GITR and CD30 are T cell costimulatory members of the TNFR superfamily known to regulate T cell responses. Elucidating the mechanisms whereby these receptors modulate T cell responses is crucial for maximizing their potential for immunotherapy. In this thesis, I examine the role of GITR and CD30 on CD8 T cell responses to influenza virus. I show that CD8 T cell intrinsic GITR is required for both maximal primary and secondary CD8 T cell expansion to influenza, while in contrast, CD30 is dispensable for anti-influenza CD8 T cell responses. GITR does not impact on CD8 T cell proliferation or homing, however, it mediates CD8 T cell survival signaling. GITR induces TRAF2/TRAF5 dependent, but TRAF1 independent, NF-κB activation, resulting in the upregulation of the pro-survival molecule Bcl-xL. Furthermore, I show that GITR on CD8 T cells can augment viral clearance and confer protection from death upon severe influenza infection of mice. Similarly, CD30 also elicits protection from death upon severe influenza infection, although the cells responsible for this effect remain to be elucidated. In this thesis, I also show that in unimmunized mice GITR expression is upregulated to higher than basal levels on a population of CD8 memory phenotype cells in the bone marrow. In contrast, CD8 memory phenotype T cells in the spleen and LN have GITR levels similar to that on naïve T cells. The upregulation of GITR in the bone marrow is IL-15 dependent and therefore, GITR serves as a marker for cells that have recently received an IL-15 signal. Furthermore, GITR is required for the persistence, but not for the homeostatic proliferation of CD8 memory phenotype T cells in the bone marrow. Therefore, GITR plays a key role for CD8 T cell intrinsic responses to influenza, as well as for the persistence of CD8 memory phenotype T cells.

GITR and CD30 are T cell costimulatory members of the TNFR superfamily known to regulate T cell responses. Elucidating the mechanisms whereby these receptors modulate T cell responses is crucial for maximizing their potential for immunotherapy. In this thesis, I examine the role of GITR and CD30 on CD8 T cell responses to influenza virus. I show that CD8 T cell intrinsic GITR is required for both maximal primary and secondary CD8 T cell expansion to influenza, while in contrast, CD30 is dispensable for anti-influenza CD8 T cell responses. GITR does not impact on CD8 T cell proliferation or homing, however, it mediates CD8 T cell survival signaling. GITR induces TRAF2/TRAF5 dependent, but TRAF1 independent, NF-κB activation, resulting in the upregulation of the pro-survival molecule Bcl-xL. Furthermore, I show that GITR on CD8 T cells can augment viral clearance and confer protection from death upon severe influenza infection of mice. Similarly, CD30 also elicits protection from death upon severe influenza infection, although the cells responsible for this effect remain to be elucidated. In this thesis, I also show that in unimmunized mice GITR expression is upregulated to higher than basal levels on a population of CD8 memory phenotype cells in the bone marrow. In contrast, CD8 memory phenotype T cells in the spleen and LN have GITR levels similar to that on naïve T cells. The upregulation of GITR in the bone marrow is IL-15 dependent and therefore, GITR serves as a marker for cells that have recently received an IL-15 signal. Furthermore, GITR is required for the persistence, but not for the homeostatic proliferation of CD8 memory phenotype T cells in the bone marrow. Therefore, GITR plays a key role for CD8 T cell intrinsic responses to influenza, as well as for the persistence of CD8 memory phenotype T cells.

