Das humane Genom besteht zu ungefähr 8 % aus humanen endogenen Retroviren (HERVs), jedoch sind viele aufgrund von Mutationen oder Deletionen nicht mehr funktionell. Trotzdem wurden funktionelle HERV-Proteine gefunden, welche offene Leserahmen (ORFs) besitzen und für funktionelle Hüll-Glykoproteine wie z.B. Syncytin-1, Syncytin-2 und HML-2 kodieren. Diese HERV-Hüllproteine beinhalten eine suppressive Domäne (SU) und induzieren möglicherweise eine Immunsuppression diverser Immunzellen während einer gesunden Schwangerschaft. In dieser Arbeit wurden spezifisch die modulatorischen Eigenschaften verschiedener HERVHüllproteine (Syncytin-1, -2 und HML-2) auf Immunzellen untersucht. Wir konnten zeigen, dass die HERV-Bindungsrezeptoren ASCT-1, -2 und MFSD2A auf der Oberfläche von T-Zellen und DCs exprimiert werden. Für funktionelle Experimente wurden HERV-Hüllproteine transgen in CHO-Zellen exprimiert, die als Effektorzellen in Ko-Kultur- Systemen verwendet wurden. Es konnte keine Hemmung der PMA/Ionomycin-stimulierten T-Zell-Proliferation durch die Effektorzellen gefunden werden. Darüber hinaus beeinträchtigten die Effektorzellen nicht die Expression von Reifungsmarkern auf DCs nach LPS-Aktivierung, induzierten jedoch die Produktion der pro-inflammatorischen Zytokine IL- 12 und TNF-α. Dagegen inhibierten die konstitutiv HERV-Hüllprotein-exprimierenden Chorionkarzinom-Zelllinien BeWo und JEG die PMA/Ionomycin-stimulierte T-Zell- Proliferation sehr effektiv. Die Chorionkarzinom-Zelllinien hatten ebenfalls keinen Einfluss auf die phänotypische LPS-DC-Reifung, modulierten aber die LPS-DC-Zytokin-Antwort sehr effektiv zu einem suppressiven Profil durch eine Inhibition der pro-inflammatorischen Zytokine IL-12 und TNF-α sowie einen Anstieg von anti-inflammatorischem IL-10. BeWound JEG-Zellen, aber auch HERV-Hüllprotein-exprimierende Effektorzellen verändern die durch LPS-DC-stimulierte allogene T-Zell-Proliferation. Dies war mit einer verringerten Bildung von DC/T-Zell-Konjugaten sowie mit einer Hemmung der IFN-γ-Sekretion und der Ca2+-Mobilisation dieser T-Zellen assoziiert. Des Weiteren wurden eine reduzierte p-Tyrosin- Akkumulation und kein Ausschluss des F-Aktin-Signals in der immunologischen Synapse, der Kontaktstelle dieser DC/T-Zell-Konjugate, gefunden. Zusammenfassend lassen diese Ergebnisse vermuten, dass HERV-Hüllproteine die T-Zell- Proliferation nicht direkt beeinflussen, sich aber modulierend auf DCs auswirken und dadurch mit deren allogene T-Zell-Proliferation interferieren. / Human endogenous retroviruses (HERVs) comprise about 8 % of the human genome but many are not functional due to mutations or deletions. However, some HERVs have been reported to harbor open reading frames (ORFs) and are coding for functional envelope (env) glycoproteins (Syncytin-1, Syncytin-2 and HML-2). These HERV env glycoproteins have suppressive domains (SU) and may modulate immune cells especially in the placenta where they are highly expressed. In this work, we investigated the ability of different HERV envs (Syncytin-1, -2 and HML-2) to modulate immune cell function. We showed that HERV binding receptors ASCT-1, -2 and MFSD2A are expressed by T-cells and DCs. For functional analysis, HERV envs were transiently expressed in CHO-cells which were then used as effector cells for co-culture assays. We demonstrated that HERV env expressing CHO effector cells did not significantly inhibit PMA/Ionomycin stimulated T-cell proliferation, and did not prevent phenotypic maturation of DCs in response to LPS. However, they did strongly augment the release of the pro-inflammatory cytokines IL-12 and TNF-α from LPS-DCs. In contrast, BeWo and JEG choriocarcinoma cell-lines constitutively expressing HERV env proteins reduced the T-cell proliferation very effectively. These celllines did not prevent phenotypic maturation of