Chemerin e seu receptor (ChemR23) têm sido amplamente associados à disfunção endotelial, inflamação e resistência à insulina. No entanto, é ainda desconhecido se chemerin influencia diretamente a sinalização da insulina na vasculatura. A hipótese deste estudo é de que chemerin diminui a sinalização vascular da insulina, e que o uso de antagonista de ChemR23 (CCX 832) em um modelo de diabetes do tipo 2 relacionado à obesidade melhora as respostas vasculares a insulina. Mecanismos moleculares e vasculares foram investigados em artérias mesentéricas e células de músculo liso vascular em cultura (CMLV) de camundongos C57BL/6J, db/m (controles, não obesos, não diabéticos) e db/db (diabéticos, obesos), assim como em células endoteliais (CE) de humanos em cultura. Nossos resultados mostraram que chemerin diminui a vasodilatação induzida por insulina em camundongos C57BL/6J, efeito mediado por ChemR23, PI3K/Akt e estresse oxidativo. Em CMLV, chemerin, através de mecanismos dependentes de estresse oxidativo e ChemR23, diminui a fosforilação de IRS-1, PI3K e Akt e a translocação de GLUT4 para a membrana, induzidas por insulina. Chemerin também diminui a captação de glicose induzida por insulina via estresse oxidativo e ativação de AMPK e PI3K/Akt. Em CE, chemerin diminui a sinalização de óxido nítrico (NO) ativada pela insulina, novamente via ChemR23, estresse oxidativo e PI3K/Akt. CCX 832 diminui a massa corporal (sem alterar a ingestão de ração), os níveis de insulina e glicose (sem alterar a tolerância à glicose) e estresse oxidativo em aorta e rim de camundongos db/db. CCX 832 restaura parcialmente a disfunção vascular observada em camundongos db/db, sem modificar parâmetros estruturais destas artérias. Adicionalmente, CCX 832 diminui marcadores pró-inflamatórios em tecido adiposo perivascular (PVAT) e melhora a sinalização da insulina em aorta de camundongos db/db. Nossos achados destacam o sistema chemerin/ChemR23 como um novo e promissor alvo terapêutico para limitar a resistência à insulina e as complicações vasculares associadas ao diabetes relacionado à obesidade. / Chemerin and its G protein-coupled receptor (ChemR23) have been associated with endothelial dysfunction, inflammation and insulin resistance. Whether chemerin directly influences insulin signaling in the vasculature is unknown. We hypothesized that chemerin impairs vascular insulin signaling in obesity-related type 2 diabetes, effect that would be improved by the novel ChemR23 antagonist (CCX 832). Molecular and vascular mechanisms were probed in mesenteric arteries and cultured vascular smooth muscle cells (VSMC) from C57BL/6J, non-diabetic lean db/m and diabetic obese db/db mice as well as in human microvascular endothelial cells (EC). Chemerin decreased insulin-induced vasodilatation in C57BL/6J mice, effect mediated by ChemR23, PI3K/Akt and oxidative stress. In VSMC, chemerin, via oxidative stress- and ChemR23-dependent mechanisms, decreased insulin-induced IRS-1, PI3K and Akt phosphorylation, GLUT4 translocation to the membrane. In addition, chemerin decreases insulin-induced glucose uptake via oxidative stress and AMPK and PI3K/Akt activation. In EC, chemerin decreased insulin-activated nitric oxide (NO) signaling via ChemR23, oxidative stress and PI3K/Akt signaling pathway. CCX 832 decreases body weight (without altering food intake), insulin and glucose levels (without altering glucose tolerance) and oxidative stress in aorta and kidney from db/db mice. CCX 832 partially restored vascular dysfunction in db/db mice without modifying structural parameters. Additionally, CCX 832 decreases proinflammatory markers in perivascular adipose tissue (PVAT) and improves insulin signaling in aorta from db/db mice. Our findings highlight chemerin/ChemR23 system as a promising new therapeutic target to limit insulin resistance and vascular complications associated with obesity-related diabetes.

