B cells have a central role in the pathogenesis of collagen-induced arthritis (CIA), an animal model of the autoimmune disease rheumatoid arthritis. In this report, a specific subset of an innate type of B cells, B-1 B cells, have been studied for the involvement in CIA. The B-1 B cells were shown to produce small amounts of collagen-specific antibodies upon stimulation in vitro, suggesting that they play a minor role in the development of CIA. This report also includes how marginal zone B cells, another innate type of B cells with natural collagen-reactivity, can be identified in the medullary sinuses of lymph nodes of collagen-immunized mice, implying involvement in auto antigen trapping.

Base teórica: Artrite reumatoide (AR) é uma doença autoimune que afeta as articulações e progride de maneira simétrica e erosiva. Além dos achados articulares, pode ocorrer de perda muscular e síndrome da caquexia. Atualmente, não existe um marcador que sirva de preditor da síndrome de caquexia reumatoide. Estudos metabolômicos em pacientes com AR demonstram uma complexidade em encontrar um biomarcador para caquexia. Ademais, não há modelo experimental de caquexia descrito na literatura, mas o modelo de artrite induzida por colágeno (CIA) possui potencial de ser modelo de caquexia reumatoide. A partir deste modelo, pode-se fazer a busca por biomarcadores de caquexia reumatoide via metabolômica. Objetivo: Avaliar o modelo de CIA como modelo experimental de caquexia reumatoide. Avaliar o perfil metabólico da urina no modelo de CIA e correlacionar com parâmetros clínicos de caquexia reumatoide em busca de possíveis biomarcadores. Métodos: Camundongos machos DBA/1J foram induzidos (CIA; n=13) no dia zero e receberam reforço 18 dias após, e grupo mantidos saudáveis sem indução (CO; n=11). Nos dias 0, 18, 25, 35, 45, 55 e 65 após a indução, foram realizados: coleta de urinas; teste de desempenho físico; teste de locomoção espontânea; teste de força; medida do volume do edema da pata traseira; avaliação do escore clínico; pesagem; e avaliação da ingestão alimentar. Após os 65 dias, os animais foram eutanasiados e tecidos musculares (gastrocnêmio – GA; e tibial anterior – TA) foram dissecados para pesagem e realização da razão sarcoplasmática. Os dados foram analisados por ANOVA de duas vias, seguido de Bonferroni, ou teste t de Pearson, com significância a partir de um p<0,05. A urina coletada foi submetida à ressonância nuclear magnética (1D e 2D J-res). Os metabolitos foram identificados via Chenomx (1D) e pelo Birmingham Metabolite Library (BML; 2D J-res). Utilizou-se a o modelo estatístico de PCA, PLSDA e PLSR para criar ranqueamento de metabolitos (significância a partir de um p<0,05). Analizou-se as rotas metabólicas via Metaboanalyst a partir do ranqueamento de metabólitos obtidos. Os metabólitos obtidos foram filtrados para rotas metabólicas que ocorrem no músculo para identificação de potenciais biomarcadores de perda muscular. Resultados: O grupo CIA apresentou redução de até 24% na locomoção espontânea, de até 66% na força e de até 24% no teste de desempenho físico após 35 dias da indução, bem como redução no peso do GA (24%) e TA (25%), e relação sarcoplasmática (22 e 23%, respectivamente) em relação ao grupo CO. Os modelos estatísticos de PCA, PLSDA e PLSR, e o filtro pelas rotas metabólicas relacionadas com o músculo geraram uma lista de 28 metabólitos e relacionados com o desenvolvimento da doença, sendo eles: 3-metilhistidina, 4-aminobutirato, acetilcolina, arginina, aspartato, carnosina, creatina, creatinina, glutamina, histamina, histidina, isoleucina, leucina, metionina, lisina, mio-inositol, dimetilglicina, acetilalanina, acetilmetionina, pantotenato, fenilalanina, fosfocolina, fosfocreatina, piridoxina, sarcosina, succinilacetona, tiamina, e urocanato. Conclusão: Em concordância com os resultados de redução nos parâmetros de: massa muscular, locomoção espontânea, força e desempenho físico, somando-se a ausência de anorexia bem como mudança no peso, o modelo animal de CIA representa um modelo experimental próprio para caquexia reumatoide. A análise do perfil metabólico deste modelo permite sugerir 28 metabólitos relacionados ao processo de