Regulatory T cells (Tregs), expressing the transcription factor Foxp3, form a key component of peripheral immune tolerance, guarding against auto-aggressive immune responses. Multiple Sclerosis is an inflammatory and demyelinating disease of the central nervous system (CNS) which is largely believed to be mediated by immune components reacting to the self myelin antigens that insulate the nerve fibres. Recent investigations have reported that regulatory T cells are dysfunctional in MS patients; therefore enhancing the regulatory T cell responses in MS is an attractive therapeutic target. Using the mouse model of MS, experimental autoimmune encephalomyelitis (EAE) we have attempted to develop disease-relevant Treg-based therapies to prevent disease induction. This required an understanding of the antigenic-reactivity of Tregs during disease. Results described in this thesis show that a proportion of Tregs in the draining lymph nodes and CNS were reactive to the disease initiating antigen(s) and could suppress in vitro responses of naïve T cells bearing transgenic T cell receptors, recognising the same antigen. Adoptive transfer of antigen-reactive Tregs suppressed disease induced with the same antigen, but also reduced disease induced with a distinct myelin antigen. Peptide-based tolerance using a high affinity MHC binding peptide analogue expanded and maintained antigen-reactive T cells which were tolerant to antigenic restimulation, although these cells did not express Foxp3. Peptide-treated mice showed reduced incidence of disease relapses during EAE induced against a distinct myelin antigen. Thus, while EAE and MS will involve a polyclonal effector T cell response to many antigens, therapeutic targeting of Tregs reactive against one CNS component may be sufficient to reduce disease. Endogenous expression of myelin autoantigen did not grossly alter the response of antigenreactive Tregs in the periphery. However, expression of endogenously derived viral superantigen enhanced the proportion of superantigen-reactive Foxp3+ Tregs in the periphery. This observation was extended using exogenous superantigen, suggesting that prolonged exposure to low dose (super)antigen tips the balance of the immune system in favour of regulation. This has implications for the ability to successfully fight infection, as well as for the limitation of autoaggressive responses and may contribute to the understanding of the hygiene hypothesis.

A esclerose múltipla é uma doença inflamatória crônica desencadeada por células T autorreativas contra antígenos proteicos da mielina. A encefalomielite experimental autoimune é o modelo murino mais utilizado para o estudo da EM. As tubas uterinas e o útero de camundongos fêmeas são órgãos ricos em células mesenquimais que são pouco utilizadas em estudos. Dessa forma, no presente projeto, caracterizamos a obtenção dessa população e avaliamos sua capacidade imunossupressora utilizando o modelo de EAE. Observamos que o tratamento é capaz de modular o perfil de linfócitos T CD4+ durante sua ativação nos linfonodos, induzindo o direcionamento para a subpopulação Tr1 e atenuando as Th1 e Th17. Assim, houve uma diminuição do número de células infiltrantes no SNC associado a uma menor ativação de células da microglia. Em conjunto, demostraramos que as meMSC utilizadas como tratamento são capazes de atrasar o desenvolvimento da EAE e, portanto, evidenciando o caráter imunomodulador das MSCs derivadas do endométrio, chamando a atenção para seu potencial terapêutico. / Multiple sclerosis is a chronic inflammatory disease triggered by autoreactive T cells against myelin protein. Experimental Autoimmune Encephalomyelitis is the most commonly used murine model for the study of MS. The uterine tubes and uterus of female mice are organs rich in mesenchymal cells which are rarely used. Thus, in the present work, we characterized the extraction of this population and evaluated its immunosuppressive capacity using the EAE model. We observed that the meMSC treatment is capable of modulating the CD4 T lymphocyte profile during its activation in the lymph nodes, inducing the expansion of the Tr1 subpopulation and attenuating Th1 and Th17. Consequently, there was a decrease in the number of infiltrating cells in the CNS associated with a reduction of microglial activation. Taken together, our results demonstrated that the meMSCs used as treatment are capable of delaying the development of EAE, therefore, evidencing its immunomodulatory features drawing attention to its therapeutic potential.

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The anti-inflammatory and restorative effects of the flavonoid-enriched fraction AF4 were examined in a mouse model of experimental autoimmune encephalomyelitis (EAE). Relative to EAE mice that received vehicle (water, 10 ml/kg/day), oral administration of AF4 (25 mg/kg/day) beginning 24 hours after the onset of clinical signs reduced disease progression that was accompanied by diminished pro-inflammatory cytokine gene expression (cerebellum and spinal cord) and protein concentrations in the plasma. LPS-induced release of TNF-? from the whole blood of EAE mice that received AF4 was reduced at peak disease severity (day 18) but not once central inflammation had declined (day 31) indicative of unique immune modulator properties. Lastly, the expression of myelin-associated genes (PGC-1?, SCD1, and MBP) suggestive of remyelination was enhanced in the spinal cord of EAE mice that received AF4. These findings suggest that AF4 reduces EAE severity by selectively inhibiting autoimmunity and enhancing the expression of genes necessary for remyelination.

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The project described in this thesis began with a broad analysis of the changes to amino acid and biogenic amine concentrations in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of Multiple Sclerosis (MS). That study identified deficits in specific neurotransmitters during EAE that I targeted pharmacologically using the antidepressant drug phenelzine. Phenelzine administration substantially influenced the concentrations of amino acids and biogenic amines in EAE mice in a manner likely to be therapeutic. In the final experiment, I treated EAE mice chronically with phenelzine; This treatment was associated with significant improvements in motor abilities compared to vehicle treated animals. In an open field, improvements were also observed in behavioural indices of depression, physical sickness and anxiety. The results of this thesis may offer new insights into the pathogenesis of EAE and MS and indicate the disease-modifying potential of phenelzine treatment in MS.

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Made available in DSpace on 2014-06-11T19:24:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-06Bitstream added on 2014-06-13T19:51:42Z : No. of bitstreams: 1 pezavento_sfgz_me_botfm.pdf: 924376 bytes, checksum: e516f8ff865bbc2e7b75d6fc6e8d7450 (MD5) / A única vacina disponível contra a tuberculose (TB) é o bacilo de Calmette- Guérin (BCG), que é constituída por uma cepa de Mycobacterium bovis vivo atenuado, mas que possui variabilidade de proteção de 0 a 80%. Portanto é necessário o desenvolvimento de novas vacinas para o controle dessa infecção. Dentre as novas formulações profiláticas em teste para TB destacam-se as vacinas gênicas, sendo a DNAhsp65 a vacina investigada por nosso grupo. A DNAhsp65 é uma construção que contém o gene que codifica a proteína de choque térmico de 65KDa do M. leprae. Em virtude da elevada homologia entre a hsp65 micobacteriana e a hsp60 dos mamíferos, pode ocorrer mimetismo antigênico, ou seja, reatividade cruzada entre anticorpos e células T específicas para hsp65 micobacteriana e hsp60 humana. Nesse contexto, nosso grupo tem estudado o possível efeito colateral da DNAhsp65 no desenvolvimento de doenças autoimunes. Neste projeto foi investigado o efeito (proteção, exacerbação ou inocuidade) de diferentes formulações vacinais para TB na encefalite autoimune experimental (EAE). Os objetivos foram caracterizar a resposta imune específica induzida pela DNAhsp65 em ratos Lewis e avaliar o efeito imunomodulador tanto da estratégia clássica de imunização (3 doses de DNAhsp65 por via im) quanto da estratégia prime-boost BCG / DNAhsp65, nas características clínicas, imunológicas e histológicas da EAE. Quanto à imunogenicidade da DNAhsp65, foram testadas duas doses da vacina administradas via intramuscular (100 e 300 μg), sendo que somente a maior dose induziu IgG2b específica para hsp65 e produção de IFN- em cultura de células esplênicas estimuladas in vitro com rhsp65. Para avaliar o efeito da imunização com DNAhsp65 ou do prime-boost BCG /DNAhsp65 no desenvolvimento da EAE, os animais foram previamente imunizados com 3 doses de 300 μg de DNAhsp65 ou... / BCG is the only accepted vaccine against tuberculosis (TB) but its protective ability is very limited. Many new vaccines are therefore being evaluated. Our group has been working with DNAhsp65 that is a genetic construction containing the gene of hsp65 from Mycobacterium leprae. In previous experimental work we demonstrated that both, DNAhsp65 alone or associated with BCG, in a prime-boost regimen, were effective to control TB. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 alone or associated with BCG in a rat model of multiple sclerosis. Female Lewis rats were immunized with 3 doses of DNAhsp65 or primed with BCG followed by 2 DNAhsp65 boosters. The animals were then immunized with myelin associated with Complete Freund Adjuvant to develop experimental autoimmune encephalomyelitis (EAE). The following parameters were evaluated: weight loss, clinical score, inflammation at the central nervous system (CNS) and immune response against myelin. No deleterious effect was associated with these immunizations schedules. Immunized animals equally lost weight, the clinical scores were similar and inflammation at the CNS did not increase. Interestingly, both procedures determined decreased inflammation in the brain and lumbar spinal cord. This was concurrent with a modulatory effect over cytokine production by peripheral lymphoid organs. Cell cultures from spleen and lymph nodes in vitro stimulated with myelin produced less IFN- and IL-10, respectively. This phenomenon was more clear in rats immunized with the genetic vaccine alone than with the prime-boost strategy. Together the results suggest that these strategies for TB prophylaxis would not accelerate or aggravate MS, being therefore... (Complete abstract click electronic access below)

