Étude de la fonction ovarienne chez les souris déficientes des enzymes hyaluronidases

Les mammifères femelles naissent avec un très grand nombre de follicules ovariens primordiaux (104-106); par contre, la grande majorité (99%) de ces follicules n’atteignent jamais la maturité et subissent l’atrésie, principalement par l’apoptose des cellules de la granulosa. Notre laboratoire a démontré que les hyaluronidases des mammifères induisent l’apoptose des cellules de la granulosa et sont impliquées dans l’atrésie des follicules mais que cet effet apoptotique ne serait pas dû à leur activité enzymatique. Notre modèle propose que les hyaluronidases aient un rôle dans les follicules non destinés à ovuler. Le but de la présente étude est d’évaluer la folliculogénèse et la fertilité des souris déficientes de ces enzymes. Les résultats montrent que la délétion de Hyal-3 ne semble pas affecter la fonction ovarienne des souris mais qu’il pourrait y avoir un effet compensatoire par Hyal-1 chez les souris déficientes de Hyal-3 étant donné que son expression est augmentée chez ces souris. La délétion de Hyal-1 a pour effet d’augmenter le nombre des follicules primordiaux, primaires et secondaires, particulièrement chez les souris de bas âge, et de diminuer le niveau d’apoptose des cellules de la granulosa. Afin d’évaluer la fonction de Hyal-1, -2 et -3 sans effet compensatoire entre elles, nous avons voulu créer une souris déficiente des ces 3 hyaluronidases spécifiquement dans les gonades en utilisant le système Cre/loxP. Un vecteur contenant la séquence Cre sous le contrôle du promoteur de Inhibin-α, qui conduit l’expression des gènes en aval chez les cellules somatiques des gonades, a été construit avec succès. En conclusion, cette étude nous révèle que Hyal-3 ne semble pas affecter la fonction ovarienne mais que la délétion de Hyal-1 augmente la folliculogénèse et diminue l’apoptose des cellules de la granulosa.Female mammals are born with a large number of ovarian primordial follicles, though the vast majority of these never reach the preovulatory stage and undergo atresia, mainly through granulosa cell apoptosis. Our laboratory has established that mammalian hyaluronidases induce apoptosis of ovarian granulosa cells and that they are involved in follicular atresia but that their apoptotic effect is not due to their enzymatic activity. Our model suggests that mammalian hyaluronidases might have a role in follicles not destined to ovulate. The aim of this study was to evaluate the folliculogenesis and fertility of mice devoid of these enzymes. Our results showed that Hyal-3 KO mice have normal folliculogenesis, which could be explained by a compensatory effect of Hyal-1 since its expression is upregulated in these mice. In contrast, Hyal-1 KO mice had increased numbers of primordial, primary and secondary follicles, particularly in young mice, and lower levels of granulosa cell apoptosis. In order to investigate the effect of the three hyaluronidases, Hyal-1, -2 and -3, without a compensatory effect by one another, we decided to create a transgenic mouse deficient in all these three hyaluronidases but only in the gonads by using the Cre/loxP system. We successfully created a plasmid containing the Cre sequence under the control of Inhibin-α promoter, which conducts gene expression in somatic cells of the gonads. In conclusion, the present work demonstrates that Hyal-3 does not have any effect on ovarian function, but that deletion of Hyal-1 in mice promotes increased folliculogenesis and lowers granulosa cell apoptosis

Differential expression of RANK, RANK-L, and osteoprotegerin by synovial fluid neutrophils from patients with rheumatoid arthritis and by healthy human blood neutrophils