perda muscular, que podem vir a ser biomarcadores de caquexia reumatoide, objetivando prognóstico, diagnóstico e acompanhamento da síndrome. Destes metabólitos, os principais são pertencentes ao metabolismo de: histidina; arginina e prolina; glicina, serina e treonina; fosfocreatina, bem como outros aminoácidos e vitaminas do complexo B. / Background: Rheumatoid Arthritis (RA) is an autoimmune disease that affects the joints and has a symmetric development and it is erosive. Besides joint damage, it can develop muscle loss progress into cachexia syndrome. Currently, there is no marker that can predict it development in rheumatoid patients. Metabolomics in RA have shown to be complex to find out a biomarker for this syndrome. Also, there is no experimental model of cachexia described in literature yet; however the collageninduced arthritis (CIA) animal model seems to be a feasible model for rheumatoid cachexia. With this model, the research for a biomarker of rheumatoid cachexia can be done by metabolomics. Objectives: It will be evaluated if the CIA animal model can be also an animal model of rheumatoid cachexia. Afterwards, it will be evaluated a metabolic profile from urine of this animal model and correlate with clinical signs of rheumatoid cachexia to find out plausible biomarkers of it. Methods: Male DBA/1J mice were submitted to CIA (n=13), immunization occurred at day zero and a booster was performed 18 days after, and a healthy group with no induction (CO; n=11). At the 0,18, 25, 35, 45, 55 and 65 days after the first injection, it was done: urine collection; physical performance test; free exploratory locomotion test; strength test; hindpaw edema volume measurement; follow up disease development; weighted; and food intake. After the 65 days, animals were euthanized and muscle (gastrocnemious – GA; and tibial anterior – TA) were dissected, and weighted for sarcoplasmic ratio. Data were analyzed by two-way ANOVA followed by Bonferroni post hoc, and t-test of Pearson, and statistical critical limit was set for p<0.05. The collected urine was used for nuclear magnetic resonance (1D and 2D J-res). Metabolites were identified by Chenomx (1D) and by the Birmingham Metabolite Library (BML; 2D J-res). Statistical model were performed using PCA, PLSDA and PLSR to create a ranking list of the metabolites (statistical critical limit was set for p<0.05). It was analyzed the metabolic pathway by Metaboanalyst from the data of metabolite ranking list. Then, the metabolite list was filtered by the metabolic pathways that take place in muscle tissue, in order to identify plausible biomarkers of muscle loss. Results: CIA group has shown reduction in up to 24% of free locomotion fatigue, up to 66% of strength and up to 24% of endurance physical performance after 35 days of the induction, as well as a decrease in GA (24%) and TA (25%) weight, and sarcoplasmic ratio also reduced (22 and 23%, respectivamente) related to CO group. The PCA, PLSDA and PLSR statistical models, and the filter by metabolic pathways related to muscle provided a list of 28 metabolites related to disease development, as can be listed: 3-methylhistidine, 4-aminobutyrate, acetylcholine, arginine, aspartate, carnosine, creatine, creatinine, glutamine, histamine, histidine, isoleucine, leucine, methionine, lysine, myo-inositol, dimethylglycine, acetylalanine, acetylmethionine, pantothenate, phenylalanine, phosphocholine, phosphocreatine, pyridoxine, sarcosine, succinylacetone, thiamine, and urocanate. Conclusions: Accordingly with the data with reduction of: muscle mass, spontaneous locomotion, strength and physical performance, added with absence of anorexia as well as weight change, CIA animal model is a feasible experimental model for rheumatoid cachexia. Concerning the metabolic profile from this model, it can be suggested 28 metabolites related to muscle loss in which can be tested for biomarker of rheumatoid cachexia, targeting prognosis, diagnosis, and syndrome follow up. From those metabolites, the main ones are engaged to metabolism of: histidine; arginine and proline; glycine, serine and threionine; phosphorcreatine, as well as other amino acids and vitamins from B complex.

