No description available.

Chlamydia trachomatis/HSV-2 vaginal co-infections are seen clinically, suggesting that these sexually transmitted pathogens may interact. We previously established an intravaginal Chlamydia muridarum/HSV-2 super-infection model and observed that chlamydial pre-infection protects mice from a subsequent lethal HSV-2 challenge. However, the mechanism of protection remains unknown. The type I interferon, IFN-β, binds to the type I interferon receptor (IFNR), elicits a host cellular antiviral response and inhibits HSV replication in vitro and in vivo. Previous studies have demonstrated that C. muridarum infection stimulates genital tract (GT) IFN-β production; therefore, we hypothesized that chlamydial pre-infection protects mice from HSV-2 challenge via the IFN-β/IFNR-induced antiviral response. To test this prediction, we quantified IFN-β levels in vaginal swab samples. Detection of IFN-β in C. muridarum singly infected, but not in mock-infected animals, prompted the use of the super-infection model in IFNR knockout (IFNR-/-) mice. We observed that C. muridarum pre-infection reduces HSV-2-induced mortality by 40% in wild-type mice and by 60% IFNR-/-mice. Severity of HSV-2 disease symptoms and viral shedding was also similarly reduced by C. muridarum pre-infection. These data indicate that, while chlamydial infection induces GT production of IFN-β, type I IFN-induced antiviral responses are likely not required for the observed protective effect.

Cardiovascular collapse is the major factor contributing to the mortality of trauma-hemorrhage (T-H) patients. Toll-like receptors (TLRs) play a critical role in T-H-induced cardiac dysfunction. This study evaluated the role of TLR9 agonist, CpG-oligodeoxynucleotide (ODN) 1826, in cardiac functional recovery after T-H. Trauma-hemorrhage was induced in a murine model by soft tissue injury and blood withdrawals from the jugular vein to a mean arterial pressure of 35 ± 5 mmHg. Mice were treated with CpG-ODN 1826 (10 μg/30 g body weight) by intraperitoneal injection 1 h before T-H (n = 5-8/group). Hemodynamic parameters were measured before, during hemorrhage, and at 60 min after T-H. Trauma-hemorrhage significantly decreased the mean arterial pressure and left ventricular pressure compared with sham controls. In contrast, CpG-ODN administration significantly attenuated the decrease in arterial pressure and left ventricular pressure due to T-H. Trauma-hemorrhage markedly decreased myocardial levels of phosphorylated Akt by 57.9%. However, CpG-ODN treatment significantly blunted the decrement in phospho-Akt by activating the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY294002 partially abolished CpG-induced cardioprotection, indicating that additional signaling pathways are involved in the protective effect of CpG-ODN after T-H. We observed that CpG-ODN treatment also significantly attenuated the decrease in myocardial phospho-ERK levels after T-H. Inhibition of ERK by U0126 also partially abolished the cardioprotective effect of CpG-ODN after T-H. Our data suggest that CpG-ODN significantly attenuates T-H-induced cardiac dysfunction. The mechanisms involve activation of both PI3K/Akt and ERK signaling pathways. The TLR9 agonist, CpG-ODN 1826, may provide a novel treatment strategy for preventing or managing cardiac dysfunction and enhancing recovery in T-H patients.