A esclerose múltipla é uma doença inflamatória crônica desencadeada por células T autorreativas contra antígenos proteicos da mielina. A encefalomielite experimental autoimune é o modelo murino mais utilizado para o estudo da EM. As tubas uterinas e o útero de camundongos fêmeas são órgãos ricos em células mesenquimais que são pouco utilizadas em estudos. Dessa forma, no presente projeto, caracterizamos a obtenção dessa população e avaliamos sua capacidade imunossupressora utilizando o modelo de EAE. Observamos que o tratamento é capaz de modular o perfil de linfócitos T CD4+ durante sua ativação nos linfonodos, induzindo o direcionamento para a subpopulação Tr1 e atenuando as Th1 e Th17. Assim, houve uma diminuição do número de células infiltrantes no SNC associado a uma menor ativação de células da microglia. Em conjunto, demostraramos que as meMSC utilizadas como tratamento são capazes de atrasar o desenvolvimento da EAE e, portanto, evidenciando o caráter imunomodulador das MSCs derivadas do endométrio, chamando a atenção para seu potencial terapêutico. / Multiple sclerosis is a chronic inflammatory disease triggered by autoreactive T cells against myelin protein. Experimental Autoimmune Encephalomyelitis is the most commonly used murine model for the study of MS. The uterine tubes and uterus of female mice are organs rich in mesenchymal cells which are rarely used. Thus, in the present work, we characterized the extraction of this population and evaluated its immunosuppressive capacity using the EAE model. We observed that the meMSC treatment is capable of modulating the CD4 T lymphocyte profile during its activation in the lymph nodes, inducing the expansion of the Tr1 subpopulation and attenuating Th1 and Th17. Consequently, there was a decrease in the number of infiltrating cells in the CNS associated with a reduction of microglial activation. Taken together, our results demonstrated that the meMSCs used as treatment are capable of delaying the development of EAE, therefore, evidencing its immunomodulatory features drawing attention to its therapeutic potential.

Experimental autoimmune encephalomyelitis (EAE) is the rodent model of multiple sclerosis (MS), a chronic autoimmune neuroinflammatory disease that has a devastating impact on various neurological functions of the patients. The hallmarks of both, MS and EAE, are neuroinflammation, demyelination and neuroaxonal degeneration. Various types of lymphoid and myeloid cells were shown to infiltrate the central nervous system and to participate in disease pathology. However, the role of eosinophil granulocytes has been less explored thus far. An early study showed that eosinophils infiltrate into the spinal cord of EAE mice and suggested their role in the disease progression. Recently, it was reported that eosinophils can play a protective role against EAE when mice are treated with an extract from helminths. Furthermore, it was shown that EAE development is not altered in mice deficient for interleukin-5, an important eosinophil pro-survival factor. Taken together, the role of eosinophils in EAE is currently unclear and needs to be investigated in detail. In the present study, we use the active model of EAE, whereby we immunized the C57BL/6 mouse strain with MOG35-55 peptide emulsified in the complete Freund’s adjuvant, in order to study a possible contribution of eosinophils to the disease pathology. Using the flow cytometry and RT-qPCR analysis of the spinal cord, we show that eosinophils infiltrate into the tissue in the course of EAE. The infiltration is likely driven by eosinophil chemoattractants, such as eotaxin-1, as the concentration of the latter was increased in the spinal cord during EAE, as shown on mRNA and protein level. Moreover, detailed flow cytometry analysis of spinal cord eosinophils revealed that they show signs of activation, namely an increase in CD11b and decrease in CCR-3 surface expression. Furthermore, we observed signs of degranulation of spinal cord eosinophils in EAE which was measured as a decrease of the side scatter parameter and an upregulation of CD63 surface expression. These data suggest a potential role of eosinophils in the pathology of EAE. In order to elucidate whether eosinophils are important for the disease development, eosinophil-deficient mice were subjected to EAE and the clinical development of the disease was observed. For this purpose, we used two independent models of eosinophil deficiency - ΔdblGATA1 and interleukin-5-depleted mice. ΔdblGATA1 mice are a genetically manipulated mouse strain bearing a deletion in GATA1 promoter that causes a specific depletion of eosinophils. Interestingly, clinical development of EAE was not affected in these mice when compared to their wild-type controls. As a next step, we depleted eosinophils by injecting wild-type mice with an antibody against the eosinophil pro-survival factor interleukin-5 in order to reduce eosinophil numbers in the effector phase of EAE. In accordance with the result from the experiment with ΔdblGATA1 mice, EAE progression was not altered in the eosinophil-depleted mice when compared to mice that were injected with an isotype control antibody. Further, we analyzed the neuroinflammation and demyelination in the spinal cord of 4ΔdblGATA1 mice subjected to EAE. Specifically, the infiltration of inflammatory cell populations, including CD4 and CD8 T cells, neutrophils and macrophages, was assessed by flow cytometry. In agreement with the unchanged clinical EAE development, inflammatory cell infiltration was not affected in ΔdblGATA1 mice. Furthermore, we analyzed expression of pro-inflammatory cytokines in the spinal cord of ΔdblGATA1 mice subjected to EAE in order to better characterize the inflammatory status. No significant changes were detected further confirming that eosinophils do not contribute to neuroinflammation in EAE. Finally, we assessed the demyelination in the spinal cord of ΔdblGATA1 EAE mice using luxol fast blue staining to detect myelin. In accordance with the unaffected clinical development and inflammatory status, we did not observe any difference in the spinal cord demyelination in ΔdblGATA1 mice when compared to their wild-type littermates. Taken together, although eosinophils infiltrate into the spinal cord of EAE mice and are activated and degranulate therein, they are dispensable for EAE development.