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.

La paraparésie spastique tropicale ou la myélopathie associée à HTLV-1 (TSP/HAM) est une maladie neurologique chronique caractérisée par le développement de paralysies spastiques des membres inférieurs et de déficits sensoriels divers. Une infiltration périvasculaire souvent observée dans le système nerveux central des patients atteints de TSP/HAM correspondant essentiellement à des lymphocytes T CD4+, cibles préférentielles du HTLV-1 in vivo. Cependant, le facteur déclencheur du processus inflammatoire de la TSP/HAM est toujours méconnu. De ce fait, nous nous sommes intéressés à l'étude de l'implication d'une nouvelle population inflammatoire des T CD4+, les TH17, dans cette pathologie. Une quantification de l'expression de l'ARNm d'IL-17, la cytokine inflammatoire sécrétée par les TH17, a été menée sur les cellules du sang périphérique issues de patients infectés ainsi que sur des lignées cellulaires chroniquement infectées par HTLV-1. L'expression élevée de l'IL-17 détectée dans les lignées cellulaires est corrélée avec celle de la protéine transactivatrice du HTLV-1, Tax. Par ailleurs, Tax induit l'expression du régulateur transcriptionnel clé des TH17, RORγ, par l'intermédiaire de la cytokine pro-inflammatoire Ostéopontine. Finalement, nous avons pu montrer l'existence d'une relation dynamique entre l'expression de l'ARNm de Tax, OPN, RORγ, IL-17 et IL-22 chez les patients asymptomatiques et TSP/HAM avec une expression d'IL-17 et d'IL-22 plus élevée chez le groupe des TSP/HAM. Nos résultats suggèrent que l'infection par HTLV-1 in vivo induirait une réponse TH17 qui pourrait avoir un rôle majeur dans la pathogenèse de la TSP/HAM. / HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological inflammatory disease of the central nervous system characterized by a chronic, progressive inflammatory demyelinating myelopathy. It is thought that the pathogenesis of this disease involves a predominant infiltration of CD4+ T cells which are the main subset of in vivo infected cells with HTLV-1. However, until now, the identity of the triggering factor which promotes the inflammatory process in HAM/TSP remains unclear. Therefore, we investigated the implication of the new CD4+ T cells inflammatory lineage TH17 in this disease. We quantified the mRNA expression levels of IL-17, a cytokine associated with the TH17 response, in peripheral blood from 10 HAM/TSP patients, 6 healthy asymotomatic carriers (HCs) and 4 normal uninfected controls as well as in HTLV-1-infected T-cell lines. Elevated production of IL-17 observed in HTLV-1-infected T-cell lines was correlated with the expression of Tax, the major HTLV-1 regulatory protein. Thus, we established that Tax increases the expression of RORγ, the TH17-lineage specific transactivator, by inducing Osteopontin expression, an inflammatory cytokine known to promote TH17 response. Finally, we demonstrated a dynamic relationship between the expression of Tax, Osteopontin, RORγ, IL-17 and IL-22 mRNAs in HCs and HAM/TSP patients, where higher expression of IL-17 and IL-22 were observed in HAM/TSP cases. These findings suggest that in vivo infection by HTLV-1 may lead to a deleterious deviation of CD4+ T Helper response to TH17, that could play a major role in HAM/TSP pathogenesis.

