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

Platelet-Activating Factor Acetylhydrolase (PAFAH) is a monocyte-derived phospholipase A2 that catalyzes the hydrolysis of platelet-activating factor (PAF) and has been implicated in atherosclerosis. Although PAF and other proinflammatory stimuli are postulated to induce the enzyme, mechanisms controlling PAFAH expression are largely unknown at present. We have shown that PAFAH induction in monocytes is increased in response to oxidized phospholipids. The PAFAH 5' flanking region has at least 10 putative Stat elements, and IL-6 has been shown to be downstream from the prostaglandin receptor, EP2, which has been shown to bind oxidized phospholipids, prompting the hypothesis that Stat proteins might regulate its expression. To test this hypothesis, we treated human monocytes with IL-6, a monocyte-derived cytokine that activates Stat3, IL-8, a monocyte-derived cytokine induced by Stat3, and oxidized 1-palmitoyl-2-arachidonoyl-sn-3-phosphocholine (oxPAPC), a major component of the oxidized LDL particle. Two monocyte-derived cell types, macrophages (MO) and dendritic cells (DC) were prepared from primary human monocytes. The cells were treated with various doses of IL-6, IL-8, or oxPAPC for various time frames in the absence of serum. Culture supernatants from the cytokine-treated cells were harvested and screened for PAFAH protein and activity and cell monolayers were assessed for PAFAH mRNA by quantitative real time PCR (qPCR). Cells treated with oxPAPC were further analyzed for secreted IL-6 using ELISA and activation of Stat3 using Western Blot. Both IL-6 and IL-8 induced PAFAH expression in a dose-dependent manner. Although both MO and DC responded to the cytokines, preliminary experiments suggested that induction of PAFAH is more robust in DC than MO. Cytokine-treated cells exhibited increased PAFAH activity in their culture supernatants that correlated with increased PAFAH protein levels. Treatment with oxPAPC induced IL-6 secretion and subsequent Stat3 activation in DC. Together, these data support the hypothesis that PAFAH expression is regulated by oxidized phospholipids and proinflammatory cytokines in developing atheromas.

Temperature has been known to be an important factor for in vitro studies where human cell cultures are infected with HRV (human Rhinovirus). The mechanisms behind the temperature effect on the struggle between virulence and cellular defense, are still largely unknown and may be a crucial part in finding a treatment to the common cold. In this study we focused on a few cellular key elements in this struggle and observed behavior changes in regards to the pre-infection growth temperature and the temperature during the viral infection. Past studies have focused mainly on the temperature post inoculation, but here we also wanted to correlate virulence to the growth temperatures preceding the viral infection. We found that the growth temperature of the cell did indeed affect its response to the HRV. If the cells had been growing in an optimal body temperature of 37°C before getting virally infected at 33°C, the viability of the cells did decrease in comparison to cells that had been growing in 33°C from before the viral infection. We could also observe a significant temperature dependence regarding IL-8 release upon HRV inoculation. HRV strive to block induction of inflammatory cytokines such as interferons and IL-1. It may be that impaired IL-8 release at lower temperatures will prevent important danger signals alerting the immune system when cytokine signaling is otherwise hampered by viral intervention.

