A hiporreatividade das células T observada na resposta imune a antígenos de P. brasiliensis de pacientes com paracoccidioidomicose ativa deve contribuir para o não controle da doença, levando à disseminação do fungo. É, na maioria das vezes, reversível com tratamento antifúngico. Os mecanismos que levam a esta hiporreatividade não são bem conhecidos. No entanto, foram demonstrados em resultados prévios em nosso laboratório que células mononucleares de pacientes frente a gp43 apresentam níveis elevados de apoptose. Para tentarmos explicar esse mecanismo, nossa primeira hipótese foi de avaliar se a ativação celular desses pacientes estavam sendo prejudicada por uma ativação inadequada induzida pela expressão alterada de moléculas co-estimulatórias como CD80, CD86, CD28, CD152, ICOS e PD-1. A expressão dessas moléculas foi avaliada em células T e monócitos de pacientes com doença ativa (n = 7...) e controles curados (n = 2...) de um episódio prévio de PCM, mantidas em cultura com antígeno metabólico de Candida albicans (CMA), gp43 ou sem estímulo após 4 dias em cultura. Os resultados obtidos demonstraram que a expressão do CD28 foi comparável entre doentes e controles, e que a expressão de CD152, PD-1 e ICOS, que preferencialmente exercem um papel negativo na sinalização celular, foi maior em células T de pacientes, estimuladas ou não, quando comparadas com células de indivíduos controles. Em paralelo, foram realizados experimentos com a adição dos respectivos anticorpos bloqueadores, que, no entanto, não restabeleceu a proliferação celular dos pacientes. A expressão das moléculas CD80 e CD86 na superfície dos monócitos foi similar em pacientes e controles. Já a expressão dessas moléculas na superfície de linfócitos foi maior nos pacientes tanto em células estimuladas como não estimuladas. O bloqueio com os respectivos anticorpos no dia 0 inibiu a resposta tanto com gp43 como com CMA, porém de forma diferenciada. Nos pacientes e controles a inibição da molécula CD86 diminuiu a resposta tanto para gp43 como para CMA e a inibição da molécula CD80 diminuiu a resposta proliferativa apenas para gp43, e somente no grupo controle, sugerindo que os diferentes antígenos exigem diferentes moléculas durante o processo de apresentação antigênica. A adição desses anticorpos no 4o dia da cultura não modificou a resposta linfoproliferantiva dos pacientes e controles. Nossos dados favorecem a hipótese, derivada de outros modelos de exposição crônica a antígenos exógenos, de que a exposição repetida a antígenos de P. brasiliensis por um longo período in vivo, verificada nos pacientes com paracoccidioidomicose, levam as células T a um estado de tolerância adaptativa, que dificilmente é revertida in vitro. A partir desses resultados analisamos a participação da apoptose de células T nesse provável estado de tolerância nas células dos pacientes. Observamos que a expressão da molécula anti-apoptótica Bcl-2 está diminuída nas células T de pacientes previamente estimuladas comparadas com as células dos controles, e mesmo após o reestímulo in vitro a diminuição da expressão dessa molécula persiste. Desta forma, a diminuição da expressão da molécula Bcl-2 ex vivo nas células T de pacientes sugere fortemente que essas células estão vulneráveis a apoptose. Para corroborar esta hipótese, analisamos a expressão das caspases 8 e 9 na forma ativa. Inicialmente, analisamos a expressão destas moléculas em células mononucleares de pacientes e controles mantidas em cultura por 4 dias com e sem estímulo de CMA e gp43 e observamos que as células dos controles expressam maiores níveis de ambas moléculas em relação as células dos pacientes. Esses resultados foram surpreendentes uma vez que o aumento da expressão de moléculas que estariam direcionando as células para apoptose era esperado em células de pacientes e não de controles. Para explicar este resultado sugerimos a possibilidade (hipótese já apareceu várias vezes) de que as células dos pacientes poderiam estar entrando em apoptose num estágio mais inicial, antes do quarto dia. Por isso realizamos experimentos adicionais em que analisamos a expressão dessas caspases ex vivo. Com essa análise observamos que células TCD3+ de pacientes expressam altos níveis tanto de caspase 8 como caspase 9 comparadas às células de controles. Esses resultados podem ajudar a explicar porque nos ensaios para a análise da resposta proliferativa de pacientes