A desnutrição é uma condição nutricional que ainda constitui um grande problema de saúde publica e acomete grande parte da população mundial. Atualmente, sabe-se que vários aspectos da resposta imunológica podem estar alterados na decorrência da desnutrição, dentre eles alterações funcionais como redução da migração celular, fagocitose, atividade bactericida, bem como alterações na produção de espécies reativas do oxigênio e nitrogênio, além de comprometimento da expressão de importantes receptores de reconhecimento de patógenos e alteração na produção de citocinas pró-inflamatórias, dentre elas o TNF-α. O TNF-α é uma citocina, produzida principalmente por macrófagos, que participa de uma ampla gama de atividades biológicas, incluindo os processos de inflamação, crescimento, diferenciação e apoptose, essa citocina exerce seus efeitos através da ligação a dois receptores distintos, TNFR-I e TNFR-II. A ativação via receptor TNFR-I, no entanto, é responsável pela maioria dos efeitos do TNF-α, e desencadeia uma série de eventos intracelulares que resultam, principalmente, na ativação do fator de transcrição NFκB. Considerando a relevância dessa via de sinalização mediada por TNF-α, avaliamos em um modelo de desnutrição protéica a via de sinalização de ativação do fator de transcrição NFκB mediada pelo TNF-α via TNFR-I. Sendo assim, utilizamos camundongos Balb/c, machos adultos, submetidos à desnutrição protéica, após perda de aproximadamente 20% do peso corpóreo, foram coletadas macrófagos peritoneais e cultivados ou não com TNF-α, o sobrenadante foi utilizado para avaliação da produção de citocinas (IL-1α, IL-1β, IL-6, IL-12, IL-10) e as células utilizadas para avaliação da expressão do TNFR-I, bem como a expressão de proteínas da via de sinalização (TRADD, TRAF, RIP, IKK, IKBα, p IKBα, NFκB e p NFκB). Os resultados obtidos mostraram que os animais desnutridos apresentaram anemia, leucopenia, diminuição da celularidade medular e da cavidade peritoneal. Os resultados demonstraram redução da expressão de TNFR-I, além de diminuição da expressão das porções fosforiladas de IKBα e NFκB, associado a uma diminuição na produção de IL-1β e IL-12. Nesse trabalho compreendemos aspectos relacionados a resposta imune inata e nosso dados permitem inferir que o estado nutricional interfere no estado de ativação de macrófagos e na capacidade de resposta dessas células. / Malnutrition is a nutritional condition that consists in a major public health issue and affects a considerable part of the global population. Currently, It is known that many aspects of the immunological response can be altered due to the malnutrition impairment, among them functional changes, for instance, cell migration reduction, phagocytosis, bactericidal response, as well as changes in the reactive oxygen and nitrogen species production, besides, substantially cell receptors expression impaired, responsible for the recognition of pathogens and changes in the production of proinflammatory cytokines, such as TNF-α. This cytokine is primarily produced by macrophages, and it is associated to a wide range of biological activities, including the inflammation processes, growing, differentiation, and apoptosis. The TNF-α act through the linkage to distinct receptors, TNFR-I and TNFR-II. The TNFR-I receptor activation, however, is responsible for most TNF-α effects, and triggers a series of intracellular events that mainly result in the activation of the NFκB transcription factor. Regarding the relevance of this signaling pathway mediated by the TNF-α, we evaluated through a protein malnutrition model the signaling pathway for the NFκB transcription factor mediated by the TNF-α through TNFR-I. Adult male Balb/c mice, were submitted to a protein malnutrition and after a loss of nearly 20% body weight, peritoneal macrophages were collected and cultivated, or not with TNF-α. The supernatant was collected for cytokine production evaluation (IL-1α, IL-1β, IL-6, IL-12, and IL-10) and the cell for TNFR-I expression evaluation, as well as the proteins of the signaling pathway (TRADD, TRAF, RIP, IKK, IKBα, p IKBα, NFκB, and p NFκB). The compiled results highlights that the malnourished animals present signs of anemia, leukopenia, and decrease of bone marrow cellularity and peritoneal cavity. The results presented reduction of the TNFR-I expression, in addition to the phosphorylated portions of the IKBα and the NFκB expression, associated to a decrease in the production of IL-1β and IL-12. In this essay we perceive the aspects related to the innate immune response, and the outcome data allowed us to conclude that the nutritional status interferes on the macrophages activation and the response capabilities of these cells.

The tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were initially discovered as proteins that inducibly interact with the intracellular region of TNF receptors (TNFRs). Because the TNFRs lack intrinsic catalytic activity, the TRAFs are hypothesized to orchestrate signaling activation downstream of the TNFR superfamily, however their mechanism of activation remains unclear (Inoue et al., 2000; Bishop, 2004). Originally, the TRAFs were compared to the signal transducers and activators of transcription (STAT) protein family, due to their sequence homology, and the presence of multiple RING- and zinc-finger domains, suggesting that their function may be to regulate transcriptional activity (Rothe et al., 1994; Hu et al., 1994; Sato et al. 1995; Cheng et al., 1995). However, subsequent research focused predominantly on their cytoplasmic functions, and more recently on their function as E3 ubiquitin ligases (Pineda et al., 2007). In my research, I analyzed the subcellular localizations of the TRAFs following CD40 ligand (CD40L)-stimulation, and found that TRAF2 and 3 rapidly translocate into the nucleus of primary neurons and Neuro2a cells. Interestingly, similar analysis conducted in pre-B lymphocytes (Daudi cells) revealed a different response to CD40L-stimulation, with TRAF2 and 3 being rapidly degraded within 5-minutes of stimulation. These findings are significant because they demonstrate for the first time that the TRAFs translocate into the nucleus and suggest that they may function within the nucleus in a cell-specific manner. I next analyzed the ability of TRAF2 and 3 to bind to DNA, and found that they both bind to chromatin and the NF-kappaB consensus element in Neuro2a cells, following CD40L-stimulation. Similar analyses of the chromatin binding of TRAF2 and 3 in Daudi cells revealed that they were rapidly degraded, similar to the results from my analysis of their subcellular localization. These findings show for the first time that the TRAFs interact with DNA, and therefore support the hypothesis that the TRAFs may function within the nucleus as transcriptional regulators. Finally, I analyzed the ability of the TRAFs to regulate transcriptional activity by luciferase assay. Previous studies showed that overexpression of TRAF2 and 6 could induce NF-kappaB transcriptional activity; however researchers have not been able to determine the mechanism by which they do so. In my studies, I found that every TRAF can directly regulate transcriptional activity either as co-activators or co-repressors of transcription, in a cell- and target protein-specific manner. Additionally, I found that TRAF2 can act as a transcriptional activator, and that its ability to regulate transcription is largely dependent upon the presence of its RING-finger domain. In conclusion, these studies have revealed an entirely novel function for the TRAFs as immediate-early transcriptional regulators. Future research into the genes that are regulated by the specific TRAF complexes will further elucidate how the TRAFs regulate TNFR signaling, as well as whether dysfunctions in TRAF signaling may be associated with known disorders. If specific TRAF complexes are found to regulate specific genes, then pharmacological targeting of the individual TRAF complexes may allow for the highly specific inhibition of signaling events downstream of the TNFRs, without compromising overall receptor signaling, transcription factor pathways, or cellular systems.

A desnutrição é uma condição nutricional que ainda constitui um grande problema de saúde publica e acomete grande parte da população mundial. Atualmente, sabe-se que vários aspectos da resposta imunológica podem estar alterados na decorrência da desnutrição, dentre eles alterações funcionais como redução da migração celular, fagocitose, atividade bactericida, bem como alterações na produção de espécies reativas do oxigênio e nitrogênio, além de comprometimento da expressão de importantes receptores de reconhecimento de patógenos e alteração na produção de citocinas pró-inflamatórias, dentre elas o TNF-α. O TNF-α é uma citocina, produzida principalmente por macrófagos, que participa de uma ampla gama de atividades biológicas, incluindo os processos de inflamação, crescimento, diferenciação e apoptose, essa citocina exerce seus efeitos através da ligação a dois receptores distintos, TNFR-I e TNFR-II. A ativação via receptor TNFR-I, no entanto, é responsável pela maioria dos efeitos do TNF-α, e desencadeia uma série de eventos intracelulares que resultam, principalmente, na ativação do fator de transcrição NFκB. Considerando a relevância dessa via de sinalização mediada por TNF-α, avaliamos em um modelo de desnutrição protéica a via de sinalização de ativação do fator de transcrição NFκB mediada pelo TNF-α via TNFR-I. Sendo assim, utilizamos camundongos Balb/c, machos adultos, submetidos à desnutrição protéica, após perda de aproximadamente 20% do peso corpóreo, foram coletadas macrófagos peritoneais e cultivados ou não com TNF-α, o sobrenadante foi utilizado para avaliação da produção de citocinas (IL-1α, IL-1β, IL-6, IL-12, IL-10) e as células utilizadas para avaliação da expressão do TNFR-I, bem como a expressão de proteínas da via de sinalização (TRADD, TRAF, RIP, IKK, IKBα, p IKBα, NFκB e p NFκB). Os resultados obtidos mostraram que os