LPS-DCs, but shifted their cytokine response towards an immune suppressive profile by inhibiting the pro-inflammatory cytokines IL-12 and TNF-α and enhancing the release of the anti-inflammatory cytokine IL-10. The choriocarcinoma cell-lines and CHO effector cells inhibited the LPS-DC induced allogenic Tcell- proliferation. This was associated with a loss of DC/T-cell conjugate frequency as well as with an impairment of IFN-γ release and Ca2+ mobilization in T-cells. In addition, we observed an aberrant pattern of p-tyrosine and F-actin signal in the interface of these DC/Tcell conjugates. Altogether, these findings suggest that HERV proteins do not target T-cell activation directly, but modulate the DCs' ability to promote T-cell expansion.

Objectives: Human Endogenous Retroviruses (HERVs) were originally derived from germ cell infection by exogenous retroviruses and comprise around eight per cent of the human genome. HERVs are highly expressed in the placenta, where HERV-W (syncytin 1) has been demonstrated to perform a fusogenic function. Due to their retroviral origin, placental syncytin 1 has been suggested to also be involved in modulating the maternal immune system. The placenta constantly sheds microvesicles (MV) into the maternal circulation, demonstrated to cause innate immune cell activation associated with normal pregnancy. In pre-eclampsia, there is both increased placental MV shedding and a heightened pro-inflammatory immune response. It was therefore hypothesised that HERVs shed via placental MV play a role in feto-maternal immune interactions and thus may be an important factor in the pathogenesis of preeclampsia (PE). More specifically, it was hypothesised that syncytin 1-positive MV activate monocytes through toll-like receptor 4 (TLR-4). The aim of this study was to determine if syncytin 1 is released from the placenta via MV and exerts an immunological effect. Methods: HERV mRNA and protein expression was measured in placenta and the BeWo choriocarcinoma cell line by qPCR, western blotting (WB) and immunostaining. Glycosylation of syncytin 1 protein was determined by PNGase F treatment followed by WB. MV shed by first trimester, term normal and PE placental explants as well as BeWo cells were isolated by ultracentrifugation. Morphology of these microvesicles was examined by electron microscopy. Syncytin 1 protein and RNA was detected in microvesicles by WB and PCR. Activation and priming of PBMCs to respond to lipopolysaccharide (LPS) by syncytin 1-positive MV and recombinant syncytin 1 was examined through cytokine production by ELISA and multiplex. Antagonism of TLR-4 by LPS-RS was used to determine involvement of the receptor. The role of syncytin 1 in MV activation was examined by siRNA knockdown. Results: HERVs are highly expressed in placental tissue. Syncytin 1 is a glycosylated protein and its expression is altered in PE. MV shed from the BeWo choriocarcinoma cell line and from first trimester and term placental explants, express HERV protein and RNA. Syncytin 1 positive MV and recombinant syncytin protein cause activation of PBMCs. Greatest activation is stimulated by PE MV. Normal MV exhibit a neutral or suppressive effect on subsequent LPS challenge to PBMCs. PE MV exacerbate the response to LPS. Antagonism of TLR-4 on PBMCs and knockdown of syncytin 1 content in MV reduces activation by placental MV.Conclusions: The findings of this thesis suggest that syncytin 1 protein expressed by the placenta is shed into the maternal circulation via MV, and can activate immune cells through TLR-4. Syncytin 1-positive microvesicles may play a role in endotoxin tolerance of innate immune cells in pregnancy. The increased activation by PE MV implies that in addition to the increased microvesicle load in this pathology, a factor intrinsic to PE MV is responsible for increased inflammation. These studies implicate microvesicle-bound syncytin 1 in the regulation of immunotolerance during pregnancy.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24524号 / 医博第4966号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 萩原 正敏, 教授 山崎 渉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