com acréscimo de anticorpos bloqueadores de moléculas coestimulatórias, não houve reconstituição da resposta especifica a gp43: essas células estariam pré-ativadas e pré-programadas para entrarem apoptose, e, portanto, refratárias a tratamentos in vitro, como já descrito em células em estado de tolerância adaptativa. / The T-cell hypoproliferative reactivity observed in the immune response to P. brasiliensis antigens of patients with active paracoccidioidomycosis probably contributes to the failure of the host in controlling the infection, leading to a disseminated disease. It is, however, largely reversible with treatment in most patients. The mechanisms leading to this hyporresponsiveness are not well known. We have previously demonstrated that patients\' mononuclear cells in presence of gp43 exhibit enhanced apoptotic levels. I an attempt to explain such findings, we hypothethized that these cells were inadequately activated due to altered costimulatory molecules expression, such as CD80, CD86, CD28, CD152, ICOS e PD-1. Expression of these molecules were evaluated on T-cells and monocytes of the peripheral blood of patients with active, disseminated PCM (n = 7...), and healthy individuals with a past history of treated and cured PCM (n = 2...). These cells were cultured in presence of a Candida albicans metabolic antigen (CMA), gp43, or kept without exogenous stimuli for 4 days. Our results show tgat the expression of CD28 was comparable between patients an controls\' cells, and that CD152, PD-1 e ICOS, all of which known to deliver negative costimulatory signaling and to arrest cell cycle entry, were overexpressed in patients\' T-cells. In parallel, we performed additional experiments where the respective costimulatory signalings were blocked by addition of blocking antibodies specific to each of these molecules. Whatever the blocking antibody used, there was no reversal of the hypoproliferative state of patients\' T-cells. However, while the expression of the CD80 and CD86 molecules on monocytes was similar between controls and patients, their expression on T-cells was significantly higher in patients. Adding the respective blocking antibodies at day zero of the culture, we could observe that both the gp43 and the CMA responses were inhibited, but differentially according to the antibody employed. In both patients and controls the blocking CD86 signaling decreased the response to gp43 and CMA of patients and controls, while blocking of CD80 signaling decreased only the response to gp43, and only in the control group. These data suggest that different antigens may have different costimulatory requirements for antigen presentation. Addition of the antibodies at the ay 4 of culture did not restore the lymphoproliferative response or modified the response of the controls. Our results suggest that the hypothesis, raised from other models of prolonged foreign antigen exposure, that repetitive and persistent in vivo exposure to fungal antigens, which is described in patients with PCM, lead the T-cells to a adaptive tolerant state, which is hardly reverted in vitro. We then investigated the fate of such putatively tolerized patients\' cells, by analyzing the role that apoptosis may have in this tolerant state. We observed that expression of the antiapoptotic molecule Bcl-2 was lower in patients\' cells, even when the cells were in vitro reestimulated with CMA and gp43, suggesting that the cells are more susceptible to undergo apoptosis. When then analyzed the expression of the active form of the caspase 8 and 9 molecules. We first analyzed their expression on cells kept in cultures for 4 days with or without stimuli. Unexpectedly, we observed that controls\' cells, and not patients\' cells, exhibited higher levels of expression of both molecules. To explore further these data, we tested the hypothesis that the patients\' cells were already undergoing apoptosis at an earlier than 4 days stage. Caspases expression were therefore analyzed ex vivo. In fact, we observed that TCD3+ cells exhibited markedly enhanced caspase 8 and expression as compared to controls\' cells. These findings may help to explain why we failed to redress the proliferative responses to gp43 in the experiments where blocking antibodies were added: these cells would be committed to apoptotic death, thus refractory to in vitro manipulations, as described for adaptively tolerant T-cells.

