L’infection à VIH-1 est associée à une forte déplétion des lymphocytes T CD4+ à polarisation Th17 au niveau des tissus lymphoïdes associés aux muqueuses intestinales (GALT, gut-associated lymphoid tissues). Ceci conduit à la translocation microbienne, qui est une cause d’activation immunitaire chronique et de progression de la maladie. Les cellules épithéliales (CE) jouent un rôle critique dans le maintien de l’intégrité et de l’homéostasie au niveau des muqueuses intestinales via le recrutement des cellules de l’immunité innée (e.g., neutrophiles) et adaptative (e.g., cellules Th17). Les neutrophiles produisent des molécules antivirales (e.g., défensines-) et ont la capacité de limiter la réplication virale au niveau des muqueuses. Les cellules Th17 jouent un double rôle lors de l’infection à VIH. Elles contribuent d’une part à la défense contre différents pathogènes opportunistes en augmentant, via la production d’IL-17, la capacité des CE à attirer les cellules Th17 et les neutrophiles. D’autre part, les cellules Th17 jouent un rôle délétère en tant que cibles de réplication virale et sources de cytokines pro-inflammatoires. La fréquence des cellules Th17 est diminuée dans les GALT mais pas dans les poumons des patients infectés par le VIH, suggérant qu’il existe des mécanismes différents par lesquels les cellules Th17 sont recrutées vers ces sites anatomiques. Nous avons testé l’hypothèse selon laquelle le VIH interfère avec la capacité des CE intestinales et non pas pulmonaires à produire des chimiokines (CK) responsables de l’attraction des cellules Th17 et des neutrophiles. Nous avons démontré que les CE intestinales et pulmonaires produisent des CK spécifiques pour les cellules Th17 (CCL20) et les neutrophiles (CXCL8) en réponse à des stimuli pro-inflammatoires tels que l’IL-1 et le TNF-. Le TNF- agit en synergie avec l’IL-17, un « signal de danger » récemment identifié, et augmente la capacité des CE intestinales mais pas pulmonaires à produire la chimiokine CCL20. Cette synergie s’explique par l’augmentation préférentielle de l’expression du récepteur à l’IL-17 à la surface des CE intestinales suite à la stimulation par le TNF-. L’exposition au VIH n’affecte pas la production de CCL20 et de CXCL8 par les CE intestinales, mais altère la capacité des CE alvéolaires à produire ces chimiokines en accord avec la permissivité sélective de ces dernières à l’infection par le VIH. En conclusion, nos résultats démontrent que (i) le VIH n’interfère pas directement avec la capacité des CE intestinales à recruter des cellules Th17 et des neutrophils et que (ii) la production de CCL20 par ces cellules est dépendantes de la synergie entre le TNF- et l’IL-17. Ainsi, la déplétion des cellules Th17 et la pénurie en IL-17 dans les GALT des sujets infectés pourrait causer de façon préférentielle des altérations fonctionnelles au niveau des CE intestinales, se traduisant par l’altération du recrutement des cellules Th17 en réponse au CCL20. / The HIV-1 infection is associated with a severe loss of CD4+ T-cells with Th17 polarization from the gut-associated lymphoid tissues (GALT). These alterations lead to microbial translocation, which is a cause of chronic immune activation and disease progression in HIV-infected subjects. Epithelial cells (EC) play a critical role in maintaining mucosal integrity and homeostasis in the GALT by mechanisms including recruitment of innate (e.g., neutrophils) and adaptive immunity cells (e.g., Th17 cells). Neutrophils produce antiviral molecules (e.g., -defensins) that may limit HIV replication at mucosal sites. Th17 cells play a dual role in HIV pathogenesis. Th17 cells contribute to the defence against different opportunistic pathogens by increasing the ability of epithelial cells to attract neutrophils in an IL-17-dependent manner. On the other hand, Th17 cells play a deleterious role in HIV pathogenesis as they are sites of productive viral replication and a source of pro-inflammatory cytokines. The frequency of Th17 cells is decreased in the GALT but not in the lungs of HIV-infected individuals, suggesting distinct mechanisms of Th17 recruitment in these anatomic sites in the context of HIV pathogenesis. In this manuscript we tested the hypothesis that HIV differentially interfere with the ability of intestinal but not pulmonary EC to produce chemokines that attract Th17 cells and neutrophils. We demonstrated that both intestinal and pulmonary EC produce chemokines that specifically attract Th17 cells (CCL20) and neutrophils (CXCL8) upon stimulation with the pro-inflammatory cytokines IL-1 and TNF- . TNF-α acted in synergy with IL-17, a recently identified « danger signal », and increases the capacity of intestinal but not pulmonary EC to produce CCL20. This synergistic effect can be explained by the preferential upregulation of IL-17 receptor expression on intestinal EC upon TNF- stimulation. The exposure of intestinal EC to HIV did not affect their ability to produce CCL20 and CXCL8; however, exposure to HIV altered the production of these chemokines by alveolar EC, consistent with their selective permissiveness to infection. In conclusion, our results demonstrate that (i) HIV does not interfere directly with the ability of intestinal EC to attract Th17 cells and neutrophils and that (ii) the ability of intestinal EC to recruit the Th17 cells via CCL20 production is selectively dependent on the synergy between TNF- and IL-17. Thus, the depletion of Th17 cells and the shortage in IL-17 in the GALT of HIV-infected subjects may preferentially lead to functional alterations of the intestinal barrier resulting by the alteration of Th17 recruitment in response to CCL20.