animais desnutridos apresentaram anemia, leucopenia, diminuição da celularidade medular e da cavidade peritoneal. Os resultados demonstraram redução da expressão de TNFR-I, além de diminuição da expressão das porções fosforiladas de IKBα e NFκB, associado a uma diminuição na produção de IL-1β e IL-12. Nesse trabalho compreendemos aspectos relacionados a resposta imune inata e nosso dados permitem inferir que o estado nutricional interfere no estado de ativação de macrófagos e na capacidade de resposta dessas células. / Malnutrition is a nutritional condition that consists in a major public health issue and affects a considerable part of the global population. Currently, It is known that many aspects of the immunological response can be altered due to the malnutrition impairment, among them functional changes, for instance, cell migration reduction, phagocytosis, bactericidal response, as well as changes in the reactive oxygen and nitrogen species production, besides, substantially cell receptors expression impaired, responsible for the recognition of pathogens and changes in the production of proinflammatory cytokines, such as TNF-α. This cytokine is primarily produced by macrophages, and it is associated to a wide range of biological activities, including the inflammation processes, growing, differentiation, and apoptosis. The TNF-α act through the linkage to distinct receptors, TNFR-I and TNFR-II. The TNFR-I receptor activation, however, is responsible for most TNF-α effects, and triggers a series of intracellular events that mainly result in the activation of the NFκB transcription factor. Regarding the relevance of this signaling pathway mediated by the TNF-α, we evaluated through a protein malnutrition model the signaling pathway for the NFκB transcription factor mediated by the TNF-α through TNFR-I. Adult male Balb/c mice, were submitted to a protein malnutrition and after a loss of nearly 20% body weight, peritoneal macrophages were collected and cultivated, or not with TNF-α. The supernatant was collected for cytokine production evaluation (IL-1α, IL-1β, IL-6, IL-12, and IL-10) and the cell for TNFR-I expression evaluation, as well as the proteins of the signaling pathway (TRADD, TRAF, RIP, IKK, IKBα, p IKBα, NFκB, and p NFκB). The compiled results highlights that the malnourished animals present signs of anemia, leukopenia, and decrease of bone marrow cellularity and peritoneal cavity. The results presented reduction of the TNFR-I expression, in addition to the phosphorylated portions of the IKBα and the NFκB expression, associated to a decrease in the production of IL-1β and IL-12. In this essay we perceive the aspects related to the innate immune response, and the outcome data allowed us to conclude that the nutritional status interferes on the macrophages activation and the response capabilities of these cells.

The redox state in the cell plays a major role in determining vital functions and its major imbalance can lead to severe cell injury or death. Redox active proteins and cytokines involved in this process includes thioredoxin (Trx), protein disulfide isomerase (PDI), and tumor necrosis factor (TNF) superfamilies. Trx is a multipotent protein and key regulator of cellular redox balance operating in synergy with Trx reductase and NADPH (the Trx system). Trx has gene regulatory activity of several transcription factors. It also controls in a fascinating way redox-sensitive “on-off” decisions for apoptotic or hypertrophic pathways. Trx protects against H2O2 and TNFmediated cytotoxicity, a pathway in which TNF receptor-binding generates ROS. TNF is an autocrine growth factor and survival factor in vitro and in vivo for B-type of chronic lymphocytic leukemia (B-CLL) cells. The overall aim of this study was to investigate the importance of redox active proteins and cytokines in inflammation and cancer. We focused on: i) the role of Trx, TrxR, and selenium in carcinogenesis and in resistant cancer cells. ii) the importance of Trx in cancer cells and the redox regulation of TNF and its receptors TNFR1 and TNFR2. iii) the potential role of Trx as a key regulator in cellular redox balance, in the pathogenesis of cardiac dysfunction; its relationship to stress response parameters. iv) whether unmutated CLL (UCLL) responses to PKC and ROS pathways were different from mutated CLL (M-CLL) responses. Our results demonstrate pronounced selective selenium-mediated apoptosis in therapy resistant cells and suggest that redox regulation through the Trx system is an important target for cancer therapy. Trx was strikingly elevated in heart failure cases compared with controls signifying an adaptive stress response that is higher the more severe the disease. TNF autocrine release was redox modulated and the TNF receptors interacted at the cell surface membrane with the redox-active PDI, which excerted a stringent redox-control of the TNFR signaling. The proliferative response as well as increase of autocrine TNF and Trx were higher in U-CLL than in M-CLL. The overall conclusion of the four papers included in this thesis is that redox-active proteins and cytokines plays an important role in control and regulation of cancer and inflammation. Furthermore, redox regulation via thioredoxin by selenium, may offer novel treatment possibilities for resistant tumors disease.