Les protéines IFITM (« InterFeron Inducible TransMembrane proteins »), et en particulier les membres 1, 2 et 3, sont des facteurs de restriction antiviraux dont l’expression est induite par le système immunitaire inné en réponse à une infection virale. Elles inhibent la réplication de nombreux virus pathogènes pour l’homme parmi lesquels figurent le virus de la grippe A, le VIH (Virus de l’Immunodéficience Humaine) de type 1 ou encore le virus de l’hépatite C. Ces virus entrent dans la cellule hôte, soit par fusion directe avec la membrane plasmique, soit par la voie de l’endocytose. Il est à présent communément admis que les protéines IFITM, localisées au sein des membranes plasmiques et endolysosomales, agissent en inhibant la fusion des membranes virales et cellulaires, empêchant par conséquent l’entrée du virus dans la cellule et donc sa réplication. D’autre part, dans le cas du VIH, leur incorporation dans les particules virales produites par la cellule hôte diminuerait la capacité de ces particules à infecter de nouvelles cellules cibles. Cependant, les mécanismes moléculaires par lesquels les protéines IFITM interfèrent avec le cycle viral ne sont pas encore clairement définis.Parmi les membres de la famille IFITM, IFITM3 est celui qui présente l’effet antiviral le plus systématique selon les différentes études. Il constitue donc un modèle de référence pour étudier la famille IFITM.Déterminer la structure ainsi que la topologie membranaire d’IFITM3 sous sa forme active rendrait alors possible la réalisation d’études fonctionnelles, dont les résultats contribueraient sans nul doute à élucider le(s) mécanisme(s) par le(s)quel(s) IFITM3 exerce son activité antivirale.C’est pourquoi, nous nous sommes tout d’abord attelés à reconstituer IFITM3 au sein de membranes artificielles (liposomes, nanodisques), car contrairement aux micelles de détergent, ces membranes artificielles peuvent mimer l’environnement natif des protéines membranaires et par conséquent, offrir de plus grandes chances de les y étudier sous leur forme active. Nous avons ensuite procédé à la caractérisation biochimique et biophysique d’IFITM3 et avons mis en évidence la formation de dimère de la protéine ainsi que de plus grandes espèces oligomériques. L’analyse structurale d’IFITM3 reconstituée en nanodisques par RMN nous a quant à elle permis d’identifier une courte région hélicoïdale dans la région N-terminale extramembranaire d’IFITM3 encore jamais décrite auparavant et pouvant correspondre à un motif d’internalisation. Nous avons en outre observé, par microscopie électronique à coloration négative, de potentiels effets d’IFITM3 sur la courbure de la membrane de liposomes qui pourraient être à l’origine de son action inhibitrice sur la fusion virale. Et enfin, nous avons montré au travers d’expériences TEVC que lorsqu’IFITM3 est présente dans l’environnement extracellulaire d’ovocytes de xénope, celle-ci est capable d’engendrer des fuites ioniques au travers de la membrane des ovocytes qui pourraient résulter soit, d’une déstabilisation de la membrane par IFITM3 soit, d’une formation de pores membranaires par la protéine. / The host cell first line of defence against viral infections induces the production of interferons. These interferons are then released in the surrounding medium where they bind to target cells and induce the expression of hundreds of genes so called interferon-stimulated genes (ISGs). The interferon inducible transmembrane proteins IFITM are part of the products of these ISGs. IFITM1, 2 and 3 are antiviral factors able to restrict the replication of a broad variety of enveloped viruses, such as influenza virus, HIV-1 (Human Immunodeficiency Virus) and Hepatitis C virus. These viruses enter in the host cell either by direct fusion with the cell membrane or by endocytosis. IFITM proteins contain two membrane regions for insertion or interaction with plasma and endolysosomal membranes where they block the fusion of virus particles with cellular membranes by a mechanism which is still undefined. In addition, their incorporation into new HIV virions, in virus producing cells, has been correlated with decreased infectivity.Among the IFITM protein family members, IFITM3 is the one showing the most recurrent antiviral effect in the different studies. Therefore it represents a good model to study the whole IFITM family.The determination of its structure and membrane topology is crucial to be able to clarify, through structure-based functional studies, the mechanism(s) by which IFITM3 interfere with the viral cycle.Here we characterized and we studied IFITM3 structure and membrane topology in a lipidic environment close to its native environment such as liposomes and nanodiscs. We demonstrated that IFITM3 can self-associate to form at least a dimer. Some higher order associations of IFITM3 have been observed after its reconstitution into liposomes and big size nanodiscs. We discovered by NMR in solution that the N-terminal region of IFITM3 contains a small helical region, never described until now, which could correspond to an internalization motif. We also observed by negative staining electron microscopy some liposomal membrane curvature changes that could be assigned to the presence of IFITM3 in these liposomes. And we discovered through TEVC experiments that IFITM3 addition in the extracellular environment of xenopus oocytes produces ion leaks through the oocyte membrane which could result either from membrane destabilization or from a pore formation.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18627号 / 生博第318号 / 新制||生||42(附属図書館) / 31527 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 米原 伸, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24749号 / 生博第490号 / 新制||生||65(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 石川 冬木, 教授 高田 穣, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Background Immune-stimulatory agents, like agonists of the innate immune receptor RIG-I, are currently tested in clinical trials as an intratumoral treatment option for patients with unresectable melanoma, aiming to enhance anti-tumor T cell responses. Switching of melanoma toward a dedifferentiated cell state has recently been linked to T cell and therapy resistance. It remains to be determined whether RIG-I agonists affect melanoma differentiation, potentially leading to T cell resistance. Methods Patient metastases-derived melanoma cell lines were treated with the synthetic RIG-I agonist 3pRNA, and effects on tumor cell survival, phenotype and differentiation were determined. Transcriptomic data sets from cell lines and metastases were analyzed for associations between RIG-I (DDX58) and melanoma differentiation markers and used to define signaling pathways involved in RIG-I- driven dedifferentiation. The impact of 3pRNA-induced melanoma dedifferentiation on CD8 T cell activation was studied in autologous tumor T cell models. Results RIG-I activation by 3pRNA induced apoptosis in a subpopulation of melanoma cells, while the majority of tumor cells switched into a non-proliferative cell state. Those persisters displayed a dedifferentiated cell phenotype, marked by downregulation of the melanocytic lineage transcription factor MITF and its target genes, including melanoma differentiation antigens (MDA). Transition into the MITFlow/MDAlow cell state was JAK-dependent, with some cells acquiring nerve growth factor receptor expression. MITFlow/MDAlow persisters switched back to the proliferative differentiated cell state when RIG-I signaling declined. Consistent with our in vitro findings, an association between melanoma dedifferentiation and high RIG-I (DDX58) levels was detected in transcriptomic data from patient metastases. Notably, despite their dedifferentiated cell phenotype, 3pRNA-induced MITFlow/ MDAlow persisters were still efficiently targeted by autologous CD8 tumor-infiltrating T lymphocytes (TILs). Conclusions Our results demonstrate that RIG-I signaling in melanoma cells drives a transient phenotypic switch toward a non-proliferative dedifferentiated persister cell state. Despite their dedifferentiation, those persisters are highly immunogenic and sensitive toward autologous TILs, challenging the concept of melanoma dedifferentiation as a general indicator of T cell resistance. In sum, our findings support the application of RIG-I agonists as a therapeutic tool for the generation of long-term clinical benefit in non-resectable melanoma.