abstract: Obesity impairs skeletal muscle maintenance and regeneration, a condition that can progressively lead to muscle loss, but the mechanisms behind it are unknown. Muscle is primarily composed of multinucleated cells called myotubes which are derived by the fusion of mononucleated myocytes. A key mediator in this process is the cellular fusion protein syncytin-1. This led to the hypothesis that syncytin-1 could be decreased in the muscle of obese/insulin resistant individuals. In contrast, it was found that obese/insulin resistant subjects had higher syncytin-1 expression in the muscle compared to that of the lean subjects. Across the subjects, syncytin-1 correlated significantly with body mass index, percent body fat, blood glucose and HbA1c levels, insulin sensitivity and muscle protein fractional synthesis rate. The concentrations of specific plasma fatty acids, such as the saturated fatty acid (palmitate) and monounsaturated fatty acid (oleate) are known to be altered in obese/insulin resistant humans, and also to influence the protein synthesis in muscle. Therefore, it was evaluated that the effects of palmitate and oleate on syncytin-1 expression, as well as 4E-BP1 phosphorylation, a key mechanism regulating muscle protein synthesis in insulin stimulated C2C12 myotubes. The results showed that treatment with 20 nM insulin, 300 µM oleate, 300 µM oleate +20 nM insulin and 300 µM palmitate + 300 µM oleate elevated 4E-BP1 phosphorylation. At the same time, 20 nM insulin, 300 µM palmitate, 300 µM oleate + 20 nM insulin and 300 µM palmitate + 300 µM oleate elevated syncytin-1 expression. Insulin stimulated muscle syncytin-1 expression and 4E-BP1 phosphorylation, and this effect was comparable to that observed in the presence of oleate alone. However, the presence of palmitate + oleate diminished the stimulatory effect of insulin on muscle syncytin-1 expression and 4E-BP1 phosphorylation. These findings indicate oleate but not palmitate increased total 4E-BP1 phosphorylation regardless of insulin and the presence of palmitate in insulin mediated C2C12 cells. The presence of palmitate inhibited the upregulation of total 4EB-P1 phosphorylation. Palmitate but not oleate increased syncytin-1 expression in insulin mediated C2C12 myotubes. It is possible that chronic hyperinsulinemia in obesity and/or elevated levels of fatty acids such as palmitate in plasma could have contributed to syncytin-1 overexpression and decreased muscle protein fractional synthesis rate in obese/insulin resistant human muscle. / Dissertation/Thesis / Masters Thesis Biology 2017

Les syncytines sont des gènes d'enveloppes rétrovirales (env) capturés qui sont essentiels pour l'établissement du placenta chez les mammifères. Il a été proposé que la diversité des syncytines capturées explique pourquoi le placenta est l'organe le plus variable chez les mammifères. Ici nous avons employé deux approches pour étudier le lien entre la capture d'env et l'émergence et la diversité des structures placentaires. D'abord, nous avons étudié la placentation des Hyaenidae, les seuls carnivores à présenter un placenta très invasif hémochorial, comme l'humain. Comme tous les carnivores, les hyènes expriment la syncytin-Car1 précédemment décrite, mais nous avons identifié une nouvelle env, capturée uniquement chez ces dernières, que nous avons nommée Hyena-Env2. Ce nouveau gène est présent au même locus chez toutes les hyènes, ayant été capturé pendant la radiation de la famille. Il est non-fusiogène mais a néanmoins été conservé