Functional links between bone remodeling and the immune system in chronic inflammatory arthritis are mediated, in part, by the ligand of receptor activator of nuclear factor-kappa-B (RANK-L). Because neutrophils play a crucial role in chronic inflammation, the goal of this study was to determine whether proteins of the RANK/RANK-L pathway are expressed by synovial fluid (SF) neutrophils from patients with rheumatoid arthritis (RA) and to characterize this pathway in normal human blood neutrophils. The expression of RANK-L, osteoprotegerin (OPG), RANK, and tumor necrosis factor receptor-associated factor 6 (TRAF6) was determined by polymerase chain reaction, enzyme-linked immunosorbent assay, Western blotting, and cytofluorometry. RANK signaling was analyzed by the degradation of inhibitor of kappaB-alpha (I-κB-α). SF neutrophils from patients with RA express and release OPG and express the membrane-associated forms of RANK-L and RANK. In contrast, normal blood neutrophils express only the membrane-associated form of RANK-L. They do not express the mRNAs encoding OPG and RANK. SF neutrophils from RA patients and normal blood neutrophils release no soluble RANK-L. They express the mRNA for TRAF6. The expression of OPG and RANK by normal human blood neutrophils, however, can be induced by interleukin-4 + tumor necrosis factor-alpha and by SFs from patients with RA. In contrast, SFs from patients with osteoarthritis do not induce the expression of OPG and RANK. Moreover, the addition of RANK-L to normal blood neutrophils pretreated by SF from patients with RA decreased I-κB-α, indicating that RANK signaling by neutrophils stimulated with SF is associated with nuclear factor-kappa-B activation. In summary, RANK-L is expressed by inflammatory and normal neutrophils, unlike OPG and RANK, which are expressed only by neutrophils exposed to an inflammatory environment. Taken together, these results suggest that neutrophils may contribute to bone remodeling at inflammatory sites where they are present in significantly large numbers

The mitochondrial protease HtrA2 restricts the NLRP3 and AIM2 inflammasomes.

Activation of the inflammasome pathway is crucial for effective intracellular host defense. The mitochondrial network plays an important role in inflammasome regulation but the mechanisms linking mitochondrial homeostasis to attenuation of inflammasome activation are not fully understood. Here, we report that the Parkinson\u27s disease-associated mitochondrial serine protease HtrA2 restricts the activation of ASC-dependent NLRP3 and AIM2 inflammasomes, in a protease activity-dependent manner. Consistently, disruption of the protease activity of HtrA2 results in exacerbated NLRP3 and AIM2 inflammasome responses in macrophages ex vivo and systemically in vivo. Mechanistically, we show that the HtrA2 protease activity regulates autophagy and controls the magnitude and duration of inflammasome signaling by preventing prolonged accumulation of the inflammasome adaptor ASC. Our findings identify HtrA2 as a non-redundant mitochondrial quality control effector that keeps NLRP3 and AIM2 inflammasomes in check

