Orientador: Beatriz Maria Machado de Medeiros / Banca: Dagmar Ruth Stach Machado / Banca: Alexandrina Sartori / Resumo: As proteínas Yops. formam uma família de proteínas secretadas por Yersinia spp que incluem efetores intracelulares (seis efetores foram identificados: YopE, YopH, YopM, YpkA/YopO, YopJ/YopP e YopT) e vários componentes do aparato de secreção-translocação que são liberado pela bactéria sob quelação de Ca2+. As .Yops. efetoras têm sido relacionadas a uma série de propriedades de virulência, incluindo resistência à fagocitose, citotoxicidade e desfosforilação de proteínas do hospedeiro. Porém, a interação das .Yops. de Yersinia com a resposta imune específica do hospedeiro não está bem esclarecida. O objetivo deste estudo foi investigar o papel imunomodulador das "Yops" secretadas por amostras de Yersinia pseudotuberculosis sobre a produção de anticorpos e autoanticorpos por linfócitos B. Para tanto camundongos foram infectados com uma amostra selvagem de Y. pseudotuberculosis (YpIII) e com amostras mutantes, defectivas na secreção de determinadas .Yops. efetoras (YopH, YopE, YopM, YpkA e YopJ). Foram obtidas células esplênicas destes animais, e as células secretoras de imunoglobulinas inespecíficas e específicas (anti-Yersinia e anti-.Yops.) foram quantificadas através do teste ELISPOT. A presença de anticorpos específicos anti-Yersinia e anti-.Yops. no soro dos animais infectados foi analisada através do teste ELISA. A presença de auto-anticorpos séricos foi analisada através do teste DOT-BLOT. Não se observou nenhuma diferença entre o número de células secretoras de imunoglobulinas (Igs) inespecíficas dos animais inoculados com a amostra selvagem, YpIIIpIB102 (wt), em relação aos controles. Já a amostra YpIII pIB522, embora defectiva na secreção de YopE, provocou uma redução dos linfócitos B secretores de IgG2a, IgM e IgA. A única ativação observada ocorreu para o isotipo IgG2a (aumento de 1,7 vezes)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The "Yops" proteins form a protein family secreted by Yersinia spp that includes intracellular effectors (six effectors had been identified: YopE , YopH, YopM, YpkA/YopO, YopJ/YopP and YopT) and some components of the secretion-translocation apparatus that are released by the bacteria under Ca2+ quelation. The "Yops" effectors have been related to a series of virulence properties, including resistance to phagocytosis, citotoxicity and desfosforilation of host proteins. However, the interaction of the Yersinia "Yops" with the host specific immune response is not well defined. The objective of this study was to investigate the immunomodulatory role of the "Yops" secreted by strains of Yersinia pseudotuberculosis on the production of antibodies and autoantibodies by splenic B lymphocytes. To this end, mice were infected with wild-type Y. pseudotuberculosis (YpIII) or with mutant strains, unable to secrete specific "Yops" (YopH, YopE, YopM, YpkA and YopJ). Spleen cells were obtained, and the cells secreting nonspecific and specific immunoglobulins (anti-Yersinia and anti-"Yops") was quantified by the ELISPOT technique. The presence of anti-Yersinia and anti- .Yops. specific antibodies in infected mice serum was investigated by ELISA and the presence of autoantibodies by DOT-BLOT assay. It was not observed neither difference between the number of nonspecific Igs-secreting cells of the animals infected with YpIIIpIB102 (wt) in relation to the controls. The strain YpIII pIB522, although defective in YopE secretion, provoked a reduction in the B lymphocytes secreting IgG2a, IgM and IgA. The unique activation observed was that of IgG2a isotype (an increase of 1.7-fold) on the 7th day post infection. The YopH- strain, YpIII pIB29, provoked an increase in the number of IgG1-, IgG2a- and IgG3- secreting cells (between 1.4 to 2.4-fold), on the 7th and 14th days post infection... (Complete abstract, click electronic address below) / Mestre

Made available in DSpace on 2014-06-11T19:30:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-06-19Bitstream added on 2014-06-13T19:18:55Z : No. of bitstreams: 1 maia_jml_me_arafcf.pdf: 491989 bytes, checksum: 76c75c0de1431ea6c8ce3ff67f4446d3 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / As proteínas Yops. formam uma família de proteínas secretadas por Yersinia spp que incluem efetores intracelulares (seis efetores foram identificados: YopE, YopH, YopM, YpkA/YopO, YopJ/YopP e YopT) e vários componentes do aparato de secreção-translocação que são liberado pela bactéria sob quelação de Ca2+. As .Yops. efetoras têm sido relacionadas a uma série de propriedades de virulência, incluindo resistência à fagocitose, citotoxicidade e desfosforilação de proteínas do hospedeiro. Porém, a interação das .Yops. de Yersinia com a resposta imune específica do hospedeiro não está bem esclarecida. O objetivo deste estudo foi investigar o papel imunomodulador das Yops secretadas por amostras de Yersinia pseudotuberculosis sobre a produção de anticorpos e autoanticorpos por linfócitos B. Para tanto camundongos foram infectados com uma amostra selvagem de Y. pseudotuberculosis (YpIII) e com amostras mutantes, defectivas na secreção de determinadas .Yops. efetoras (YopH, YopE, YopM, YpkA e YopJ). Foram obtidas células esplênicas destes animais, e as células secretoras de imunoglobulinas inespecíficas e específicas (anti-Yersinia e anti-.Yops.) foram quantificadas através do teste ELISPOT. A presença de anticorpos específicos anti-Yersinia e anti-.Yops. no soro dos animais infectados foi analisada através do teste ELISA. A presença de auto-anticorpos séricos foi analisada através do teste DOT-BLOT. Não se observou nenhuma diferença entre o número de células secretoras de imunoglobulinas (Igs) inespecíficas dos animais inoculados com a amostra selvagem, YpIIIpIB102 (wt), em relação aos controles. Já a amostra YpIII pIB522, embora defectiva na secreção de YopE, provocou uma redução dos linfócitos B secretores de IgG2a, IgM e IgA. A única ativação observada ocorreu para o isotipo IgG2a (aumento de 1,7 vezes)... / The Yops proteins form a protein family secreted by Yersinia spp that includes intracellular effectors (six effectors had been identified: YopE , YopH, YopM, YpkA/YopO, YopJ/YopP and YopT) and some components of the secretion-translocation apparatus that are released by the bacteria under Ca2+ quelation. The Yops effectors have been related to a series of virulence properties, including resistance to phagocytosis, citotoxicity and desfosforilation of host proteins. However, the interaction of the Yersinia Yops with the host specific immune response is not well defined. The objective of this study was to investigate the immunomodulatory role of the Yops secreted by strains of Yersinia pseudotuberculosis on the production of antibodies and autoantibodies by splenic B lymphocytes. To this end, mice were infected with wild-type Y. pseudotuberculosis (YpIII) or with mutant strains, unable to secrete specific Yops (YopH, YopE, YopM, YpkA and YopJ). Spleen cells were obtained, and the cells secreting nonspecific and specific immunoglobulins (anti-Yersinia and anti-Yops) was quantified by the ELISPOT technique. The presence of anti-Yersinia and anti- .Yops. specific antibodies in infected mice serum was investigated by ELISA and the presence of autoantibodies by DOT-BLOT assay. It was not observed neither difference between the number of nonspecific Igs-secreting cells of the animals infected with YpIIIpIB102 (wt) in relation to the controls. The strain YpIII pIB522, although defective in YopE secretion, provoked a reduction in the B lymphocytes secreting IgG2a, IgM and IgA. The unique activation observed was that of IgG2a isotype (an increase of 1.7-fold) on the 7th day post infection. The YopH- strain, YpIII pIB29, provoked an increase in the number of IgG1-, IgG2a- and IgG3- secreting cells (between 1.4 to 2.4-fold), on the 7th and 14th days post infection... (Complete abstract, click electronic address below)

A sepse é a Síndrome da Resposta Inflamatória Sistêmica decorrente de uma infecção por gram positivos/negativos, fungos ou vírus. É caracterizada por alta liberação de mediadores inflamatórios podendo levar à morte. As células B-1 são encontradas em cavidades peritoneal e pleural de camundongos e sua origem e função ainda não são completamente conhecidas. Apresentam marcadores de superfície de linhagem mielóide e linfóide e migram para focos inflamatórios comportando-se como macrófagos. Objetivo: investigar o papel da célula B-1 na resposta inflamatória após estímulo com lipopolissacáride (LPS) in vitro e in vivo. Métodos: TNF-, IL-6, IL-10 (ELISA) e nitrito (Griess) foram dosados em sobrenadante de cultura celular (106 cel./ml). As células em cultura receberam por 24h de estímulo com 10 g/mL de LPS de Escherichia coli (026:B6 Sigma®). Foram realizados os seguintes grupos cultura de célula B-1 (Balb/c), cultura de macrófagos de linhagem (RAW 264.7) coculturas (macrófagos de linhagem RAW 264.7 e células B-1 (Balb/c, C57BL/6 e C57BL/6 IL-10 -/-), e células peritoneais de camundongos Balb/c e Balb/Xid (imunodeficiente em célula B-1) A endotoxemia foi induzida com injeção de LPS 15 mg/kg (i.p.) em camundongos Balb/c e Balb/Xid. Foram quantificados, TNF-, IL-6, IL-10 e nitrito em soro, pulmão e intestino dos animais após 1,5, 4 e 6 horas após a injeção de LPS. Ensaios de inoculação de células B-1 (Balb/c) em camundongos Balb/Xid foram realizados, e curva de sobrevida foi analisada após indução de endotoxemia. Resultados: Após o estímulo com LPS, células B-1 produziram IL-10 e a presença destas células em cocultura com macrófago promoveu a diminuição na produção de TNF-, IL-6, Nitrito e aumento de IL-10. Contudo, célula B-1 (IL-10 -/-) em cocultura com macrófagos, não inibem a produção de mediadores pro inflamatórios. Análise com macrófagos peritoneais de camundongo Balb/Xid e Balb/c após estímulo com LPS em cultura mostrou reprodução do fenômeno encontrado com os experimentos com cultura de célula imortalizada, isto é, maior produção de TNF-, IL-6 e NO em Balb/Xid (B-1 deficiente). Os estudos in vivo mostraram 60% de mortalidade em camundongo Balb/Xid comparando com Balb/c (0%) após 16 horas de injeção de LPS. Nos animais Balb/Xid encontramos padrão pro inflamatório exacerbado com maiores concentrações de TNF-, IL-6 e menores concentrações de IL-10 no plasma e tecidos quando comparamos com Balb/c. Conclusões: Nossos dados mostraram que a presença de células B-1 promoveram diminuição de mediadores pro inflamatórios e aumento de IL-10 em coculturas com macrófagos e que a modulação da resposta inflamatória pode ser devida a secreção de IL-10 pela célula B-1. Este padrão de resposta pro inflamatória se repete in vivo e é a possível causadora da maior taxa de mortalidade em camundongos da linhagem Balb/Xid. / Sepsis syndrome is caused by inappropriate immune activation due to bacteria and bacterial components released during infection. This syndrome is the leading cause of death in intensive care units. Specialized B-lymphocytes located in the peritoneal and pleural cavities are known as B-1 cells. These cells produce IgM and IL-10, both of which are potent regulators of cell-mediated immunity. It has been suggested that B-1 cells modulate the systemic inflammatory response in sepsis. In this study, we conducted in vitro and in vivo experiments in order to investigate a putative role of B-1 cells in a murine model of LPS-induced sepsis. Macrophages and B-1 cells were studied in monocultures and in co-cultures. The B-1 cells produced the anti-inflammatory cytokine IL-10 in response to LPS. In the B-1 cell-macrophage co-cultures, production of proinflammatory mediators (TNF-, IL-6 and nitrite) was lower than in the macrophage monocultures, whereas that of IL-10 was higher in the co-cultures. Co-culture of B-1 IL-10/ cells and macrophages did not reduce the production of the proinflammatory mediators (TNF-, IL-6 and nitrite). After LPS injection, the mortality rate was higher among Balb/Xid mice, which are B-1 cell deficient, than among wild-type mice (65.0% vs. 0.0%). The Balb/Xid mice also presented a proinflammatory profile of TNF-, IL-6 and nitrite, as well as lower levels of IL-10. In the early phase of LPS stimulation, B-1 cells modulate the macrophage inflammatory response, and the main molecular pathway of that modulation is based on IL-10-mediated intracellular signaling.

A sepse é causada por microorganismos tais como: bactérias Gram-negativas, Gram-positivas, fungos e vírus. A sepse grave e o choque séptico estão associados a taxas de mortalidade de 40 a 60%. O evento mais importante para a evolução do quadro séptico é a apoptose das células efetoras do sistema imune. A eliminação de um grande número de células do sistema imune compromete a defesa efetiva do hospedeiro. Para estudarmos o papel das células imunes na sepse utilizamos o modelo de tolerância ao LPS. Em nossos estudos induzimos tolerância ao LPS em camundongos Balb-C e analisamos os padrões da expressão gênica nos linfócitos do baço. Para o mapeamento dos genes, utilizamos o macroarray, identificamos o grupo funcional de genes que tem maior relevância na proteção induzida pela tolerância, e em seguida confirmamos os resultados encontrados através do RT-PCR, Western Blotting, atividade de caspase 1 e citometria de fluxo. Encontramos uma importante redução na expressão de genes, como heat shock proteins, óxido nítrico e apoptose. Após análise da membrana contendo os genes integradores da resposta produzida pela tolerância, optamos por enfatizar inicialmente os genes envolvidos no processo apoptótico devido à relevância deste processo no quadro séptico conforme mostraram os trabalhos encontrados na literatura. Demonstramos que animais tolerantes ao LPS apresentam diminuição dos eventos apoptóticos, com redução na expressão dos genes ligados às caspases 7, 8 e 11, assim como a redução dos genes ligados ao Bid e Apaf-1. Ao analisarmos o RNAm através do RT-PCR, encontramos redução na Caspase 3, Bax e Bcl2, resultado que se confirmou ao analisarmos a expressão protéica através do Western- Blotting. Realizamos citometria de fluxo para avaliarmos a ocerrência de apoptose nos linfócitos dos animais submetidos à ligadura cecal. Confirmamos uma redução da apoptose e necrose em linfócitos dos animais tolerantes em relação aos controles. Com estes resultados podemos propor que a tolerância seria benéfica na redução da apoptose e controle do quadro séptico, além disso, a diminuição na expressão do gene ligado à caspase 11 estaria contribuindo para a diminuição do quadro inflamatório, pois a caspase 11 é um mediador essencial na resposta ao choque séptico. / Sepses are caused by microorganisms such as: Gram-negative bacteria, Gram-positive bacteria, fungus and virus. Several sepses and the septic shock have been associated with the mortality rates from 40 to 60%. One of the most important events for the septic evolution is the apoptosis cells of the immune system. The cells immune system elimination compromises the host. We induced LPS tolerance in Balb-C mice and analyzed genes expressions in spleens; we found an important reduction in the genes expressions like: heat shock proteins, nitric oxide and apoptosis. After analysis of the membrane containing the sepses genes produced by tolerance, we emphasized initially the genes involved in the apoptosis. Demonstrated that animals LPS tolerants presented decrease in apoptotic events, with reduction in the genes expression related caspases 7, 8, 11, Bid and Apaf-1. In the mRNA by RT-PCR, we found a reduction in Caspase 3, Bax and Bcl2, and these results were confirmed by Western-blot. We realized flow cytometry and we showed that the results are maintained, presenting reduction in both the apoptotic and necrotic events in tolerants animals. These results showed that the tolerance would be favorable in the apoptosis reduction and control of the sepsis, moreover, the expression reduction to caspase 11 genes would be contributing to the inflammatory reduction because Caspase 11 is an essential mediator in the septic shock.

Introdução: A Imunodeficiência Comum Variável (ICV) é a imunodeficiência primária mais prevalente na prática clínica. Suas manifestações vão desde infecções de repetição (que são as mais prevalentes) à neoplasias e doenças autoimunes. Admite-se que o quadro clínico da ICV esteja associado a alterações na diferenciação de linfócitos B (LB), mas também em linfócitos T, monócitos e células dendríticas, além de mutações em genes relacionados a interações entre linfócitos T e B ou homeostasia de linfócitos B. Isto determina uma grande complexidade na classificação destes pacientes, sendo a correlação entre características clínicas e fenotípicas essencial para o melhor entendimento da doença e acompanhamento dos pacientes. Objetivo: Descrever o fenótipo clínico e das subpopulações de LB da coorte de pacientes com ICV acompanhados no Ambulatório de Imunodeficiências Primárias do Hospital das Clínicas e verificar o modelo de classificação que melhor se adequa a estes pacientes. Materiais e Métodos: Após a separação de PBMC e imunofenotipagem dos subtipos de LB de amostras de 70 pacientes, estes foram classificados segundo os critérios das classificações de Freiburgo, Paris e EUROclass. Também foram realizadas a análise das manifestações clínicas; a correlação entre as células de memória e as concentrações séricas das imunoglobulinas e a correlação entre os subtipos de LB e as principais manifestações clínicas desta coorte. Resultados: Esta coorte apresentou um grande distúrbio na distribuição das subpopulações de LB, sendo que todos os pacientes apresentaram redução na frequência de LB de memória com troca de isotipo, mas apenas as pacientes mulheres apresentaram aumento estatisticamente significante nas frequências de LB naive e LB CD21low e redução na frequência de plasmoblastos. Os subtipos de LB transitório e LB de zona marginal não apresentaram diferenças significativas entre pacientes e controles. Não observamos correlação entre as células B de memória e os níveis séricos de imunoglobulinas. A análise clínica revelou diferenças na prevalência de algumas complicações clínicas, entretanto, a correlação entre os subtipos de LB e estas manifestações mostrou associação apenas entre linfadenopatia e redução de plasmoblastos. Por fim, a classificação quanto aos três modelos propostos, revelou que nenhum deles se adequou para esta coorte de pacientes brasileiros. Conclusão: verificamos uma diversidade tanto nas subpopulações de LB quanto na prevalência de algumas manifestações clínicas e por conta destas alterações, até o momento nenhuma classificação se mostrou apropriada para esta coorte. Estes achados reforçam o conceito de heterogeneidade da doença e confirmam a necessidade de um novo modelo de classificação que compreenda outros marcadores. / Introduction: Common Variable Immunodeficiency (CVID) is the primary antibody deficiency most often seen in clinical practice. Patients present different clinical features ranging from recurrent infections (most prevalent) to malignancies and autoimmune disorders. It is accepted that this clinical picture is associated with alterations in the differentiation of B cells but also in T cells, monocytes and dendritic cells, besides mutations in genes related to interactions between T and B cells or in B cell homeostasis. This makes the classification of CVID patients very difficult, on the other hand the association between clinical and immunological phenotype is essential for better understanding of the disease and follow-up of patients. Objective: To describe the clinical and immunological phenotype of a Brazilian cohort of CVID patients followed at the Division of Clinical Immunology and Allergy of University of São Paulo Medical School and to classify them and analyze which is the most appropriated classification for this cohort. Methods and Results: Seventy patients were studied and flow cytometric B cell phenotyping was performed in PBMCs, using a mixture of monoclonal antibodies. The samples were classified according to the Freiburg, Paris and EUROclass classifications. We also analyzed the major clinical manifestations and correlated them to B cell phenotypes, besides correlating memory B cell frequency and serum IgA, IgG and IgM levels. Comparing patients and controls, we found a disturbed frequency of B cell subtypes in this cohort. Both male and female patients presented a reduction on switched memory B cells. However, only female patients had a significant increase on naïve B cells, CD21low B cells and reduction of plasmablasts. Marginal zone and transitory B cells showed no statistical differences between groups. There was no correlation between memory B cell frequency and serum immunoglobulin levels. Clinical analysis revealed a disturbed prevalence in some features but the correlation between B cell subtypes and clinical manifestations revealed only an association between lymphadenopathy and plasmablast reduction. Finally, the classification of this cohort according to the three proposed models revealed that none of them were appropriated to this Brazilian cohort. Conclusion: We observed a diversity in B cell subtypes frequency and the profile of some clinical complications. Because of these findings in both clinical and B cell subtypes, until now no proposed classification is appropriated for this cohort. These findings reinforce the concept of CVID heterogeneity and confirm the necessity of a new classification model that involves other markers

A resposta imune ao P. chabaudi se caracteriza pela ativação policlonal de linfócitos B na fase aguda da doença, que resulta em uma intensa produção de anticorpos IgM e IgG de baixa afinidade e autoanticorpos. Apesar de esses anticorpos contribuírem para o controle da infecção, pode retardar a geração de anticorpos de alta afinidade que garantem a destruição dos parasitas e a proteção contra infecções subseqüentes. O nosso objetivo foi caracterizar os mecanismos envolvidos na ativação policlonal dos linfócitos B na infecção pelo P. chabaudi AS. Os nossos resultados mostraram que a cooperação T-B e importante nessa fase da infecção. Os ensaios in vitro mostraram que os linfócitos T da fase aguda da infecção são capazes de estimular linfócitos B naives, aumentando a expressão do CD69 e induzindo proliferação e produção de anticorpos. A molécula de classe II do MHC é importante na ativação policlonal dos linfócitos B, aumentando a expressão do CD69 e induzindo a proliferação de anticorpos, devido ao seu envolvimento na ativação dos linfócitos T. Assim, moléculas como CD28, CD40L e ICOS são importantes na ativação, proliferação e produção de anticorpos. / Polyclonal B cell activation is a feature of the early spleen B cell response to human and murine malaria, which results in intense production of parasite low-affinity IgM and IgG and autoantibodies. Although this response may contribute for the initial parasite control, it may also hinder the generation of high-affinity IgG that guarantees parasite elimination and protection against secondary infections. To characterize the mechanism involved in the polyclonal B cell response to P. chabaudi AS, we have analyzed in vivo and in vitro the role of several molecules known to be involved in antibody production. According to our in vivo results, this response depends on the cooperation of CD4+ T cells with B cells. The in vitro assays shows that the expression of MHC class II molecules is crucial for B cell activation in terms of CD69 expression, proliferation and antibody production, apparently due to their requirement for T cell activation. Signaling pathways mediated by the co-stimulatory receptor CD28, CD40L and ICOS has a central role for B cell proliferative response and antibody production. This study may help to clarify the molecular basis of polyclonal B cell activation induced during acute malaria.

Les maladies cardiovasculaires sont la première cause de mortalité dans le monde et voient leur incidence augmenter avec l’expansion de leurs principaux facteurs de risque, tels que le vieillissement, l’obésité et le diabète. Parmi ces maladies l’anévrisme de l’aorte abdominale (AAA), dont on sait aujourd’hui que le système immunitaire favorise le développement. Il est parmi les pathologies les plus courantes de l’aorte abdominale et représente un problème de santé publique. Le rôle des macrophages dans l’AAA a été récemment mis en évidence. Ces cellules inflammatoires sont les plus représentées dans l’adventice anévrismale, et ont tendance à s’accumuler au cours du temps dans l’AAA. Des données récentes montrent que leur accumulation est particulièrement accrue dans les premiers jours de la formation de l’AAA chez la souris, et que l’inhibition de la voie CCR2/MCP1 ou la déplétion précoce des monocytes circulants ont un effet protecteur. Il semble donc essentiel de comprendre les mécanismes d’infiltration des monocytes lors de l’initiation et du développement de l’AAA. Mon travail de thèse se focalise sur le rôle des sous-types monocytaires et l’impact de leur trafic sur le développement de l’AAA. En utilisant un modèle d’induction d’anévrisme chez des souris ApoE-/-, nous avons montré que l’angiotensine-II (Ang II) entraine la mobilisation des monocytes ‘Ly-6Chigh’, et des monocytes ‘Ly-6Clow’ à un moindre degré, à partir de la rate, puis leur recrutement au niveau de l’aorte. La splénectomie ou la déficience en lymphocyte B inhibe cette mobilisation monocytaire et protège de l’anévrisme. En revanche, la reconstitution de souris immuno-déficientes Apoe-/-Rag2-/- par des splénocytes totaux, contrairement aux splénocytes déplétés en lymphocytes B, restaure la mobilisation monocytaire et restitue la susceptibilité de ces souris à l’AAA. Cette thèse apporte de nouveaux indices sur les événements précoces impliqués dans la formation d’AAA, en mettant l’accent sur le rôle du réservoir monocytaire splénique dans ce processus. Elle identifie également une fonction jusque-là méconnue des lymphocytes B dans la mobilisation rapide et transitoire des monocytes en réponse à l’Ang II. Il conviendra par la suite d’identifier les mécanismes moléculaires sous-jacents à ces interactions, ce qui devrait permettre d’envisager de nouvelles voies thérapeutiques visant à moduler l’effet de ces types cellulaires dans l’AAA. / Cardiovascular diseases are the first cause of mortality around the industrialized world with a continuous increase in their incidence along with the expansion of the major risk factors such as aging, obesity and diabetes. In abdominal aortic aneurysm (AAA), the presence of inflammatory infiltrates, and particularly monocytes/macrophages, has underscored the contribution and importance of immuno-inflammatory responses in aneurysmal degeneration. It is one of the most common diseases of the abdominal aorta and presents a major health problem. The importance of macrophages has recently been highlighted by a number of evidence, which are the main population observed within the site of aneurysm, are believed to derive from circulating monocytes although no direct evidence has been provided to date. Recent evidence has shown that their accumulation is particularly enhanced in the early onset of AAA in mice, and that the inhibition of CCR2/ MCP1 signaling as well as the early depletion of circulating monocytes, are protective. Hence, it seems crucial to understand the mechanisms of monocyte recruitment in the initiation and progression of AAA. In this PhD project with a particular interest in abdominal aortic aneurysm, we were particularly interested in understanding the trafficking behavior of monocyte subsets in AAA and their role in disease pathogenesis. Using a mouse model of aneurysm induction in ApoE-/- mice, we showed that Ang II triggered the mobilization of Ly-6Chigh, and to a lesser extent Ly-6Clow monocytes, from the spleen and their consequent recruitment in the aorta. Spleen removal or B lymphocyte deficiency in Apoe-/- mice similarly impaired early monocyte mobilization in response to Ang II and protected against AAA development, independently of blood pressure. Reconstitution of Apoe-/- Rag-/- mice with total splenocytes but not with B-Cell depleted splenocytes restored monocyte mobilization in response to Ang II and enhanced susceptibility to AAA. Taken together, this study provides novel mechanistic insights on the early events involved in AngII-induced AAA formation. It highlights the role of the splenic monocyte reservoir in this process and identifies an intriguing role for B lymphocytes in mediating AngII-induced early and transient mobilization of splenic monocytes. Nevertheless, further understanding of the molecular mechanisms that underlie such interactions is likely to lead to the identification of effective therapeutic targets.