pendant plus de 10 millions d'années et est exprimé à l'interface materno-fœtale du placenta, ce qui en fait un gène candidat pour expliquer le passage à la placentation hémochoriale qui a eu lieu chez les Hyaenidae. Ensuite, nous avons cherché des gènes syncytine dans le genre non-mammifère Mabuya, des lézards vivipares présentant un type rare de placenta très complexe et proche de celui des mammifères. Nous avons identifié une env qui a été capturée et conservée dans ce genre depuis sa radiation, il y a 25 millions d'années. Ce gène, que nous avons appelé syncytin-Mab1, est capable d'induire la fusion cellule-cellule et est exprimé dans une couche de cellules fusionnées à l'interface materno-fœtale du placenta, deux propriétés canoniques de syncytine. Nous avons aussi identifié le récepteur de syncytin-Mab1, MPZL1, et avons montré que leur interaction induit son activation et sa phosphorylation. L'activation de MPZL1 a été liée à la migration et à l'invasion cellulaire, indiquant que cette interaction env-récepteur pourrait jouer un rôle dans l'invasion placentaire du tissu maternel observée chez les Mabuya. Pour conclure, la caractérisation de ces deux nouvelles env indique que les gènes de type syncytine ont pu jouer un rôle à la fois dans l'émergence du placenta de Mabuya et dans la structure atypique du placenta des hyènes, supportant la notion que la capture d'env est une force évolutive majeure. / Syncytins are captured retroviral envelope genes (env) that are essential for the establishment of placental structures in mammals. The syncytins present in different mammalian families are highly diverse, resulting from distinct capture events, and it has been suggested that this might play a role in making the placenta the most diverse structure in mammals. Here we used two different approaches to investigate the links between env capture and emergence and diversity of placental structures. First, we investigated placentation in Hyaenidae, the only carnivorans that present a highly invasive hemochorial placenta, as is also found in humans. Hyenas express the previously identified syncytin-Car1 gene, as do all carnivorans, but we identified a new hyena-specific captured env that we named Hyena-Env2. This new gene is present at the same locus in all hyenas, having been captured during the radiation of this family. It is non-fusiogenic but still conserved over at least 10 million years of evolution and expressed at the materno-fetal interface in the hyena placenta, making it a candidate gene for explaining the endotheliochorial to hemochorial placental transition that occurred in Hyeanidae. Second, we searched for syncytin-like genes in the non-mammalian Mabuya lizards, which are viviparous and present a rare type of highly complex placenta that is very reminiscent of mammalian placentas. We identified an env gene that was captured and conserved in this genus since its radiation 25 million years ago. This gene, that we named syncytin-Mab1, is able to mediate cell-cell fusion in vitro and is expressed in a fused cell layer at the materno-fetal interface of the placenta in vivo, characteristic features of canonical mammalian syncytin genes. We also identified the cellular gene MPZL1 as the cognate receptor of syncytin-Mab1 and showed that their interaction induces activation and phosphorylation of the former. MPZL1 activation has been linked with cell migration and invasion, indicating that this env-receptor interaction could play a role in the placental invasion of maternal tissues observed in Mabuya. In conclusion, the characterization of these two novel env genes indicates that syncytin-like env might have played a role both in the emergence of the Mabuya placenta and the atypical placental structure of hyenas, reinforcing the notion that env capture is a major driving force in evolution.

