OBJETIVO: Avaliar a expressão dos perfis de citocinas Th1, Th2 e Th17 em pacientes com LES e verificar possíveis associações com os níveis séricos de vitamina D. MÉTODOS: Estudo transversal com inclusão de 172 pacientes acompanhados no ambulatório de reumatologia do Hospital de Clínicas de Porto Alegre. Os níveis de vitamina D foram medidos por quimiluminescência. Níveis séricos <20 ng/ml foram considerados como deficiência de vitamina D e níveis ≥20 ng/ml foram considerados normais. As citocinas foram medidas no soro após o descongelamento das amostras em uma única ocasião, usando Kit CBA (cytometric beads array) Th1/Th2/Th17. RESULTADOS: Cento e sessenta e um (94%) pacientes eram mulheres e 128 (74,4%) foram classificados como euro-descendentes. A idade média foi de 40,5±13,8 anos e a idade média no momento do diagnóstico foi de 31,5±13,4 anos. Na entrada do estudo, os pacientes tiveram mediana (intervalo interquartil) de atividade da doença (SLEDAI- systemic lupus erythematosus disease activity index) de 2 (1-4) e cronicidade (SLICC damage index- systemic lupus international collaborating clinics) de 0 (0-1). O nível médio de vitamina D foi de 25,4±11,04 ng/ml. Cinquenta e nove (34,3%) pacientes apresentavam deficiência de vitamina D e 113 (65,7%) tinham níveis considerados normais. Nenhuma associação e correlação estatisticamente significativa foram encontradas. Os níveis de INF-α e SLEDAI mostraram uma correlação positiva fraca (rs=0,22, p=0,04). Análise de regressão linear foi realizada para controlar possíveis fatores de confusão. CONCLUSÃO: A deficiência de vitamina D é prevalente em pacientes com LES, entretanto, não foram encontradas correlações e associações entre níveis de vitamina D e perfil de citocinas. Confirmamos a correlação existente entre o IFN-α e SLEDAI, conforme a literatura. Efeito in vitro de vitamina D no perfil de citocinas não foi reproduzido no presente estudo. Estudos longitudinais podem ajudar a esclarecer a influência da vitamina D na fisiopatogenia do LES. / OBJECTIVES: To evaluate the expression of Th1, Th2 and Th17 cytokine profiles in SLE patients and verify possible associations with serum vitamin D levels. METHODS: Cross-sectional study with 172 SLE patients, followed at the outpatient clinic of rheumatology at Hospital de Clínicas de Porto Alegre were included. The levels of vitamin D were measured by chemiluminescence. Serum levels <20 ng/ml were considered as vitamin D deficiency. Normal vitamin D levels were defined as ≥20ng/ml. Cytokines were measured in serum after thawing the samples on a single occasion, using Kit CBA (cytometric beads array) Th1/Th2/Th17. RESULTS: One hundred sixty one (94%) patients were women and 128 (74.4%) were classified as European derived. The mean age of patients was 40.5±13.8 years and the mean age at diagnosis was 31.5±13.4 years. At the time of study entry, patients had median (IQR) of active disease (SLEDAI- systemic lupus erythematosus disease activity index) of 2 (1-4) and chronicity (SLICC damage index- systemic lupus international collaborating clinics) of 0 (0-1). Mean vitamin D levels were 25.4±11.04 ng/ml. Fifty-nine (34.3%) patients had vitamin D deficiency and 113 (65.7%) had normal levels. No association and statistically significant correlations was found. The levels of INF-α and SLEDAI showed a weak positive correlation (rs=0.22, p=0.04). Linear regression analysis was performed to control for possible confounding factors. CONCLUSION:Vitamin D deficiency is prevalent in patients with SLE, however, no statistically significant correlations and associations between vitamin D levels and cytokine profile were found. We confirm the correlation between IFN-α and SLEDAI, according to the literature. In vitro effect of vitamin D on the cytokine profile was not reproduced in this study. Longitudinal studies may help clarify the influence of vitamin D in the pathogenesis of SLE.

