No description available.

Exposure to spores and hyphae of Alternaria alternata, an airborne ubiquitous fungus, is clinically associated with allergic airway disorders including allergic rhinitis, asthma, and chronic rhinosinusitis. Dendritic cells are known as the type of antigen presenting cells most often associated with allergic inflammation. In this study, we used mouse bone marrow-derived dendritic cells (BMDCs) as a model to study the ability of A. alternata spores and different components of the spore cell wall to stimulate innate immune responses. We found that BMDCs were highly sensitive to A. alternata spores, chitin and the major allergen Alt a 1. Following stimulation with these molecules, the expression of MHC II and other co-stimulators, like CD40, CD86, and OX40L, were highly up regulated. In order to determine how different cell wall components affect the T cells, we conducted co-culture experiments of BMDCs and lymphocytes in this study. Both spores and Alt a1 did not induce IL-4 in mixed lymphocytes reactions. Interestingly, we found that Alt a 1 induced the switching of the CD4+ T cell population to the Th17 type, with a major increase in IL-17A secretion. This study reveals that A. alternata components may balance the innate immune responses between Th2 and Th17 pathways, and/or contributes to the development and exacerbation of more serve neutrophilic forms of asthma. / Master of Science

Experiências vivenciadas durante o período pré-natal são determinantes para a saúde do feto. A ocorrência de infecções maternas e a consequente ativação do sistema imune da mãe ocasionam uma série de alterações estruturais e funcionais no cérebro da prole, podendo predispor o indivíduo a transtornos psiquiátricos na vida pós-natal, como esquizofrenia e autismo. No entanto, estudos que investigam as alterações imunes na prole ainda são escassos na literatura. Dessa forma, o objetivo do presente estudo foi avaliar, na prole, o impacto da ativação imune materna sobre a atividade imune periférica, a resposta imune-inflamatória no sistema nervoso central (SNC), e sobre o desenvolvimento da encefalomielite autoimune experimental (EAE), o modelo murino de Esclerose Múltipla. Camundongos fêmeas prenhes receberam uma administração de salina ou lipopolissacarídeo (LPS) ao final da gestação (dia gestacional 17) e, quando adulta, a prole foi submetida a 3 experimentos principais, analisando: (1) produção de citocinas, atividade de células da periferia e desenvolvimento da hipersensibilidade do tipo tardia; (2) produção de mediadores inflamatórios por células residentes do SNC e; (3) desenvolvimento dos sintomas clínicos e da resposta imune no decorrer da EAE. Nossos resultados mostraram que a ativação imune materna provocou na prole alterações imunes periféricas, como aumento da produção de Interleucina(IL)- 12 e exacerbação da resposta de hipersensibilidade do tipo tardia; potencialização da produção das citocinas IL-1β e IL-6 em cultura primária de células residentes do SNC e; piora na severidade dos sintomas clínicos causados pela EAE, que coincide com aumento do infiltrado de linfócitos e macrófagos no SNC e ativação imuneinflamatória das células da glia. Tomados em seu conjunto, os dados do presente trabalho sugerem que condições inflamatórias durante a gestação, particularmente durante o final da gestação, podem predispor o feto a distúrbios autoimunes e neurodegenerativos na vida adulta. / Prenatal period experiences are crucial for the fetal health. The occurrence of maternal infections and subsequent maternal immune system activation cause a number of structural and functional changes in the brain of the offspring that may predispose individuals to psychiatric disorders in post-natal life, such as schizophrenia and autism. However, studies investigating offspring´s immune alterations are still scarce in the literature. The aim of this study was to evaluate, in mice offspring taken from LPS-treated dams, the impact of maternal immune activation on peripheral immune cell activity, central nervous system (CNS) inflammatory response, and development of experimental autoimmune encephalomyelitis (EAE), the murine model of multiple sclerosis. Pregnant female mice received a dose of either saline or lipopolysaccharide (LPS) during late gestation (gestational day 17), and offspring were used in three experiments to analyze: (1) cytokine production and activity by peripheral immune cells and development of delayed type hypersensitivity, (2) production of inflammatory mediators by resident CNS cells and, (3) development of clinical symptoms and immune response during the course of EAE. Our results showed that maternal immune activation resulted in immune alterations in the offspring, such as increased peripheral production of interleukin (IL) -12 and exacerbated response of delayedtype hypersensitivity; enhancement of IL-1β and IL-6 productions in primary CNS resident cells culture and; increased severity of EAE clinical symptoms, which is positively correlated with the increased lymphocytes and macrophages infiltration within the CNS and also with the immune-inflammatory activation of glial cells. Taken together, the data from this study suggest that inflammatory conditions during pregnancy, especially during the late pregnancy, may predispose the fetus to autoimmune and neurodegenerative disorders in adulthood.