O câncer de mama é um dos tumores malignos mais comuns e que afeta um grande número de mulheres da população mundial. O processo inflamatório gerado juntamente com o crescimento descontrolado das células tumorais promove um estresse metabólico no microambiente tumoral levando ao acúmulo de adenosina extracelular decorrente da hipóxia tecidual. A adenosina gerada e seus efeitos mediados via receptor A2A (A2AR) interfere em vários subtipos celulares. Neste trabalho, avaliamos o papel do receptor A2A na indução de uma resposta de células Th17 durante o carcinoma mamário experimental, visto que estas desempenham um importante papel no crescimento e na progressão do tumor de mama invasivo. Para isso, utilizamos o modelo de carcinoma mamário murino que desenvolve (4T-1) e o que não desenvolve metástase (67NR). Nossos resultados mostraram que o tumor mamário 4T-1 apresenta alta expressão do receptor A2A comparado com o tumor 67NR. A deficiência do receptor A2a preveniu o crescimento do tumor mamário 4T- 1 e de colônias de células tumorais em sítios secundários da doença, concomitante com a diminuição da resposta de perfil Th17 e do recrutamento de neutrófilos para o sitio primário. Ainda, que as células tumorais 4T-1 apresentaram uma alta expressão dos receptores de adenosina e das ectonucleotidases (CD73 e CD39), sugerindo que a via de sinalização de adenosina exerce um efeito direto nessas células. A administração de adenosina ou AMP (trifosfato de adenosina) em culturas de células tumorais 4T-1 induziu xvi um aumento da expressão de IL-6, CCL20 e CXCL1, mediadores importantes para o recrutamento de linfócitos T produtores de IL-17 e de neutrófilos. Além disso, durante o crescimento do tumor 4T-1, observamos uma alta expressão da enzima ciclooxigenase 2 (Cox-2) comparado com o tumor 67NR. No entanto, os animais deficientes geneticamente do receptor A2A apresentaram uma redução significativa da expressão de Cox-2 no microambiente tumoral comparados com os animais BALB/c. A inibição da enzima Cox-2 com indometacina ou celecoxicib (inibidor seletivo) em animais com tumor 4T-1 preveniu o crescimento do tumor primário e, consequentemente, resultou na redução da expressão de moléculas relacionadas como o perfil de resposta de células Th17 tais como IL-6, CCL20, IL-17A e Ror?t. Esses resultados indicam que o bloqueio da via de sinalização do receptor A2A interfere na indução de resposta de células Th17, abrindo novas perspectivas para o desenvolvimento de terapias alternativas para o controle de tumores invasivos / Breast cancer is one of the most common and aggressive malignant tumors, affecting a large number of women in the worldwide population. The inflammatory process generated along with the uncontrolled growth of tumor cells promotes metabolic stress in the tumor microenvironment leading to the accumulation of extracellular adenosine due to the generation of tissue hypoxia. The generated adenosine and its effects mediated by A2A (A2AR) interfere in several cell subtypes. In this study, we evaluated the role of A2A receptor in the induction of a Th17 cell response in experimental breast carcinoma, given that these cells play an important role in invasive breast tumor growth and progression. For this, we employed the murine mammary metastatic (4T-1) and nonmetastatic carcinoma (67NR) model. Our results showed that there is high expression of adenosine A2A receptor in breast tumor 4T-1 compared to 67NR. The A2AR deficiency prevented the growth of metastatic breast tumor 4T-1 and tumor cell colonies in secondary disease sites, together with a decrease in Th17 response profile and in the neutrophils recruitment to the primary site. The tumor cells 4T-1 presented high expression of adenosine receptors and ectonucleotidases (CD73 and CD39), suggesting that the adenosine signaling pathway has a direct effect on these cells. Adenosine or AMP (adenosine triphosphate) administration in tumoral 4T-1 cell cultures induced an increase in IL-6, CCL20 and CXCL1 expression, important mediators in the recruitiment of IL-17 producing T lymphocytes and neutrophils. Besides that, during 4T-1 tumor growth we observed high expression of cyclooxygenase 2 (Cox-2) compared to 67RN tumor. However A2A deficient mice showed a significative reduction in Cox-2 expression in tumoral microenvironment compared to BALB/c mice. Cox-2 inhibition with indometacine or celecoxib (selective inhibitor) in mice with 4T-1 tumor prevented primary tumor growth and, consequently, resulted in reduction of the expression of molecules related to the Th17 profile, as IL-6, CCL20, IL-17A and Ror?t. These results indicate that the blockade of the A2A signaling pathway interfere on the induction of Th17 cells response, opening new perspectives for the development of alternative therapies to the control of invasive tumors

Introdução: O Papilomavírus Humano está muito bem associado com diversos tipos de cânceres humanos, como câncer anogenital e oral. Alguns estudos demonstram que o aparecimento de lesões e a progressão para o câncer estão relacionados ao tipo de resposta imune do hospedeiro. Deste modo, evidências indicam que a resposta imune do hospedeiro tem um papel muito importante para o curso da infecção pelo HPV. Objetivo: Avaliar a resposta imune específica in vitro ao Papilomavírus Humano (HPV) em homens com lesões causadas por HPV e sem lesão por HPV. Material e Métodos: Foram recrutados 31 pacientes e 11 voluntários, que formaram 4 grupos de estudo; sendo 12 pacientes no Grupo A (HIV +/ HPV +); 09 pacientes no Grupo B (HIV-/HPV+); 10 pacientes no Grupo C (HIV+/ HPV-); e 11 indivíduos saudáveis no Grupo D (HIV-/HPV-). Foram realizados ensaios de cultura celular para mensurar a resposta celular específica \"in vitro\" do tipo Th1/Th2/Th17 (INF-y, IL-2, TNFalfa, IL-4, IL-10 e IL-17) sob o estímulo da vacina quadrivalente do HPV (HPV 6, 11, 16 e 18) e à proteína E7 de HPV-16. Resultados: O grupo coinfectado (HIV +/ HPV+) apresentou níveis mais elevados de citocinas, principalmente do perfil Th2, comparando-se com os dados dos demais grupos de estudo. O grupo coinfectado apresentou níveis elevados de IL-6 e IL-10 (Perfil Th2) em relação ao grupo controle (HIV-/HPV-), com significância estatística (p < 0.0001 e p < 0.0001, respectivamente). Conclusão: Foi demonstrada uma elevada produção de citocinas no grupo HPV+/HIV+, sugerindo uma forte imunomodulação pela coinfecção HIV/HPV. Entretanto, novos estudos devem ser realizados para comprovar estes dados. Além de apresentar um perfil essencialmente Th2 do grupo coinfectado, principalmente pelos níveis elevados de IL-6 e IL-10 apresentados, sugerindo que estas duas citocinas possam servir como biomarcadores para persistência viral, uma vez que, os pacientes soropositivos para HIV apresentam maior persistência de HPV, e monitorar a progressão para lesões mais graves / Introduction: Human Papillomavirus is associated with different types of human cancers, such as anogenital and oral cancer. Some studies show that the appearance of lesions and progression to cancer are related to the type of host immune response. Thus, evidence indicates that the host immune response has a role key in the course of HPV infection. Objective: To evaluate the specific immune response in vitro to HPV in men with lesions caused by HPV and without injury caused by HPV. Methods: We recruited 31 patients and 11 volunteers, who formed four groups, with 12 patients in Group A (HIV+/HPV+); 09 patients in Group B (HIV-/HPV+); 10 patients in Group C (HIV+/HPV-) and 11 healthy subjects in Group D (HIV-/HPV-). Cells culture assay was performed to measure the specific immune response \"in vitro\" Th1/Th2/Th17 (IFN-y, IL-2, TNF-alfa, IL-4, IL-10 and IL-17) under the stimulation of quadrivalent HPV vaccine (HPV 6, 11, 16 and 18) and the E7 protein of HPV-16. Results: The coinfected group (HIV+/HPV+) had higher levels of cytokines, especially Th2 profile, compared with data from the other study groups. The coinfected group showed high levels of IL-6 and IL-10 (Th2 profile) compared to the control Group (HIV- /HPV-), with statistical significance (p < 0.0001 and p < 0.0001, respectively). Conclusion: This study demonstrated a high production of cytokines in the coinfected group, suggesting a strong immunomodulation by coinfection HIV/HPV. However, further studies should be conducted to confirm these data. In addition to presenting essentially a Th2 profile, especially by high levels of IL-6 and IL-10 presented, suggesting that these two cytokines may serve as biomarkers for viral persistence, since HIV seropositive patients have a higher persistence of HPV, and monitor the progression to more serious injuries

