Inflammatory Role of Toll-Like Receptors in Human and Murine Adipose Tissue

It was recently demonstrated that TLR4 activation via dietary lipids triggers inflammatory pathway and alters insulin responsiveness in the fat tissue during obesity. Here, we question whether other TLR family members could participate in the TLR-mediated inflammatory processes occurring in the obese adipose tissue. We thus studied the expression of TLR1, TLR2, TLR4, and TLR6 in adipose tissue. These receptors are expressed in omental and subcutaneous human fat tissue, the expression being higher in the omental tissue, independently of the metabolic status of the subject. We demonstrated a correlation of TLRs expression within and between each depot suggesting a coregulation. Murine 3T3-L1 preadipocyte cells stimulated with Pam3CSK4 induced the expression of some proinflammatory markers. Therefore, beside TLR4, other toll-like receptors are differentially expressed in human fat tissue, and functional in an adipocyte cell line, suggesting that they might participate omental adipose tissue-related inflammation that occurs in obesity

Identification of a Variable Number of Tandem Repeats Polymorphism and Characterization of LEF-1 Response Elements in the Promoter of the IDO1 Gene

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step of the kynurenine pathway that is an important component of immunomodulatory and neuromodulatory processes. The IDO1 gene is highly inducible by IFN-γ and TNF-α through interaction with cis-acting regulatory elements of the promoter region. Accordingly, functional polymorphisms in the IDO1 promoter could partly explain the interindividual variability in IDO expression that has been previously documented.A PCR-sequencing strategy, applied to DNA samples from healthy Caucasians, allowed us to identify a VNTR polymorphism in the IDO1 promoter, which correlates significantly with serum tryptophan concentration, controlled partially by IDO activity, in female subjects, but not in males. Although this VNTR does not appear to affect basal or cytokine-induced promoter activity in gene reporter assays, it contains novel cis-acting elements. Three putative LEF-1 binding sites, one being located within the VNTR repeat motif, were predicted in silico and confirmed by chromatin immunoprecipitation. Overexpression of LEF-1 in luciferase assays confirmed an interaction between LEF-1 and the predicted transcription factor binding sites, and modification of the LEF-1 core sequence within the VNTR repeat motif, by site-directed mutagenesis, resulted in an increase in promoter activity.The identification of a VNTR in the IDO1 promoter revealed a cis-acting element interacting with the most downstream factor of the Wnt signaling pathway, suggesting novel mechanisms of regulation of IDO1 expression. These data offer new insights, and suggest further studies, into the role of IDO in various pathological conditions, particularly in cancer where IDO and the Wnt pathway are strongly dysregulated

Interleukin-7 Regulates Adipose Tissue Mass and Insulin Sensitivity in High-Fat Diet-Fed Mice through Lymphocyte-Dependent and Independent Mechanisms

Although interleukin (IL)-7 is mostly known as a key regulator of lymphocyte homeostasis, we recently demonstrated that it also contributes to body weight regulation through a hypothalamic control. Previous studies have shown that IL-7 is produced by the human obese white adipose tissue (WAT) yet its potential role on WAT development and function in obesity remains unknown. Here, we first show that transgenic mice overexpressing IL-7 have reduced adipose tissue mass associated with glucose and insulin resistance. Moreover, in the high-fat diet (HFD)-induced obesity model, a single administration of IL-7 to C57BL/6 mice is sufficient to prevent HFD-induced WAT mass increase and glucose intolerance. This metabolic protective effect is accompanied by a significant decreased inflammation in WAT. In lymphocyte-deficient HFD-fed SCID mice, IL-7 injection still protects from WAT mass gain. However, IL-7-triggered resistance against WAT inflammation and glucose intolerance is lost in SCID mice. These results suggest that IL-7 regulates adipose tissue mass through a lymphocyte-independent mechanism while its protective role on glucose homeostasis would be relayed by immune cells that participate to WAT inflammation. Our observations establish a key role for IL-7 in the complex mechanisms by which immune mediators modulate metabolic functions

Preadipocyte response and impairment of differentiation in an inflammatory environment.

Recent reports suggest the potential role of toll-like receptor 4 (TLR4) in initiation of inflammatory responses and fatty acid-induced insulin resistance. We describe here the synthesis of pro-inflammatory products in 3T3-L1 preadipocyte cell line after stimulation with lipopolysaccharide (LPS), a TLR4 agonist. Expression profiles of mRNA coding for IL6, CCL2, CCL5, CCL11, NOS2, and PTGS2 demonstrated a higher responsiveness to LPS of these transcripts in preadipocytes than in fully differentiated adipocytes, confirming inflammatory features of preadipocytes. IL6, CCL2, CCL5 and CCL11 were secreted in 3T3-L1 supernatants within 4 h after LPS stimulation. In addition, continuous exposure to LPS during adipocyte differentiation impaired this process as was demonstrated by analysis of mRNA profiles of lipogenesis enzymes (FABP4, GPD1, LPL), adipokines (adiponectin, resistin, visfatin, leptin), and of the transcription factor PPARgamma. This suggests that toll-like receptor mediated activation could regulate maintenance of preadipocyte status, and inflammatory environment encountered in inflamed white adipose tissue