La maladie de Crohn (MC) et la rectocolite hémorragique (RCH) sont les deux principales formes cliniques des maladies inflammatoires chroniques de l’intestin (MICI) responsables d’une atteinte inflammatoire de la paroi du tube digestif avec des ulcérations extensives. Ce sont des maladies fréquentes en Europe et en Amérique du Nord avec plus de 2.5 millions de malades. Du fait de l’augmentation importante de leur prévalence, de leur morbidité, du retentissement sur la qualité de vie des malades et du coût de leur prise en charge médicale, les MICI sont devenues un problème majeur de santé publique. Au cours de ces maladies, l’inflammation intestinale peut être contrôlée par les traitements médicamenteux ou la chirurgie sans pour autant obtenir de guérison complète et définitive. Bien que leur origine reste mal connue, l’hypothèse actuelle présente les MICI comme des maladies multifactorielles, secondaires à une réponse immunitaire muqueuse anormale dirigée contre la flore intestinale, survenant chez des individus génétiquement prédisposés et entrainant une inflammation intestinale. Le but du travail était d’explorer les mécanismes à l’origine de cette inflammation intestinale associée au développement des MICI en étudiant plus particulièrement certains facteurs environnementaux, moléculaires et microbiens. Nous avons étudiés tout d’abord l’aluminium comme facteur environnemental en démontrant qu’il pouvait participer au développement et à l’aggravation de l’inflammation intestinale sur des modèles de colite chez la souris. Nous avons ensuite étudié un facteur moléculaire important pour l’apoptose des cellules, la caspase-8. Nous avons montré que cette caspase-8 maintenait l’homéostasie intestinale des cellules épithéliales intestinales et que sont absence entraînait une inflammation intestinale ressemblant à la maladie de Crohn. Finalement nous nous sommes intéressés à un facteur microbien, Saccharomyces cerevisiae CNCM I-3856 qui est une levure. Nous avons démontré que cette levure était capable d’induire un effet anti-inflammatoire et analgésique chez l’animal en activant PPARγ dans le colon. Chez l’homme nous avons montré dans une étude randomisée que Saccharomyces cerevisiae CNCM I-3856 réduisait les douleurs abdominales chez les patients atteints du syndrome de l’intestin irritable. En conclusion, l’exploration de ces trois facteurs environnementaux, moléculaires et microbiens permet de mieux comprendre le développement de l’inflammation intestinale. La perspective de ce travail est le développement dans un futur proche des nouvelles thérapeutiques ciblées permettant de lutter contre l’inflammation intestinale. / Crohn's disease (CD) and ulcerative colitis (UC) are the two main clinical forms of chronic inflammatory bowel disease (IBD) responsible for intestinal inflammation with extensive ulceration of the mucosa. These are common diseases in Europe and North America with over 2.5 million patients. Due to the significant increase in their prevalence, their morbidity, the impact on quality of life of patients and the cost of their medical care, IBD has become a major public health problem. In these diseases, intestinal inflammation may be controlled by drug treatment or surgery without obtaining a complete and final cure. Although their origin remains unclear, the current hypothesis presents IBD as multifactorial diseases secondary to an abnormal mucosal immune response directed against the intestinal flora, occurring in genetically predisposed individuals and causing intestinal inflammation. The aim of this work was to explore the mechanisms behind this intestinal inflammation associated with the development of IBD studying some particular environmental, molecular and microbial factors. We studied first the aluminum as an environmental factor and demonstrated that he could participate in the development and exacerbation of intestinal inflammation in models of colitis in mice. We then studied an important factor in molecular cell apoptosis, caspase-8. We have shown that caspase-8 was maintaining intestinal homeostasis in intestinal epithelial cells and that absence of caspase-8 leads to intestinal inflammation mimicking Crohn's disease. Finally we studied a microbial factor, Saccharomyces cerevisiae CNCM I-3856 which is yeast. We demonstrated that this yeast was capable of inducing an anti-inflammatory and analgesic effect in animals by activating PPARgamma in the colon. In humans we have shown in a randomized study that Saccharomyces cerevisiae CNCM I-3856 reduced abdominal pain in patients with irritable bowel syndrome. In conclusion, the exploration of these three environmental molecular and microbial factors helps to better understand the development of intestinal inflammation. The perspective of this work is the development in the near future of new targeted therapies directed against intestinal inflammation.