Neutrophile sind Zellen des Immunsystems von Säugetieren. Ihre zerstörerische Kraft spielt eine essentielle Rolle bei der Bekämpfung von Mikroorganismen, birgt aber auch das Potential erheblicher Kollateralschäden. Um chronische Entzündungen zu vermeiden, müssen diese Zellen streng reguliert werden. Die Neutrophilen selber nehmen an dieser Regulierung durch das Freisetzen von pro- und antiinflammatorischen Signalen Teil, unter anderem produzieren sie Zytokine oder initiieren rechtzeitig die Apoptose. Ein Eckpfeiler der Regulierung dieser Funktionen ist der oxidative Burst, bei dem Neutrophile reaktive Sauerstoffspezies (ROS) bilden. Die molekularen Ziele von ROS, welche diese Mechanismen regulieren, sind nicht alle identifiziert. Wir haben ataxia-telangiectasia mutated (ATM) Kinase, ein Regulator der DNA-Schadensantwort (DDR), als einen ROS-abhängigen Modulator von Neutrophilen identifiziert. Mutationen in ATM führen zu der Erkrankung Ataxia Telangiectasia (AT). AT Patienten leiden nicht nur unter den Folgen der fehlerhaften DNA-Reparatur sondern zeigen auch inflammationsassoziierte Krankheitserscheinigungen. Diese Beobachtung veranlasste uns, die Neutrophilen von AT Patienten genauer zu untersuchen. Wir zeigen, dass Neutrophile von AT Patienten erhöhte Menge an Zytokinen produzieren und Apoptose verzögern. Wir zeigen auch, dass DNA Schaden die Zytokinproduktion unterdrückt und Apoptose durch einen Mechanismus, der ATM, p38, und Chk2 verwendet initiiert. ROS sind notwendig für die endogene Regulierung dieser Prozesse. Diese Arbeit enthüllt einen neuartigen Mechanismus der Regulierung von Neutrophilen und etabliert die DDR als ein Ziel der ROS-gesteuerten Immunmodulation. Im Zusammenhang wird auch gezeigt, dass dysregulierte Neutrophilenaktivitäten einem inflammatorischen Phänotyp in AT zugrundeliegen könnte. Wir glauben, dass Entzündung eine treibende Kraft hinter Teilen der Pathologie von AT sein könnte und somit ein Ziel für klinische Intervention darstellt. / Neutrophils are cells of the mammalian innate immune system whose inflammatory functions are essential for microbial clearance but cause collateral tissue damage. Inflammation is regulated by both pro- and anti-inflammatory signals, including cytokine production and initiation of apoptosis. A cornerstone of the regulation of these functions is the oxidative burst, by which neutrophils generate reactive oxygen species (ROS). The downstream targets of ROS responsible for regulating these functions are not fully identified. We have identified ataxia telangiectasia mutated (ATM) kinase, a master regulator of the DNA damage response (DDR), as a ROS-dependent modulator of neutrophil responses. Mutations in ATM cause the disease Ataxia-telangiectasia (AT). In addition to disorders resulting from defective DNA repair, AT patients suffer from symptoms linked to inflammation, leading us to examine their neutrophil responses. We report that neutrophils from AT patients overproduce pro-inflammatory cytokines and delay apoptosis. We further show that DNA damage in neutrophils suppresses cytokine production and can initiate apoptosis via a mechanism involving ATM, p38, and Chk2. Furthermore, the oxidative burst was required for activation of ATM to regulate these processes.. This work reveals a novel mechanism for the regulation of neutrophil functions, establishing the DDR as a mediator of immune regulation by ROS. Furthermore, it indicates that neutrophil dysregulation may underlie chronic inflammation in AT patients. We propose that inflammation may be a driving force behind some of the pathology of AT, providing a potential target for clinical intervention for some symptoms of this currently untreatable disease.