Aging is a risk factor for adipose tissue dysfunction, which is associated with inflammatory innate immune mechanisms. Since the adipose tissue/liver axis contributes to hepatosteatosis, we sought to determine age-related adipose tissue dysfunction in the context of the activation of the innate immune system fostering fatty liver phenotypes. Using wildtype and immune-deficient mice, we compared visceral adipose tissue and liver mass as well as hepatic lipid storage in young (ca. 14 weeks) and adult (ca. 30 weeks) mice. Adipocyte size was determined as an indicator of adipocyte function and liver steatosis was quantified by hepatic lipid content. Further, lipid storage was investigated under normal and steatosis-inducing culture conditions in isolated hepatocytes. The physiological age-related increase in body weight was associated with a disproportionate increase in adipose tissue mass in immune-deficient mice, which coincided with higher triglyceride storage in the liver. Lipid storage was similar in isolated hepatocytes from wildtype and immune-deficient mice under normal culture conditions but was significantly higher in immune-deficient than in wildtype hepatocytes under steatosis-inducing culture conditions. Immune-deficient mice also displayed increased inflammatory, adipogenic, and lipogenic markers in serum and adipose tissue. Thus, the age-related increase in body weight coincided with an increase in adipose tissue mass and hepatic steatosis. In association with a (pro-)inflammatory milieu, aging thus promotes hepatosteatosis, especially in immune-deficient mice.

Vaccination against intracellular pathogens requires generation of pool of memory T cells, which can respond upon infection and mediate immune responses by either killing of infected host cells or induce killing mechanisms in infected cells. T cell-inducing vaccines aim to deliver the antigen to antigen presenting cells (APCs) by presenting on MHC molecules thus bridging innate and adaptive immunity. The intracellular pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), which is a devastating disease in catfish industry. E. ictaluri can survive in professional phagocytes and use them as an infection source. Two new live attenuated vaccine (LAV) strains, EiDELTAevpB and ESC-NDKL, were developed by our group. However, the role of LAVs in phagocytosis, bacterial killing, and antigen presentation is unexplored. Therefore, further research is necessary to determine immune responses in channel catfish against LAVs. The long-term goal of this project is to identify immunological APC-dependent mechanisms that underscore E. ictaluri pathogenesis to enable development of effective control strategies for ESC. The overall goal of this project is to assess the role of three professional APCs, dendritic cells (DCs), macrophages and B cells in the LAV-induced innate and adaptive immune responses in catfish. The central hypothesis is that efficacious LAV strains will enhance phagocytosis and microbial killing, and promote the generation of T cells that regulate and control protective B cell-mediated immunity. The rationale for this research is that more detailed knowledge about phenotype and function of catfish APCs will not only help gain insight into the evolution of vertebrate adaptive immune system but will provide valuable information for development and optimization of immunotherapies and vaccination protocols for aquaculture use. In this study, we first identified DC-like cells in immune-related organs of catfish and assessed their expression patterns in lymphoid organs of catfish in E. ictaluri infection. Although WT strain induces the functional inability of DC-like cells in migration and maturation, LAVs strains promote the migration and maturation of DC-like cells for antigen presentation. Two LAVs enhanced the phagocytosis and killing activity in catfish macrophages and B cells. Also, LAVs induce high expression of T cell-related genes without causing inflammation.