L'indoléamine 2,3-dioxygénase et la différenciation, maturation des cellules dendritiques

La voie des kynurénines est l’une des trois voies de biosynthèse du NAD, qu’elle produit à partir de la dégradation du plus rare des acides aminés essentiels, le tryptophane. Cette voie catabolique est initiée par trois enzymes distinctes, dont l’indoléamine 2,3-dioxygénase 1 (IDO-1). L’IDO-1 est la seule des trois enzymes dont l’expression est induite par des stimuli pro-inflammatoires tels que l’IFN-γ, le TNF-α ainsi que des produits bactériens et viraux. De plus, son expression est induite de manière particulièrement importante au cours de la maturation des cellules dendritiques (DC) qui jouent un rôle clé, tant dans les réponses immunes innées qu’adaptatives. Par conséquent, outre la production d’un cofacteur essentiel du métabolisme cellulaire, le catabolisme du tryptophane participe également à la régulation de la réponse immune. En effet, la fonction d’agent antimicrobien fut la première attribuée à l’IDO-1 parce qu’en réponse aux stimuli inflammatoires, celle-ci dégrade le tryptophane et limite la prolifération d’organismes pathogènes auxotrophes pour cet acide aminé. Ensuite, l’inhibition de l’activité catalytique de l’IDO-1 par une approche pharmacologique permit de mettre en évidence la contribution de cette enzyme au phénomène de tolérance maternelle pour les fœtus semi-allogéniques qu’elle porte. Enfin, sa participation à la tolérance périphérique fut proposée étant donné sa capacité à induire le développement de Treg ou à leur servir de mécanisme effecteur. Cependant, ces deux dernières fonctions ne sont pas très claires puisque les souris invalidées pour le gène de l’IDO-1 (IDO-1-/-) ne présentent ni défaut reproducteur, ni signe d’auto-immunité spontanée.Comme deux publications récentes suggéraient que l’inhibition pharmacologique de l’IDO-1 affecte la maturation des moDC humaines, nous avons effectué une analyse détaillée du compartiment immunitaire des souris IDO-1-/-, avec une attention toute particulière pour les DC. Au début, nous avons également constaté un important défaut de la maturation des BMDC IDO-1-/- générées in vitro en présence de GM-CSF. Cependant, le défaut se révéla extrêmement dépendant des conditions de culture, puisqu’un changement de substrat de culture ou de facteur de croissance suffit à restaurer une maturation normale de ces cellules. De même, l’analyse de la maturation des DC spléniques démontra de manière claire que l’IDO-1 n’est certainement pas essentielle à la maturation des DC in vivo. Nous avons ensuite montré que l’expression fonctionnelle d’IDO-1 protège les cellules et potentiellement les souris qui l’expriment lorsqu’elles sont soumises à des stress oxydatifs, suggérant que l’IDO-1 puisse consommer les anions O2- afin d’assurer son activité catalytique. Contre toute attente, nous avons ensuite constaté que les souris IDO-1-/- survivent plus longtemps aux infections par la forme pléiomorphe du parasite Trypanosoma brucei brucei. Bien que la levée de l’inhibition de la lymphoprolifération chez les souris IDO-1-/- soit l’explication la plus évidente de l’augmentation de leur survie, nous suggérons plutôt que c’est la perte de la fonction antioxydante de l’IDO-1-/- qui leur confère cette résistance. En conclusion, l’IDO-1 ne semble pas jouer un rôle important dans la différenciation et maturation des cellules dendritiques. Nos observations préliminiares indiquent cependant que cette enzyme pourrait jouer un rôle anti-oxydant, et protége donc les cellules dendritiques d’un stress oxydant potentiellement causé lors des réponses innées anti-microbiennes.Doctorat en sciences, Spécialisation biologie moléculaireinfo:eu-repo/semantics/nonPublishe

Normal development and function of dendritic cells in mice lacking IDO-1 expression.