Base teórica: Artrite reumatoide (AR) é uma doença autoimune que afeta as articulações e progride de maneira simétrica e erosiva. Além dos achados articulares, pode ocorrer de perda muscular e síndrome da caquexia. Atualmente, não existe um marcador que sirva de preditor da síndrome de caquexia reumatoide. Estudos metabolômicos em pacientes com AR demonstram uma complexidade em encontrar um biomarcador para caquexia. Ademais, não há modelo experimental de caquexia descrito na literatura, mas o modelo de artrite induzida por colágeno (CIA) possui potencial de ser modelo de caquexia reumatoide. A partir deste modelo, pode-se fazer a busca por biomarcadores de caquexia reumatoide via metabolômica. Objetivo: Avaliar o modelo de CIA como modelo experimental de caquexia reumatoide. Avaliar o perfil metabólico da urina no modelo de CIA e correlacionar com parâmetros clínicos de caquexia reumatoide em busca de possíveis biomarcadores. Métodos: Camundongos machos DBA/1J foram induzidos (CIA; n=13) no dia zero e receberam reforço 18 dias após, e grupo mantidos saudáveis sem indução (CO; n=11). Nos dias 0, 18, 25, 35, 45, 55 e 65 após a indução, foram realizados: coleta de urinas; teste de desempenho físico; teste de locomoção espontânea; teste de força; medida do volume do edema da pata traseira; avaliação do escore clínico; pesagem; e avaliação da ingestão alimentar. Após os 65 dias, os animais foram eutanasiados e tecidos musculares (gastrocnêmio – GA; e tibial anterior – TA) foram dissecados para pesagem e realização da razão sarcoplasmática. Os dados foram analisados por ANOVA de duas vias, seguido de Bonferroni, ou teste t de Pearson, com significância a partir de um p<0,05. A urina coletada foi submetida à ressonância nuclear magnética (1D e 2D J-res). Os metabolitos foram identificados via Chenomx (1D) e pelo Birmingham Metabolite Library (BML; 2D J-res). Utilizou-se a o modelo estatístico de PCA, PLSDA e PLSR para criar ranqueamento de metabolitos (significância a partir de um p<0,05). Analizou-se as rotas metabólicas via Metaboanalyst a partir do ranqueamento de metabólitos obtidos. Os metabólitos obtidos foram filtrados para rotas metabólicas que ocorrem no músculo para identificação de potenciais biomarcadores de perda muscular. Resultados: O grupo CIA apresentou redução de até 24% na locomoção espontânea, de até 66% na força e de até 24% no teste de desempenho físico após 35 dias da indução, bem como redução no peso do GA (24%) e TA (25%), e relação sarcoplasmática (22 e 23%, respectivamente) em relação ao grupo CO. Os modelos estatísticos de PCA, PLSDA e PLSR, e o filtro pelas rotas metabólicas relacionadas com o músculo geraram uma lista de 28 metabólitos e relacionados com o desenvolvimento da doença, sendo eles: 3-metilhistidina, 4-aminobutirato, acetilcolina, arginina, aspartato, carnosina, creatina, creatinina, glutamina, histamina, histidina, isoleucina, leucina, metionina, lisina, mio-inositol, dimetilglicina, acetilalanina, acetilmetionina, pantotenato, fenilalanina, fosfocolina, fosfocreatina, piridoxina, sarcosina, succinilacetona, tiamina, e urocanato. Conclusão: Em concordância com os resultados de redução nos parâmetros de: massa muscular, locomoção espontânea, força e desempenho físico, somando-se a ausência de anorexia bem como mudança no peso, o modelo animal de CIA representa um modelo experimental próprio para caquexia reumatoide. A análise do perfil metabólico deste modelo permite sugerir 28 metabólitos relacionados ao processo de perda muscular, que podem vir a ser biomarcadores de caquexia reumatoide, objetivando prognóstico, diagnóstico e acompanhamento da síndrome. Destes metabólitos, os principais são pertencentes ao metabolismo de: histidina; arginina e prolina; glicina, serina e treonina; fosfocreatina, bem como outros aminoácidos e vitaminas do complexo B. / Background: Rheumatoid Arthritis (RA) is an autoimmune disease that affects the joints and has a symmetric development and it is erosive. Besides joint damage, it can develop muscle loss progress into cachexia syndrome. Currently, there is no marker that can predict it development in rheumatoid patients. Metabolomics in RA have shown to be complex to find out a biomarker for this syndrome. Also, there is no experimental model of cachexia described in literature yet; however the collageninduced arthritis (CIA) animal model seems to be a feasible model for rheumatoid cachexia. With this model, the research for a biomarker of rheumatoid cachexia can be done by metabolomics. Objectives: It will be evaluated if the CIA animal model can be also an animal model of rheumatoid cachexia. Afterwards, it will be evaluated a metabolic profile from urine of this animal model and correlate with clinical signs of rheumatoid cachexia to find out plausible biomarkers of it. Methods: Male DBA/1J mice were submitted to CIA (n=13), immunization occurred at day zero and a booster was performed 18 days after, and a healthy group with no induction (CO; n=11). At the 0,18, 25, 35, 45, 55 and 65 days after the first injection, it was done: urine collection; physical performance test; free exploratory locomotion test; strength test; hindpaw edema volume measurement; follow up disease development; weighted; and food intake. After the 65 days, animals were euthanized and muscle (gastrocnemious – GA; and tibial anterior – TA) were dissected, and weighted for sarcoplasmic ratio. Data were analyzed by two-way ANOVA followed by Bonferroni post hoc, and t-test of Pearson, and