Wir nutzten in dieser Studie den apikomplexen Parasiten Eimeria falciformis als Modell. Unsere Ergebnisse zeigen, dass das in infizierten Wildtypmäusen dominierende Zytokin IFN-γ für Immunschutz und für die Entwicklung der Darmpathologie entbehrlich war. E. falciformis-infizierte IFN-γR-/- and IFN-γ-/- Mäuse zeigten extremen Körpergewichtsverlust und starke Pathologie im Darm. Die Entwicklung des Parasiten in diesen Mäusen war überraschenderweise reduziert. Diese Beobachtungen gingen mit einer drastisch erhöhten Produktion von parasiten-spezifischem IL-17A und IL-22 durch CD4+ T Zellen einher. Gleichzeitige Neutralisierung von IL-17A und IL-22 in E. falciformis-infizierten IFN-γR-/- Mäusen verringerte den Körpergewichtsverlust und die Darmpathologie, und führte zu einer erhöhten Ausscheidung von Parasiten. Die Behandlung einer E. falciformis-infizierten intestinalen Epithelzelllinie mit IL-17A oder IL-22 führte zu einer signifikant reduzierten Entwicklung von E. falciformis in vitro. Diese Daten demonstrieren erstmalig einen anti-parasitären Effekt von IL-22 im Darm und deuten auf redundante Rollen von IL-17A und IL-22 im Hinblick auf die Förderung von Darmpathologie in Abwesenheit von IFN-γ hin. Um E. falciformis als Modellsystem weiter zu entwickeln, haben wir die Transfektion von E. falciformis Sporozoiten mit verschiedenen Plasmiden die den Reporter YFP und den Resistenzmarker DHTS enthalten etabliert. Rektal in Mäuse injizierte Sporozoiten entwickelten sich erfolgreich zu Oocysten, wenn auch mit geringerer Effizienz im Vergleich zur oralen Infektion mit Oozysten. Wiederholte in vivo Selektion YFP-exprimierender Oozysten führte zu Populationen mit maximal 34 % YFP-exprimierenden Parasiten. Wir demonstrieren in dieser Arbeit zum ersten Mal die Transfektion von E. falciformis und zeigen Perspektiven im Hinblick auf die Etablierung einer stabil transgenen Parasitenlinie auf. / The roles of Th1 and Th17 responses as mediators of host protection and pathology in the intestine are the subjects of intense research. Here we investigated a model of intestinal inflammation driven by the intracellular apicomplexan parasite Eimeria falciformis. Although IFN-γ was the predominant cytokine during E. falciformis infection in wild type mice, it was found to be dispensable for host defence and the development of infection-driven intestinal inflammation. E. falciformis-infected IFN-γR-/- and IFN-γ-/- mice developed dramatically exacerbated body weight loss and intestinal pathology, but surprisingly harboured fewer parasites. This was associated with a striking increase in parasite-specific IL-17A and IL-22 production in the mesenteric lymph nodes and at the site of infection. Concurrent neutralisation of IL-17A and IL-22 in E. falciformis infected IFN-γR-/- mice resulted in a reduction in infection induced body weight loss and inflammation and significantly increased parasite shedding. Taken together these data demonstrate for the first time an anti-parasitic effect of IL-22 during an intestinal infection and suggest that IL-17A and IL-22 have redundant roles in driving intestinal pathology in the absence of IFN-γ signalling. To further develop E. falciformis as a model system, we established transfection of E. falciformis sporozoites using various plasmids that contain the fluorescent reporter YFP and the resistance marker DHTS. Sporozoites applied rectally to mice were shown to complete their life cycle, albeit with a lower efficiency in comparison to oral infection with oocysts. Repeated in vivo selection using pyrimethamine and/or FACS and manual sorting led to a maximum percentage of 34 % YFP-expressing oocysts. Taken together, we demonstrate for the first time transfection of E. falciformis and provide perspectives for further work on the establishment of a stable transgenic parasite line.