L’oncologie actuelle est encore confrontée à la résistance et à la progression rapide des cancers. Les mécanismes de résistance intrinsèque développés par les cellules tumorales peuvent compromettre l’efficacité des chimiothérapies et des immunothérapies. Il est maintenant admis que l’état de la réponse immunitaire de l’hôte détermine en partie l’issue thérapeutique des patients. L’objectif de notre équipe de recherche est donc de caractériser cette réponse et d’étudier l’impact des thérapies conventionnelles sur celle-ci dans le but d’identifier les mécanismes liés à un échappement futur de la tumeur. Dans ce contexte, nous avons montré qu’une chimiothérapie (5-FU, oxaliplatine, anti-VEGF (« Vascular Endothelium Growth Factor » : FOLFOX-bevacizumab) provoque chez certains patients une chute des gMDSC (cellules myéloïdes immunosuppressives granulocytaires) périphériques qui est associée à une meilleure réponse thérapeutique. Comme chez la souris, cet effet sur les gMDSC provoque néanmoins une élévation des Th17, une population pro-angiogénique, qui limite l’efficacité de la chimiothérapie. La suite de notre travail a eu pour objectif de tester l’effet « anti-Th17 » de l’activation de l’histone désacétylase SIRT1. SIRT1 est une enzyme capable de perturber l’acétylation de STAT3, un facteur essentiel à la différenciation des Th17. Nous avons montré que l’utilisation d’agonistes pharmacologiques de SIRT1 (resvératrol, SRT1720, metformine) inhibe la polarisation des Th17 par la désacétylation de STAT3 et que cet effet permet de limiter la croissance tumorale dans un modèle de cancer colique et de mélanome chez la souris (B16F10, CT26). Nous avons validé ce concept chez l’homme, ce qui suggère qu’il est possible de cibler les Th17 par cette stratégie en complément de la chimiothérapie. Le dernier volet de ce travail est consacré à la comparaison du profil immunologique périphérique de volontaires sains à celui d’une cohorte prospective de cancers bronchiques non à petites cellules. Cette étude nous a permis de mettre en lumière les altérations immunitaires induites par la tumeur et de lier ces altérations à la réponse au nivolumab (anti-PD-1). Un premier modèle prédictif de réponse a pu être généré grâce aux données d’un panel d’analyse des cellules myéloïdes. Ce modèle révèle une fois encore que les cellules gMDSC ont un rôle prédictif défavorable, alors que les populations présentatrices d’antigènes (cellules dendritiques et monocytes) exprimant PD-L1 ont un bon rôle prédictif. Les données présentées dans cette partie sont préliminaires et devront être confirmées avec la cohorte de validation qui est en cours d’inclusion. L’ensemble de ce travail a permis de montrer qu’il est essentiel de cibler spécifiquement les cellules myéloïdes immunosuppressives et les Th17 pour favoriser l’efficacité des chimiothérapies et de l’immunothérapie dans le cancer. / Actual oncology is still facing resistance and rapid progression of cancer. Intrinsic resistance mechanisms developed by tumor cells determine chemotherapy and immunotherapy efficacy. It is now recognized that the host immune response status is in part implicated in the therapeutic outcome of patients. The aim of our research team is to characterize this response and to study the impact of therapies in order to identify the mechanisms associated with future exhaust of the tumor. In this context, we have shown that chemotherapy (5-FU, oxaliplatin, anti-VEGF: FOLFOX-bevacizumab) in some patients causes a drop in devices gMDSC (granulocytic myeloid derived suppressive cells) that is associated with better therapeutic response. Nevertheless, as in mice, this effect on gMDSC causes an elevation of Th17, a pro-angiogenic population, which limits the effectiveness of chemotherapy. The result of our work was aimed to test the effect "anti-Th17" activating SIRT1 deacetylase histone. SIRT1 is an enzyme capable of disrupting the acetylation of STAT3, a key factor in the differentiation of Th17. We have shown that by using pharmacological agonists SIRT1 (resveratrol, SRT1720, metformin) inhibits Th17 polarization by deacetylation of STAT3 and that this effect can limit tumor growth in colorectal and melanoma murine models (B16F10, CT26). We validated this concept in humans, suggesting that it is possible to target Th17 cells by this strategy in addition to chemotherapy. The final component of this work is devoted to the comparison of peripheral immunological profile of healthy volunteers to a prospective cohort of non-small cell lung cancer. This study has allowed us to highlight the immune alterations induced by the tumor and to link these changes in response to nivolumab (anti-PD-1). A first response predictive model could be generated using data from a panel analysis of myeloid cells. This model proves once again that gMDSC have a negative predictive role, while antigen presenting (dendritic cells and monocytes) expressing PD-L1 has a good predictive role. Data presented in this section are preliminary and must be confirmed with the validation cohort that is currently included. All of this work has shown that it is essential to specifically target immunosuppressive myeloid cells and Th17 to promote the efficacy of chemotherapy and immunotherapy in cancer.