Le hématopoïétique protéine adaptateur spécifique SLP-76 est un élément clé des voies de transduction du signal en aval du récepteur des cellules T (TCR). Analyse des lignées de cellules T SLP-76 déficient et SLP-76 souris knock-out montré que SLP-76 est indispensable pour le développement des lymphocytes T et de réactivité des cellules T matures. Les travaux antérieurs de notre laboratoire en utilisant des lignées de cellules T a conduit à l'identification et la caractérisation d'une boucle de rétroaction négative impliquant SLP-76, qui module la signalisation TCR-dépendante et l'activation des cellules T. Cependant, la signification physiologique de ce mécanisme de régulation n'a pas été abordé jusqu'à présent. L'objectif de cette thèse est de répondre à la fonction de ce mécanisme de régulation in vivo par l'analyse de la fonction des cellules T dans une souche de souris généré récemment "knock-in", exprimant un mutant SLP-76-S376A. Basé sur les résultats précédents, était attendu ce mutant de perturber la boucle de rétroaction négative SLP-76-dépendante. Je montre ici que l'expression de SLP-76-S376A ne donne pas lieu à des modifications phénotypiques significatives de thymocytes matures ou des sous-ensembles de cellules T, ce qui suggère que ce mutant ne modifie pas de manière significative le développement des cellules T. En outre, aucune différence n'a été observée dans le taux de prolifération ou la mort cellulaire induite par activation de cellules T mutantes par rapport aux témoins, et l'expression des marqueurs d'activation de surface n'a été que légèrement affectées. Cependant, une activation accrue de plusieurs protéines de signalisation a été observée dans les cellules CD4 + et CD8 + T stimulées in vitro, conformément aux résultats précédents dans les lignées de cellules T humaines. In vitro les cellules T CD4 + activés ont montré une augmentation significative de la sécrétion de l'interleukine-4 et -5, tandis que d'autres cytokines comme l'interleukine-2 et l'interféron-γ est apparu insensible. Ces résultats suggèrent que le réglage de la signalisation TCR-dépendante par la boucle de rétroaction négative SLP-76-dépendante peut favoriser réponses de type Th2, avec des conséquences potentielles dans certaines pathologies telles que l'asthme ou les infections parasitaires. Ainsi, nous avons utilisé un modèle expérimental de l'asthme d'évaluer la réponse des cellules eosinophiles in vivo. En effet, les résultats préliminaires ont montré que l'antigène contesté souris mutantes d'asthme induisant recrutent beaucoup plus d'éosinophiles dans les poumons par rapport aux souris témoins. Pris ensemble, ces données suggèrent que les cellules SLP76-S376A T comprennent modulations dans proximale signal de transduction TCR qui peuvent générer un biais vers la production de cytokines Th2 en réponse à la stimulation du TCR. / The hematopoietic specific adapter protein SLP-76 is a key element of signal transduction pathways downstream of the T cell receptor (TCR). Analysis of SLP-76-deficient T cell lines and SLP-76 knockout mice showed that SLP-76 is indispensable for T cell development and for responsiveness of mature T cells. Previous work of our laboratory using T cell lines led to the identification and characterization of a negative-feedback loop involving SLP-76, which modulates TCR-dependent signaling and T cell activation. However, the physiological significance of this regulatory mechanism was not addressed thus far. The aim of this PhD thesis is to address the function of this regulatory mechanism in vivo by analyzing T cell function in a recently generated “knock-in” mouse strain expressing a SLP-76-S376A mutant. Based on previous results, this mutant was expected to disrupt the SLP-76-dependent negative feedback loop. I show here that expression of SLP-76-S376A does not result in significant phenotypic alterations of thymocytes or mature T cell subsets, suggesting that this mutant does not significantly affect T cell development. Furthermore, no differences were observed in proliferation rates or activation-induced cell death of mutant T cells compared to controls, and the expression of surface activation markers was only slightly affected. However, an increased activation of several signaling proteins was observed in both CD4+ and CD8+ T cells stimulated in vitro, in line with previous results in human T cell lines. In vitro activated CD4+ T cells showed a significant increase in secretion of interleukin-4 and -5, whereas other cytokines like interleukin-2 and interferon- appeared unaffected. These results, suggest that tuning of TCR-dependent signaling by the SLP-76-dependent negative feedback loop may favor Th2-type responses, with potential consequences in specific pathologies such as asthma or parasite infections. Thus, we have employed an experimental model of asthma to evaluate the eosinophil response in vivo. Indeed, preliminary results showed that asthma-inducing antigen challenged mutant mice recruit significantly more eosinophils to the lung compared to control mice. Taken together, these data suggest that SLP76-S376A T cells comprise modulations in proximal TCR signal transduction that may generate a bias towards production of Th2-cytokines in response to TCR stimulation.