Dendritic cells (DCs) have been shown to express the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO-1), a protein presently thought to exert dual and possibly contrasting effects on the immune response. Depletion of tryptophan and release of tryptophan catabolites have been shown to exert a tolerogenic influence on T cell responses, while the IDO enzymatic activity has been recently suggested to promote DC maturation. In this report, we have explored the putative role of IDO-1 in regulating DC biology by analyzing DC development and function from IDO-1 deficient mice. In keeping with previous observations, lack of IDO-1 expression was found to affect in vitro DC generation from bone mouse precursors cultured in the presence of GM-CSF. However, change in growth factor (Flt3L) and/or culture conditions (low-adherence vessels) abolished the difference observed between wt (wild type) and IDO-1-deficient, in vitro generated DCs. Moreover, IDO-1-deficient mice displayed a normal DC compartment in vivo, suggesting that IDO-1 does not play a significant role in DC development and function in vivo. Collectively, these observations suggest that despite a possible role for IDO-1 expression in regulating DC differentiation in vitro under commonly used culture conditions, IDO-1 is largely dispensable for DC development and function in vivo.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Transgenic Artifacts Caused by Passenger Human Growth Hormone

The minigene encoding human growth hormone (hGH) has been incorporated into over 300 transgenic mouse lines to improve transgene expression. However, unexpected and functional hGH expression can drastically alter physiology. We list here the mouse lines in which ectopic hGH has been confirmed, and we provide a wiki for lines awaiting analysis.status: publishe

Metabolic and Behavioural Phenotypes in Nestin-Cre Mice Are Caused by Hypothalamic Expression of Human Growth Hormone

The Nestin-Cre driver mouse line has mild hypopituitarism, reduced body weight, a metabolic phenotype and reduced anxiety. Although several causes have been suggested, a comprehensive explanation is still lacking. In this study we examined the molecular mechanisms leading to this compound phenotype. Upon generation of the Nestin-Cre mice, the human growth hormone (hGH) minigene was inserted downstream of the Cre recombinase to ensure efficient transgene expression. As a result, hGH is expressed in the hypothalamus. This results in the auto/paracrine activation of the GH receptor as demonstrated by the increased phosphorylation of signal transducer and activator of transcription 5 (STAT5) and reduced expression of growth hormone releasing hormone (Ghrh). Low Ghrh levels cause hypopituitarism consistent with the observed mouse growth hormone (mGH) deficiency. mGH deficiency caused reduced activation of the GH receptor and hence reduced phosphorylation of STAT5 in the liver. This led to decreased levels of hepatic Igf-1 mRNA and consequently postnatal growth retardation. Furthermore, genes involved in lipid uptake and synthesis, such as CD36 and very low-density lipoprotein receptor were upregulated, resulting in liver steatosis. In conclusion, this study demonstrates the unexpected expression of hGH in the hypothalamus of Nestin-Cre mice which is able to activate both the GH receptor and the prolactin receptor. Increased hypothalamic GH receptor signaling explains the observed hypopituitarism, reduced growth and metabolic phenotype of Nestin-Cre mice. Activation of either receptor is consistent with reduced anxiety.status: publishe

Metabolic and Behavioural Phenotypes in Nestin-Cre Mice Are Caused by Hypothalamic Expression of Human Growth Hormone.

The Nestin-Cre driver mouse line has mild hypopituitarism, reduced body weight, a metabolic phenotype and reduced anxiety. Although several causes have been suggested, a comprehensive explanation is still lacking. In this study we examined the molecular mechanisms leading to this compound phenotype. Upon generation of the Nestin-Cre mice, the human growth hormone (hGH) minigene was inserted downstream of the Cre recombinase to ensure efficient transgene expression. As a result, hGH is expressed in the hypothalamus. This results in the auto/paracrine activation of the GH receptor as demonstrated by the increased phosphorylation of signal transducer and activator of transcription 5 (STAT5) and reduced expression of growth hormone releasing hormone (Ghrh). Low Ghrh levels cause hypopituitarism consistent with the observed mouse growth hormone (mGH) deficiency. mGH deficiency caused reduced activation of the GH receptor and hence reduced phosphorylation of STAT5 in the liver. This led to decreased levels of hepatic Igf-1 mRNA and consequently postnatal growth retardation. Furthermore, genes involved in lipid uptake and synthesis, such as CD36 and very low-density lipoprotein receptor were upregulated, resulting in liver steatosis. In conclusion, this study demonstrates the unexpected expression of hGH in the hypothalamus of Nestin-Cre mice which is able to activate both the GH receptor and the prolactin receptor. Increased hypothalamic GH receptor signaling explains the observed hypopituitarism, reduced growth and metabolic phenotype of Nestin-Cre mice. Activation of either receptor is consistent with reduced anxiety