Chemerin e seu receptor (ChemR23) têm sido amplamente associados à disfunção endotelial, inflamação e resistência à insulina. No entanto, é ainda desconhecido se chemerin influencia diretamente a sinalização da insulina na vasculatura. A hipótese deste estudo é de que chemerin diminui a sinalização vascular da insulina, e que o uso de antagonista de ChemR23 (CCX 832) em um modelo de diabetes do tipo 2 relacionado à obesidade melhora as respostas vasculares a insulina. Mecanismos moleculares e vasculares foram investigados em artérias mesentéricas e células de músculo liso vascular em cultura (CMLV) de camundongos C57BL/6J, db/m (controles, não obesos, não diabéticos) e db/db (diabéticos, obesos), assim como em células endoteliais (CE) de humanos em cultura. Nossos resultados mostraram que chemerin diminui a vasodilatação induzida por insulina em camundongos C57BL/6J, efeito mediado por ChemR23, PI3K/Akt e estresse oxidativo. Em CMLV, chemerin, através de mecanismos dependentes de estresse oxidativo e ChemR23, diminui a fosforilação de IRS-1, PI3K e Akt e a translocação de GLUT4 para a membrana, induzidas por insulina. Chemerin também diminui a captação de glicose induzida por insulina via estresse oxidativo e ativação de AMPK e PI3K/Akt. Em CE, chemerin diminui a sinalização de óxido nítrico (NO) ativada pela insulina, novamente via ChemR23, estresse oxidativo e PI3K/Akt. CCX 832 diminui a massa corporal (sem alterar a ingestão de ração), os níveis de insulina e glicose (sem alterar a tolerância à glicose) e estresse oxidativo em aorta e rim de camundongos db/db. CCX 832 restaura parcialmente a disfunção vascular observada em camundongos db/db, sem modificar parâmetros estruturais destas artérias. Adicionalmente, CCX 832 diminui marcadores pró-inflamatórios em tecido adiposo perivascular (PVAT) e melhora a sinalização da insulina em aorta de camundongos db/db. Nossos achados destacam o sistema chemerin/ChemR23 como um novo e promissor alvo terapêutico para limitar a resistência à insulina e as complicações vasculares associadas ao diabetes relacionado à obesidade. / Chemerin and its G protein-coupled receptor (ChemR23) have been associated with endothelial dysfunction, inflammation and insulin resistance. Whether chemerin directly influences insulin signaling in the vasculature is unknown. We hypothesized that chemerin impairs vascular insulin signaling in obesity-related type 2 diabetes, effect that would be improved by the novel ChemR23 antagonist (CCX 832). Molecular and vascular mechanisms were probed in mesenteric arteries and cultured vascular smooth muscle cells (VSMC) from C57BL/6J, non-diabetic lean db/m and diabetic obese db/db mice as well as in human microvascular endothelial cells (EC). Chemerin decreased insulin-induced vasodilatation in C57BL/6J mice, effect mediated by ChemR23, PI3K/Akt and oxidative stress. In VSMC, chemerin, via oxidative stress- and ChemR23-dependent mechanisms, decreased insulin-induced IRS-1, PI3K and Akt phosphorylation, GLUT4 translocation to the membrane. In addition, chemerin decreases insulin-induced glucose uptake via oxidative stress and AMPK and PI3K/Akt activation. In EC, chemerin decreased insulin-activated nitric oxide (NO) signaling via ChemR23, oxidative stress and PI3K/Akt signaling pathway. CCX 832 decreases body weight (without altering food intake), insulin and glucose levels (without altering glucose tolerance) and oxidative stress in aorta and kidney from db/db mice. CCX 832 partially restored vascular dysfunction in db/db mice without modifying structural parameters. Additionally, CCX 832 decreases proinflammatory markers in perivascular adipose tissue (PVAT) and improves insulin signaling in aorta from db/db mice. Our findings highlight chemerin/ChemR23 system as a promising new therapeutic target to limit insulin resistance and vascular complications associated with obesity-related diabetes.

