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.

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-1β 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-1β 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.

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-1β 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-1β 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.

Une petite population de lymphocytes T exprimant les deux corécepteurs CD4 et CD8 et appelée double positive (DP), a été détectée dans le sang périphérique de donneurs sains et de patients atteints de diverses pathologies dont la sclérose en plaques (SEP). Nous avons émis l’hypothèse qu’il s’agissait de lymphocytes T hautement activés pouvant contribuer à l’inflammation chronique présente dans la SEP. Nous avons comparé les cellules T DP obtenues du sang de donneurs sains et de patients atteints de la SEP et non traités. La fréquence des cellules DP était similaire chez les patients et les donneurs sains. La proportion de lymphocytes T DP qui exprimaient les chaines du récepteur de l’interleukine-15 (IL-15) était plus élevée que pour les autres populations lymphocytaires. Des mesures d’induction de la phosphorylation du STAT5 (signal transducer and activator of transcription) ont démontré que les cellules DP ont répondu à des doses plus faibles et pour de plus longues périodes à l’IL-15 comparativement aux autres lymphocytes T. Le pourcentage de lymphocytes T DP ayant la capacité de produire l’interféron-gamma et des enzymes lytiques était élevé chez les témoins sains mais ces niveaux étaient significativement réduits chez les patients atteints de la SEP. La caractérisation phénotypique de cellules DP a suggéré que ces cellules ont des propriétés similaires aux lymphocytes T activés. Bien qu’il ne s’agisse que d’une caractérisation partielle, il semble que les lymphocytes T DP perdent une partie de leurs propriétés chez les patients atteints de la SEP. / A small population of T lymphocytes expressing both CD4 and CD8 called double positive (DP) T lymphocytes has been detected in the peripheral blood of healthy donors and patients affected by different pathologies such as multiple sclerosis (MS). We hypothesize that these cells represent a highly activated T lymphocyte subset that could contribute to the characteristic inflammation found in MS. Thus, we compared DP T cells from healthy donors to those from untreated MS patients. We found similar frequencies of DP T lymphocytes between both groups. A higher percentage of DP T lymphocytes expressed the IL-15R (interleukin 15 receptor) than other T cell populations. Moreover, IL-15 triggered the phosphorylation of STAT5 in a greater proportion of CD4+CD8+ T lymphocytes compared to other T cells. A greater percentage of CD4+CD8+ T lymphocytes can produce interferon gamma and lytic enzymes compared with other T cell subsets. However, those levels were drastically lower in MS patients. The phenotypic characterization of the DP cells suggests they have similar properties as activated T cells. Even though the characterization process is still in its infancy, it appears that the DP T cells may lose some of their properties in MS patients.

IL-1 is a pro-inflammatory cytokine which play an important role in the activation and regulation of host defence and immune responses to inflammation or injury. IL-1 is able to bind and activate IL1-RI and IL1-RII, which are found on many cells types. The role of the IL-1 signalling in the deployment of Th cell subsets, especially Th17 cells is well known. However, the specific cells which are responsible for the expression of IL-1 signalling in the immune defense and in the development of the Th cell lineage in response to infection, is still largely unclear. Therefore in this thesis, IL1-RI conditional knockout mice specifically in hematopoietic cells (IL1-RI vaviCre+) were generated. Using IL1-RI vaviCre+ mice in comparison with IL1-RI global knockout mice (IL1-RI-/-) would determine whether the expression IL-1 signalling from hematopoietic cells is responsible for the immune defense and in the development of the Th1, Th2 and Th17 cells against gastrointestinal helminth Trichuris muris (T.muris) infections. The generation of IL1-RI vaviCre+ mice have been investigated at the genomic and proteomic level in order to confirm that the Il1-rI gene is inactivated in hematopoietic cells. The characterisation of IL1-RI vaviCre + mice at the genomic level confirmed that the Il1-rI gene was obliterated successfully. At protein level the characterisation of IL1- RI vaviCre + mice confirmed that IL1-RI was dysfunctional in hematopoietic cells. Additionally, the development of the immune cells was investigated in IL1-RI vaviCre + and IL1-RI-/- mice. Our findings demonstrated that the lymphocyte development was not affected by the deletion of the IL1- RI gene. This data indicated that IL1- RI vaviCre + and IL1-RI-/- mice are vital in vivo models. In high dose infection, both IL1-RI vaviCre + and IL1-RI -/- mice were able to clear the infections due to their ability to generate a Th2 response. Both IL1-RI vaviCre + and IL1-RI -/- mice infected with low dose of T.muris were susceptible to infections and showed high levels of Th1 cytokines. Thus, we hypothesised that IL1-RI signalling in hematopoietic cells was not required for worm expulsion and the generation of Th2 and Th1 response. Interestingly, low dose T.muris infection showed a clear reduction in the Th17 cytokines IL22 and IL17 in both IL1-RI vaviCre + and IL1-RI -/- mice, suggesting that IL-1 signalling expressed from hematopoietic cells is responsible for the development of Th17 cells and secretion of IL17 and IL22. IL1- RI vaviCre + and IL1-RI -/- mice infected with low dose of T.muris also showed an increase in inflammation in the colon and decreased of goblet cell hyperplasia. It is well known that IL22 plays an important role in preventing tissue damage and repair. Thus, in this study IL22 global knockout mice (IL22 -/-) were used to determine if the change in crypt lengths and goblet cell hyperplasia in IL1-RI vaviCre + and IL1-RI -/- was due to an absence of IL22. Our finding showed that IL22 -/- mice infected with low dose of T.muris had increased crypt length and a reduction in goblet cells. The similar phenotype in crypt length and goblet cell hyperplasia between IL22 -/-, IL1-RI vaviCre + and IL1-RI -/- mice suggested that a lack of IL22 in IL1-RI vaviCre + and IL1-RI -/- mice is responsible for the change in mice phenotype. It also provides more evidence for the role of IL-1 signaling in hematopoietic cells in the generation of Th17 cells and in the production of its cytokine IL22.IL1-RII is an inhibitor of IL1-RI, thus, in this study IL1-RII global knockout mice (IL1-RII -/-) mice was used in comparison with IL1-RI -/- mice to verify the role of IL-1 signaling in the development of Th17 cells. Our finding showed an overexpression of IL17 and IL22 in IL1-RII -/- compared with IL1-RI -/- mice and a higher level of IL17 in IL1-RII -/- mice compared with IL1-RII flox/flox mice. This data confirmed that IL-1 signaling is important for the development of Th17 cells and the production of its cytokine IL17 and IL22.