HIV et exosomes : rôle de l'homéostasie lipidique du compartiment endosomal dans le maintien des réservoirs du HIV

Les exosomes sont des vésicules de 50-90 nm, générées dans le compartiment endosomal tardif, ou corps multivésiculaires, et sécrétés dans le milieu extracellulaire par les cellules vivantes. Les exosomes sont impliqués dans la communication intercellulaire, transportant à la fois des lipides bioactifs, des protéines, et des acides ribonucleiques (mRNAs, microRNAs) pouvant moduler la réponse de la cellule receveuse. Nous avons montré que les exosomes dérivés de RBL-2H3 contiennent l'ensemble des différentes phospholipases : une PLC, une PLD et des membres des trois classes de PLA2 (cPLA2, iPLA2, sPLA2) ainsi que tous les membres de la famille des Ras GTPase. Nous avons pu observer une augmentation de l'activité PLD2 et PLA2 en présence de GTP. Des analyses lipidomiques des exosomes ont révélé la présence d'acides gras libres tels que l'acide arachidonique et plusieurs prostaglandines. L'accumulation de ces vésicules dans le compartiment endosomal de cellules cibles a été observée. L'implication du compartiment endosomal dans le stockage et la dissémination du HIV, dans les cellules réservoirs comme les monocytes/macrophages, pourrait rendre compte de l'impossibilité d'éradiquer le virus, même sous thérapie antirétrovirale efficace. Les corps multivésiculaires pourraient être un site d'assemblage du HIV permettant au virus de détourner, pour sa sortie, la voie préexistante des exosomes. Ce compartiment concentre à la fois le cholestérol et l'acide lysobisphosphatidique (LBPA), dont le métabolisme fait intervenir une cPLA2 sensible au MAFP. Dans un second temps, nous avons évalué l'importance de l'homéostasie lipidique sur la production du HIV i) en modifiant la teneur et la distribution du cholestérol intracellulaire, ii) en inhibant l'activité PLA2. Ces analyses ont été réalisées sur une coculture de cellules monocytaires humains (THP-1) infectées par le HIV et non infectées, et également sur des cocultures de macrophages dérivés de monocytes primaires humains infectés et non infectés. La progestérone, hormone stéroïde, et le MAFP, inhibiteur de PLA2, ont permis d'inhiber la production de HIV dans nos conditions de culture et nous avons également pu observer l'absence de virions en périphérie de la cellule. Le LBPA pourrait être un élément commun à l'effet de la progestérone et du MAFP. Cibler les PLA2s sensibles au MAFP représente donc une stratégie visant à altérer les réservoirs de HIV, et pourrait mener au développement de nouvelles molécules pour l'éradication du HIV, complémentaires des thérapies actuelles.Exosomes are vesicles about 50-90nm, generated in the late endosome compartment or multivesicular bodies, and released in the extracellular medium. We have considered them as non infectious particles and found that mast cells -derived exosomes contain phospholipases : a phospholipase C (PLC), a PLD, 3 classes of PLA2 (cPLA2, iPLA2, sPLA2) which are activatables by GTP. They also contain free fatty acids such as arachidonic acid, and prostaglandins. Following internalization, they accumulate in late endosome compartments which are a site for storage and assembly for HIV. This compartment concentrates lipids such as cholesterol and lysobisphosphatidic (LBPA) acid for which a PLA2 intervenes in metabolism. We assessed the role of lipidic homeostasis on HIV production using human monocytes coculture, by i) modifying the rate and distribution of intracellular cholesterol,and ii) inhibiting PLA2 activity. We found that the steroid hormone progesterone, and the PLA2 inhibitor, (MAFP), allowed to lower HIV production and to reduce the release of particles from cell periphery. The LBPA could be a common element to the effect of progesterone and MAFP. Targeting MAFP sensitive PLA2s represents a strategy to alter HIV reservoir and could lead to the development of new molecules in HIV eradication

Traduire, mon coiffeur et moi

Illustration : Marlène Junius. Mon premier abonnement à Traduire remonte à l’hiver 2006. Un geste solidaire, qui visait à grossir le nombre des abonnés à LA revue de la SFT et, par extension, apporter du baume au cœur « aux copains » bénévoles affairés chaque trimestre à nourrir les voraces appétits de leurs lecteurs. Auparavant, mon aventure avec le trimestriel n’avait jamais franchi le cap de l’effet coupe-faim. Le survol de ses énoncés scientifiques présageait d’innombrables va-et-vient d..

Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA.