Mice deficient in the respiratory chain gene Cox6a2 are protected against high-fat diet-induced obesity and insulin resistance

Oxidative phosphorylation in mitochondria is responsible for 90% of ATP synthesis in most cells. This essential housekeeping function is mediated by nuclear and mitochondrial genes encoding subunits of complex I to V of the respiratory chain. Although complex IV is the best studied of these complexes, the exact function of the striated muscle-specific subunit COX6A2 is still poorly understood. In this study, we show that Cox6a2-deficient mice are protected against high-fat diet-induced obesity, insulin resistance and glucose intolerance. This phenotype results from elevated energy expenditure and a skeletal muscle fiber type switch towards more oxidative fibers. At the molecular level we observe increased formation of reactive oxygen species, constitutive activation of AMP-activated protein kinase, and enhanced expression of uncoupling proteins. Our data indicate that COX6A2 is a regulator of respiratory uncoupling in muscle and we demonstrate that a novel and direct link exists between muscle respiratory chain activity and diet-induced obesity / insulin resistance.status: publishe

<i>hGH</i> is expressed in the hypothalamus and to a lesser extent in the pituitary gland of Nestin-Cre mice.

<p>A) Schematic representation of the Cre-hGH transgene based on Tronche et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135502#pone.0135502.ref013" target="_blank">13</a>] B) The expression of <i>hGH</i> was investigated by RT-qPCR in the hypothalamus, the pituitary gland and the liver of 3-month-old male Nestin-Cre mice and control littermates (n = 4–5). C) Detection of a bicistronic Cre-hGH transcript by RT-qPCR, using a forward primer annealing to Cre and a reverse primer annealing to hGH. (n = 4–5) D) PCR was performed on hypothalamus cDNA from control and Nestin-Cre male mice (n = 4 per group), using primers in Cre and the last exon of hGH. E) hGH ELISA was performed on hypothalamus, pituitary and liver lysates from Nestin-Cre and control littermates (n = 6 for both groups). ND, not detected. Data are shown as mean ± SD, * p<0.05, **p<0.01, ***p<0.001.</p

The total body weight of Nestin-Cre mice is reduced.

<p>The total body weight of 3-month-old Nestin-Cre and wild type male mice (n = 4–5). Data are represented as mean ± SD. ***p<0.001.</p

Hypothalamic hGH expression increases STAT5 phosphorylation, induces <i>Cish</i> expression and leads to a reduction in the expression of <i>Ghrh</i>.

<p>A) Immunoblotting for STAT5 and phospho-STAT5 on hypothalamus lysates from Nestin-Cre versus control male mice. Three independent samples are shown per genotype. Actin was used as a loading control. B) Quantification of the phospho-STAT5/STAT5 ratio, **p<0.01. C) Hypothalamic <i>Cish</i> expression as quantified by RT-qPCR, n = 4–5, **p<0.01. D) The expression of <i>Ghrh</i> was investigated by RT-qPCR in the hypothalamus of 3-month-old male Nestin-Cre mice and control littermates, n = 4, *p<0.05.</p

GHD in Nestin-Cre mice leads to a decrease in STAT5 phosphorylation, lower expression of <i>Igf1</i> and an increase in the expression of <i>CD36</i> and <i>Vldlr</i> in the liver.

<p>A) Western blot analysis was performed for STAT5 and phospho-STAT5 on liver lysates from 1-month-old male Nestin-Cre mice and control littermates. B) Quantification of the phospho-STAT5/STAT5 ratio, **p<0.01. C) The expression of <i>Igf1</i>, <i>CD36</i> and <i>VLDLR</i> was investigated by RT-qPCR in the liver of 3-month-old male Nestin-Cre mice and control littermates (n = 3–4). Data are represented as mean ± SD. * p<0.05, **p<0.01, ***p<0.001.</p