Optimisation de la technique de BRET (étude de l'association des variants d'HMGA1 avec le diabète de type 2)

Les interactions protéine-protéine sont à la base de plusieurs processus biologiques dans la cellule. L étude de ces interactions est importante pour comprendre le rôle des protéines inconnues et les mécanismes les mettant en jeu. Ainsi, le suivi de ces interactions est un enjeu majeur en biologie moléculaire. La première partie de ma thèse s est concentrée sur l amélioration d une technique de suivi de ces interactions protéine-protéine : la technique de BRET.. La technique de BRET repose sur le transfert d énergie par résonance de type Förster qui s opère entre une enzyme luminescente donneuse d énergie et une protéine fluorescente acceptrice d énergie par oxydation d un substrat métabolisable par le donneur d énergie. Ce phénomène peut être utilisé en biologie moléculaire pour suivre l interaction de protéines d intérêt fusionnées au donneur ou à l accepteur d énergie. Contrairement à bon nombre de techniques d étude des interactions protéine-protéine, le BRET est non invasif et permet l étude dynamique de l interaction de deux protéines en cellules vivantes ainsi que le criblage de molécules capables de moduler cette interaction. Dans notre étude, nous avons cherché à optimiser la technique de BRET pour le criblage moléculaire en sélectionnant le meilleur couple de donneur/accepteur d énergie. Un nouveau donneur d énergie, la Rluc8, qui possède des caractéristiques lumineuses supérieures à la Rluc, a été testé en association avec deux nouveaux accepteurs d énergie, la YPet, un variant de la YFP optimisé pour le transfert d énergie, et la RGFP, une protéine fluorescente issue de Renilla reniformis qui est responsable avec la Rluc du phénomène de BRET chez cet organisme marin. Ces nouveaux donneurs/accepteurs d énergie ont été comparés au couple Rluc/YFP, habituellement utilisé en BRET1, en fusion directe et dans des tests de suivi d interactions protéine-protéine impliquant les RCPG et la βARR2 afin d évaluer leur potentiel dans l amélioration de la technique de BRET. Nous avons ainsi pu montrer que la Rluc8, la YPet et la RGFP permettaient d améliorer la sensibilité de la méthode de BRET1 en augmentant au minimum d un facteur 2 le signal de BRET mesuré. Ces résultats démontrent l intérêt à utiliser ces nouveaux donneur/accepteur d énergie en technique de BRET et laissent présager des améliorations qu elles pourraient apporter aux tests de suivi actuels des interactions protéine-protéine. Étude de l association des variants d HMGA1 avec le diabète de type 2. Le diabète de type 2 est une maladie multifactorielle commune, qui est caractérisée par deux dysfonctions majeures observées chez tous les individus atteints : un dysfonctionnement de la cellule β pancréatique et une résistance progressive des tissus à l action de l insuline. Cette pathologie ayant une forte composante héréditaire, on pensait que les études d association pangénomique sur cette maladie identifieraient des loci génétiques associés aux deux phénotypes. Cependant, la majorité des loci identifiés ont été reliés à la fonction de la cellule β pancréatique ou à des fonctions biologiques encore inconnues. Au final, ces études n ont permis d expliquer que 10% de la part due à la génétique dans la prédisposition au diabète de type 2. Il reste ainsi de nombreuses voies à explorer afin de comprendre l héritabilité de cette maladie, à l image de l implication des variants rares ou peu fréquents à effet fort dans la susceptibilité au diabète de type 2. Étant donné que le séquençage à haut débit reste une technique compliquée, onéreuse, et nécessitant un grand nombre d échantillons, nous pensions que l approche gène candidat pourrait aussi être un moyen d identifier ces variants rares ou peu fréquents à effet fort associés aux maladies communes.Il a été montré qu une diminution de l expression d HMGA1 ( High Mobility Group A1 ), causée par des mutations perte de fonction sur ce gène, pouvait être responsable du phénotype d insulinorésistance et de diabète de type 2 observés chez des sujets atteints de formes sévères d insulinorésistance. Ce gène code pour une protéine non-histonique capable de se fixer sur des régions de l ADN riches en adénosine et en thymidine pour réguler l expression de gènes cibles. HMGA1 est un régulateur positif de l expression du récepteur à l insuline (INSR) et du récepteur à l IGF (insulin-like growth factor-I), et son expression est induite par la voie Wnt/β-Catenin, voie de signalisation connue pour son rôle essentiel dans la physiopathologie du diabète de type 2. Récemment, un nouveau variant intronique (c.136-14_136-13insC) d HMGA1 a été associé au diabète de type 2, ainsi qu à une diminution de l expression des gènes HMGA1 et INSR. Ces résultats suggèrent que les variants peu fréquents sur HMGA1 pourraient être fortement associés au diabète de type 2 à travers leurs effets sur l insulinorésistance. Ainsi, la deuxième partie de ma thèse s est concentrée sur la recherche de variants d HMGA1 associés au diabète de type 2.Afin de confirmer cette hypothèse, nous avons séquencé le locus HMGA1 dans une cohorte française comprenant 368 patients atteints de diabète de type 2 et possédant des antécédents familiaux de diabète, et 372 sujets normoglycémiques sans antécédents familiaux de diabète. Cette première étape nous a permis d identifier 72 variants génétiques dans HMGA1. Parmi ces 72 variants, 3 variants peu fréquents possédaient une distribution génotypique différente entre les sujets atteints de DT2 et les sujets normoglycémiques : les rs35383185, c.136-14_136-13insC et rs41269028. Nous avons répliqué ces variants chez 4 538 patients atteints de DT2 et 4 015 individus normoglycémiques afin d évaluer leur association avec le diabète de type 2 : chez ces individus, ces 3 variants n étaient pas associés au diabète de type 2. De plus, ils n avaient aucun effet sur l expression d HMGA1 et d INSR et sur les traits métaboliques intermédiaires.En collaboration avec le consortium DIAGRAM, une méta-analyse des résultats existants sur le variant c.136-14_136-13insC et des SNP en déséquilibre de liaison avec