Les rétrovirus endogènes (ERV) sont des éléments constitutifs de la plupart des génomes eucaryotes, et représentent chez l’humain environ 400000 loci. Les HERV sont divisés en familles distinctes, composées d’éléments apparentés mais structurellement hétérogènes. Leur activité peut être néfaste, neutre, mais aussi bénéfique. La majorité des HERV semble silencieuse dans les cellules somatiques. Cependant certains présentent une forte activité en contextes physiologiques. Par ailleurs, une expression significative de HERV est fréquemment observée dans des contextes pathologiques, tels que les cancers. La mise sous silence des éléments répétés est supposée se produire principalement par la méthylation de leur ADN. Nous nous sommes donc intéressés à l’implication de la méthylation des régions régulatrices des HERV, les LTR, dans le contrôle de leur expression. D’une part cette étude nous a permis de mettre en évidence une méthylation locus- et tissu- spécifique de LTR HERV en contexte physiologique, impliquant notamment des modalités particulières de méthylation contrôlant l’expression placentaire de HERV domestiqués. D’autre part ce travail nous a permis de déterminer que six loci HERV-W, incluant un locus domestiqué, sont réactivés de manière autonome dans des tumeurs testiculaires sous l’influence d’un changement de modalité de méthylation intra-famille. Ainsi la méthylation des HERV influence leur expression, mais sous des modalités variables selon les loci et les contextes concernés / Endogenous retroviruses are constitutive elements of most eukaryotic genomes. They represent about 400,000 loci in the human genome. HERVs are divided into distinct families on the basis of phylogenetic identities but are highly heterogeneous in structures. Their activity can be detrimental, neutral, or beneficial to the host. Majority of HERVs seems silent in somatic cells. Still, some are highly expressed in physiological contexts. Besides, a significant expression of HERVs is frequently observed in pathological contexts such as cancers. Silencing of repeated elements is supposed to occur mainly through DNA methylation. We were therefore interested by the implication of HERV regulatory region (LTR) methylation in the control of their expression. First, this study identified locus and tissues –specific HERV LTR methylation in physiological context, worth noting particular methylation modalities that control domesticated HERVs placental expression. Second, we could determine a change in intra-family LTR methylation modalities in testicular tumors leading to the autonomous reactivation of six HERV-W loci, among which a domesticated one. Thus methylation clearly influences HERVs expression, but under modalities varying upon the loci and the contexts

Astronauts at are risk of losing 1.0-1.5% of their bone mass for every month they spend in space despite their adherence to high impact exercise training programs and diets high in nutrients, potassium, calcium, and vitamin D, all designed to preserve the skeletal system. This article reviews the basics of bone formation and resorption and details how exposure to microgravity or simulated microgravity affects the structure and function of osteoblasts, osteocytes, osteoclasts, and their mesenchymal and hematologic stem cell precursors. It details the critical roles that insulin-like growth factor-1 and its receptor insulin-like growth factor-1 receptor (GFR1) play in maintaining bone homeostasis and how exposure of bone cells to microgravity affects the function of these growth factors. Lastly, it discusses the potential of tumor necrosis factor-related apoptosis-inducing ligand, syncytin-A, sclerostin inhibitors and recombinant IGF-1 as a bone-saving treatment for astronauts in space and during their colonization of the Moon.

Syncytin-1 is an endogenous retroviral envelope glycoprotein specifically expressed in human placenta, where the protein was adopted for its physiological function. After interaction with specific receptors, transmembrane proteins ASCT1 and ASCT2, Syncytin-1 initiates cell-cell fusion leading to formation of multinucleated syncytiotrophoblast, which is essential for feto-maternal nutrients exchange. In this diploma thesis a new cell-cell fusion quantification assay was implemented for characterisation of Syncytin-1 fusion determinants. The assay uses Syncytin-1 and ASCT2 expressed separately with fragments of luciferase in heterologous cell-culture system. The assay enables to specifically quantify cell-cell fusions based on activity of reconstituted luciferase reporter. This study discovered new facts about the role of intracytoplasmic tail of Syncytin-1 in the process of the cell- cell fusion. This specific part of protein contains a tandem motif sensitive to changes in amino acid sequence that led to loss of fusogenic potential of Syncytin-1. It was further confirmed, that the protein Suppressyn works as an inhibitor of cell-cell fusions initiated by Syncytin-1. Suppressyn however does not bind to receptors of Syncytin-1 and the mechanism of its inhibition remains unsolved. Finally, it was demonstrated...

No description available.