Nos últimos anos, notou-se aumento da incidência de carcinoma espinocelular de orofaringe (CECOF) associado ao HPV. Sabe-se que CECOF associado ao HPV apresenta melhor prognóstico do que CECOF não infectado por HPV. Inúmeros estudos em carcinoma cervical demonstram alterações de TLRs, isto provavelmente devido às associações das oncoproteínas E6 e E7 com estes receptores. Em humanos, existem 10 TLRs identificados, os quais colaboram na resposta imune contra bactérias, fungos e vírus, bem como colaboram na promoção ou regressão do tumor. Esta influência do TLR na carcinogênese tem sido alvo de inúmeros estudos devido à ligação entre inflamação e o câncer. O presente trabalho teve como objetivo verificar diferenças na expressão e função de receptores Toll-like em carcinoma espinocelular de orofaringe (CECOF). Para tal, foram utilizados trinta e sete espécimes diagnosticados como CECOF e a expressão imuno-histoquímica das proteínas p16 e TLR4 analisadas. Duas linhagens de CECOF HPV16 + e duas CECOF HPV-. foram utilizadas para análise da expressão de TLR1-10, IL-6 e IL-8, por qPCR. A detecção dos principais TLRs (TLR1, TLR2, TLR6 e TLR4) foi feita por citometria de fluxo. Para ativação da via de sinalização de TLR2, e posterior análise da expressão de IL6 e IL8, as células foram estimuladas com peptidoglicano. Para verificar a expressão e função de TLR4, as células foram estimuladas com LPS e LPS UP para posterior análise de IL-6 e IL-8, por ELISA. Os resultados demonstraram diferenças na expressão gênica de TLR1 e TLR6 entre as linhagens HPV- e o grupo HPV+ e diferenças na expressão proteica de TLR9. TLR2 apresentou aumento da expressão proteica em todas as linhagens e demonstra desencadeamento da resposta imune, com secreção de IL6 e IL8 nas linhagens HPV- (SCC72 e SCC89) e em uma das linhagens HPV+ (SCC2). Interessantemente, TLR4 não apresentou diferenças significativas na expressão gênica e proteica. Entretanto, as linhagens HPV+ não demonstraram resposta pró-inflamatória mesmo quando estimuladas com LPS e LPS ultra puro, agonista específico de TLR4. Assim, este trabalho contribui para estabelecer o perfil da expressão dos receptores Toll-like em linhagens celulares de CECOF HPV- e HPV+, e aponta para alterações ocorridas na via de sinalização mediada por TLR4. Além disso, nossos resultados abrem portas para futuros estudos na avaliação de alterações causadas no sistema imune inato pelo HPV, em carcinomas espinocelulares de orofaringe. / The incidence of HPV- associated oropharynx squamous cell carcinoma (OPSCC) has increased in recent years. HPV- associated OPSCC has a better prognosis than OPSCC not infected with HPV. In cervical carcinoma, HPV oncoproteins E6 and E7 influence the expression of Toll-like receptors (TLR). To date, 10 TLRs have been identified in humans and many are important for the detection of bacteria, fungi and viruses, as well as regression and tumor promotion. This influence of TLR in carcinogenesis has been the subject of numerous studies, due to the connection between inflammation and cancer. This study aimed to determine differences in the expression and function of Toll-like receptor in OPSCC. Thirty-seven tumours were selected and immunohistochemistry for p16 and TLR4 was performed. Two HPV16-associated OPSCC and two OPSCC not associated to HPVcell lines were used. qPCR was performed to analyze the expression of TLR1-10, IL-6 and IL-8. The detection of the main TLRs (TLR1, TLR2, TLR6 and TLR4) was performed by flow cytometry. For activation of the TLR2-signaling pathway and subsequent analysis of IL6 and IL8 expression, cells were stimulated with peptidoglycan. To verify the expression and function TLR4 cells were stimulated with LPS and LPS UP and subsequent analysis of IL-6 and IL-8 by ELISA. The results showed differences in the expression of TLR1 and TLR6 between the HPV- group and the HPV+ group and differences in protein expression of TLR9. TLR2 has increased protein expression in all cell lines and demonstrates the trigger of the immune response, with the secretion of IL6 and IL8 in HPV- cell lines (SCC72 and SCC89) and one HPV+ cell line (SCC2). Interestingly, TLR4 showed no significant differences in gene and protein expression. However, HPV+ cell lines showed no pro-inflammatory response even when stimulated with LPS and LPS UP, a specific agonist of TLR4. This work helps to establish the profile of the expression of Toll-like receptors in OPSCC (SCC72 and SCC89) and HPV- associated OPSCC (SCC2 and SCC90), in vitro, and points to changes in the signaling pathway mediated by TLR4. In addition, our results open new avenues for future studies to assess changes in the innate immune system caused by HPV in oropharynx carcinomas.