The innate immune system is an evolutionarily conserved primary defense system against microbial infections. One of the central components of innate immunity are the pattern recognition receptors which sense infection by detecting various conserved molecular patterns of pathogens and trigger a variety of signaling pathways. In this dissertation, the signaling pathways of several classes of these receptors were dissected. In chapters II and III, the role of two NOD-like receptors, NLRP3 and NLRC4 were investigated in the context of infection with the fungal pathogen, C. albicans. C. albicans is an opportunistic pathogen that causes diseases mainly in immunocompromised humans and innate immunity is critical to control the infection. In chapters II and III, we demonstrate that a multiprotein-inflammasome complex formed by the NLR protein, NLRP3 and its associated partners, ASC and caspase-1 are critical for triggering the production of mature cytokine IL-1β in response to C. albicans. NLRC4, another inflammasome forming NLR that is activated by intracellular bacterial pathogens, was not required for this process in macrophages. Thus, our data indicates that NLRP3 inflammasome responds to fungal infections in addition to its known stimuli such as bacterial and viral infections, toxic, crystalline and metabolic signals. Interestingly, this NLRP3 dependent inflammasome response was maintained even when the pathogen is not viable, and is either formalin fixed or heat-killed (HK). Hence, in chapter III, we examined β-glucans, a structural cell wall component, as the potential immunostimulatory component of C. albicans and dissected the inflammasome responses to β -glucans. We observed that NLRP3-ASC-caspase-1 inflammasome was critical for commercially obtained particulate β-glucans similar to the case of C. albicans. β-glucan sensing C-lectin receptor dectin-1 and the complement receptor CR3 mediated inflammasome activation, IL-1β production in response to the glucan particles. Interestingly, CR3 which recognizes glucans as well as complement opsonized pathogens was strongly required for HK C. albicans induced IL-1β, and partially required for that of live C. albicans, while dectin-1 was not required. Consistent with the receptor studies, blocking of β -glucan receptors by pre-incubating cells with nonstimulatory, soluble glucans led to decreased IL-1β production in response to HK C. albicanswith no effect on IL-1β in response to the live fungus. Dectin-1, CR3 and β-glucan sensing also triggered a moderate dendritic cell death response to β-glucans and HK C. albicans. Live C. albicans induced cell death requires phagocytosis but not the inflammasome, β-glucan sensing, dectin-1 or CR3. The Drosophila caspase-8 like molecule DREDD plays an essential, nonapoptotic role in the Drosophila NF-κB pathway called the ‘IMD’ pathway. Owing to the remarkable evolutionary conservation between Drosophila and mammalian innate immune NF-κB pathways, we explored the potential role of caspase-8 in inflammasomes and in TLR signaling. Using casp8-/- Rip3-/- macrophages and dendritic cells, we observed that caspase-8, specifically augments β-glucan and HK C. albicans induced IL-1β as well as cell death in a caspase-1 independent manner, but not that of live C. albicans, in chapter III. We also found that caspase-8 differentially regulates TLR4 and TLR3 induced cytokine production (chapter IV). Caspase-8 specifically promotes TLR4 induced production of cytokines such as TNF, IL-1β in response to LPS and E. coli. On the other hand, caspase-8 negatively regulates TRIF induced IFNβ production in TLR4 and TLR3 signaling in response to LPS and dsRNA. Caspase-8 executed a similar mode of regulation of the cytokine RANTES in MEFs, in part, by collaborating with RIP3. Strikingly, caspase-8 deficiency alone triggers higher macrophage death and IL-1β production in response to TLR ligands, due to the presence of RIP3. Thus, in addition to its conventional roles in apoptosis, caspase-8 modulates TLR4 and TLR3 induced cytokine production and prevents RIP3 mediated hyper inflammation in response to TLR signals. Together, our findings provide valuable information on fungal pattern recognition and inflammasome pathways and define the contribution of β-glucan sensing to C. albicans induced inflammasome responses. In addition, we demonstrate how caspase-8 adds a layer of specificity to inflammasome as well as TLR signaling. Overall, these results also shed light on the cross talk between death signaling components and innate immune pathways to mount a specific and potentially effective innate immune response against microbial pathogens.

No description available.