Chemoattractant agents play a crucial role in the initiation of immune responses, by regulating the traffic and function of leucocyte populations. Their receptors are therefore considered as potential targets for the development of new therapies in the fields of cancer and inflammatory diseases. ChemR23, a previously orphan receptor discovered in the laboratory, is structurally related to receptors for chemoattractant agents. It is expressed on immature myeloid and plasmacytoid dendritic cells (mDCs and pDCs respectively), as well as on adipocytes, macrophages, NK and endothelial cells. Chemerin, the endogenous ligand of ChemR23, is abundant in various human samples originating from inflammatory diseases, including pleural effusions. Chemerin is secreted as an inactive precursor, prochemerin, and is activated by the removal of six or seven amino-acids from its carboxy-terminus by serine proteases, such as as cathepsin G and elastase. Chemerin acts as a chemoattractant agent of low nanomolar potency for macrophages, immature mDCs and pDCs. It is however more active on pDCs, in line with the higher expression of ChemR23 on these cells. pDCs possess important immunoregulatory properties in lung diseases, and their ability to secrete large amounts of type I interferon (IFN) upon viral infection makes them crucial players in anti-viral immunity.<p>According to these elements, and to the role of neutrophils in the physiopathology of many inflammatory lung diseases and in the generation of active chemerin, we began in 2007 to study the role of chemerin and its receptor ChemR23 in inflammatory lung diseases. We first characterized the mouse chemerin/ChemR23 system, and described that this system was very similar to the human one, in terms of distribution, pharmacology and functional properties. We then used wild type mice (WT) and mice invalidated for the receptor (ChemR23-/-) in various models of inflammatory lung diseases, including asthma, lung fibrosis, viral pneumonia, and acute lung injury. <p>Whereas the asthma and lung fibrosis models did not allow to demonstrate a significant role of the chemerin/ChemR23 system (possibly as a result of the lack of production of active chemerin in these models), infection by either the Pneumonia Virus of Mice (PVM), the mouse counterpart of human RSV, or by a murinized H1N1 influenza strain resulted in a significantly higher mortality rate in ChemR23-/- mice as compared to their WT counterparts. Using the PVM-induced pneumonia model, we observed that the excessive mortality of knock-out mice is caused by an inadequate and excessive innate immune response characterized by a massive recruitment of neutrophils to the lungs, associated with a delayed viral clearance and lower type I IFN synthesis. This latter observation suggested an impairment of pDC recruitment, according to the important contribution of pDCs to the production of type I IFNs in viral diseases, and the role of chemerin in the recruitment of these cells. We indeed confirmed a lower recruitment of pDCs in the lung of infected ChemR23-/- mice, as compared to WT mice. However, experiments of adoptive transfert and depletion of pDCs failed to proof a link between impaired pDC recruitment and the excessive morbidity and mortality observed in ChemR23-invalidated mice. <p>In parallel, we studied the role of the chemerin/ChemR23 system in the control of innate immune responses, by using a model of acute lung injury caused by the intra-tracheal instillation of bacterial lipopolysaccharide (LPS). In this model, administration of recombinant chemerin together with LPS in WT mice resulted in a significant (about 50%) reduction of neutrophil recruitment to both lung parenchyma and airways. Assessment of pro-inflammatory cytokines and chemokines in broncho-alveolar lavage fluids confirmed this anti-inflammatory effect of chemerin, which was ChemR23-dependent, as the inflammatory response of ChemR23-/- mice was unaffected by chemerin. In our hands, chemerin does not modulate macrophage functions, in contrast to data recently published by other groups, attributing anti-inflammatory effects of chemerin or chemerin-derived peptide to the modulation of macrophage activation and phagocytosis. Other hypotheses that could take our observations into account are presently investigated, including an immunomodulatory role of chemerin on lung epithelial or endothelial cells, and/or the ChemR23-dependent recruitment of subtypes of macrophages or other myeloid cells endowed with immunosuppressive properties. <p>In conclusion, our studies characterized the mouse chemerin/ChemR23 system and highlighted the role of this system in the physiopathology of some inflammatory lung diseases. Our results suggest that the chemerin/ChemR23 system might be considered as a potential therapeutic target for the development of future anti-infectious and anti-inflammatory therapies, particularly for viral pneumonia, which represent a major public health problem, as well as for acute respiratory distress syndrome (ARDS) following severe acute lung injuries.