statistical critical limit was set for p<0.05. The collected urine was used for nuclear magnetic resonance (1D and 2D J-res). Metabolites were identified by Chenomx (1D) and by the Birmingham Metabolite Library (BML; 2D J-res). Statistical model were performed using PCA, PLSDA and PLSR to create a ranking list of the metabolites (statistical critical limit was set for p<0.05). It was analyzed the metabolic pathway by Metaboanalyst from the data of metabolite ranking list. Then, the metabolite list was filtered by the metabolic pathways that take place in muscle tissue, in order to identify plausible biomarkers of muscle loss. Results: CIA group has shown reduction in up to 24% of free locomotion fatigue, up to 66% of strength and up to 24% of endurance physical performance after 35 days of the induction, as well as a decrease in GA (24%) and TA (25%) weight, and sarcoplasmic ratio also reduced (22 and 23%, respectivamente) related to CO group. The PCA, PLSDA and PLSR statistical models, and the filter by metabolic pathways related to muscle provided a list of 28 metabolites related to disease development, as can be listed: 3-methylhistidine, 4-aminobutyrate, acetylcholine, arginine, aspartate, carnosine, creatine, creatinine, glutamine, histamine, histidine, isoleucine, leucine, methionine, lysine, myo-inositol, dimethylglycine, acetylalanine, acetylmethionine, pantothenate, phenylalanine, phosphocholine, phosphocreatine, pyridoxine, sarcosine, succinylacetone, thiamine, and urocanate. Conclusions: Accordingly with the data with reduction of: muscle mass, spontaneous locomotion, strength and physical performance, added with absence of anorexia as well as weight change, CIA animal model is a feasible experimental model for rheumatoid cachexia. Concerning the metabolic profile from this model, it can be suggested 28 metabolites related to muscle loss in which can be tested for biomarker of rheumatoid cachexia, targeting prognosis, diagnosis, and syndrome follow up. From those metabolites, the main ones are engaged to metabolism of: histidine; arginine and proline; glycine, serine and threionine; phosphorcreatine, as well as other amino acids and vitamins from B complex.

Les patients atteints de polyarthrite rhumatoide (PR) ont une diminution de l'espérance de vie de 10 à 15 ans. Cette augmentation de la mortalité semble liée à un processus athéromateux accéléré. La dysfonction endothéliale (DE) joue un rôle clé dans ce processus. L'arginase est une enzyme qui régule le niveau de NO par compétition avec la NO Synthase (NOS) pour leur substrat commun, la L-arginine.Nous avons montré que la vasodilatation Acétylcholine (ACh) dépendante était altérée dans le modèle d'Arthrite Induite à Adjuvant (AIA) chez le rat Lewis, témoignant d'une DE. L'incubation des anneaux aortiques avec la nor-NOHA un inhibiteur sélectif d'arginase a amélioré la réponse vasculaire à l'ACh chez les rats AIA. L'activité et l'expression vasculaire de l'arginase se sont révélées augmentées chez les rats AIA et corrélées positivement à la sévérité de l'arthrite.Nous avons caractérisé les mécanismes impliqués dans la DE du rat AIA. Nos résultats ont montré que la DE mettait enjeu une diminution de l'activité de la NO synthase, un déficit en EDHF, une suractivité de la COX-2, ainsi qu'une production excessive des anions superoxydes. Le traitement curatif des rats AIA par la nor-NOHA pendant 3 semaines, a permis de restaurer la fonction endothéliale. L'inhibition de l'arginase n'a pas modifié l'atteinte articulaire des rats AIA.Nos travaux ont permis de mieux comprendre la physiopathologie de la DE associée à la PR et ont déterminé pour la première fois le rôle délétère de l'arginase dans cette anomalie. Ils ouvrent des perspectives quant à l'utilisation des inhibiteurs d'arginase comme futurs traitements pharmacologiques de l'atteinte vasculaire du patient PR / Patients with RA are characterized by a decrease in life expectancy of 10 to 15 years. This increase in mortality seems to be related to an accelerated atheroma process. Endothelial dysfunction (ED) has a key role in these processes. The arginase is an enzyme which regulates the level of NO by competing with the NO synthase (NOS) to their common substrate, L-arginine.We showed that acetylcholine (ACh) dependent vasodilation was altered in the model of Adjuvant Induced Arthritis (AIA) in Lewis rats, indicating a endothelial dysfunction. The incubation of aortic rings with nor-NOHA has improved the vascular response to ACh in AIA rats. The activity and expression of vascular arginase are increased in AIA rats and positively correlated with the severity of arthritis.We characterized the mechanisms involved in DE in AIA rats. Our results showed that ED involved a decrease of activity of NO synthase, a decrease of EDHF, overactiviry of COX-2, thromboxane synthase and prostacyclin synthase, as well as excessive superoxide anions. The cure of AIA rats by a selective inhibitor of arginase, nor-NOHA for 3 weeks, has restored endothelial function. In contrast, inhibition of arginase activity did not change the weight, the diameter of ankles, radiological or histological articular damage in AIA rats.Our work has led to a better understanding of pathophysiology of ED associated with rheumatoid arthritis and determined for the first time the deleterious role of arginase in this vascular anomaly. These results open prospects for the use of arginase inhibitors as future pharmacological treatment of vascular patient PR.