Introdução: O fragmento amino-terminal do pró-peptídeo natriurético do tipo B (NT-proBNP) é um forte marcador de doença cardiovascular com evidências recentes de que a inflamação também pode influenciar seus valores. A diferenciação dessa variável de confusão é de particular interesse nas doenças reumáticas. Objetivos: Avaliar o comportamento dos valores de NT-proBNP em pacientes com espondilite anquilosante (EA) pré e pós uso de bloqueadores de TNF para determinar a possível associação entre os valores de NT-proBNP e os parâmetros inflamatórios. Métodos: Quarenta e cinco pacientes consecutivos com EA sem evidência prévia ou atual de doença cardiovascular ou disfunção miocárdica sistólica e que eram elegíveis para terapia anti-TNF foram incluídos prospectivamente. Todos os pacientes receberam bloqueadores de TNF e foram avaliados para concentrações circulantes de NT-proBNP, parâmetros clínicos e laboratoriais de atividade de doença, fatores de risco cardiovasculares tradicionais e ecodopplercardiografia convencional e tecidual no momento da inclusão e após seis meses de tratamento. Resultados: No momento da inclusão, todos os pacientes tinham EA ativa, os valores de NT-proBNP tinham uma mediana de 36 (20-72) pg/mL e 11% dos valores estavam altos mesmo na ausência de alteração miocárdica sistólica. A análise de regressão linear múltipla revelou que esse peptídeo estava independentemente correlacionado com o VHS (p < 0,001), com a idade dos pacientes (p = 0,01) e com a pressão de pulso (p = 0,01) no momento da inclusão. Após seis meses, todos os parâmetros relacionados a doença de base melhoraram e os valores de NT-proBNP se reduziram significativamente [24 (16-47) pg/mL, p = 0,037] quando comparados com os valores do momento da inclusão. As mudanças nos valores de NT-proBNP correlacionaram-se positivamente com as mudanças nos valores do VHS (r = 0,41, p = 0.006). Os fatores de risco cardiovasculares avaliados permaneceram estáveis durante o seguimento. Conclusão: As elevações nos valores de NT-proBNP devem ser interpretadas com cuidado nos pacientes com EA ativa e sem evidência de doença cardiovascular. A redução no curto prazo dos valores de NT-proBNP nesses pacientes recebendo terapia anti-TNF parece refletir uma melhora do estado inflamatório / Introduction: N-terminal pro-brain natriuretic peptide (NT-proBNP) is a strong marker of cardiovascular disease with recent evidence that inflammation may also influence its levels; discrimination of this confounding variable is of particular interest in rheumatic diseases. Objectives: to evaluate NT-proBNP in ankylosing spondylitis (AS) patients pre- and post-TNF blocker to determine the possible association between NT-proBNP levels and inflammatory parameters. Methods: Forty-five consecutive AS patients without previous/current cardiovascular disease or systolic myocardial dysfunction, who were eligible to anti-TNF therapy, were prospectively enrolled. All patients received TNF blockers and they were evaluated for circulating NT-proBNP levels, clinical and laboratory parameters of disease activity, traditional cardiovascular risk factors, and conventional and tissue Doppler imaging echocardiography at baseline (BL) and six months after (6M) treatment. Results: At BL, all patients had active AS, NT-proBNP levels had a median of 36 (20-72) pg/mL and 11% were high in spite of no systolic alteration. Multiple linear regression analysis revealed that this peptide, at BL, was independently correlated with ESR (p < 0.001), age (p = 0.01) and pulse pressure (p = 0.01). After 6M, all disease parameters improved and NT-proBNP levels were significantly reduced [24 (16-47) pg/mL, p = 0.037] compared to BL. Changes in NT-proBNP were positively correlated with ESR changes (r = 0.41, p = 0.006). Cardiovascular risk factors remained stable during follow-up. Conclusion: our data suggests that elevations of NT-proBNP should be interpreted with caution in active AS patients with no other evidence of cardiovascular disease. The short-term reduction of NT-proBNP levels in these patients receiving anti-TNF therapy appears to reflect an improvement in inflammatory status

Introdução: O fragmento amino-terminal do pró-peptídeo natriurético do tipo B (NT-proBNP) é um forte marcador de doença cardiovascular com evidências recentes de que a inflamação também pode influenciar seus valores. A diferenciação dessa variável de confusão é de particular interesse nas doenças reumáticas. Objetivos: Avaliar o comportamento dos valores de NT-proBNP em pacientes com espondilite anquilosante (EA) pré e pós uso de bloqueadores de TNF para determinar a possível associação entre os valores de NT-proBNP e os parâmetros inflamatórios. Métodos: Quarenta e cinco pacientes consecutivos com EA sem evidência prévia ou atual de doença cardiovascular ou disfunção miocárdica sistólica e que eram elegíveis para terapia anti-TNF foram incluídos prospectivamente. Todos os pacientes receberam bloqueadores de TNF e foram avaliados para concentrações circulantes de NT-proBNP, parâmetros clínicos e laboratoriais de atividade de doença, fatores de risco cardiovasculares tradicionais e ecodopplercardiografia convencional e tecidual no momento da inclusão e após seis meses de tratamento. Resultados: No momento da inclusão, todos os pacientes tinham EA ativa, os valores de NT-proBNP tinham uma mediana de 36 (20-72) pg/mL e 11% dos valores estavam altos mesmo na ausência de alteração miocárdica sistólica. A análise de regressão linear múltipla revelou que esse peptídeo estava independentemente correlacionado com o VHS (p < 0,001), com a idade dos pacientes (p = 0,01) e com a pressão de pulso (p = 0,01) no momento da inclusão. Após seis meses, todos os parâmetros relacionados a doença de base melhoraram e os valores de NT-proBNP se reduziram significativamente [24 (16-47) pg/mL, p = 0,037] quando comparados com os valores do momento da inclusão. As mudanças nos valores de NT-proBNP correlacionaram-se positivamente com as mudanças nos valores do VHS (r = 0,41, p = 0.006). Os fatores de risco cardiovasculares avaliados permaneceram estáveis durante o seguimento. Conclusão: As elevações nos valores de NT-proBNP devem ser interpretadas com cuidado nos pacientes com EA ativa e sem evidência de doença cardiovascular. A redução no curto prazo dos valores de NT-proBNP nesses pacientes recebendo terapia anti-TNF parece refletir uma melhora do estado inflamatório / Introduction: N-terminal pro-brain natriuretic peptide (NT-proBNP) is a strong marker of cardiovascular disease with recent evidence that inflammation may also influence its levels; discrimination of this confounding variable is of particular interest in rheumatic diseases. Objectives: to evaluate NT-proBNP in ankylosing spondylitis (AS) patients pre- and post-TNF blocker to determine the possible association between NT-proBNP levels and inflammatory parameters. Methods: Forty-five consecutive AS patients without previous/current cardiovascular disease or systolic myocardial dysfunction, who were eligible to anti-TNF therapy, were prospectively enrolled. All patients received TNF blockers and they were evaluated for circulating NT-proBNP levels, clinical and laboratory parameters of disease activity, traditional cardiovascular risk factors, and conventional and tissue Doppler imaging echocardiography at baseline (BL) and six months after (6M) treatment. Results: At BL, all patients had active AS, NT-proBNP levels had a median of 36 (20-72) pg/mL and 11% were high in spite of no systolic alteration. Multiple linear regression analysis revealed that this peptide, at BL, was independently correlated with ESR (p < 0.001), age (p = 0.01) and pulse pressure (p = 0.01). After 6M, all disease parameters improved and NT-proBNP levels were significantly reduced [24 (16-47) pg/mL, p = 0.037] compared to BL. Changes in NT-proBNP were positively correlated with ESR changes (r = 0.41, p = 0.006). Cardiovascular risk factors remained stable during follow-up. Conclusion: our data suggests that elevations of NT-proBNP should be interpreted with caution in active AS patients with no other evidence of cardiovascular disease. The short-term reduction of NT-proBNP levels in these patients receiving anti-TNF therapy appears to reflect an improvement in inflammatory status