 1-dependent upon zymosan stimulation. Interestingly, we further evidenced PERK-mediated and CAAT/enhancer-binding protein β-dependent trans-activation of IL23A upon lipopolysaccharide treatment. Our findings uncover that the ER stress response can potently modulate cytokine expression in PAMP-stimulated human DCs