O controle de respostas imunes patológicas (autoimunidade, alergia, rejeição de transplantes) tem sido um dos principais objetivos dos imunologistas. Apesar dos avanços recentes, a maioria dos tratamentos atuais ainda procura diminuir a imunidade e inflamação em vez de restabelecer o estado saudável da tolerância imunológica. Sepse é uma doença desencadeada pela presença de bactérias e/ou produtos bacterianos como lipopolissacarídeos (LPS), componente principal da membrana externa de bactérias gram-negativas, ativando a resposta imune do hospedeiro. A caracterização do perfil de linfócitos na resposta à tolerância ao LPS são de extrema importância para a contribuição do estudo da imunodepressão na sepse. O objetivo deste estudo foi investigar se a comprovada redução de mortalidade previamente vista em modelo de sepse animal através tolerância ao LPS, pode ser associada com o aumento da população de linfócitos T CD4+ regulatórios e Th17. Camundongos machos C57/6, receberam por via subcutânea ( s.c.) injecções de LPS ( 1mg/kg ) durante 5 dias , seguido por perfuração e ligadura cecal (CLP ) . Citocinas e linfócitos marcados foram medidos durante, após a tolerância e o desafio CLP. Ambos os subtipos de células T analisados Treg e Th17 , mostrou aumento destas células no baço durante e após a tolerância. Este estudo demonstrou que a mortalidade reduzida depois de tolerância previamente constatada pode ser associada com o aumento da população de células T regulatórias e Th17 devido a imunorregulação do hiperinflamação e recrutamento de neutrófilos / The control of pathological immune responses (autoimmunity, allergy, transplant rejection) has been a major goal of immunologists. Despite recent advances, most current treatments still seeks to reduce immunity and inflammation rather than restore the healthy state of immune tolerance. Sepsis is a disease triggered by the presence of bacteria and / or bacterial products like lipopolysaccharide (LPS), the main component of the outer membrane of gram-negative bacteria, activating the immune response of the host. The characterization of lymphocyte profile in response to LPS tolerance is extremely important for the study of immunosuppression in sepsis contribution. The aim of this study was to investigate whether the proven reduction in mortality seen previously in animal sepsis model by tolerance to LPS, can be associated with the increase in population of CD4 + regulatory and Th17. Mice C57 / 6 mice received subcutaneous (s.c.) injection of LPS (1mg / kg) for 5 days, followed by cecal ligation and puncture (CLP). Cytokines and marked lymphocytes were measured during after tolerance and CLP challenge. Both subtypes of T cells Treg and Th17 analyzed showed an increase of these cells in the spleen during and after tolerance. This study demonstrated that reduced mortality after previously seen tolerance may be associated with increasing the population of regulatory T cells and Th17 because immunoregulation of the hiperinflamação and neutrophil recruitment

Recentemente demonstrou-se o papel de células T produtoras de IL-17 na patogênese da esclerosa múltipla e de seu modelo, a EAE. Através da produção desta e de outras citocinas, a população chamada Th17 promove o rompimento da barreira hematoencefálica e a conseqüente infiltração de células patogênicas para dentro do SNC. Nesse contexto, em nosso trabalho utilizamos o fenômeno da tolerância oral para avaliar a capacidade deste em suprimir a resposta imune durante o modelo de EAE, mais especificamente as células Th17. Nossos dados demonstram uma diminuição de IL-17 tanto na periferia como no SNC dos animais tolerados. Além disso, detectamos menos CCL2 e IL-6 em células extraídas do CNS dia 10 pós-imunização. Não observarmos diferença na produção de IL-4,5,10, 13, IL-12p70, TNF-<font face=\"symbol\">a, e IFN-<font face=\"symbol\">g entre os grupos. Em suma, nossos resultados mostram que o fenômeno da tolerância oral é capaz de suprimir parâmetros de EAE devido a uma menor capacidade linfoproliferativa associada a uma supressão de células patogênicas Th17 tanto na periferia como no SNC. / It has recently been shown the role of IL-17 secreting cells on the pathogenesis of multiple sclerosis and also in its model, EAE. Due to the secretion of this and other cytokines, the population so called Th17, promotes the disruption of the blood-brain barrier and the following infiltration of pathogenic cells into the CNS. In this context, in our work we used the oral tolerance phenomenon to evaluate its supressive capacity, more specifically over the Th17 cells. We showed that oral tolerated mice has a diminished production of IL-17 both in the periphery and in the CNS. Futhermore, we detected lower levels of CCL2 and IL-6 also from brain and spinal cord extracted mononucear cells at day 10th post-immunization. We were not able to detect differences on IL-4,5,10, 13, IL-12p70, TNF-<font face=\"symbol\">a, e IFN-<font face=\"symbol\">g between the groups. Thus, our results show that the oral tolerance phenomenon suppresses EAE findings, mainly due to a lower lymphoprolipherative response associated to a supression over the expansion of Th17 pathogenic T cells both in the periphery and inside the CNS.

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

Le tabagisme aggrave de nombreuses maladies inflammatoires chroniques (BPCO, maladie de Crohn, arthrite rhumatoïde, psoriasis) associées à la sous-population de lymphocytes T CD4+ (LT CD4+) inflammatoires sécrétant l’IL-17 (Th17). Le tabagisme est associé à une augmentation en nombre et en proportion, du nombre de Th17 systémiques et pulmonaires, contrastant avec la rareté habituelle de ces cellules dans les tissus périphériques-sites d’inflammation. Le tabagisme est associé à l’augmentation de la sénescence des cellules pulmonaires récemment impliquée dans la pathogénèse de la BPCO. Les mécanismes responsables de l’augmentation des Th17 chez les fumeurs sont encore inconnus, et le rôle potentiel de la sénescence dans cette augmentation et la modification des fonctions des Th17 n’a jamais été exploré.Dans ce travail, nous avons fait l’hypothèse que les Th17 présentent une susceptibilité augmentée à la sénescence induite par l’exposition à la fumée de cigarette, en comparaison des autres sous-populations de LT CD4+, qui serait responsable de l’augmentation des Th17, et contribuerait à l’aggravation de leur potentiel inflammatoire. Nous avons analysé la susceptibilité des Th17 à la sénescence induite par l’exposition au condensat de FC (CFC), et le rôle de la voie de signalisation ERK1/2 dans ce phénomène. Nous avons également analysé le rôle des espèces réactives de l’oxygène (ERO), impliquées, d’une part dans la régulation de l’activation de ERK1/2, et d’autre part dans la sénescence cellulaire.Des lymphocytes T CD4+ CCR6+ Th17 et T CD4+ CCR6- quiescents de donneurs sains ont été exposés in vitro au CFC. La production d’ERO est mesurée en analysant l’oxydation de la sonde H2DCF-DA (cytométrie de flux). La sénescence est évaluée en analysant l’expression de p16INK4a (ImmunoFluorescence). L’analyse de l’expression des cytokines (Luminex/CBA) évalue le potentiel inflammatoire des cellules.Nos résultats montrent que les LT CD4+ Th17 quiescents exposés au CFC présentent une augmentation de différents marqueurs de sénescence : activité -galactosidase, expression des inhibiteurs du cycle cellulaire p16INK4a et ATF3. L’exposition au CFC modifie le profil sécrétoire des Th17 quiescents et induit leur sécrétion d’IL-8. Nous montrons que la voie des MAPKs ERK1/2 est impliquée dans l’induction du phénotype sénescent des Th17 en réponse à une exposition au CFC. La sur-expression de p16INK4a est associée à une activation et une translocation nucléaire de ERK1/2 plus importante dans les Th17. Le traitement antioxydant par la NAC, diminue l’expression des ERO et de p16INK4a induite par l’exposition au CFC dans toutes les sous-populations de LT CD4+, mais maintient la production d’ERO et l’expression de p16INK4a plus importantes dans les Th17 en comparaison des autres sous-populations de LT CD4+. Le traitement par l’inhibiteur du complexe III de la chaîne respiratoire mitochondriale, l’antimycine, maintient la production d’ERO plus importante dans les Th17, mais l’expression de p16INK4a est diminuée à des niveaux comparables dans tous les LT CD4+. En revanche, le traitement par l’agent découplant, le FCCP, diminue l’expression de p16INK4a et la production d’ERO à des niveaux comparables dans tous les LT CD4+, et abroge les différences de production d’ERO entre les Th17 et les T CD4+ CCR6-.Nous montrons que les lymphocytes Th17 humains présentent une susceptibilité plus importante à la sénescence induite par l’exposition au CFC en comparaison des autres sous-populations de LT CD4+. Nos résultats suggèrent que l’activité mitochondriale basale plus importante dans les Th17, est responsable de la plus grande susceptibilité des Th17 à la sénescence après exposition au CFC. Enfin, nous montrons pour la première fois qu’un découplage modéré de la chaîne respiratoire mitochondriale est une solution efficace pour prévenir la sénescence des Th17 et pourrait être une stratégie anti-inflammatoire dans les maladies chroniques associées aux Th17 / Smoking worsens chronic inflammatory diseases (COPD, Crohn disease, rheumatoid arthritis, psoriasis) associated with inflammatory CD4+ IL-17 secreting lymphocytes (Th17). Smoking is associated with an absolute number and proportion increase, at both systemic and pulmonary levels, of Th17 cells. This increase of Th17 cells in smokers contrasts with their usual rarity in peripheral tissues and inflammatory sites. Most of the knowledge about the effects of cigarette smoke exposure comes from COPD studies, and, in COPD increased senescence of pulmonary cells has been associated to the pathogenesis of the disease. The mechanisms responsible for the increase of Th17 cells are still unknown, and the potential role of senescence in this increase and functional modifications of Th17 in smokers has never been explored.In this study, we hypothesized that Th17 present a higher susceptibility to cigarette smoke-induced senescence, as compared to other CD4+ T lymphocytes subsets, which could be responsible for the increased number and proportion of Th17 in smokers, and the higher inflammatory potential of these cells. We analyzed senescence susceptibility of Th17 exposed to cigarette smoke condensate (CSC), and the potential role of ERK1/2 signaling pathway in this phenomenon. We also analyzed the potential role of reactive oxygen species (ROS) known to be implicated, on one hand in ERK1/2 activation, and on another hand in cellular senescence.Quiescent CCR6+ Th17 and CCR6- CD4+ T lymphocytes of healthy donors are exposed in vitro to cigarette smoke extract (CSE). ROS production is measured by H2DCF-DA oxidation (flow cytometry). Senescence is evaluated by p16INK4a expression (ImmunoFluorescence). Expression of relevant cytokines (Luminex/CBA) evaluated inflammatory potential.Our results show that quiescent CD4+ Th17 exposed to CSE present an increase of senescence markers: -galactosidase activity and expression of cell cycle inhibitors p16INK4a and ATF3. Moreover, CSE exposure modifies Th17 secretion pattern and increases IL-8 secretion. Our results also show that ERK1/2 MAPK pathway is implicated in Th17 senescent phenotype induction upon CSE exposure. The overexpression of p16INK4a is associated with a higher activation and a nuclear translocation of ERK1/2 in Th17 cells. Treatment with the anti-oxidant NAC reduces CSE-induced ROS and p16INK4a expression in all CD4+ T cell subsets, but the higher production of ROS and higher p16INK4a expression in Th17 as compared to other CD4+ T cells are maintained. Treatment with mitochondrial complex III inhibitor, antimycine, maintains the higher production of ROS in Th17 as compared to other CD4+ T lymphocytes, whereas p16INK4a expression is reduced to the same level in all subsets. Conversely, treatment with the mitochondrial decoupling agent, FCCP, reduces p16INK4a expression to the same level in Th17 as in other CD4+ T cell subsets, and abrogates the difference of ROS production between Th17 compared to CCR6- CD4+ T lymphocytes.We show that human Th17 lymphocytes present a higher senescence susceptibility to CSE exposure as compared to other CD4+ T lymphocytes sub-populations. Moreover, our results suggest that a higher mitochondrial activity in Th17 in steady state is responsible for the Th17 higher senescence susceptibility upon CSC exposure. Finally, we show for the first time, that mild mitochondrial respiratory chain uncoupling is an effective solution to prevent Th17 senescent phenotype and could represent an anti-inflammatory strategy in Th17-associated chronic inflammatory diseases

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

O controle de respostas imunes patológicas (autoimunidade, alergia, rejeição de transplantes) tem sido um dos principais objetivos dos imunologistas. Apesar dos avanços recentes, a maioria dos tratamentos atuais ainda procura diminuir a imunidade e inflamação em vez de restabelecer o estado saudável da tolerância imunológica. Sepse é uma doença desencadeada pela presença de bactérias e/ou produtos bacterianos como lipopolissacarídeos (LPS), componente principal da membrana externa de bactérias gram-negativas, ativando a resposta imune do hospedeiro. A caracterização do perfil de linfócitos na resposta à tolerância ao LPS são de extrema importância para a contribuição do estudo da imunodepressão na sepse. O objetivo deste estudo foi investigar se a comprovada redução de mortalidade previamente vista em modelo de sepse animal através tolerância ao LPS, pode ser associada com o aumento da população de linfócitos T CD4+ regulatórios e Th17. Camundongos machos C57/6, receberam por via subcutânea ( s.c.) injecções de LPS ( 1mg/kg ) durante 5 dias , seguido por perfuração e ligadura cecal (CLP ) . Citocinas e linfócitos marcados foram medidos durante, após a tolerância e o desafio CLP. Ambos os subtipos de células T analisados Treg e Th17 , mostrou aumento destas células no baço durante e após a tolerância. Este estudo demonstrou que a mortalidade reduzida depois de tolerância previamente constatada pode ser associada com o aumento da população de células T regulatórias e Th17 devido a imunorregulação do hiperinflamação e recrutamento de neutrófilos / The control of pathological immune responses (autoimmunity, allergy, transplant rejection) has been a major goal of immunologists. Despite recent advances, most current treatments still seeks to reduce immunity and inflammation rather than restore the healthy state of immune tolerance. Sepsis is a disease triggered by the presence of bacteria and / or bacterial products like lipopolysaccharide (LPS), the main component of the outer membrane of gram-negative bacteria, activating the immune response of the host. The characterization of lymphocyte profile in response to LPS tolerance is extremely important for the study of immunosuppression in sepsis contribution. The aim of this study was to investigate whether the proven reduction in mortality seen previously in animal sepsis model by tolerance to LPS, can be associated with the increase in population of CD4 + regulatory and Th17. Mice C57 / 6 mice received subcutaneous (s.c.) injection of LPS (1mg / kg) for 5 days, followed by cecal ligation and puncture (CLP). Cytokines and marked lymphocytes were measured during after tolerance and CLP challenge. Both subtypes of T cells Treg and Th17 analyzed showed an increase of these cells in the spleen during and after tolerance. This study demonstrated that reduced mortality after previously seen tolerance may be associated with increasing the population of regulatory T cells and Th17 because immunoregulation of the hiperinflamação and neutrophil recruitment