The role of mitochondria in innate immunity

Mitochondria are dynamic cellular organelles known for their role in energy production, regulation of metabolism, calcium homeostasis and apoptosis. Recent findings emphasize the emerging role of mitochondria as critical intracellular regulators of innate immune responses to both pathogens and cell stress. Mitochondria are essential for the innate immune signaling in response to various bacterial and viral insults, by providing a platform for the assembly and subsequent initiation of immune responses. In addition, due to their bacterial origin, mitochondria present with the potential to launch an immune response by releasing immunogenic components in the form of formylated peptides and mitochondrial DNA (mtDNA). In fact, in the past few years it has been shown that mtDNA released from the mitochondrial matrix into the cytosol launches an inflammatory response through activation of the DNA receptor cGAS (cyclic guanosine monophosphate–adenosine monophosphate synthase). The objectives of this thesis strive for further understanding these two distinct contributions of mitochondria to innate immunity; as a signaling platform for antiviral signaling, and as a direct activator of the cytosolic DNA sensing machinery. The first part of this thesis examines the role of mitochondrial SUMOylation by a membrane anchored ligase called MAPL in mediating the antiviral response to dsRNA virus infection. MAPL has been previously implicated in the innate immune response to Sendai virus infection, therefore the viral-induced MAPL interactome was mapped using the proximity-dependent biotinylation screening method, termed BioID. The data reveals a requirement for SUMOylation of the dsRNA sensor RIG-I to induce a conformational change that allows RIG-I to interact with its mitochondrial antiviral signaling adaptor (MAVS) and launch the innate immune response. This work in combination with in vitro and in vivo experiments in a MAPL knockout mouse model allows us to highlight a critical role for MAPL and mitochondrial SUMOylation in regulating antiviral signaling. Additionally, with these groundbreaking data, we set the stage for a systematic analysis of the interaction landscape, providing important insights into the dynamic events occurring at the surface of mitochondria during antiviral response. A second critical contribution of mitochondria to innate immunity is the potential for released mtDNA or formylated peptides to activate the immune signaling. However, our understanding of the mechanisms that may result in the release of mtDNA into the cytosol has been limited. To address this, we adopted a cell model where exposure to ultraviolet light leads to the activation of innate immune signaling. In this system, mtDNA is observed to exit mitochondria within cargo-selected mitochondrial derived vesicles (MDV). Using a gene silencing approach and immunofluorescence, we demonstrate that mtDNA release requires core proteins previously identified for the generation of vesicles, and that this ultimately drives the innate immune response through cGAS activation. This is the first report of mtDNA as a cargo of MDVs and highlights the importance of this pathway as a direct activator of the cytosolic DNA sensing machinery. Together with the characterization of the role of SUMOylation in generating a signaling platform during viral infection, this thesis provides new insights into multiple mechanisms by which mitochondria contribute to innate immunity.Les mitochondries sont des organites cellulaires dynamiques, connues pour leur rôle dans la production d'énergie, la régulation du métabolisme, l'homéostasie du calcium et l'apoptose. Des découvertes récentes soulignent le rôle émergent des mitochondries en tant que régulateurs intracellulaires critiques des réponses immunitaires innées aux pathogènes et au stress cellulaire. Les mitochondries sont essentielles pour la signalisation immunitaire innée en réponse à diverses agressions bactériennes et virales, en fournissant une plateforme pour l'assemblage et l'initiation subséquente des réponses immunitaires. En outre, en raison de leur origine bactérienne, les mitochondries présentent la capacité de déclencher une réponse immunitaire en libérant des composants immunogènes sous forme de peptides formylés et d'ADN mitochondrial (ADNmt). En effet, au cours des dernières années, il a été montré que l'ADNmt libéré de la matrice mitochondriale dans le cytosol déclenche une réponse inflammatoire via l'activation du récepteur d'ADN cGAS (guanosine monophosphate cyclique-adénosine monophosphate synthase). Les objectifs de cette thèse visent à mieux comprendre ces deux contributions distinctes des mitochondries à l'immunité innée; en tant que plateforme de signalisation pour la signalisation antivirale, et en tant qu'activateur direct de la machinerie de détection de l'ADN cytosolique. La première partie de cette thèse examine le rôle de la SUMOylation mitochondriale par une ligase membranaire appelée MAPL dans la médiation de la réponse antivirale suite à une infection par un virus à ARNdb. MAPL a déjà été impliqué dans la réponse immunitaire innée à l'infection par le virus Sendai, par conséquent l'interactome MAPL induit par le virus a été cartographié en utilisant la méthode de criblage de biotinylation dépendant de la proximité, appelée BioID. Les données révèlent une exigence pour la SUMOylation du récepteur d'ARNdb RIG-I afin d'induire un changement de conformation qui permet à RIG-I d'interagir avec son adaptateur MAVS (mitochondrial antiviral signaling) et de lancer la réponse immunitaire innée. Ce travail en combinaison avec des expériences in vitro et in vivo dans un modèle de souris 'knockout' pour MAPL nous permet de mettre en évidence un rôle critique pour MAPL et la SUMOylation mitochondriale dans la régulation de la signalisation antivirale. De plus, avec ces données révolutionnaires, nous ouvrons la voie à une analyse intéractomique systématique, fournissant des informations importantes sur les événements se produisant à la surface des mitochondries au cours de la réponse antivirale. Une deuxième contribution cruciale des mitochondries à l'immunité innée est le potentiel qu'ont l'ADNmt et les peptides formylés d'activer la signalisation immunitaire. Cependant, notre compréhension des mécanismes qui peuvent entraîner la libération d'ADNmt dans le cytosol est encore très limitée. Pour y remédier, nous avons adopté un modèle cellulaire dans lequel l'exposition à la lumière UV entraîne l'activation de la signalisation immunitaire innée. Dans ce système, on observe que l'ADN mitochondrial est sélectionné comme cargaison des vésicules dérivées des mitochondries (MDV) et ainsi quitte les mitochondries. En utilisant le silençage génique et l'immunofluorescence, nous démontrons que la libération d'ADNmt nécessite des protéines de base précédemment identifiées pour la génération de vésicules, et que cela conduit à une réponse immunitaire innée par l'activation de cGAS. Pour la première fois l'ADN mitochondrial est identifié comme cargaison des MDVs, ce qui souligne l'importance de cette voie en tant qu'activateur direct de la machinerie de détection de l'ADN cytosolique. Combinée à la caractérisation du rôle de SUMOylation dans la génération d'une plateforme de signalisation pendant l'infection virale, cette thèse apporte de nouvelles perspectives sur les mécanismes par lesquels les mitochondries contribuent à l'immunité innée