Nos últimos anos, notou-se aumento da incidência de carcinoma espinocelular de orofaringe (CECOF) associado ao HPV. Sabe-se que CECOF associado ao HPV apresenta melhor prognóstico do que CECOF não infectado por HPV. Inúmeros estudos em carcinoma cervical demonstram alterações de TLRs, isto provavelmente devido às associações das oncoproteínas E6 e E7 com estes receptores. Em humanos, existem 10 TLRs identificados, os quais colaboram na resposta imune contra bactérias, fungos e vírus, bem como colaboram na promoção ou regressão do tumor. Esta influência do TLR na carcinogênese tem sido alvo de inúmeros estudos devido à ligação entre inflamação e o câncer. O presente trabalho teve como objetivo verificar diferenças na expressão e função de receptores Toll-like em carcinoma espinocelular de orofaringe (CECOF). Para tal, foram utilizados trinta e sete espécimes diagnosticados como CECOF e a expressão imuno-histoquímica das proteínas p16 e TLR4 analisadas. Duas linhagens de CECOF HPV16 + e duas CECOF HPV-. foram utilizadas para análise da expressão de TLR1-10, IL-6 e IL-8, por qPCR. A detecção dos principais TLRs (TLR1, TLR2, TLR6 e TLR4) foi feita por citometria de fluxo. Para ativação da via de sinalização de TLR2, e posterior análise da expressão de IL6 e IL8, as células foram estimuladas com peptidoglicano. Para verificar a expressão e função de TLR4, as células foram estimuladas com LPS e LPS UP para posterior análise de IL-6 e IL-8, por ELISA. Os resultados demonstraram diferenças na expressão gênica de TLR1 e TLR6 entre as linhagens HPV- e o grupo HPV+ e diferenças na expressão proteica de TLR9. TLR2 apresentou aumento da expressão proteica em todas as linhagens e demonstra desencadeamento da resposta imune, com secreção de IL6 e IL8 nas linhagens HPV- (SCC72 e SCC89) e em uma das linhagens HPV+ (SCC2). Interessantemente, TLR4 não apresentou diferenças significativas na expressão gênica e proteica. Entretanto, as linhagens HPV+ não demonstraram resposta pró-inflamatória mesmo quando estimuladas com LPS e LPS ultra puro, agonista específico de TLR4. Assim, este trabalho contribui para estabelecer o perfil da expressão dos receptores Toll-like em linhagens celulares de CECOF HPV- e HPV+, e aponta para alterações ocorridas na via de sinalização mediada por TLR4. Além disso, nossos resultados abrem portas para futuros estudos na avaliação de alterações causadas no sistema imune inato pelo HPV, em carcinomas espinocelulares de orofaringe. / The incidence of HPV- associated oropharynx squamous cell carcinoma (OPSCC) has increased in recent years. HPV- associated OPSCC has a better prognosis than OPSCC not infected with HPV. In cervical carcinoma, HPV oncoproteins E6 and E7 influence the expression of Toll-like receptors (TLR). To date, 10 TLRs have been identified in humans and many are important for the detection of bacteria, fungi and viruses, as well as regression and tumor promotion. This influence of TLR in carcinogenesis has been the subject of numerous studies, due to the connection between inflammation and cancer. This study aimed to determine differences in the expression and function of Toll-like receptor in OPSCC. Thirty-seven tumours were selected and immunohistochemistry for p16 and TLR4 was performed. Two HPV16-associated OPSCC and two OPSCC not associated to HPVcell lines were used. qPCR was performed to analyze the expression of TLR1-10, IL-6 and IL-8. The detection of the main TLRs (TLR1, TLR2, TLR6 and TLR4) was performed by flow cytometry. For activation of the TLR2-signaling pathway and subsequent analysis of IL6 and IL8 expression, cells were stimulated with peptidoglycan. To verify the expression and function TLR4 cells were stimulated with LPS and LPS UP and subsequent analysis of IL-6 and IL-8 by ELISA. The results showed differences in the expression of TLR1 and TLR6 between the HPV- group and the HPV+ group and differences in protein expression of TLR9. TLR2 has increased protein expression in all cell lines and demonstrates the trigger of the immune response, with the secretion of IL6 and IL8 in HPV- cell lines (SCC72 and SCC89) and one HPV+ cell line (SCC2). Interestingly, TLR4 showed no significant differences in gene and protein expression. However, HPV+ cell lines showed no pro-inflammatory response even when stimulated with LPS and LPS UP, a specific agonist of TLR4. This work helps to establish the profile of the expression of Toll-like receptors in OPSCC (SCC72 and SCC89) and HPV- associated OPSCC (SCC2 and SCC90), in vitro, and points to changes in the signaling pathway mediated by TLR4. In addition, our results open new avenues for future studies to assess changes in the innate immune system caused by HPV in oropharynx carcinomas.