In the gut-associated lymphoid tissues, Th17 cells mediate mucosal homeostasis and inflammation. During HIV infection, Th17 cells become depleted and functionally impaired, which is implicated in the pathogenesis of chronic inflammation in patients treated with highly active antiretroviral therapy. IL-7 is a cytokine that mediates homeostatic responses in T lymphocytes, such as proliferation and survival, which are dysregulated during HIV infection. Whether similar dysregulation occurs in Th17 cells has yet to be reported. IL-7 receptor α (CD127) expression and IL-7 responses were therefore measured in blood-derived Th17 cells from uninfected individuals and effectively treated, HIV-infected individuals by flow cytometry. Th17 cells from uninfected individuals expressed CD127 and, in response to IL-7, exhibited phosphorylation of STAT5, upregulation of Bcl-2, and proliferation. During HIV infection, expression of CD127 and pSTAT5 in Th17 cells was comparable to that observed in cells from uninfected individuals. Interestingly, expression of Bcl-2 was upregulated while proliferation was dramatically impaired. These findings may provide further insight into the mechanisms by which Th17 cells fail to become restored during HIV infection.

No description available.

No description available.

Em países tropicais como o Brasil, as micoses superficiais (dermatofitoses) são comumente encontradas. O Dermatófito mais comum é o Trichophyton rubrum (Tr). Mananas e galactomananas na parede do Tr podem suprimir a resposta celular ao fungo. Quanto à resposta imune antifúngica, sabe-se a importância da via Th17. Algumas lectinas do tipo C (CLRs) como o receptor de manose e/ou receptores similares a Toll (TLRs) regulam o equilíbrio entre as vias Th1 e Th17. Nossos objetivos foram obter um extrato antigênico de Tr que induza resposta imune celular; quantificar e qualificar a resposta imune de indivíduos controles com lesão branda e de pacientes com dermatofitose extensa e/ou persistente causadas pelo Tr e por fim, avaliar a expressão de CLRs em monócitos do sangue periférico nos mesmos grupos. Para tanto, produzimos 11 extratos antigênicos de Tr. Pudemos observar na eletroforese em gel de poliacrilamida proteínas com pesos moleculares de aproximadamente 70 kDa e 38 kDa para os extratos fúngicos: Extrato TCA - Meta 1, Extrato tindalizado G1 e Extrato Coca 1. Avaliamos a resposta linfoproliferativa de células mononucleares por incorporação de timidina triciada em controles e pacientes ao peptídeo YIIDTGIDID do fungo Tr (Tri R2) e aos extratos antigênicos produzidos em nosso laboratório. Utilizamos como estímulos: PWM, CMA, Tri R2, PMA/Ionomicina. Para os ensaios funcionais avaliamos quatro pacientes e 6 indivíduos controles. Para a fenotipagem das células Th17, Th17MEM, Tc17 e Tc17MEM por citometria de fluxo, utilizamos a análise Booleana no software FlowJo X. A avaliação da expressão dos CLRs: CD206 (Receptor de Manose), Dectin 1 e Dectin 2 em monócitos do sangue periférico de controles e pacientes foi efetuada por citometria de fluxo. Dos 11 extratos produzidos de Tr, 7 se mostraram bons estimuladores para pelo menos um dos controles analisados, expressos como ponto máximo de índice de estimulação (p.m.I.E.). Dentre eles destacamos: Extrato TCA - Meta 1 (p.m. I.E. = 14,49 em 5 ug/mL), Extrato Tindalizado G1 (p.m.I.E. = 23,00 em 2,5 ug/mL) e Extrato Coca 1 (p.m.I.E. = 173,36 em 0,31 ug/mL). Na avaliação da expressão dos receptores das células Th17 e Tc17 (Th17R e Tc17R, respectivamente) após seis dias de estímulo por: Tri R2, extrato Coca 1 e o extrato TCA - Meta 1, o extrato Coca se mostrou o melhor estimulador para as populações Th17, com a frequência de 8,40% (controle) e 12,30% (paciente 1). Na avaliação da expressão de Th17R e Tc17R por 6 horas ao estímulo por PMA/Iono, todos os controles (n=3) se mostraram responsivos e no grupo de pacientes (n=3) pudemos observar maior frequência para o paciente 1 nas populações Th17 (1,64%) e Th17MEM (3,27%), e para as células Tc17 (10,70%) e Tc17MEM (3,58%). Observamos redução da expressão de CLRs nos pacientes: CD206: média 60,24% (controles) e 21,27% (pacientes), Dectin 1: 22,42% (controles) e 12,06% (pacientes) e Dectin 2: 20,26% (controles) e 4,99% (pacientes). Controles (n=6) e pacientes (n=3). A inovação na produção de extrato antigênico Extrato TCA - Meta 1 encoraja o estudo dos extratos fúngicos, para se obter melhores condições de avaliações imunológicas em pacientes com dermatofitose. Caracterizamos e qualificamos a resposta imune celular frente ao peptídeo TriR2 e aos extratos antigênicos, além de avaliarmos a expressão dos CLRs nesse grupo especial de pacientes / In tropical countries like Brazil, superficial fungal infections (dermatophytosis) are commonly found. The most common dermatophyte is Trichophyton rubrum (Tr). Mannans and galactomannans of Tr cell wall can suppress cellular responses to the fungus. Regarding the antifungal immune response, the importance of Th17 pathway is warranted. Some C-type lectins (CLRs) as the mannose receptor and / or Toll-like receptors (TLRs) regulate the balance between Th1 and Th17 pathways. Our objectives were to obtain an antigenic extract of Tr to induce cellular immune response; to quantify and classify the immune response of control subjects with mild injury and patients with extensive and / or persistent dermatophytosis caused by Tr, and finally evaluating the expression of CLRs in peripheral blood monocytes in the same groups. Therefore, we produced 11 antigenic extracts of Tr. Proteins with molecular weights of approximately 70 kDa and 38 kDa were evidenced in polyacrylamide gel electrophoresis for the following fungal extracts: extract TCA - Target 1, Tindalized extract G1 and extract Coca 1. We assessed the lymphoproliferative response of mononuclear cells by tritiated thymidine incorporation in the controls and patients, stimulated by YIIDTGIDID peptide fungus Tr (Tri R2) and the antigenic extracts produced in our laboratory. We used as stimuli: PWM, CMA, Tri R2, and PMA/Iono. For functional assays we evaluated four patients and 6 control individuals. For the phenotyping of Th17 cells, Th17MEM, Tc17 and Tc17MEM by flow cytometry, we used a Boolean analysis performed by FlowJo X software. Evaluation of the expression of CLRs: CD206 (mannose receptor), Dectin 1 and Dectin 2 in peripheral blood monocytes from patients and controls was performed by flow cytometry. Of the 11 extracts produced from Tr, seven proved to be able to stimulate proliferation of peripheral blood mononuclear cells of at least one of the analyzed controls, expressed as peak stimulation index (p.m.I.E.). Among them, were included: extract TCA - Target 1 (pmIE = 14.49 at 5 ug /mL), Tindalized G1 Extract (pmIE = 23,00 at 2.5 ug /mL) and extract Coca 1 (pmIE = 173.36 at 0.31 ug /mL). In the evaluation of the expression of receptors of Th17 cells and Tc17 (Th17R and Tc17R, respectively) after six days of stimulation by: Tri R2, Coca extract and the extract TCA 1 - Meta 1, Coca extract showed to be the best stimulator for Th17 populations, with the frequency of 8.40% (control) and 12.30% (patient 1). In the evaluation of the expression of Th17R and Tc17R after 6 hours of stimulation by PMA / Iono, all controls (n = 3) responded and in the group of patients (n = 3) we observed response more frequently for the patient #1, in Th17 populations (1.64%), Th17MEM (3.27%), Tc17 cells (10.70%) and Tc17MEM (3.58%). We observed a reduction of expression of CLRs in patients: CD206: average 60.24% (controls) and 21.27% (patients), Dectin 1: 22.42% (controls) and 12.06% (patients) and Dectin2: 26% (controls) and 4.99% (patients). Controls (n = 6) and patients (n = 3). Innovation in the production of antigenic extract extract TCA - Target 1 encourages the study of fungal extracts to obtain better conditions of evaluation of the immune response in patients with dermatophytosis. We characterized and qualified the cellular immune response to the TriR2 peptide, to antigen extracts, and evaluated the expression of CLRs in this special group of patients

Les cellules T auxiliaires TH17 sont caractérisées par l’expression de la cytokine IL-17A, ainsi que le facteur de transcription RORɣt. Elles sont connues pour jouer un rôle clé dans la pathogenèse de la sclérose en plaques. Ces cellules existent sous deux formes : les cellules régulatrices, immunomodulatrices, et les cellules pathogènes qui sont critiques pour l'inflammation. Il est donc important de comprendre le mécanisme qui sous-tend la différenciation des cellules TCD4+ naïves en ces deux types cellulaires. J'ai trouvé que le facteur de transcription Ikaros est un répresseur indirect de la transcription des gènes pathogéniques (Il3, Csf2, Ifng, Stat4…) dans les cellules TCD4+ naïves murines, cultivées pour induire une polarisation vers le phénotype TH17 régulateur. De plus, en absence d’Ikaros et en conditions de culture régulatrice, l’ajout d’IL-6 seul augmente l’expression de GM-CSF, facteur clé dans l’induction des maladies auto-immunes, suggérant un rôle d’Ikaros dans la régulation de cette voie. En conclusion, nos résultats suggèrent que Ikaros est nécessaire pour polariser correctement les cellules TCD4+ naïves dans le programme TH17. / TH17 cells are characterized by the expression of the cytokine IL-17A, as well as the transcription factor RORɣt. They are known to play key role in the pathogenesis of the multiple sclerosis. These cells exist in two forms: the regulating cells, immunomodulatory, and the pathogenic cells which are critical for the inflammation. Thus it is important to understand the mechanism which underlies the differentiation of naïve CD4+ T cells in these two cellular types. I found that the transcription factor Ikaros is an indirect repressor of the transcription of pathogenic genes (Il3, Csf2, Ifng, Stat4…) in naïve CD4+ T cells, cultured to induce a polarization toward regulatory TH17 cells. Moreover, in absence of Ikaros and in regulatory condition of culture, adding IL-6 alone increases the expression of GM-CSF, key factor to induce auto-immune diseases, suggesting a role of Ikaros in this pathway. In conclusion, our results suggest that Ikaros is necessary to polarize correctly naïve CD4+ T cells in TH17 cells.

Psoriasiform diseases are a group of cutaneous disorders that are characterized by impaired keratinocyte maturation leading to epidermal hyperplasia and thickening of skin. This group of disorders includes psoriasis, seborrheic dermatitis (SD) and mycosis fungoides (MF). Psoriasis has been recently shown to be mediated by the pro-inflammatory T helper cell subset, namely Th17 cells, whereas the pathogenesis of SD and MF are still poorly understood. SD is characterized by inflamed skin that primarily manifests on areas populated with sebaceous glands. Interestingly, SD is very common amongst immunosuppressed patients such as those with HIV-AIDS, suggesting the importance of an immune response in the development of SD. Because SD shares common clinical and histopathological features with psoriasis, a disease in which Th17 axis cytokines is known to be involved, and given that Th17 cells and their related cytokines have been implicated in the pathogenesis of a wide range of autoimmune and inflammatory disorders, it is possible that Th17 axis cytokines play a role in the pathogenesis of SD. We explored the involvement of Th17 axis cytokines in a D2C mouse model of psoriasiform disease that shows a high degree homology to the clinicopathological characteristics of human seborrheic dermatitis. IL-6 and IL-23, which are important for the differentiation of Th17 cells, and IL-17 and IL-22, which are the Th17 effector molecules, were measured at both protein and mRNA levels in sera and lesional skin from D2C mice. An immunohistochemical analysis was also performed to detect the presence of IL-17 in D2C lesional skin relative to normal skin from DBA/2 controls. Our data demonstrated significantly elevated levels of IL-6, IL-17 and IL-22 in sera from diseased D2C mice compared to controls and/or convalescent mice. There were no significant differences in IL-23 protein levels in sera from D2C mice compared to those from wild type mice or convalescent D2C mice. RT-PCR revealed a significant increase in IL-23 and IL-17 gene expression in D2C lesional skin relative to normal skin. Gene expression levels of IL-22, but not IL-6, were statistically significant elevated in D2C skin lesions compared to controls, by real time PCR. Our IHC study of IL-17 expression showed an abundance of positively stained mononuclear cells in D2C lesional skin relative to DBA/2 normal skin. Altogether, our data demonstrate that Th17 axis cytokines are elevated locally at mRNA levels for IL-23, IL-17, and IL-22 and systematically at protein levels for IL-6, IL-17, and IL-22. This data lay the foundation for further studies investigating a role for Th17 axis cytokines in the cutaneous inflammatory disease seen in our mouse model of SD and, ultimately, in the development of human SD. Mycosis fungoides (MF) is the most common type of cutaneous T cell lymphoma (CTCL). The etiology of MF is unknown, but there is substantial evidence suggesting a potential role