Stress, either physical or psychological, can have a dramatic impact on the immune system. Little progress, however, has been made in understanding stress-induced immune suppression. We report here that mice subjected to chronic 12-hour daily physical restraint for two days significantly increased the expression of Toll-like receptor 4 (TLR4). Interestingly, TLR4-deficient mice are resistant to stress-induced lymphocyte reduction. In addition, restraint stress caused dramatic decrease in T help 1 (Th1) cytokine IFN-γ and IL-2 levels but increase in Th2 cytokine IL-4 in wild type mice. Moreover, the restraint stress significantly inhibits changes of Th1 and Th2 cytokines in TLR4-deficient mice compared with the wild type mice. Therefore, stress modulates the immune system through a TLR4-dependent mechanism.

Lymphatic metastasis in breast cancer (BC) is one of the most important prognostic factors for patient survival. The escaped tumor cells reach distant vital organs and their unopposed expansion in these organs may cause mortality to patient. Tumor cells are transported to lymph node (LN) exclusively by tumor lymphatic vessels (LV). Increased tumor lymphangiogenesis, i.e., the formation of new LV is currently thought to be promoted by soluble factors such as VEGF-C and –D that activate VEGFR-3 expressed in lymphatic endothelial cells (LEC). These factors are secreted by malignant, tumor-infiltrating immune and stromal cells and create a favorable environment for formation of new vessels. However, emerging evidence suggests that tumor lymphangiogenesis is also promoted by Myeloid-derived Lymphatic Endothelial Cell Progenitors (M-LECP). We recently showed that M-LECP are abundant in mouse and human breast tumors and that their density strongly correlates with both lymphatic formation and nodal metastasis. Characterization of M-LECP showed that nearly all these cells express typical markers of the M2-type of macrophages such as CD163, CD204, and CD209. These cells are consider to be strongly immunosuppressive as exemplified by their inhibition of mobilization, activation, and survival of the key defenders against cancer cells, cytotoxic CD8+ T lymphocytes. Here, we compare the in vitro differentiation of M-LECP derived from bone marrow (BM) myeloid precursors primed with CSF-1 followed by secondary stimulants such as LPS, an immunomodulatory ligand for TLR4, and IL-4, IL-13, and IL-10 downstream targets of this receptor that are known to promote M2-macrophage development. Expression of these stimulants was analyzed by qPCR, flow cytometry, and ELISA during M-LECP differentiation. Our study describes the expression and functionality of these Th2 cytokines and their receptors during M-LECP differentiation. We found that each of the Th2 pathways singularly promotes M-LECP differentiation but there is an absent additive effect. We also found that IL-10 but no other Th2 cytokines is upregulated along with its receptor and contributes to the expression of the lymphatic properties similarly to LPS. To our knowledge, the role of IL-10 in development of lymphatic phenotype through differentiation of M-LECP has not been reported previously. Lastly, we show recruitment of M-LECP in a mouse BC model and the co-expression of the Th2 cytokine receptors in these cells. These studies have a potential to identify new regulators of M-LECP production in the bone marrow that could serve as biomarkers and targets for inhibiting tumor lymphatic formation, and by extension, lymph node metastasis.