<p> <p><p>Les agents chimioattractants jouent un rôle fondamental dans l’initiation des réponses immunes en régulant le trafic et la fonction des populations leucocytaires. Leurs récepteurs constituent dès lors des cibles d’intérêt pour le développement de traitements contre les maladies inflammatoires et le cancer. Le laboratoire d’accueil a identifié le récepteur ChemR23, exprimé à la surface des cellules dendritiques myéloïdes (mDCs) et plasmacytoïdes (pDCs) immatures, des macrophages, des cellules NK, des adipocytes, et des cellules endothéliales. Le ligand endogène du récepteur ChemR23, la chémérine, est présent en abondance dans divers échantillons pathologiques d’origine inflammatoire. La chémérine est produite sous la forme d'un précurseur inactif, la prochémérine, qui nécessite pour devenir active le clivage protéolytique de six ou sept acides aminés à son extrémité carboxy-terminale. La chémérine induit le chimiotactisme des macrophages et des DCs immatures, et en particulier des pDCs immatures en accord avec l’expression plus importante de ChemR23 par les pDCs. Les pDCs jouent un rôle immunorégulateur important en pathologie pulmonaire, en particulier dans la physiopathologie des pneumonies virales, par leur capacité à produire d’importantes quantités d’interféron (IFN) de type I.<p>Compte tenu de ces éléments et du rôle des polynucléaires neutrophiles dans de nombreuses pathologies pulmonaires, ainsi que dans la génération de chémérine active à partir de son précurseur, nous avons débuté en octobre 2007, l’étude du rôle de la chémérine et de son récepteur ChemR23 dans le contrôle des pathologies pulmonaires inflammatoires. Nous avons tout d’abord caractérisé le système chémérine/ChemR23 chez la souris et avons montré que ce système présentait des caractéristiques similaires à celles décrites chez l’homme, en termes de distribution, de pharmacologie et de propriétés fonctionnelles. <p>Ensuite, nous avons comparé des souris sauvages et invalidées pour le récepteur ChemR23 (ChemR23-/-) dans divers modèles de pathologies pulmonaires. Les modèles d’asthme et de fibrose pulmonaire induite par instillation de bléomycine ou de silice n’ont pas permis de mettre en évidence un rôle important du couple chémérine/ChemR23, peut-être en raison de l’absence de génération de forme active de chémérine dans ces modèles. En revanche, l’administration de deux agents viraux différents, le PVM (Pneumonia Virus of Mice), l’équivalent murin du RSV humain, et un virus de l’influenza H1N1 murinisé, a résulté en un taux de mortalité 40% plus élevé pour les souris ChemR23-/- par rapport à leurs homologues sauvages. En utilisant le modèle de pneumonie induite par le PVM, nous avons montré que cette différence de mortalité est causée par une réponse immune inappropriée et excessive, associée à une réduction de l’élimination du virus, ainsi qu’à un déficit de synthèse d’IFN de type I. Les pDCs, dans un contexte d’infection virale, sont capables de synthétiser d’importantes quantités d’IFN de type I, et nous avons mis en évidence un déficit relatif de recrutement en pDCs chez les souris ChemR23-/- infectées. Néanmoins, les expériences de transfert adoptif et de déplétion de pDCs n’ont pas permis de lier ce défaut de recrutement à l’excès de morbidité et de mortalité observé chez les souris ChemR23-/- infectées. <p>En parallèle, le rôle de ce couple ligand-récepteur dans le contrôle des réponses immunitaires innées a été étudié dans un modèle de pneumopathie aiguë induite par instillation intra-trachéale de lipopolysaccharide (LPS). Dans ce modèle, l’administration simultanée de chémérine recombinante avec le LPS entraîne chez les souris sauvages une diminution significative (environ 50%) du nombre de polynucléaires neutrophiles recrutés dans les voies aériennes et dans le parenchyme pulmonaire, ainsi qu’une importante diminution de synthèse de cytokines pro-inflammatoires. Cet effet anti-inflammatoire de la chémérine est dépendant de ChemR23, et ne semble pas être secondaire à un effet de la chémérine sur l’activation des macrophages, contrairement à certaines données publiées récemment par d’autres groupes. D’autres hypothèses permettraient cependant de prendre en compte ces observations, notamment un effet de la chémérine sur les cellules épithéliales et/ou endothéliales pulmonaires, ainsi que sur le recrutement de sous-populations de macrophages ou d’autres cellules myéloïdes possédant des propriétés immunosuppressives. Des expériences complémentaires ont été initiées afin de tester ces hypothèses. <p>En conclusion, après avoir caractérisé le système chémérine/ChemR23 chez la souris, nos études ont permis de mettre en évidence le rôle de ce couple ligand/récepteur dans la physiopathologie de certaines pneumopathies inflammatoires, ouvrant ainsi de nouvelles perspectives thérapeutiques, en particulier pour le traitement des pneumopathies virales, qui constituent un problème de santé publique majeur, ainsi que des syndromes de détresse respiratoire aiguë (ARDS). / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished