RESUME Depuis leur découverte en 1973 par Steinman et Cohn, le rôle des cellules dendritiques dans l’initiation des réponses immunitaires a largement été documenté. En effet, les cellules dendritiques constituent les cellules présentatrices d’antigènes professionnelles capables de détecter des molécules microbiennes et inflammatoires afin d’activer le système immunitaire. Outre leur implication dans l’induction des réponses immunes, de plus en plus d’études suggèrent que les cellules dendritiques interviennent dans le contrôle des réponses immunitaires via la sécrétion de cytokines anti-inflammatoires et/ou l’activation ou l’induction de lymphocytes T régulateurs. Ceux-ci incluent les cellules T régulatrices issues naturellement du thymus et les cellules T régulatrices induites en périphérie. Des résultats obtenus au sein de notre laboratoire ont mis en évidence l’importance des cellules T régulatrices dans le contrôle des réponses de type Th1 induites à l’aide de cellules dendritiques matures chargées avec des antigènes étrangers. Nous avons, dès lors, étudié le rôle du récepteur CTLA-4 exprimé constitutivement à la surface des cellules T régulatrices dans le contrôle des réponses immunitaires induites à l’aide de cellules dendritiques matures et dans un modèle d’inflammation intestinale. L’injection d’anticorps anti-CTLA-4 induit in vitro et in vivo une inhibition de la production d’IFNγ et protège les souris de la colite pro-Th1 induite par l’instillation de TNBS. Cette protection corrèle étroitement avec l’induction de lymphocytes T régulateurs exprimant fortement la molécule ICOS et sécrétant de l’interleukine 10. De plus, nos résultats suggèrent que l’interleukine 10 et l’indoléamine 2, 3 dioxygénase seraient impliquées dans la fonction régulatrice des lymphocytes T ICOShigh. Nous avons également analysé les mécanismes impliqués dans le contrôle des réponses de type Th1 par les lymphocytes T régulateurs naturels. Nos résultats suggèrent une régulation différente des réponses Th1 en présence et en absence de cette population régulatrice. En effet, les réponses Th1 sont dépendantes de l’interleukine 12 en présence de lymphocytes T régulateurs naturels, alors qu’en leur absence, la molécule CD70 est requise. En conclusion, nos résultats suggèrent que les lymphocytes T régulateurs naturels et induits contrôlent les réponses immunes de type Th1. Au cours de ce travail, nous avons mis en évidence des stratégies distinctes par lesquelles ces deux populations régulatrices contrôlent la réponse immune. Ces résultats complètent la compréhension des mécanismes de régulation du système immunitaire et ouvrent de nouvelles perspectives d’approche immunothérapeutique.

The organic mercury compound ethylmercurithiosalicylate (thimerosal), an antiseptic and a preservative, has recently raised public health concern due to its presence in vaccines globally. Thimerosal dissociates in the body to thiosalicylate and ethyl mercury (EtHg), which is partly converted to inorganic mercuric mercury (Hg2+). The immunosuppressive, immunostimulatory, and de novo autoimmunogen effect of thimerosal in mice, as well as the accelerating/aggravating effect on spontaneous systemic autoimmunity including dose-response aspects were the subject of this thesis. Thimerosal perorally (590 μg Hg/kg body weight (bw)/day) to genetically susceptible (H-2s) mice caused immunosuppression during the first week with reduction of the total number of splenocytes, T- and B-cells. The suppression lasted 2 weeks for CD4+ cells, but was superseded by a strong immunostimulation/proliferation including T- as well as B-cells, and polyclonal B-cell activation (PBA). Antinuclear antibodies targeting the 34-kDa nucleolar protein fibrillarin (AFA) appeared after 10 days, followed by renal mesangial and systemic vessel wall immune-complex (IC) deposits. The Lowest Observed Adverse Effect Level (LOAEL) was in the order AFA = glomerular and splenic vessel wall deposits < hyperimmunoglobulinemia < PBA. The LOAEL for AFA was 118 μg Hg/kg bw/day. The LOAEL for the different parameters of this thimerosal-induced systemic autoimmune condition (HgIA) was 3-11-fold higher compared with HgIA induced by HgCl2. The thimerosal-induced HgIA shared with HgCl2 a significant dose-response relationship, and requirement for: T-cells, the costimulatory factor CD28, the IFN-γ/IFN-γ-receptor pathway,but not IL-4. The mRNA expression in lymph nodes of IL-2, IFN-γ, IL-4, and IL-15 was significantly increased but not delayed compared with HgCl2. Treatment with the ubiquitous organic Hg compound methyl Hg using equimolar doses of Hg (533 μg Hg/kg bw/day) caused a transient immunosuppression, followed by a weak immunostimulation and AFA. The IgG AFA isotypes induced by the organic Hg compounds MeHg and EtHg were stable and dominated by a Th1-like pattern over a broad time- and dose range. Treatment with inorganic HgCl2 caused a dose- and time-dependent pattern of IgG AFA isotypes. Low doses favored a Th1-like pattern, a high dose a balanced or Th2-like pattern. Middle-range doses showed initially a Th1-like pattern which gradually evolved into a balanced or Th2-like pattern. The qualitative difference in IgG AFA isotypes between organic and inorganic Hg may be due to differences in activation and/or suppression of T-helper cell subsets or factors influencing the Th1/Th2-function. Speciation of the renal Hg2+ concentration and comparison with the threshold dose for induction of AFA by HgCl2 showed that even with the lowest doses of thimerosal and MeHg used in this thesis, the AFA response might from a dose threshold point of view have been caused by conversion of the organic Hg species to Hg2+. Primary treatment with inorganic Hg (HgCl2) accelerates/aggravates murine systemic autoimmunity, both spontaneous (genetic) and induced by other means. This capacity was assessed for thimerosal over a broad dose range using the (NZB X NZW)F1 hybrid mouse model. Significantly increased antinuclear antibodies (ANA) was seen after 4-7 weeks treatment (LOAEL 147 μg Hg/kg bw/day), and the response was dose-dependent up to 13 weeks. Renal mesangial and systemic vessel walls deposits similar to those in de novo HgIA were present after 7 weeks treatment. Twenty-two to 25 weeks treatment with thimerosal caused, in a dose-dependent fashion (LOAEL 295 μg Hg/kg bw/day), relocalization of the spontaneously developing glomerular IC deposits from the capillary vessel walls to the mesangium, which attenuated histological kidney damage and proteinuria, and increased survival. Thimerosal caused systemic vessel wall IC-deposits over a broad dose range: the Low Observed Adverse Effect Level (LOAEL) for renal and splenic vessel wall IC deposits was 18 and 9 μg Hg/kg bw/day, respectively. The No Observed Adverse Effect Level (NOAEL) could not be determined for the latter, since deposits were present even with the lowest dose used. Thimerosal causes in genetically susceptible mice an initial, transient immunosuppression which is superseded by a strong immunostimulation and systemic autoimmunity, sharing many characteristics with the HgIA induced by inorganic HgCl2. The IgG AFA isotype pattern is however qualitatively different, and the threshold dose substantially higher. In contrast, long-term treatment with thimerosal induces systemic vessel wall IC-deposits also using doses below those needed to induce HgIA de novo in H-2s mice.

Atopic asthma is characterized by excessive T helper 2 (Th2)-like immunity to allergens in the bronchial mucosa. The Th2-cytokine interleukin (IL)-4 induces IgE production, while the Th2-cytokine IL-5 promotes eosinophilic inflammation in the airways of asthmatics. Most asthmatics are atopic, but a subgroup is non-atopic. We hypothesize that allergen-induced Th2, particularly IL-5, responses can be observed in peripheral blood in both atopic and non-atopic asthmatic children but not in healthy control children. The aim of the present study was to determine IL-4, IL-5, IL-9, IL-10, IL-13 and IFN-γ secretion induced from peripheral blood mononuclear cells (PBMC) by a broad panel of inhalant allergens (timothy, cat, birch, dog and house dust mite) in asthmatic children with and without sensitization. The study included 13 atopic asthmatic, 5 non-atopic asthmatic, and 12 non-atopic non-asthmatic children. PBMC were stimulated with allergens and cytokine production was measured with enzyme-linked immunosorbent assay (ELISA). Higher levels of cat and dog antigen-induced IL-5 release were more commonly observed in both atopic and non-atopic asthmatics than in controls. Children with atopic, but not non-atopic, asthma produced higher levels of allergen-induced IL-4 and IL-9 than controls. Non-atopic asthmatics produced more IL-10 than atopic asthmatics after cat stimulation. High levels of eosinophilia-associated IL-5 responses are induced by cat and dog allergen in both atopic and non-atopic asthmatic children. The Th2 cytokines IL-4 and IL-9 were associated only with atopic asthma, probably due to their IgE-inducing properties.

We aimed to prospectively investigate the paternal antigen-induced cytokine secretion by peripheral blood mononuclear cells (PBMCs) in response to hormone treatment in women undergoing in vitro fertilisation (IVF) and to examine the predictive value of the cytokine secretion profile in the outcome of IVF treatment, in a pilot study. Twenty-five women were included and IVF treatment was successful for six and unsuccessful for 19 women. Blood samples were collected before IVF treatment, on four occasions during IVF and four weeks after embryo transfer. The numbers of Th1-, Th2- and Th17-associated cytokine-secreting cells and cytokine levels in cell supernatants were analysed by enzyme-linked immunospot-forming (ELISpot), enzyme-linked immune-sorbent (ELISA) or Luminex assay. None of the cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, TNF and GM-CSF) had any predictive value regarding IVF outcome. The majority of the cytokines reached their peak levels at ovum pick-up, suggesting an enhancing influence of the hormonal stimulation. Pregnancy was associated with a high number of IL-4-, IL-5- and IL-13-secreting cells four weeks after ET. In conclusion, the results do not support our hypothesis of a more pronounced peripheral Th1 and Th17 deviation towards paternal antigens in infertile women with an unsuccessful IVF outcome, although this is based on a small number of observations. A larger study is required to confirm this conclusion. Higher numbers of Th2-associated cytokine-secreting cells in pregnant women four weeks after ET do corroborate the hypothesis of a Th2 deviation during pregnancy.

Allergens are complex mixes of proteins and other compounds that have innate signaling capacity leading to Th2 inflammation. Understanding the role of each of these signals is essential to determining what separates allergens from innocuous proteins. Here, we examine two models for Th2 inflammation: infection with the helminth Trichinella spiralis and footpad immunization with papain, a cysteine protease structurally similar to proteases found in many common allergens including grass pollen and dust mites and helminth-secreted proteases secreted. Together, these studies highlight previously unappreciated effector roles of accessory cells during Th2 inflammation.

Thesis (Ph.D.). / Written for the Dept. of Medicine. Title from title page of PDF (viewed 2008/05/09). Includes bibliographical references.

Thesis (Ph.D.)--Australian National University, 2004.

Thesis (Ph. D.)--University of Chicago, Committee on Immunology, December 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D. in Clinical Sciences) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 140-156). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2016-08-12T16:15:37Z No. of bitstreams: 1 Dissertação_ICS_ Joanemile Pacheco de Figueiredo.pdf: 786075 bytes, checksum: e932bb435d8f92e49c6cdcf62892ceac (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2016-08-23T15:31:26Z (GMT) No. of bitstreams: 1 Dissertação_ICS_ Joanemile Pacheco de Figueiredo.pdf: 786075 bytes, checksum: e932bb435d8f92e49c6cdcf62892ceac (MD5) / Made available in DSpace on 2016-08-23T15:31:26Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Joanemile Pacheco de Figueiredo.pdf: 786075 bytes, checksum: e932bb435d8f92e49c6cdcf62892ceac (MD5) / CAPES; CNPq; FAPESB; National Institute of Health; / A prevalência de infecção por helmintos é negativamente associada à prevalência de alergia, e alguns trabalhos têm demonstrado uma inversa associação entre infecções por helmintos e gravidade de asma. Neste trabalho, foi avaliada a resposta imune específica para alérgeno 1 de Dermatophagoides pteronyssinus (Der p 1) em asmáticos infectados com helmintos de uma área endêmica no município do Conde-Bahia (Grupo 1), comparando-se com a resposta em asmáticos não infectados de uma área não endêmica, Salvador-Bahia (Grupo 2). Os indivíduos foram selecionados utilizando-se questionário ISAAC modificado e exames parasitológicos de fezes por meio da técnica de Kato-Katz. Foram realizados testes cutâneos de leitura imediata com aeroalérgenos, dosagem de IgE específica para Der p 1, coleta de amostras de poeira domiciliar para estudo da fauna acarina, além da avaliação da resposta imunológica pela produção de citocinas por células mononucleares do sangue periférico estimuladas, in vitro, com Der p 1. A dosagem de citocinas no sobrenadante das culturas foi realizada pelo método de ELISA, a expressão de IL-4 pela técnica de RT-PCR e a fonte produtora de IL-10 foi avaliada utilizando-se citometria de fluxo. Foi observada menor positividade aos testes cutâneos de leitura imediata com aeroalérgenos nos asmáticos infectados por helmintos, incluindo S. mansoni (grupo 1), quando comparados com asmáticos não infectados. Embora a positividade aos testes cutâneos tenha sido baixa no grupo de asmáticos infectados, em 40,6% dos indivíduos foi observada IgE específica para o Der p 1 acima de 0,70 KU/L (classe II). Com relação à produção de citocinas, observou-se que células de asmáticos infectados pelo S. mansoni produziram baixos níveis de IL-5 em culturas estimuladas com Der p 1, quando comparados à produção desta citocina por células de indivíduos não infectados (p<0,0001). Da mesma forma, a razão da densitometria de IL-4/HPRT no grupo de infectados foi mais baixa do que no grupo de não infectados (p<0,05). Em contraste, os níveis de IL-10 foram altos em cultura de células de asmáticos infectados por S. mansoni e baixos ou indetectáveis no grupo controle (p=0,0018). Observou-se uma associação positiva entre carga parasitária de S. mansoni e níveis de IL-10 em culturas estimuladas com Der p 1 (= 0,3212, p=0,012). A adição de IL-10 recombinante humana (rhIL-10) às culturas de asmáticos não infectados resultou em diminuição na produção de IL-5 (p<0,05). Os indivíduos do grupo 1 foram tratados com anti-helmínticos e houve redução da produção de IL-10 específica para Der p 1 (p<0,05), associada à piora dos sintomas de asma após o tratamento. Por fim, os dados mostraram que, em asmáticos infectados com S. mansoni, a IL-10 foi produzida principalmente por células T CD8+ e monócitos e, no grupo de não infectados, esta citocina foi produzida basicamente por monócitos. Este estudo sugere que a IL-10 é uma citocina supressora da resposta imune inflamatória do tipo Th2 em asmáticos infectados por helmintos, incluindo S. mansoni e que as células T CD8+ constituem uma importante fonte produtora desta citocina nos indivíduos infectados.