L’aide fournie aux lymphocytes B par les lymphocytes T CD4+ est cruciale pour une réponse humorale de qualité. Les lymphocytes T helper folliculaires (Tfh) jouent un rôle majeur dans l’aide apportée aux lymphocytes B au sein des centres germinatifs et peu de choses sont connues sur la capacité d’aide des autres populations de cellules T CD4+. Le but de notre étude est d’évaluer la capacité d’aide aux lymphocytes B des lymphocytes T helper de type 2 (Th2), une population considérée, à l’origine, comme responsable de l’aide apportée aux lymphocytes B in vivo ainsi que d’analyser le rôle de l’axe IL6 / STAT3 dans la différenciation des lymphocytes Tfh et leur plasticité phénotypique. Nous montrons que les lymphocytes Th2 co-expriment des formes actives des facteurs de transcription STAT6 et STAT3 et que l’expression de STAT3 est requise pour la fonction d’aide aux lymphocytes B des lymphocytes Th2. Nous avons également pu montrer que la voie de signalisation IL-6 / STAT3 durant le développement des lymphocytes Tfh s’oppose à l’expression des gènes associés au programme de différenciation Th2. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24760号 / 生博第501号 / 新制||生||67(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 豊島 文子, 教授 野田 岳志, 教授 木村 郁夫 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Experiências vivenciadas durante o período pré-natal são determinantes para a saúde do feto. A ocorrência de infecções maternas e a consequente ativação do sistema imune da mãe ocasionam uma série de alterações estruturais e funcionais no cérebro da prole, podendo predispor o indivíduo a transtornos psiquiátricos na vida pós-natal, como esquizofrenia e autismo. No entanto, estudos que investigam as alterações imunes na prole ainda são escassos na literatura. Dessa forma, o objetivo do presente estudo foi avaliar, na prole, o impacto da ativação imune materna sobre a atividade imune periférica, a resposta imune-inflamatória no sistema nervoso central (SNC), e sobre o desenvolvimento da encefalomielite autoimune experimental (EAE), o modelo murino de Esclerose Múltipla. Camundongos fêmeas prenhes receberam uma administração de salina ou lipopolissacarídeo (LPS) ao final da gestação (dia gestacional 17) e, quando adulta, a prole foi submetida a 3 experimentos principais, analisando: (1) produção de citocinas, atividade de células da periferia e desenvolvimento da hipersensibilidade do tipo tardia; (2) produção de mediadores inflamatórios por células residentes do SNC e; (3) desenvolvimento dos sintomas clínicos e da resposta imune no decorrer da EAE. Nossos resultados mostraram que a ativação imune materna provocou na prole alterações imunes periféricas, como aumento da produção de Interleucina(IL)- 12 e exacerbação da resposta de hipersensibilidade do tipo tardia; potencialização da produção das citocinas IL-1&#946; e IL-6 em cultura primária de células residentes do SNC e; piora na severidade dos sintomas clínicos causados pela EAE, que coincide com aumento do infiltrado de linfócitos e macrófagos no SNC e ativação imuneinflamatória das células da glia. Tomados em seu conjunto, os dados do presente trabalho sugerem que condições inflamatórias durante a gestação, particularmente durante o final da gestação, podem predispor o feto a distúrbios autoimunes e neurodegenerativos na vida adulta. / Prenatal period experiences are crucial for the fetal health. The occurrence of maternal infections and subsequent maternal immune system activation cause a number of structural and functional changes in the brain of the offspring that may predispose individuals to psychiatric disorders in post-natal life, such as schizophrenia and autism. However, studies investigating offspring´s immune alterations are still scarce in the literature. The aim of this study was to evaluate, in mice offspring taken from LPS-treated dams, the impact of