ce variant nous a permis de confirmer qu il n était pas associé au diabète de type 2 dans la population européenne, contrairement aux précédentes observations de Chiefari et collègues. En conclusion, notre étude montre que les variants peu fréquents d HMGA1 ne sont pas associés au diabète de type 2 dans la population européenne, et souligne l importance d analyser de grands échantillons d individus pour correctement évaluer l association de ce type de variant avec cette pathologie.The protein-protein interactions are involved in many biological processes in the cell. The study of these interactions is important for understanding the role of unknown proteins their mechanisms. Thus, the monitoring of these interactions is a major issue in molecular biology. The first part of my thesis focused on the improvement of a technique to study these protein-protein interactions: the BRET approach. The BRET technique is based on resonance energy transfer has been described by Förster and is based on a luminescent donor enzyme and a complementary acceptor fluorophore upon oxidation of a donor enzyme substrate. This phenomenon can be used in molecular biology to assess protein protein interactions by linking proteins of interest to the donor or acceptor. Unlike many techniques for the study of protein-protein interactions, the BRET is non-invasive and allows the dynamic study of interaction between two proteins in living cells and the screening of molecules modulating this interaction. In our study, we sought to optimize the BRET technique for screening assay by selecting the best pair of donor / acceptor. Rluc8, a new donor, with superior light capabilities than Rluc, was tested in combination with two new acceptors, YPET, a variant of YFP optimized for energy transfer, and RGFP, a fluorophore from Renilla reniformis which is involved in the BREAT signal when in contact with Rluc. These new donors / acceptors were compared to the original Rluc / YFP combination, usually used in BRET1, in fusion proteins and in follow-up studies of protein-protein interactions involving GPCRs and βARR2 in order to assess their capacity to improve the BRET technique. We were able to show that Rluc8, YPET and RGFP increased the sensitivity of the original BRET1 method by increasing at least 2-fold of the BRET signal. These results demonstrate the interest of these new donors / acceptors in BRET technique and may help improving current follow-up studies of protein-protein interactions.Study of the association of variants of HMGA1 with type 2 diabetesType 2 diabetes is a common multifactorial disease, which is characterized by two major dysfunctions observed in any affected individual: a pancreatic β cell defect and a resistance to insulin action. Since this pathology has a strong hereditary component, it was thought that genome-wide association studies on this disease would identify genetic loci associated with both phenotypes. However, most loci identified have been linked to pancreatic β cell defects or unknown biological functions. So far, these studies explain less than 10% of the overall estimated genetic contribution to type 2 diabetes predisposition. Other ways are being explored to find other heritable factors, such as rare or low frequency variants with strong effect on the susceptibility to type 2 diabetes. Since high-throughput sequencing remains complicated, expensive and requires a large number of individuals, we thought that the candidate gene approach could be another way to identify these rare or low frequency variants with strong effect associated with common diseases .It was shown that decreased expression of the HMGA1 gene (\\\\\\\\\\\\\\\"High Mobility Group A1\\\\\\\\\\\\\\\"), caused by loss-of-function mutations, could be responsible of the insulin resistance phenotype and type 2 diabetes observed in subjects with severe forms of insulin resistance. This gene encodes a non-histone protein which can bind to DNA regions rich in adenosine and thymidine in order to regulate the expression of target genes. HMGA1 is a positive regulator of the insulin receptor (INSR) and IGF receptor expressions and its expression is induced by the Wnt / β-catenin pathway, known to be involved in the pathophysiology of type 2 diabetes. Recently, a new intronic variant (c.136-14_136-13insC) of HMGA1 was associated with type 2 diabetes and decreased expressions of HMGA1 and INSR. These results suggest that low frequency variants of HMGA1 could be strongly associated with type 2 diabetes through their effects on insulin resistance. Thus, the second part of my thesis focused on the search for variants in HMGA1 associated with type 2 diabetes.To confirm this hypothesis, we sequenced the HMGA1 locus in a French cohort including 368 subjects with type 2 diabetes and with a family history of diabetes and 372 normoglycemic with no family history of diabetes. We first identified 72 genetic variants in HMGA1. Out of 72 variants, three were differentially distributed between T2D and normoglycemic subjects: the rs35383185, c.136-14_136-13insC and rs41269028. We then genotyped these variants in 4,538 T2D patients and 4,015 normoglycemic individuals to assess their association with type 2 diabetes: in these individuals, these three variants were not associated with type 2 diabetes. In addition, they had no effect on HMGA1 and INSR expressions and on intermediate metabolic traits. In collaboration with the DIAGRAM consortium, a meta-analysis of between results on the c.136-14_136-13insC variant and polymorphisms in linkage disequilibrium confirmed that this low frequency variant is not associated with type 2 diabetes in the European population. Our results are in contradiction with what was reported by Chiefari and colleagues. In conclusion, our study shows that low frequency variants in HMGA1 gene are not associated with type 2 diabetes in the European population and emphasizes the need to analyze a large number of subjects to reliably assess the association of low frequency variants with the disease.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Glutathione S-Transferases of 28kDa as Major Vaccine Candidates against Schistosomiasis