Microbiota modification of Mytilus edulis larvae in response to the use of a new probiotic, the marennine, in aquaculture

In bivalve hatcheries, opportunistic pathogens have been associated with important mass mortality events of larvae and important economic lost for producers. New alternatives to the use of antibiotics, such as probiotics, have been proposed to limit the occurrence of such events in bivalve hatcheries and to stabilize bivalve production. Probiotics are microorganisms, or natural molecules, that are associated with beneficial effects for the host at different levels, especially to enhance their resistance to external stressors such as bacterial pathogens. It is know recognized that the composition of the host microbiota influences host health status and could be a target of probiotics. The aim of this study is to highlight the protective effect of a new probiotic, the marennine, on Mytilus edulis larvae during bacterial challenges in relation with a potential modification of the microbiota of the marennine-treated larvae. The main hypothesis is that the addition of marennine during larvae rearing processes could modify the conditions prevailing in the rearing medium and, as a consequence, the composition of the larvae microbiota, leading to a better resistance to bacterial infections. The marennine is a blue pigment originating from the diatom Haslea ostrearia, that have demonstrated a positive effect on larvae survival at a final concentration of 500 µg L-1. In this study, D-larvae (9 days old) and post-larvae (29 days old) were exposed for 96h to Vibrio splendidus with and without mareninne at a final concentration of 500 µg L-1. Our results demonstrated that at this concentration, the marennine have no direct antimicrobial effect on Vibrio splendidus growth kinetics. In addition, the presence of marennine did not modify the abundance of bacteria in the rearing medium, suggesting no direct antimicrobial effect of marennine on the bacterial load to which larvae were exposed during the experiments. Nevertheless, the presence of marennine increased the survival of D-larvae exposed to the pathogen but have no effect on post-larvae survival. The undergoing molecular analyses and the future metagenomics analyses of the larvae microbiota diversity will allow us to demonstrate if a modification in the larval microbiota’s richness might explain the increase of the survival rate during the production of blue mussels in aquaculture. Ultimately, our work will enable us to shed light on the importance of the larval microbiota in pathogen-resistance during bivalves rearing process

Effect of Vasectomy on Gene Expression in the Epididymis of Cynomolgus Monkey1

International audienc

Reduction of bacterial biofilm formation using marine natural antimicrobial peptides

There is an important need for the development of new “environmentally-friendly” antifouling molecules to replace toxic chemicals actually used to fight against marine biofouling. Marine biomass is a promising source of non-toxic antifouling products such as natural antimicrobial peptides produced by marine organisms. The aim of this study was to demonstrate the efficiency of antimicrobial peptides extracted from snow crab (SCAMPs) to reduce the formation of marine biofilms on immerged mild steel surfaces. Five antimicrobial peptides were found in the snow crab hydrolysate fraction used in this study. SCAMPs were demonstrated to interact with natural organic matter (NOM) during the formation of the conditioning film and to limit the marine biofilm development in terms of viability and bacterial structure. Natural SCAMPs could be considered as a potential alternative and non-toxic product to reduce biofouling, and as a consequence microbial induced corrosion on immerged surfaces. -- Keywords : Antimicrobial peptides ; Antifouling ; Bacterial diversity ; Conditioning film ; Marine biofilm