for a yet unidentified infectious agent in the pathogenesis of MF. Many studies have claimed that there is an association between MF and the Human T cell Lymphotorpic Virus Type 1 (HTLV-I); however, the involvement of this virus in the etiology of MF is a controversial topic. In our study, we used nested PCR to explore the association between HTLV-I infection and MF by screening genomic DNA from 114 skin biopsies for the presence of HTLV-I provirus. We also utilized a ViroChip and high-throughput sequencing (HTS), as a case study, to attempt to detect novel virus-specific oligonucleotides that may be associated with CTCL. Our data showed no evidence for HTLV-I proviral integration in the 114 MF samples that were screened using nested-PCR. The ViroChip and HTS results also did not reveal any signature sequence for known or unknown infectious agent in the CTCL case study. Collectively, this data argue against the involvement of HTLV-I provirus in the pathogenesis of MF.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. It is estimated that less than 1% of the individuals infected with M. leprae develop the disease. Several authors suggest that the genetic pattern and variations in the mechanisms of the patient's immune response influence the susceptibility or resistance to disease. The most recent studies have established the role of Th1, Th2 and Treg cell responses in immunopathogenesis of leprosy. However, several mechanisms of the immune response that act in clinical evolution still lack clarification, such as the role of Th17 cells, and the innate immune response. The objective of this study was to evaluate the role of the immune response in the clinical presentation of leprosy and the use of M. leprae recombinant antigens as a perspective for the development of prognostic and immunoprophylaxis tools. To investigate the involvement of immune response in the pathogenesis of leprosy, we analyzed the cytokine profile in lesions, in serum, and peripheral blood mononuclear cells (PBMC) stimulated with M. leprae antigens in leprosy patients and household contactants (HHC). CD4+IL-17+ T cells expressing IL-17A, IFN-γ and IL-10 were evaluated by confocal microscopy in lesions from patients with tuberculoid (TT, n = 09) and lepromatous leprosy (LL, n = 08). Inflammatory cytokines were measured in serum samples from 23 paucibacillary (PB) patients, 28 multibacillary (MB) and 23 HHC, using the Luminex technique. The phenotype of lymphocytes producing IL-17A and IFN-γ was determined by flow cytometry. In addition, PBMC from leprosy patients and HHC were stimulated with crude M. leprae (MLCS) and M. tuberculosis (PPD) and a recombinant antigen of M. leprae (ML2028), and the cytokine profile and the CD4+ and CD8+ multifunctional T cells (producing IFN-γ, IL-2 or TNF-α) of effector and central memory were analyzed. We observed that TT lesions expressed more CD4+IL-17A+ cells than LL. Higher levels of IFN-γ were detected in PB patients, but also in MB patients who presented leprosy reactions (LR) at the time of evaluation (MB LR+). Significantly, higher concentrations of IL-17A and IL-1β were observed in serum from PB than in from MB patients. Ex vivo cell analysis by flow cytometry revealed higher frequency of Th17 cells in TT than LL patients, and it is not high in LL patients with LR. These results indicate that the Th17 cells are associated with an effective inflammatory response that occurs in PB presentation of leprosy but were not associated with the inflammatory response in LR. Th1 response was also associated with PB presentation. However, high levels of IFN-γ were also associated with LR. Multiparameter analyzes by flow cytometry revealed a higher frequency of multifunctional T cells specific for M. leprae antigens in HHC than in leprosy patients, and it might explain the absence of disease in these individuals. These data indicate that these antigens are capable of inducing a more effective immune response and multifunctional T cell memory against M. leprae infection, and open perspectives for the future development of immunoprophylaxis with M. leprae antigens. Additionally, this study suport the attempt to induce a Th1 and Th17 response in individuals at risk of acquiring the disease, even considering this could induce only a partial protection, because it would protect against the most severe MB forms of leprosy, and reduce