Background: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein known as the main initiator of blood coagulation. TF is over-expressed on many malignant cells and apart from increasing the risk of thrombosis, the presence of TF/FVIIa also promotes the progression of cancer and metastasis by intracellular signaling. TF expressing microparticles (MP) are, moreover, often found in the circulation of cancer patients. Aim: The aim of this thesis was to study different aspects of TF activity, e.g. the importance of procoagulant MP and TF-induced intracellular signaling pathways, with focus on cell migration (chemotaxis) and apoptosis. Results: The TF signaling complexes were shown to prevent apoptosis induced by serum starvation and TRAIL in cancer cells by reduced activation of caspase-8 in a PI3k/AKT-dependent manner. FVIIa also decreased transcription of pro-apoptotic genes in cancer cells treated with TRAIL. Simvastatin triggered apoptosis by transcriptional reduction of BCL-2 due to cytosolic retention of NFκB. Simvastatin also inactivated the PI3k/AKT pathway and reduced the production of the MP-like prostasomes which, respectively, impaired the anti-apoptotic signaling by TF and reduced the procoagulant activity in the vicinity of prostate cancer cells. Intracellular events conducted by the TF/FVIIa complex selectively enhanced PDGF-BB induced chemotaxis which was partly explained by the TF/FVIIa-induced transactivation of the PDGFβ-receptor. This was dependent on Src-family members and engagement of PAR2. Conclusions: The results presented in this thesis extend the current knowledge of TF-mediated signaling. We report the TF complexes to govern the extrinsic pathway of apoptosis, present data on FVIIa-dependent regulation of apoptosis-related genes, and exclude known surface proteins as transmitters of the anti-apoptotic signals. We moreover describe TF/FVIIa to transactivate the PDGFβ-receptor and play a decisive role in the potentiated chemotaxis toward PDGF-BB in a number of cell types. Finally, we explain the mechanism behind simvastatin-induced apoptosis in cancer cells and how statins interfere with TF-dependent signaling and coagulation.

京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第13351号 / 論生博第22号 / 新制||生||58(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 井垣 達吏, 教授 垣塚 彰, 教授 影山 龍一郎 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Yersinia pestis (Y. pestis), as the causative agent of plague, has caused deaths estimated to more than 200 million people in three historical plague pandemics, including the infamous Black Death in medieval Europe. Although infection with Yersinia pestis can mostly be limited by antibiotics and only 2000-5000 cases are observed worldwide each year, this bacterium is still a concern for bioterrorism and recognized as a category A select agent by the Centers for Disease Control and Prevention (CDC). The investigation into the host-pathogen interactions during Y. pestis infection is important to advance and broaden our knowledge about plague pathogenesis for the development of better vaccines and treatments. Y. pestis is an expert at evading innate immune surveillance through multiple strategies, several mediated by its type three secretion system (T3SS). It is known that the bacterium induces rapid and robust cell death in host macrophages and dendritic cells. Although the T3SS effector YopJ has been determined to be the factor inducing cytotoxicity, the specific host cellular pathways which are targeted by YopJ and responsible for cell death remain poorly defined. This thesis research has established the critical roles of caspase-8 and RIP kinases in Y. pestis-induced macrophage cell death. Y. pestis-induced cytotoxicity is completely inhibited in RIP1-/- or RIP3-/-caspase-8-/- macrophages or by specific chemical inhibitors. Strikingly, this work also indicates that macrophages deficient in either RIP1, or caspase-8 and RIP3, have significantly reduced infection-induced production of IL-1β, IL-18, TNFα and IL-6 cytokines; impaired activation of NF-κB signaling pathway and greatly compromised caspase-1 processing; all of which are critical for innate immune responses and contribute to fight against pathogen infection. Y. pestis infection causes severe and often rapid fatal disease before the development of adaptive immunity to the V bacterium, thus the innate immune responses are critical to control Y. pestis infection. Our group has previously established the important roles of key molecules of the innate immune system: TLR4, MyD88, NLRP12, NLRP3, IL-18 and IL-1β, in host responses against Y. pestis and attenuated strains. Yersinia has proven to be a good model for evaluating the innate immune responses during bacterial infection. Using this model, the role of caspase-8 and RIP3 in counteracting bacterial infection has been determined in this thesis work. Mice deficient in caspase-8 and RIP3 are very susceptible to Y. pestis infection and display reduced levels of pro-inflammatory cytokines in spleen and serum, and decreased myeloid cell death. Thus, both in vitro and in vivo results indicate that caspase-8 and RIP kinases are key regulators of macrophage cell death, NF-κB and caspase-1 activation in Yersinia infection. This thesis work defines novel roles for caspase-8 and RIP kinases as the central components in innate immune responses against Y. pestis infection, and provides further insights to the host-pathogen interaction during bacterial challenge.