Leishmaniose visceral (LV) é uma doença crônica e potencialmente fatal causada pelas espécies Leishmania infantum/chagasi e Leishmania donovani. O desenvolvimento da resposta Th1 é classicamente associado à proteção contra esses parasitos, mas também há uma correlação positiva entre a produção de citocinas relacionadas com o padrão Th17 e a proteção contra LV por L. donovani em seres humanos. No entanto, a participação de Th17 durante a infecção por L. infantum/chagasi permanece desconhecida. O objetivo desse estudo foi avaliar a participação de Th17 e citocinas relacionadas, além do mecanismo pelo qual tais células operam durante a resposta imune do hospedeiro contra L. infantum/chagasi. Os resultados mostraram que o parasito é capaz de induzir grandes quantidades de TGF-, IL-1, IL-6 e IL-23 por células dendríticas derivadas da medula óssea (BMDC), citocinas envolvidas na indução e/ou manutenção do perfil Th17. Assim, co-cultivando células do baço de camundongos C57BL/6 naves com BMDCs infectadas com L. infantum/chagasi observou-se uma significativa produção de IL-17 por células T. Esses achados foram confirmados por experimentos in vivo onde se constatou a produção de IL-17 no fígado e no baço de camundongos WT infectados, sendo o pico de produção dessa citocina observado na 4ª e 6ª semanas após a infecção. O padrão de resposta do tipo Th17 é crítica para a imunidade protetora contra L. infantum/chagasi, uma vez que camudongos IL-17R-/-, IL-23p19-/- e IL-6-/- mostraram aumento da carga parasitária nos órgãos alvo da infecção, sendo que a susceptibilidade observada em camundongos IL-17R-/- foi associada com o aumento da produção de IL-10 por linfócitos, sugerindo que a IL-17 regula negativamente a produção de IL-10 levando ao controle da infecção causada pelo parasito. Ainda, a ausência da sinalização via IL-17R gerou uma diminuição da inflamação hepática, decorrente de uma menor capacidade proliferativa de linfócitos frente ao estímulo com conA. De maneira interessante, na ausência de IL-10 houve potencialização na produção de IL-17 por camundongos infectados, e esses foram mais resistentes à infecção, apresentando números reduzidos parasitos no baço e no fígado. Além de promover proteção através da modulação de IL-10, a IL-17 foi capaz de potencializar a produção de NO in vitro e in vivo. Juntos, nossos resultados demonstram que a L. infantum/chagasi é capaz de desencadear o padrão Th17 de resposta imune, o qual promove proteção do hospedeiro durante a infecção. / Visceral leishmaniasis (LV) is a chronic and potentially fatal disease caused by Leishmania donovani and Leishmania infantum/chagasi. The development of Th1 response is classically associated with protection against these parasites, but recent data also show that there is a positive correlation between the Th17-related cytokines production and the protection against LV by L. donovani in humans. However, the role of this CD4+ T cells subset during L. infantum/chagasi infection remains unknown. In this study, our aim was to evaluate the Th17 and related cytokines participation, besides the mechanism by which these cells playing during the L. infantum/chagasi infection.The results showed that the parasite induces high amounts of TGF-, IL-1, IL-6, and IL-23 by bone marrow-derived dendritic cells (BMDC), cytokines involved with Th17 induction and/or maintenance. Accordingly, naïve-C57BL/6 spleenocytes co-cultured with L. infantum/chagasi-infected BMDC produced significant amounts of IL-17 by TCD4+. Interestingly, IL-17 was produced in high amounts in the liver and spleen of WT infected-mice, being peaked at 4th and 6th weeks after infection. The Th17 is critical for protective immunity against L. infantum/chagasi, since that IL-17R-/-, IL-23p19-/- and IL-6-/- infected-mice showed increasing of parasite load associated with enhancement of IL-10 production in IL-17R-/- compared to WT infected-mice. Strictly, in the absence of IL-17 signaling, a smaller inflammatory infiltrate was observed in the liver. Interestingly, IL-17 production was potentiated in IL-10-/- infected-mice, and they were more resistant to infection, showing reduced parasites numbers in the spleen and liver. In addition to promoting protection through the downmodulation of IL-10, IL-17 enhanced the NO production. Together, our results demonstrate that L. infantum/chagasi trigger Th17 response that promotes the host protection during infection.