the disease transmission. This Thesis includes one paper accepted for publication, about the role of Th1 and Th17 cells in subjects with different clinical forms of leprosy, and another paper submitted about the immune response to M. leprae crude and recombinant antigens. / A Hanseníase é uma doença infecciosa crônica e de evolução lenta causada pelo Mycobacterium leprae. A literatura sugere que menos de 1% dos indivíduos infectados pelo bacilo evolui com a doença. O padrão genético dos indivíduos e diferenças nos mecanismos da resposta imune do paciente influenciam na susceptibilidade ou resistência à infecção e apresentação clínica da doença. Os estudos mais recentes estabeleceram o papel das respostas de células Th1, Th2 e Treg na imunopatogênese da hanseníase. No entanto, diversos mecanismos das células Th17 e o papel da resposta imune inata na doença ainda não estão bem estabelecidos. Diante disso, este estudo teve como objetivo avaliar o papel da resposta imune na apresentação clínica da hanseníase e o uso de antígenos brutos e recombinante de M. leprae como perspectiva para o desenvolvimento de ferramentas de prognóstico e imunoprofilaxia. Para investigar o envolvimento das células da resposta imune na patogênese da hanseníase, analisamos o perfil de citocinas em lesões, nos soros, e em células mononucleares do sangue periférico (PBMC) estimuladas com antígenos de M. leprae em pacientes com hanseníase e controles contactantes sadios (CCS). As células T CD4+IL-17+ e que expressam IL-17A, IFN- e IL-10 foram avaliadas por microscopia confocal em biópsias de lesões de pacientes com hanseníase tuberculóide (HT, n = 9) e virchowiana (HV, n = 8). As citocinas inflamatórias foram dosadas em amostras de soro de 23 paucibacilares (PB), 28 multibacilares (MB) e em 23 CCS, pela técnica de Luminex. O fenótipo de linfócitos produtores de IL-17A e IFN-γ foi determinado por citometria de fluxo. Além disso, PBMC de pacientes com hanseníase e de CCS foram estimuladas com os antígenos brutos de M. leprae (MLCS), M. tuberculosis (PPD) e recombinante de M. leprae, (ML2028), e o perfil de citocinas e o fenótipo das células T CD4+ e CD8+ multifuncionais (produtoras de IFN-γ, IL-2 ou TNF-α) de memória efetora e central foram analisados. Observamos que as lesões de HT expressaram mais células CD4+IL-17A+ do que as de HV. Níveis mais elevados de IFN-γ sérico foram detectados em pacientes com as formas HT e em MB que apresentavam reações hansênicas (MB RH+). Concentrações mais elevadas de IL-17A e IL-1β foram observadas nos soros de pacientes PB do que em MB. As análises das células ex vivo por citometria de fluxo revelaram maior frequência de células Th17 nos pacientes com hanseníase tuberculóide (HT) em comparação com aqueles com hanseníase virchowiana (HV). Estes resultados indicam que a resposta Th17 está associada a uma resposta inflamatória efetiva que se apresenta nas formas PB, mas não estão associadas à resposta inflamatória durante as reações hansênicas. A resposta Th1 também está associada às formas PB, entretanto altos níveis de IFN-γ foram associados também aos episódios de reação hansênica. As análises multiparamétricas por citometria de fluxo revelaram maior frequência de células T multifuncionais antígeno-específicas em CCS, do que em pacientes com hanseníase. Nossos dados indicam que controles contactantes, quando estimulados com antígenos brutos e com o ML2028 recombinante, produziram mais células T multifuncionais e isto sugere que estas células proporcionam uma resposta imunológica mais eficaz contra a infecção por M. leprae, podendo explicar a ausência de doença nesses indivíduos. Estes dados sugerem que esses antígenos são capazes de induzir uma resposta protetora e indutora de células T multifuncionais de memória e abrem perspectivas para o desenvolvimento futuro de imunoprofilaxia com estes antígenos de M. leprae. Além disso, o estudo dá suporte à busca de induzir uma resposta Th1 e Th17 em indivíduos em risco de adquirir a doença, mesmo que haja uma proteção parcial, pois neste caso haveria uma proteção contra formas mais graves MB, e reduziria também a transmissão da doença. Esta tese é composta por um artigo aceito para publicação sobre a resposta de células Th17 e Th1 nas formas clínicas da hanseníase e outro submetido sobre a resposta a antígenos recombinantes de M. leprae na indução de células T multifuncionais.