For the development of vaccine strategies to generate efficient protection against chronic infections such as parasitic diseases, and more precisely schistosomiasis, controlling pathology could be more relevant than controlling the infection itself. Such strategies, motivated by the need for a cost-effective complement to existing control measures, should focus on parasite molecules involved in fecundity, because in metazoan parasite infections pathology is usually linked to the output of viable eggs. In numerous animal models, vaccination with glutathione S-transferases of 28kDa has been shown to generate an immune response strongly limiting the worm fecundity, in addition to the reduction of the parasite burden. Recent data on acquired immunity directed to 28GST in infected human populations, and new development to draw adapted vaccine formulations, are presented

The Aryl Hydrocarbon Receptor in Asthma: Friend or Foe?

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that has emerged as an important player in asthma control. AhR is responsive to environmental molecules and endogenous or dietary metabolites and regulates innate and adaptive immune responses. Binding of this receptor by different ligands has led to seemingly opposite responses in different asthma models. In this review, we present two sides of the same coin, with the beneficial and deleterious roles of AhR evaluated using known endogenous or exogenous ligands, deficient mice or antagonists. On one hand, AhR has an anti-inflammatory role since its activation in dendritic cells blocks the generation of pro-inflammatory T cells or shifts macrophages toward an anti-inflammatory M2 phenotype. On the other hand, AhR activation by particle-associated polycyclic aromatic hydrocarbons from the environment is pro-inflammatory, inducing mucus hypersecretion, airway remodelling, dysregulation of antigen presenting cells and exacerbates asthma features. Data concerning the role of AhR in cells from asthmatic patients are also reviewed, since AhR could represent a potential target for therapeutic immunomodulation

Induction of TDO2 and IDO2 in Liver by High-Fat Feeding in Mice: Discrepancies with Human Obesity

Low-grade and chronic inflammation is elicited in white adipose tissue in human obesity. The presence of inflammatory molecules leads to an increased tryptophan catabolism through the induction of indoleamine-2,3-dioxygenase-1 (IDO1). In order to characterize the mechanisms underlying this dysregulation, we have studied 2 mouse models of obesity. Unexpectedly, we did not detect any IDO1 expression in obese or lean mice adipose tissue. In a previous study, we did not find any significant difference in the liver for IDO2 and tryptophan-2,3-dioxygenase (TDO2) gene expression between normal weight and obese patients. IDO2 and TDO2 expression was increased in the liver of high-fat fed mice, but not in ob/ob mice, and was strongly correlated with hydroxysteroid-(11-beta) dehydrogenase-1 (HSD11B1) expression, an enzyme that generates active cortisol within tissues. In conclusion, despite a dysregulation of tryptophan metabolism, obese mice display discrepancies with human obesity metabolism, rendering them inappropriate for further investigations in this animal model