maternal immune activation on peripheral immune cell activity, central nervous system (CNS) inflammatory response, and development of experimental autoimmune encephalomyelitis (EAE), the murine model of multiple sclerosis. Pregnant female mice received a dose of either saline or lipopolysaccharide (LPS) during late gestation (gestational day 17), and offspring were used in three experiments to analyze: (1) cytokine production and activity by peripheral immune cells and development of delayed type hypersensitivity, (2) production of inflammatory mediators by resident CNS cells and, (3) development of clinical symptoms and immune response during the course of EAE. Our results showed that maternal immune activation resulted in immune alterations in the offspring, such as increased peripheral production of interleukin (IL) -12 and exacerbated response of delayedtype hypersensitivity; enhancement of IL-1&#946; and IL-6 productions in primary CNS resident cells culture and; increased severity of EAE clinical symptoms, which is positively correlated with the increased lymphocytes and macrophages infiltration within the CNS and also with the immune-inflammatory activation of glial cells. Taken together, the data from this study suggest that inflammatory conditions during pregnancy, especially during the late pregnancy, may predispose the fetus to autoimmune and neurodegenerative disorders in adulthood.

Die Infiltration von T Zellen in das Zentrale Nervensystem (ZNS) ist ein Charakteristikum neuroinflammatorischer Erkrankungen wie der Multiplen Sklerose (MS) und ihrem Tiermodell der experimentellen autoimmunen Enzephalomyelitis (EAE), und führt zur Aktivierung intrinsischer Hirnmakrophagen, den Mikrogliazellen, zu axonaler Schädigung sowie zum Zusammenbruch der Blut-Hirnschranke. Die T Zellen, welche als erste im Gehirn erscheinen, sind vom Subtyp Th1, spezifisch für Bestandteile der Myelinscheide, wie das myelinbasische Protein (MBP), produzieren inflammatorische Zytokine und rekrutieren andere unspezifische T Zellen und Makrophagen. Da sich diese Zellen des Immunsystems gegen körpereigene Bestandteile richten, spricht man von autoreaktiven T Zellen und einer autoimmunen Erkrankung. Im ersten Teil meiner Dissertation habe ich den Einfluss dieser autoreaktiven T Zellen auf den Aktivierungszustand von Mikrogliazellen mit Hilfe muriner Schnittkulturpräparate von Hippocampus und entorhinalem Kortex untersucht, welche den myelinisierten Fasertrakt Tractus perforans mit seinen Ursprungsneuronen und Zielzellen enthielten. Gering aktivierte MBP-spezifische T Zellen induzierten die Expression der Aktivitätsmarker MHC-II und ICAM-1 auf den Mikroglia und die damit verbundene axonale Schädigung (Phagozytose) im gleichen Maße wie hochaktivierte unspezifische T Zellen. Nur Th1 Zellen konnten Mikroglia aktivieren. MBP-spezifische Th2 Zellen hingegen reduzieren die Th1 induzierte Mikrogliaaktivierung (ICAM-1) auf Kontrollniveau. MBP-spezifische Th1 Zellen konnten die Expression von B7 auf Mikrogliazellen modulieren, während die MBP-spezifischen Th2 Zellen diese Eigenschaft nicht besaßen. Durch diese Befunde kann die prominente Rolle von autoreaktiven Th1 Zellen beim Auslösen neuroinflammatorischer Prozesse auf ihre einmalige Fähigkeit, Mikrogliazellen zu aktivieren und deren kostimulatorische Moleküle zu modulieren, zurückgeführt werden. Gleichzeitig bieten die Daten eine mögliche Erklärung für die protektive Rolle von Th2 Zellen bei MS und EAE. Es ist bekannt, dass autoreaktive T Zellen, wie die MBP-spezifischen Th1 Zellen, auch im gesunden Zustand im humanen und murinen T-Zell-Repertoire vorhanden sind. Die physiologische Funktion dieser Zellen ist unklar. Untersuchungen am Nervus opticus sowie im Rückenmark in vivo belegen, dass autoreaktive T Zellen und Makrophagen die Reorganisationsprozesse im ZNS nach traumatischer Schädigung positiv