La connaissance du système immunitaire est une étape clé dans la compréhension et la prévention des infections bactériennes, virales, des maladies auto-immunes et du cancer. Les lymphocytes B (LB) sont spécialisés dans la fonction de présentation de l'antigène et sont responsables de la production d'anticorps de haute affinité qui permettent l'élimination des agents infectieux. Après l'engagement du récepteur des cellules B (BCR), les LB polarisent rapidement leur centre organisateur des microtubules (MTOC) et les molécules du CMH de classe II vers l'interface BCR-Antigen (Ag), ce qui déclenche la libération de protéases lysosomales permettant l'extraction de l’Ag immobilisé à la surface des macrophages ou cellules dendritiques, un processus essentiel pour les LB à acquérir leur fonction de présentation antigénique. Toutefois, la signalisation par le BCR n'est pas le seul facteur déterminant dans l'activation des cellules B périphériques. La reconnaissance des motifs moléculaires de virus et bactéries par les Toll Like Receptors (TLRs) est importante dans la perte de tolérance des cellules B. Les TLR sont divisés en deux familles: les TLR1, 2, 4, 5, 6 et 11, qui détectent la présence de protéines et lipides bactériens et sont exprimés à la membrane plasmique, et les TLR3, 7-8 et 9, localisés dans les endosomes et qui détectent la présence d’ARN simple brin, ou double brin et ADN non méthylé respectivement. Compte tenu de leur localisation endosomale, les TLR3, 7, 8 et 9 sont des acteurs clés dans la discrimination entre le soi non-infectieux et le non-soi infectieux. Dans les cellules dendritiques, les TLR7 et 9 sont retenus dans le réticulum endoplasmique avec une molécule chaperonne, UNC93B1. Suite à leur stimulation, ils migrent vers les compartiments lysosomaux où ils subissent un clivage protéolytique par l'AEP, une asparagine endopeptidase, pour être fonctionnels. D'autre part, il a été récemment montré que les molécules MHC de classe II intracellulaires sont capables de promouvoir l’activation complète des TLR3, 4 et 9 via une interaction directe avec la tyrosine kinase Btk dans les macrophages et les cellules dendritiques. Notre travail a porté sur l’identification de nouvelles molécules régulant la signalisation des TLR7 et 9 dans les cellules B. Nos résultats, jusqu'à présent, montrent que la signalisation du TLR7 mais pas celle du TLR9 semble être régulée par la molécule chaperone associée au CMHII, la chaîne invariante ou Ii. En effet, dans les cellules B, au repos et après stimulation, le TLR7 réside dans les lysosomes avec la chaîne invariante. Après stimulation, la chaîne invariante interagit spécifiquement avec TLR7 et sa molécule adaptatrice MyD88 favorisant la sécrétion des cytokines pro-inflammatoires telles que L’IL-6 et le TNF-α. Étonnamment, en absence de Ii ou lorsque l'expression de Ii est diminuée suite à une infection virale, TLR7, mais pas TLR9, relocalise dans le réticulum endoplasmique (RE) conduisant ainsi à une exacerbation des fonctions innées du TLR7 telles que la production des cytokines, mais pas les fonctions adaptatives du TLR7 telles que la présentation croisée associée aux molécules de classe I. Ces résultats suggèrent un nouveau rôle pour la chaîne invariante en agissant comme un régulateur négatif de la signalisation du TLR7. / Knowledge of the immune system is a key step in the understanding and prevention of bacterial, viral infections, autoimmune diseases and cancer. B Lymphocytes (BL) are specialized in the function of antigen presentation and are responsible for the production of high affinity antibodies that allow the elimination of infectious agents. After the engagement of the B Cell Receptor (BCR) , BL rapidly polarize their microtubule organizing center (MTOC) and MHC class II molecules towards the BCR- Antigen (Ag) interface, which triggers the release of lysosomal proteases allowing the extraction of immobilized Ag present on the surface of macrophages or dendritic cells. This process is essential for LB to acquire their antigen presenting function. However, the BCR signaling is not the only determining factor in the activation of peripheral B cells. Recognition of molecular patterns from viruses and bacteria by Toll Like Receptors (TLRs) is important in the loss of B cell tolerance. TLRs are divided into two families: TLR 1 , 2, 4 , 5, 6 and 11, which detect the presence of bacterial proteins and lipids and are expressed at the plasma membrane, and TLR3 , 7-8 and 9 , located in the endosomes and detect the presence of single-stranded RNA, double-stranded RNA and unmethylated DNA respectively. Given their endosomal localization, TLR3, 7, 8 and 9 are key players in the discrimination between the non-infectious self and the infectious non- self. In dendritic cells, TLR7 and 9 are retained in the endoplasmic reticulum with a chaperone molecule, UNC93B1. Following their stimulation, they migrate to the lysosomal compartments where they undergo a proteolytic cleavage by AEP, an asparagine endopeptidase, to be functional. On the other hand, it has recently been shown that MHC class II molecules are able to promote intracellular full activation of TLR3, 4 and 9 through a direct interaction with the Bruton’s tyrosine kinase Btk in macrophages and dendritic cells. Our work has focused on the identification of new signaling molecules that regulate TLR7 and 9 function in B cells. Our results, so far, show that TLR7 and not TLR9 signaling is regulated by the chaperone molecule associated with MHC class II, the invariant chain Ii. Indeed, in resting and stimulated B cells, TLR7 resides in lysosomes with the invariant chain. Upon stimulation, the invariant chain interacts specifically with TLR7 and its adaptor molecule MyD88 promoting the secretion of proinflammatory cytokines such as IL-6 and TNF-α. Surprisingly, in the absence of Ii or when the expression of Ii is reduced due to a viral infection, TLR7, but not TLR9, relocates in the endoplasmic reticulum (ER) leading to an exacerbation of the innate functions of TLR7 such as the production of cytokines, but not TLR7 adaptive responses such as cross-presentation associated with MHC class I molecules. These results suggest a new role for the invariant chain by acting as a negative regulator of TLR7 signaling in B cells.