Plantas do gênero Alternanthera (Amaranthaceae) vêm sendo estudadas por suas propriedades antiparasitária, antibacteriana e antiviral. No Brasil a planta Alternanthera tenella Colla, objeto de nossa investigação, é utilizada na medicina popular por possuir atividade antiinflamatória. Considerando a importância do sistema imunológico em infecções e em doenças auto-imunes sistêmicas que apresentam intensa reação inflamatória, o objetivo do atual estudo foi investigar a ação imunomoduladora de extratos de A. tenella no sistema imune de camundongos BALB/c e sua atividade em modelo de artrite experimental induzida pelo óleo mineral pristane (2,6,10,14-tetrametilpentadecano) em camundongos AIRmax, obtidos por seleção genética para reação inflamatória aguda máxima. Extratos brutos orgânicos (etanólico e hexânico), aquosos, frações e flavonóides foram inoculados via intraperitoneal em camundongos BALB/c imunizados ou não com eritrócitos de carneiro (EC). Efeitos imunomoduladores e imunotóxicos foram avaliados através da determinação do peso corporal e dos órgãos linfóides, celularidade do baço e de ensaios funcionais como enumeração de células formadoras de placas (PFC, plaque forming cells), produção de anticorpos anti-EC e edema de pata induzido por carragenina. Posteriormente foram avaliados os efeitos dos extratos aquosos nas subpopulações de linfócitos esplênicos (CD3, CD4, CD8 e CD19), na expressão de marcadores de ativação de linfócitos (CD25, CD40, CD45RB e CD69) e na indução de apoptose nessas células. Os extratos avaliados não induziram alterações no peso dos animais e dos órgãos (baço, timo e fígado) após 4 e 14 dias. Animais imunizados com EC e tratados com o flavonóide 2-O--L-ramnopiranosilvitexina (15 mg/kg), isolado de extrato bruto etanólico (E) ou com o extrato aquoso extraído a frio (AF - 100 mg/kg) apresentaram aumento significativo (p<0,05) no número de PFC anti-EC em comparação aos controles. Os extratos E e AF induziram aumento no título de anticorpos circulantes anti-EC das classes IgG e IgM. Estes resultados sugerem atividade imunoestimulante. Ambos os extratos aquosos, AF e extraído a quente (AQ), apresentaram atividade antiinflamatória no edema de pata induzido por carragenina, principalmente AF que demonstrou efeito dose-dependente (50% e 61% de inibição do edema nas doses de 200 mg/kg e 400 mg/kg, respectivamente). Estes extratos em camundongos BALB/c normais não induziram apoptose, alterações nas subpopulações de linfócitos e não modificaram a expressão de marcadores de ativação em linfócitos T e B. Com base nestes resultados, o extrato AF foi selecionado para utilização nos experimentos em camundongos AIRmax, para investigação de suas possíveis atividades moduladora e/ou terapêutica na artrite induzida por pristane. Os animais que foram tratados com seis doses de 200 mg/kg do extrato AF antes das injeções do pristane (G1, n=15) apresentaram menor incidência de artrite em comparação ao grupo controle positivo, composto por animais que receberam apenas pristane (G4, n=15) (54,5% e 70%, respectivamente). A porcentagem de animais que apresentaram deformidade nas articulações, também foi menor no grupo G1 (18,2%) em comparação ao G4 (30%). Os animais que receberam apenas o extrato (G3, n=14) não apresentaram artrite. Adicionalmente, AF conferiu atividade protetora ao desenvolvimento de ascite, processo inflamatório que também pode ser induzido pelo óleo mineral. As taxas de incidência de ascite nos animais tratados previamente (G1) bem como nos animais tratados após (G2, n=16) as injeções de pristane foram menores do que a do grupo controle positivo (G1=18,2%, G2=6,7% e G4= 50%). Ressalta-se que os índices de sobrevivência nos grupos de animais que receberam o extrato foram superiores ao grupo controle positivo (G1= 86,7%; G2= 93,7%; G4= 60,0%). A taxa de sobrevivência do grupo G3 foi de 100% ao final do experimento. Esses efeitos moduladores do extrato no processo da artrite parecem não ser dependentes da modulação de marcadores de ativação de linfócitos T, nem de alterações nas subpopulações dessas células (CD4+, CD8+, T regulatórias). Tampouco dependeram da indução de apoptose nos linfócitos esplênicos, conforme avaliado pelas técnicas da anexina V e análise da fragmentação de DNA. Entretanto, aumentos na porcentagem de células B/CD69+ sugerem possível participação destas células no processo de modulação da doença. Analisados conjuntamente, os resultados apresentados sugerem que alguns dos produtos vegetais avaliados podem modular a função de linfócitos B, além de apresentarem importante atividade antiinflamatória em edema de pata induzido por carragenina. Adicionalmente, o extrato AF apresentou ação moduladora na artrite induzida por pristane. Esses resultados fornecem subsídios para o entendimento das atividades biológicas da Alternanthera tenella e para a validação científica do seu uso popular / Antiparasitic, antibacterial and antiviral activities in plants of the Alternanthera genus (Amaranthaceae) have been studied. In Brasil, Alternanthera tenella Colla are used in popular medicine as an anti-inflammatory agent. Considering the importance of the immune system in infectious and systemic autoimmune diseases showing intense inflammatory reaction, the objective of this study was to investigate the immunomodulatory activity of A. tenella extracts in BALB/c mice. The plant extracts were also tested in a mineral pristane oil (2,6,10,14-tetramethylpentadecane) induced arthritis model in AIRmax mice, genetically selected for maximal acute inflammatory reactions. Organic solvent crude extracts (ethanol and hexane), aqueous fractions and isolated flavonoids were intraperitoneally inoculated in BALB/c mice immunized or not with sheep red blood cells (SRBC). Immunomodulatory and immunotoxic effects were evaluated by determining body and lymphoid organ weights, splenic cellularity and through functional assays like plaque-forming cells (PFC), antibody anti-SRBC production and carrageenan-induced paw edema. The effects of aqueous extracts on splenic lymphocyte subtypes (CD3, CD4, CD8 and CD19) and apoptosis detection in these cells were further evaluated. The extracts tested did not induce changes in body and organ (spleen, thymus and liver) weights 4 and 14 days after administration. PFC numbers were significantly increased (p<0,05) in SRBC immunized animals treated with 15mg/kg 2-O--L-ramnopiranosilvitexina, a flavonoid isolated from the etanolic (E) crude extract or with the cold aqueous extract (CAE 100 mg/kg) when compared to the controls. The E and CAE extracts induced increased anti-SRBC IgG and IgM circulating antibody titers, suggesting immunostimulatory activity. Aqueous extracts, CAE and hot aqueous extract (HAE), had significant anti-inflammatory activity in the carrageenan paw edema, especially CAE, which showed a dose-related effect (50% and 61% edema inhibition in dosages of 200 mg/kg and 400 mg/kg, respectively). In normal BALB/c mice the extracts did not induce apoptosis or changes in lymphocyte subtypes and T and B activation markers. Based on these results, CAE was selected for tests of modulatory and /or therapeutic activity in a model of pristane induced arthritis in AIRmax mice. Animals (G1, n=15) treated with six doses of CAE (200 mg/kg) before pristane injections showed smaller arthritis incidence when compared to the control positive group receiving pristane only (G4, n=15) (54,4% and 70%, respectively). Percentage of animals showing joint deformities was smaller in G1 (18,2%) in comparison to G4 (30%). The animals receiving extract only (G3, n=14) did not show signs of arthritis. In addition, CAE showed protective activity against ascites development, an inflammatory process that may be induced by the mineral oil. The arthritis incidence index, both in CAE previously treated animals (G1) and in animals treated after pristane injections (G2, n=16), was smaller than in the positive control group (G1=18,2% and G2=6,7% x G4= 50%). It is noteworthy that extract- treated animals, in both groups, also had a higher survival index when compared to the positive control group (G1= 81,9% and G2= 90,8% x G4= 40,2%). The survival index in the G3 group was 100% up to the end of the experiments. The extract modulatory effects in arthritis do not seem to be dependent on the modulation of T lymphocyte activation markers, or on changes in T cells subtypes (CD4+, CD8+, regulatory T cells). Also, they did not depend on apoptosis induction in splenic lymphocytes as evaluated by annexin V and analysis of DNA degradation techniques. However, the percentage increase of B/CD69+ cells suggest their participation in the modulatory process. Together, the results suggest that some of the evaluated plant-derived products may modulate B lymphocyte functions, besides showing important anti-inflammatory activity in carrageenan paw edema. In addition, CAE showed modulatory action in pristane induced arthritis. These results contribute to the understanding of Alternanthera tenella biological activities and provide scientific validation to its popular use.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that leads to degradation of cartilage and bone mainly in peripheral joints. In collagen-induced arthritis (CIA), a mouse model for RA, activation of autoimmune CD4+ T cells depends on a molecular recognition system where T-cell receptors (TCRs) recognize a complex between the class II MHC Aq protein and CII259-273, a glycopeptide epitope from type II collagen (CII). Interestingly, vaccination with the Aq/CII259-273 complex can relieve symptoms and cause disease regression in mice. This thesis describes the use of modified glycopeptides to explore interactions important for binding to the Aq protein and recognition by autoimmune T-cell hybridomas obtained from mice with CIA. The CII259-273 glycopeptide was modified by replacement of backbone amides with different amide bond isosteres, as well as substitution of two residues that anchor the glycopeptide in prominent pockets in the Aq binding site. A three-dimensional structure of the Aq/glycopeptide complex was modeled to provide a structural basis for interpretation of the modified glycopeptide’s immunological activities. Overall, it was found that the amide bond isosteres affected Aq binding more than could be explained by the static model of the Aq/glycopeptide complex. Molecular dynamics (MD) simulations, however, revealed that the introduced amide bond isosteres substantially altered the hydrogen-bonding network formed between the N-terminal 259-265 backbone sequence of CII259-273 and Aq. These results indicated that the N-terminal hydrogen-bonding interactions follow a cooperative model, where the strength and presence of individual hydrogen bonds depended on the neighboring interactions. The two important anchor residues Ile260 and Phe263 were investigated using a designed library of CII259-273 based glycopeptides with substitutions by different (non-)natural amino acids at positions 260 and 263. Evaluation of binding to the Aq protein showed that there was scope for improvement in position 263 while Ile was preferred in position 260. The obtained SAR understanding provided a valuable basis for future development of modified glycopeptides with improved Aq binding. Furthermore, the modified glycopeptides elicited varying T-cell responses that generally could be correlated to their ability to bind to Aq. However, in several cases, there was a lack of correlation between Aq binding and T-cell recognition, which indicated that the interactions with the TCRs were determined by other factors, such as presentation of altered epitopes and changes in the kinetics of the TCR’s interaction with the Aq/glycopeptide complex. Several of the modified glycopeptides were also found to bind well to the human RA-associated DR4 protein and elicit strong responses with T-cell hybridomas obtained from transgenic mice expressing DR4 and the human CD4 co-receptor. This encourages future investigations of modified glycopeptides that can be used to further probe the MHC/glycopeptide/TCR recognition system and that also constitute potential therapeutic vaccines for treatment of RA. As a step towards this goal, three modified glycopeptides presented in this thesis have been identified as candidates for vaccination studies using the CIA mouse model.

Le costimulateur inductible (ICOS) est un récepteur costimulatoire des cellules T crucial pour l’immunité humorale. Chez l’humain, une déficience de ICOS est associée à des infections récurrentes dû à des défauts de différentiation des lymphocytes T auxiliaires folliculaires (Tfh) et un manque d’anticorps protecteurs. Paradoxalement, certains patients déficients en ICOS développent des signes d’autoimmunité causés par des autoanticorps. Ces observations sont en accord avec des résultats suggérant que ICOS joue un double rôle dans la promotion des fonctions de cellules T effectrices et de cellules T régulatrices. L’arthrite rhumatoïde (RA) est une maladie autoimmune caractérisée par l’inflammation articulaire et la destruction progressive des os. Dans la première étude présentée au Chapitre 2, nous avons démontré que ICOS est requis pour l’initiation et la maintenance de l’arthrite induite par le collagène (CIA), un modèle murin de la RA. Nous avons démontré que des événements clés de l’initiation de la CIA incluant la production d’anticorps contre le collagène et la prolifération de cellules T inflammatoires sont dépendants de la voie de signalisation ICOS-PI3K. Aussi, nous avons trouvé que la signalisation par ICOS-PI3K promouvoit le maintien de la CIA. De plus, nous avons établi que l’inhibition de la glycolyse réduit la sévérité de la CIA, ce qui suggère un chevauchement entre la signalisation ICOS-PI3K et le métabolisme du glucose dans la pathogenèse de l’arthrite autoimmune. Dans la deuxième étude présentée au Chapitre 3, nous avons utilisé des souris dont les cellules T régulatrices sont déficientes en ICOS (ICOS FC) pour évaluer l’impact de ICOS dans les cellules Tfr. Nous avons trouvé que les souris ICOS FC possèdent un taux réduit de cellules Tfr, mais aucune diminution des cellules T régulatrices (Treg) précurseurs. De plus, les souris ICOS FC ont un taux élevé de cellules B du centre germinatif (GC) non-spécifiques ainsi qu’une production accrue d’anticorps anti-nucléaires. Nous avons aussi observé une perturbation des réponses anti-virales et de la production d’anticorps spécifiques dans les souris ICOS FC ce qui suggère des rôles non-régulateurs pour les cellules Tfr. Nous avons effectué une analyse de transcriptome de cellule unique avec des Tregs et nous avons observé une accumulation de précurseurs de Tfr dans les souris ICOS FC, ce qui suggère un défaut de la transition Treg à Tfr. Nos données suggèrent que ICOS participe à la différentiation des cellules Tfr en régulant KLF2 et NFAT2, ce qui contribue à l’établissement de traits folliculaires. En résumé, nous avons démontré que ICOS promouvoit le développement de l’arthrite autoimmune en soutenant les fonctions des cellules T inflammatoires par la voie de signalisation ICOS-PI3K. De plus, nos résultats prouvent que ICOS peut prévenir la production d’autoanticorps en supportant la différentiation des cellules Tfr. Notre travail contribue à l’avancement des connaissances en ce qui concerne le rôle double de ICOS dans l’immunité cellulaire et humorale et fournit des paramètres importants à considérer lors de la recherche de nouvelles cibles thérapeutiques. / The inducible costimulator (ICOS) is a T cell costimulatory receptor crucial for humoral immunity. In humans, ICOS deficiency is associated with recurrent infections due to defects in T follicular helper (Tfh) differentiation and lack of protective antibodies. Paradoxically, some ICOS-deficient patients were found to exhibit signs of antibody-mediated autoimmunity. These observations are congruent with findings suggesting that ICOS plays a dual role in promoting T effector and T regulatory cell functions. Due to this ambivalence, the role of ICOS in autoimmunity remains unresolved. Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and progressive bone destruction. The number of ICOS+ T cells in the synovial tissues of RA patients was found to be elevated, suggesting a potential involvement of ICOS signaling in the pathogenesis of RA. In the first study presented in Chapter 2, we showed that ICOS is required for the initiation and maintenance of collagen-induced arthritis (CIA), a murine model of RA. We found that key CIA initiation events such as anti-collagen antibody production and inflammatory T cell proliferation were dependent on ICOS-PI3K signaling. Further, we found that ICOS-PI3K signaling promotes maintenance of established CIA. Additionally, we show that glycolysis inhibition ameliorates CIA, thus suggesting potential overlaps between ICOS-PI3K signaling and glucose metabolism in the pathophysiology of autoimmune arthritis. The initiation of autoimmunity depends on the action of autoimmune effector cells, but also on a failure of regulatory cells. Amongst the latter, T follicular regulatory (Tfr) cells are thought to prevent autoantibody production. In the second study presented in Chapter 3, we used regulatory T cell-specific ICOS knockout (ICOS FC) mice to investigate the impact of ICOS signaling in Tfr cells. We found that ICOS FC mice display a significant reduction in Tfr cell numbers, but no depletion of their T regulatory (Treg) precursors. Further, ICOS FC mice exhibited a rise of extraneous germinal center (GC) B cells numbers and increased production of anti-nuclear antibodies. We also observed disruptions of anti-viral responses and antigen-specific antibody production in ICOS FC mice, suggesting non-regulatory roles for Tfr cells. We performed single-cell transcriptome analysis of regulatory T cells and observed an accumulation of Tfr precursors in ICOS FC mice suggestive of a Treg-to-Tfr transition defect. Mechanistically, we found that ICOS participates in Tfr differentiation by regulating KLF2 and NFAT2, thereby contributing to the establishment of follicular T cell traits. In sum, we demonstrate that ICOS promotes the development of autoimmune arthritis by fostering inflammatory T cell responses in a PI3K-dependent manner. In addition, our work shows that ICOS can prevent autoantibody production by supporting Tfr differentiation. Thus, we contribute insights into the dual role of ICOS in the cellular and humoral arms of autoimmunity, providing important parameters to be considered when searching for novel therapeutic targets.