beeinflussen. Diese bei neuroinflammatorischen Erkrankungen so destruktiv wirkenden autoreaktiven T Zellen verhindern nach einem experimentell gesetzten Primärschaden im ZNS das Fortschreiten der Schädigung und es kommt zu einer fast vollständigen Regeneration des Gewebes. Im zweiten Teil meiner Promotionsarbeit habe ich versucht, die Mechanismen, welche hinter dieser Protektion stecken aufzuspüren. Dazu habe ich ebenfalls das in vitro Hirnschnittmodell benutzt. Für diese Fragestellungen wurden Akutschnitte verwendet, die ein Modell für primäre Schädigung im ZNS darstellen. MBP-spezifische Th2 Zellen hatten ein größeres protektives Potential als MBP-spezifische Th1 Zellen. Die nicht ZNS-spezifischen Th1 und Th2 Zellen benötigten ihr Antigen (OVA-Peptid), um signifikant protektiv zu wirken. Durch eine Superstimulation der OVA- und MBP-spezifischen T Zellen wurde eine Neuroprotektion auf gleichem Niveau erreicht. Die Neuroprotektion nach primärer Schädigung von ZNS Gewebe ist somit antigen- und stimulationsabhängig und wird hauptsächlich von Th2 Zellen unterstützt. / The invasion of T cells into the central nervous system (CNS) is a hallmark of neuro inflammatory diseases like multiple sclerosis (MS) and its rodent model, experimental autoimmune encephalomyelitis (EAE), leading to activation of intrinsic macrophages, the microglia, axonal damage and break down of the blood brain barrier. The initial invading T cells are of the Th1 subtype and specific for parts of the myelin sheet like myelin basic protein (MBP). They produce inflammatory cytokines and recruit peripheral non-specific T cells and macrophages. Because these T cells are directed against a self antigen, they are called auto reactive T cells and the phenomenon an autoimmune disease. In the first part of my study I investigated the influence of auto reactive T cells on microglial cells' utilizing an organotypic slice culture system of hippocampus and entorhinal cortex. The slice culture contains a myelinated fibre tract - the tractus perforans - with its original and target neurons. Low activated MBP-specific T cells induced the expression of the activation markers ICAM-1 and MHC-II on microglia as well as microglial phagocytosis in the same manner as highly activated non-specific T cells. Only Th1 cells were able to activate microglia, while Th2 cells reduced the Th1 induced activation (ICAM-1 expression). MBP-specific Th1 cells could modulate the expression of co-stimulatory molecules B7-1 and B7-2, whereas MBP-specific Th2 cells could not. These findings could show why Th1 cells are responsible for EAE induction while Th2 cells can be protective. Auto reactive T cells like MBP-specific T cells have been found in the normal human and murine T cell repertoire. The physiological function of these cells is still unclear. Studies using the models of optic nerve crush or spinal cord crush have shown that macrophages and auto reactive T cells are involved in reorganisation and regeneration after CNS trauma. These auto reactive T cells, which are usually known to be destructive, could prevent CNS tissue from secondary degeneration. In the second part of my study I tried to identify the mechanisms involved in this phenomenon. I also used the organotypic slice culture system. Immediately after preparation causing the primary injury the slices were cultivated with T cells. Th2 cells were found to be more potent to prevent form secondary damage than Th1 cells. The non-CNS specific OVA Th1 and Th2 cells required their antigen to be fully protective. When over stimulated, MBP- and OVA-specific Th1 and Th2 cells proved to be protective to the same extend. Neuroprotection after primary injury depends on the T cell s state of activation and their antigen specificity. Among the cells examined I found Th2 cells were most effective in preventing CNS tissue from secondary injury.