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS

Effect of prolonged treatment with tyramine on glucose tolerance in streptozotocin-induced diabetic rats

The biogenic amine tyramine has been reported to stimulatein vitro glucose transport in adipocytes, cardiomyocytes and skeletal muscle, and to improvein vivo glucose utilization in rats. These effects were dependent on amine oxidation, since they were blocked by inhibitors of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO). We thus tested in this work whether a prolonged treatment with tyramine could improve glucose tolerance in streptozotocin-induced diabetic rats. First, tyramine content of standard rodent chow was determined by HPLC and daily tyramine intake of control rats was estimated to be around 26 μmol/kg body weight. Then, tyramine was administred during 3 weeks in streptozotocin-induced diabetic rats at 29 μmol/kg by daily i.p. injection alone or together with vanadate 0.02 μmol/kg. In another group of diabetic rats, tyramine was subcutaneously delivered at 116 μmol/kg/day by osmotic minipumps. All tyramine treatments resulted in a decrease of the hyperglycemic responses to an i.p. glucose load. Adipocytes isolated from either untreated or treated diabetic rats were sensitive to the stimulation of glucose uptake by tyramine. However, diabetic animals receiving tyramine for three weeks did not recover from their hyperglycemia, hypoinsulinemia and glucosuria. These results show that the improvement of glucose tolerance induced by prolonged tyramine administration occurs in an insulin-depleted model and probably results from peripheral insulin-like actions of the oxidation of MAO/SSAO substrates, such as the stimulation of glucose uptake into adipocytes

Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner.

The innate immune response to cytosolic DNA involves transcriptional activation of type I interferons (IFN-I) and proinflammatory cytokines. This represents the culmination of intracellular signaling pathways that are initiated by pattern recognition receptors that engage DNA and require the adaptor protein Stimulator of Interferon Genes (STING). These responses lead to the generation of cellular and tissue states that impair microbial replication and facilitate the establishment of long-lived, antigen-specific adaptive immunity. Ultimately this can lead to immune-mediated protection from infection but also to the cytotoxic T cell-mediated clearance of tumor cells. Intriguingly, pharmacologic activation of STING-dependent phenotypes is known to enhance both vaccine-associated immunogenicity and immune-based anti-tumor therapies. Unfortunately, the STING protein exists as multiple variant forms in the human population that exhibit differences in their reactivity to chemical stimuli and in the intensity of molecular signaling they induce. In light of this, STING-targeting drug discovery efforts require an accounting of protein variant-specific activity. Herein we describe a small molecule termed M04 that behaves as a novel agonist of human STING. Importantly, we find that the molecule exhibits a differential ability to activate STING based on the allelic variant examined. Furthermore, while M04 is inactive in mice, expression of human STING in mouse cells rescues reactivity to the compound. Using primary human cells in ex vivo assays we were also able to show that M04 is capable of simulating innate responses important for adaptive immune activation such as cytokine secretion, dendritic cell maturation, and T cell cross-priming. Collectively, this work demonstrates the conceivable utility of a novel agonist of human STING both as a research tool for exploring STING biology and as an immune potentiating molecule

Exosomes as intercellular signalosomes and pharmacological effectors.

International audienceCell secretion is a general process involved in various biological responses. Exosomes are part of this process and have gained considerable scientific interest in the past five years. Several steps through investigations across the last 20 years can explain this interest. First characterized during reticulocyte maturation, they were next evidenced as a key player in the immune response and cancer immunotherapy. More recently they were reported as vectors of mRNAs, miRNAs and also lipid mediators able to act on target cells. They are the only type of vesicles released from an intracellular compartment from cells in viable conditions. They appear as a vectorized signaling system operating from inside a donor cell towards either the periphery, the cytosol, or possibly to the nucleus of target cells. Exosomes from normal cells trigger positive effects, whereas those from pathological ones, such as tumor cells or infected ones may trigger non-positive health effects. Therefore regulating the biogenesis and secretion of exosomes appear as a pharmacological challenge to intervene in various pathophysiologies. Exosome biogenesis and molecular content, interaction with target cells, utilisation as biomarkers, and functional effects in various pathophysiologies are considered in this review

Apport de l’immunologie à la prise en charge diagnostique et thérapeutique des glomérulonéphrites extramembraneuses

International audienceLa glomérulonéphrite extra-membraneuse idiopathique peut désormais être qualifiée de maladie autoimmune glomérulaire. Un certain nombre d’autoanticorps a été décrit contre des composants glomérulaires ou contre des protéines alimentaires usuelles. Certains de ces autoanticorps, tel que les anticorps anti-PLA2R et les anticorps anti-THSD7A sont utiles pour suivre l’activité de la maladie et la réponse au traitement. Il existe certainement une susceptibilité génétique pour l’apparition de la maladie. La meilleure connaissance de la physiopathologie est le support rationnel à de nouvelles approches thérapeutiques en cours d’évaluation