Impacto do envelhecimento do sistema imunitário nas diferentes formas de progressão de esclerose múltipla: estudo da função tímica

Dissertação de mestrado em Ciências da SaúdeMultiple sclerosis (MS) is an autoimmune disease from the central nervous system (CNS) characterized by progressive demyelination of neurons. It is a very heterogeneous disease with three main forms of progression: the relapsing-remitting MS, the primary progressive MS and the secondary progressive MS. The lesions in the CNS constitute the hallmark of the disease and are associated with an autoimmune response. In MS, the immune response has been attributed to myelin-autoreactive T lymphocytes. T lymphocytes are produced in the thymus, an immune system primary organ that is very active during the first year of life and involutes progressively with age. Thymic involution contributes to the aging process of the immune system, refered as immunosenescence. Existing data suggest that individuals with RRMS have decreased thymic function when compared to age-matched healthy individuals. The main objective of this study is to investigate how thymic function and immune system aging correlate with the different forms of progression of MS. We hypothesize that the thymic function and the immune system aging may have an essential role for the pathophysiology and progression of the disease. To address this hypothesis, blood samples from MS patients with different forms of disease progression and from healthy individuals were analyzed. Thymic function and immune system aging were then evaluated through quantification of recent thymic emigrants (RTEs) and quantification of the naïve to memory T lymphocytes’ ratio, respectively. Our results suggest that the percentage of RTEs and the naïve to memory T lymphocytes’ ratio correlate to individual’s age similarly on healthy vs. MS treatment-naïve individuals. Interestingly, MS individuals on treatment seem to present an increased accumulation of memory subsets in the periphery and decreased thymic function. Altogether, our results suggest that alterations on thymic function and T lymphocytes subsets reported in previous studies are characteristic of MS patients on treatment but not of naïve-treatment individuals. To support our conclusion it is essential, however, to evaluate other surrogates of thymic function (e.g. T cell receptor excision circles [TRECs]) and of immunosenescence (e.g. relative length of telomere repeats) and extend this study to an increased number of MS patients. So far, we could optimize the flow-FISH technique to quantify the length of telomere repeats on T lymphocytes, introducing several modifications to the protocol previously described by others. Furthermore, although not yet optimized we could verify some progresses on the optimization of the Nested-PCR to TRECs quantification.A esclerose múltipla (EM) é uma doença autoimune do sistema nervoso central (SNC) caracterizada pela desmielinização progressiva dos neurónios. Trata-se de uma doença muito heterogénea com três formas principais de progressão: a forma de surto-remissão, a forma primária progressiva e a forma secundária progressiva. Com o tempo, os efeitos cumulativos da doença juntamente com a falta de tratamentos eficazes levam a défices motores, cognitivos e emocionais. A doença carateriza-se pela ocorrência de lesões no SNC resultantes de uma resposta auto-imune. Na EM, a inflamação tem sido atribuída à existência de linfócitos T auto-reactivos para a mielina. Os linfócitos T são produzidos pelo timo, um órgão primário do sistema imunitário muito ativo nos primeiros anos de vida e que involui de forma progressiva com a idade. A involução do timo contribui para o processo de envelhecimento do sistema imunitário, referido como imunosenescência. Os dados existentes sugerem que indivíduos com forma de surto-remissão possuem menor função tímica quando comparados com indivíduos saudáveis com a mesma idade. O principal objetivo deste estudo é investigar de que forma a função tímica e o envelhecimento do sistema imunitário se correlacionam com a manifestação das diferentes formas de progressão da doença. Colocamos a hipótese de que a função tímica e o envelhecimento do sistema imunitário poderão ter um papel essencial para a patofisiologia e progressão da EM. Para investigar esta hipótese foram analisadas amostras de sangue de indivíduos saudáveis ou doentes com diferentes formas de progressão de EM. A função tímica assim como o envelhecimento do sistema imunitário foram posteriormente avaliados através da quantificação de células recentemente exportadas do timo e da quantificação do rácio de linfócitos T naive sobre linfócitos T de memória, respetivamente. Os nossos resultados sugerem que a percentagem de células recentemente exportadas do tímo e o rácio naive/memória dos linfócitos T se correlacionam com a idade dos indivíduos de forma semelhante em indivíduos saudáveis e com EM sem tratamento. Curiosamente, indivíduos com EM e em tratamento parecem apresentar uma maior acumulação das subpopulações de memória na periferia e diminuição da função tímica. Sucintamente, os nossos resultados sugerem que as alterações na função tímica e nas subpopulações de linfócitos T reportadas em estudos anteriores são características de doentes com EM em tratamento mas não de individuos sem tratamento. Para sustentar os nossos resultados é essencial, no entanto, avaliar outros parâmetros para a função tímica (por exemplo os círculos de excisão do recetor das células T [CERT]) e para a imunosenescência (por exemplo o tamanho relativo dos telómeros) e alargar este estudo a um maior número de doentes com EM. Até ao momento fomos capazes de otimizar o flow-FISH para a quantificação do tamanho das repetições teloméricas em linfócitos T, introduzindo várias modificações ao protocolo previamente descrito por outros. Não obstante, apesar de ainda não otimizada fomos capazes de fazer progressos na otimização do Nested-PCR para a quantificação de CERTs.The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, ICVS/3B’s – PT Government Associate Laboratory, Braga/Guimarães, Portugal. This work was developed under the scope of the project NORTE-01-0246- FEDER-000012, supported by the Northern Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER, through the Competitiveness Factors Operational Program (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI 01-0145-FEDER-00703

Diferenciação das células T como um componente fundamental da patogénese da esclerose múltipla

Tese de doutoramento em Envelhecimento e Doenças CrónicasFalha na manutenção da tolerância, particularmente das células T, é descrita como subjacente à patogénese da esclerose múltipla (EM). As células T são produzidas no timo e estudos anteriores mostraram que pessoas com EM remitente-recorrente (pcEMRR) têm uma função tímica reduzida, um sistema imunitário envelhecido e células reguladoras disfuncionais. No entanto, pouco se sabe sobre as alterações imunitárias presentes no início da doença, o que é crucial para entender a etiologia da EM e encontrar novos alvos de diagnóstico e/ou terapêuticos. As terapias modificadoras da doença (TMD) atuam nas células T e têm sido eficazes na prevenção da progressão da EMRR, mas são ineficazes em pessoas com EM progressiva primária (pcEMPP), uma forma menos comum de EM. Alterações observadas nas células imunitárias de pcEMPP sugerem o envolvimento destas células nessa forma da doença. Neste projeto, pretendemos fornecer uma avaliação integrada da função tímica, da homeostasia das células T periféricas e das células reguladoras em pcEMRR recém-diagnosticadas e sem tratamento prévio e em pcEMPP, em comparação com controlos saudáveis controlados para idade e sexo. Além disso, pretendemos fornecer uma visão integrada sobre o efeito de diferentes TMD em pcEMRR em comparação com pcEMRR sem tratamento prévio. Observamos um empobrecimento das células T de memória tanto em pcEMRR quanto em pcEMPP em comparação com controlos saudáveis. Além disso, pcEMRR têm uma percentagem maior de células T reguladoras naïve e pcEMPP um número menor de células T reguladoras ativadas HLA-DR+. PcEMRR têm uma percentagem maior de células NK imaturas que expressam recetores inibitórios, e tanto pcEMRR quanto pcEMPP têm uma percentagem maior de células NK maduras a expressar recetores ativadores. PcEMPP têm uma percentagem maior de células NKT que expressam recetores ativadores e inibitórios em comparação com controlos saudáveis. Em relação ao efeito das TDM nas células imunitárias periféricas de pcEMRR, dois grupos foram formados devido ao efeito das TDM em subpopulações de células T. Em contraste com pcEMRR tratadas com teriflunomida, natalizumab ou IFNb (cluster 2), pcEMRR tratadas com fumarato de dimetilo, alemtuzumab ou fingolimod (cluster 1) mostram uma diminuição do número da maioria das subpopulações de células T em comparação com pcEMRR sem tratamento. O efeito dos TDM no número de células NK variou amplamente entre as TDM. No geral, este estudo esclarece sobre as características imunitárias presentes nas diferentes formas de EM e contribui para a sua melhor compreensão. Também elucida sobre o efeito de diferentes TDM nas células do sistema imunitário periférico. Estudos adicionais sobre os efeitos dessas alterações imunitárias podem esclarecer o seu papel na fisiopatologia da EM.Failure to maintain tolerance, particularly of T cells, has been described to underlie the pathogenesis of multiple sclerosis (MS). T cells are produced in the thymus, and previous studies have shown that people with (pw) MS have reduced thymic function, an aged adaptive immune system and impaired regulatory cell activity. However, little is known about the immune changes that occur at the onset of the disease, which could be crucial to understanding the aetiology of MS and provide new diagnostic and/or therapeutic targets. Disease-modifying therapies (DMTs) that target T cells have been effective in delaying the progression of relapsing-remitting MS (RRMS), but are ineffective in people with primary progressive MS (pwPPMS), a less common form of MS. However, changes in immune system cells in pwPPMS have been observed, suggesting their involvement in this form of MS. In this project, we aimed to provide an integrated assessment of thymic function, peripheral T cells and regulatory cells homeostasis in treatment-naïve newly diagnosed pwRRMS and in pwPPMS, compared with age- and sex-matched healthy controls. In addition, we aimed to provide an integrated view regarding the effect of different DMTs in pwRRMS compared with treatment-naïve newly diagnosed pwRRMS. We observed an impoverishment of peripheral memory T cells in both pwRRMS and pwPPMS compared to healthy controls. In addition, pwRRMS have a higher percentage of naive regulatory T cells and pwPPMS have a lower number of activated HLA-DR regulatory T cells. PwRRMS have a higher percentage of immature NK cells expressing inhibitory receptors, and both pwRRMS and pwPPMS have a higher percentage of mature NK cells expressing activating receptors. PwPPMS have a higher percentage of NKT cells expressing activating and inhibitory receptors compared to healthy controls. Regarding the effect of different DMTs on peripheral immune cells of pwRRMS, two clusters were formed based mainly on the effect of the DMTs on T cell subsets. In contrast to pwRRMS treated with teriflunomide, natalizumab or IFNb (cluster 2), pwRRMS treated with dimethyl fumarate, alemtuzumab or fingolimod (cluster 1) show a decrease in the numbers of most of the T cell subsets evaluated compared to treatment-naïve pwRRMS. The effect of DMTs on NK cell numbers varied widely between DMTs. Overall, this study provides insights into the immune features that characterise different progression forms of MS and contributes to a better understanding of the pathogenesis of MS. It also elucidates the effect of different DMTs on the cells of the peripheral immune system. Further studies of the effects of these immune changes may clarify their role in the pathophysiology of MS.The work presented in this thesis was performed at the Life and Health Sciences Research Institute (ICVS), University of Minho. Financial support was provided by grants from the Foundation for Science and Technology (FCT, Portugal)/FEDER (PD/BD/137433/2018 and COVID/BD/152629/2022), by the project NORTE-01-0145-FEDER-000039, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020

People with Primary Progressive Multiple Sclerosis Have a Lower Number of Central Memory T Cells and HLA-DR+ Tregs

The importance of circulating immune cells to primary progressive multiple sclerosis (PPMS) pathophysiology is still controversial because most immunotherapies were shown to be ineffective in treating people with PPMS (pwPPMS). Yet, although controversial, data exist describing peripheral immune system alterations in pwPPMS. This study aims to investigate which alterations might be present in pwPPMS free of disease-modifying drugs (DMD) in comparison to age- and sex-matched healthy controls. A multicentric cross-sectional study was performed using 23 pwPPMS and 23 healthy controls. The phenotype of conventional CD4+ and CD8+ T cells, regulatory T cells (Tregs), B cells, natural killer (NK) T cells and NK cells was assessed. Lower numbers of central memory CD4+ and CD8+ T cells and activated HLA-DR+ Tregs were observed in pwPPMS. Regarding NK and NKT cells, pwPPMS presented higher percentages of CD56dimCD57+ NK cells expressing NKp46 and of NKT cells expressing KIR2DL2/3 and NKp30. Higher disease severity scores and an increasing time since diagnosis was correlated with lower numbers of inhibitory NK cells subsets. Our findings contribute to reinforcing the hypotheses that alterations in peripheral immune cells are present in pwPPMS and that changes in NK cell populations are the strongest correlate of disease severity

People with primary progressive multiple sclerosis have a lower number of central memory T cells and HLA-DR+ Tregs

The importance of circulating immune cells to primary progressive multiple sclerosis (PPMS) pathophysiology is still controversial because most immunotherapies were shown to be ineffective in treating people with PPMS (pwPPMS). Yet, although controversial, data exist describing peripheral immune system alterations in pwPPMS. This study aims to investigate which alterations might be present in pwPPMS free of disease-modifying drugs (DMD) in comparison to age- and sex-matched healthy controls. A multicentric cross-sectional study was performed using 23 pwPPMS and 23 healthy controls. The phenotype of conventional CD4+ and CD8+ T cells, regulatory T cells (Tregs), B cells, natural killer (NK) T cells and NK cells was assessed. Lower numbers of central memory CD4+ and CD8+ T cells and activated HLA-DR+ Tregs were observed in pwPPMS. Regarding NK and NKT cells, pwPPMS presented higher percentages of CD56dimCD57+ NK cells expressing NKp46 and of NKT cells expressing KIR2DL2/3 and NKp30. Higher disease severity scores and an increasing time since diagnosis was correlated with lower numbers of inhibitory NK cells subsets. Our findings contribute to reinforcing the hypotheses that alterations in peripheral immune cells are present in pwPPMS and that changes in NK cell populations are the strongest correlate of disease severity.This work has been funded by a research grant from the Academic Clinical Center of the Hospital of Braga; by national funds through the Foundation for Science and Technology (FCT) (UIDB/50026/2020 and UIDP/50026/2020); and by the project NORTE-01-0145-FEDER-000039, supported by the Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). J.C.-G. has been supported by FCT PhD grants (PD/BD/137433/2018 and COVID/BD/152629/2022). C.S. has been supported by an FCT PhD grant (PD/BDE/142976/2018)

Thymic Function as a Predictor of Immune Recovery in Chronically HIV-Infected Patients Initiating Antiretroviral Therapy

International audiencePoor immunological responders (PIR) are HIV-infected patients with virologic suppression upon antiretroviral therapy (ART) but persistently low CD4+ T cell counts. Early identification of PIR is important given their higher morbimortality compared to adequate immune responders (AIR). In this study, 33 patients severely lymphopenic at ART onset, were followed for at least 36 months, and classified as PIR or AIR using cluster analysis grounded on their CD4+ T cell count trajectories. Based on a variety of immunological parameters, we built predictive models of PIR/AIR outcome using logistic regression. All PIR had CD4+ T cell counts consistently below 500 cells/μL, while all AIR reached this threshold. AIR showed a higher percentage of recent thymic emigrants among CD4+ T cells; higher numbers of sj-TRECs and greater sj/β TREC ratios; and significant increases in thymic volume from baseline to 12 months of ART. We identified mathematical models that correctly predicted PIR/AIR outcome after 36 months of therapy in 77–87% of the cases, based on observations made until 2–6 months after ART onset. This study highlights the importance of thymic activity in the immune recovery of severely lymphopenic patients, and may help to select the patients that will benefit from closer follow-up or novel therapeutic approaches

Vaccination With Recombinant Filamentous fd Phages Against Parasite Infection Requires TLR9 Expression

Recombinant filamentous fd bacteriophages (rfd) expressing antigenic peptides were shown to induce cell-mediated immune responses in the absence of added adjuvant, being a promising delivery system for vaccination. Here, we tested the capacity of rfd phages to protect against infection with the human protozoan Trypanosoma cruzi, the etiologic agent of Chagas Disease. For this, C57BL/6 (B6) and Tlr9−/− mice were vaccinated with rfd phages expressing the OVA257–264 peptide or the T. cruzi-immunodominant peptides PA8 and TSKB20 and challenged with either the T. cruzi Y-OVA or Y-strain, respectively. We found that vaccination with rfd phages induces anti-PA8 and anti-TSKB20 IgG production, expansion of Ag-specific IFN-γ, TNF-α, and Granzyme B-producing CD8+ T cells, as well as in vivo Ag-specific cytotoxic responses. Moreover, the fd-TSKB20 vaccine was able to protect against mortality induced by a high-dose inoculum of the parasite. Although vaccination with rfd phages successfully reduced both parasitemia and parasite load in the myocardium of WT B6 mice, Tlr9−/− animals were not protected against infection. Thus, our data extend previous studies, demonstrating that rfd phages induce Ag-specific IgG and CD8+ T cell-mediated responses and confer protection against an important human parasite infection, through a TLR9-dependent mechanism

Padrões de saúde e segurança no trabalho e extrativismo: o caso de comunidades rurais da Amazônia brasileira

Este artigo é resultado de pesquisa de campo realizada junto aos coletores de sementes e frutos oleaginosos - Produtos Florestais Não Madeireiros (PFNMs) - em áreas rurais de Salvaterra e Bragança, no estado do Pará. O objetivo é contribuir com os estudos empíricos sobre saúde e segurança ocupacional no extrativismo, ainda incipientes no Brasil, por falta de amparo técnico-científico relacionado à atividade extrativista e por falta de regulamentação específica para a área. A pesquisa, realizada a partir da coleta de dados primários e observacionais, aponta os riscos de saúde e segurança aos quais os coletores estão expostos e os métodos de prevenção de acidentes, a fim de identificar possíveis melhorias nas condições de trabalho em um contexto onde a regulamentação de padrões trabalhistas para autônomos, informais e extrativistas na Amazônia é praticamente inexistente. A metodologia se caracteriza como estudo de caso em profundidade com coleta de dados primários com adoção de métodos mistos para sua sistematização. Os resultados demonstram que novas práticas e normas precisam ser adotadas para que os riscos à saúde e à segurança dos coletores sejam minimizados, além de garantir fiscalização, incentivo e monitoramento de práticas de segurança adequadas à atividade e específicas para cada região.This article is the result of a field research conducted among collectors of oleaginous seeds and fruits - Non-Timber Forest Products (NTFP) - in the rural areas of Salvaterra and Bragança, in the state of Pará, Brazil. It aimed to contribute to empirical studies on health and occupational safety in extractivism, still incipient in Brazil due to the lack of technical-scientific support and the lack of specific regulation on the area. The research was conducted by the collection of primary and observational data that point out the risks to health and safety to which collectors are exposed to and the methods to prevent accidents in order to identify possible improvements to working conditions in a context in which labor regulation standards for autonomous, informal and extractive workers in the Amazon is practically inexistent. This paper’s methodology is characterized as an embedded case study along with the collection of quantitative and qualitative data with the adoption of mixed methods for their systematization. The results show that new practices and norms should be adopted so the risks to health and safety of collectors are minimized, besides ensuring the inspection, incentives and monitoring of safety practices that are adequate to the activity and specific to each region