CD40 signals are required for productive immune responses but also play a role in autoimmune disease pathogenesis. The major goal of this research was to investigate the contribution of two receptors to the development of autoimmune disease: (1) LMP1, an oncogenic EBV-encoded mimic of CD40 and (2) a naturally-occurring polymorphism in CD40, P227A, which appears to confer LMP1-like properties to the CD40 receptor. Interestingly, hCD40-P227A is overrepresented in individuals of Mexican and South American descent. Although this allele is not directly associated with SLE incidence in Hispanic populations, patients of Hispanic ethnicity have a tendency toward increased severity of SLE symptoms, particularly lupus nephritis. This work reports the initial genetic description of CD40-P227A and characterizes its gain-of-function signaling properties in mouse and human B cells. In comparison to Wt-CD40 signaling, CD40-P227A signaling results in increased binding of TRAF3, TRAF5, and Act1, as well as enhanced secretion of IL-6, TNF-α, and Ig due to a selective hyperactivation of the JNK pathway. Whereas TRAF3 is normally a negative regulator of Wt-CD40 signaling, TRAF3 is a required positive regulator of CD40-P227A signaling as demonstrated in TRAF3-deficient B cells. LMP1 is an EBV-encoded CD40 mimic which signals in an amplified and sustained manner. Although EBV is latent in >90% of humans, EBV infection is associated with autoimmunity, particularly SLE. Upon flares of autoimmunity, EBV reactivates and LMP1 is expressed, yet the contribution of LMP1 to exacerbation of disease is unknown. LMP1 transgenic mice generated in our lab have an autoreactive phenotype but do not die early due to autoimmune disease. To test the hypothesis that LMP1 cooperates with other genes in the host to exacerbate autoimmunity, mCD40-LMP1 transgenic mice were bred to two lupus-prone strains of mice. LMP1 signaling was able to enhance the autoimmune phenotype of the B6.Sle1, but not the B6.Sle3 strain. These data suggest that LMP1 is redundant with genes within the Sle3 interval, but acts cooperatively with genes within the Sle1 interval to exacerbate autoimmunity. Together, the research foci of this dissertation examine how the CD40 pathways of B cell activation can be amplified and dysregulated, by either a viral mimic of CD40, a polymorphism in its signaling domain, or its cooperation with additional gene products. Differential usage of TRAF3 as a positive, rather than a negative, regulator of signaling appears to be one common mechanism by which this occurs. In conditions where enhanced CD40 signaling may be desirable such as during chronic infections, manipulation of TRAF3-CD40 signaling may serve to enhance immune responses. It is hoped that these studies can additionally reveal important information about the normal regulation of this powerful activation pathway.

This thesis proposes a mechanism for the induction of peripheral tolerance to protein antigens. I have investigated the mechanism of tolerance induction to soluble protein antigens by targeting an antigen to small, resting B cells. For this purpose I have used a rabbit antibody directed at the IgD molecule found on the surface of most small, resting B cells but missing or lowered on activated B cells. Intravenous injection of normal mice with 100 μg of an ultracentrifuged Fab fragment of rabbit anti-mouse IgD (Fab anti-δ) makes these mice profoundly tolerant to challenge with nonimmune rabbit Fab (Fab NRG) fragments. This tolerance is antigen specific since treated mice make normal responses to an irrelevant antigen, chicken immunoglobulin (Ig). Fab fragments of rabbit Ig (rabbit Fab) not targeted to B cells do not induce tolerance as well as Fab anti-δ. Evidence suggests that the B cells must remain in a resting state for tolerance to be induced, since injection of F(ab)'2 anti-δ does not induce tolerance. Investigation of the mechanisms of the tolerance, by adoptive transfer, have shown that rabbit Fab specific B cell function has been impaired. The major effect however is in helper T cell function, as shown by adoptive transfer and lack of help for a hapten response. In vitro proliferation experiments show that the T cell response has not been shifted toward activation of different T cell subsets which do not help Ig production, nor is there any change in the Ig isotypes produced. Suppression does not appear to be the major cause of the helper T cell defect as shown by cell mixing experiments. This work shows that an antigen targeted to small B cells can induce tolerance to a soluble protein antigen, and suggests a role for small B cells in tolerance to self-proteins not presented in the thymus.