O tabagismo é o principal fator de risco para o desenvolvimento da doença pulmonar obstrutiva crônica (DPOC). A importância da imunidade adaptativa para este processo não está totalmente esclarecida, entretanto a inflamação persistente está associada à ocorrência de infecções e exacerbação da DPOC. O nosso objetivo foi desenvolver um modelo experimental de exacerbação da DPOC utilizando a exposição à fumaça de cigarro e instilação de lipopolissacarídeo (LPS), visando investigar os aspectos imunológicos. Camundongos machos (C57BL/6), foram divididos em 4 grupos. Grupo controle salina (CSAL) e grupo controle LPS (CLPS) que foram expostos ao ar filtrado durante 12 semanas e receberam duas instilações intratraqueais (50µl) de salina ou LPS (1mg/kg) com intervalo de 15 dias entre cada instilação e o grupo fumo salina (FSAL) e grupo fumo LPS (FLPS) que foram expostos à fumaça de cigarro e também receberam duas instilações intratraqueais de salina ou LPS com o mesmo intervalo de tempo entre as instilações e a mesma dosagem. Após 3 dias da última instilação os animais foram anestesiados, traqueostomizados e acoplados a um ventilador para pequenos animais para avaliação da mecânica respiratória. Em seguida, fizemos a coleta do lavado broncoalveolar (LBA) para avaliar o perfil inflamatório e posteriormente, os pulmões foram removidos e feitos cortes para análise do intercepto linear médio (Lm) subpleural e peribronquial, além da espessura epitelial. Também avaliamos a densidade de macrófagos, neutrófilos, células T CD4+, CD8+ e células T regulatória (Treg), células positivas para Stat3/5 e FosfoStat3/5 no parênquima pulmonar por imuno-histoquímica. Além disso, foram analisados por ELISA no homogenato pulmonar os fatores quimiotáticos (KC/CXCL1 e MIP-2/CXCL2) e interleucina (IL) (-17, -6, -10) e interferon gama (IFN-y). Observamos alteração da mecânica do sistema respiratório com aumento da resistência tecidual (Gtis) e elastância tecidual (Htis) nos animais expostos á fumaça de cigarro e desafiados com LPS. Nos grupos FSAL e FLPS observamos aumento de Lm (regiões subpleurais), e no grupo FLPS também observamos aumento de Lm nos espaços peribrônquicos e espessamento epitelial. A associação da fumaça de cigarro e o LPS, além de ter causado dano ao parênquima pulmonar mais difuso tanto em regiões subpleurais quanto em espaços peribrônquicos intensificou a resposta inflamatória com aumento de neutrófilos, macrófagos, células T CD4+, células positivas para Stat3, FosfoStat5 e CXCL2. A densidade das células Treg, os níveis de IL-17 e IL-6 aumentaram em ambos os grupos LPS, enquanto o nível de IL-10 aumentou apenas no grupo CLPS. O aumento das células positivas para Stat3 -5, FosfoStat3 -5, corrobora com valores mais elevados para as células IL-17 e Treg. Assim, nosso estudo demonstrou que embora a associação da fumaça de cigarro e o LPS tenha induzido a diferenciação das células Th17 e Treg, não observamos aumento da expressão de IL-10 e sim da expressão de IL-17 sugerindo que uma falha na produção de IL-10 desempenha um papel fundamental na exacerbação do processo inflamatório / The tobacco smoking is the main risk factor for the development of Chronic Obstructive Pulmonary Disease (COPD). The importance of adaptive immunity for this process is not completely clear, however there are studies attesting the association of infection with COPD exacerbation. We propose an experimental model of COPD exacerbation using a traditional method of cigarette smoke (CS) combining with lipopolysaccharide (LPS) instillations, focusing on the adaptive immunity response. C57BL/6 male mice were exposed to room air or to CS and after 3 months, they received two instillations of saline or LPS (Control/SAL; Control/LPS; CS/SAL and CS/LPS groups). Animals were anesthetized to perform the respiratory mechanics and inflammatory profile in broncho alveolar lavage (BAL) and lungs were removed to evaluate the mean linear intercept (Lm), the density of macrophages, neutrophils, CD4+ and CD8+ cells, regulatory T cells (Treg), signal transducer and activator of transcription (Stat) 3, 5 and phosphoStat 3, 5. The chemotactic factors (CXCL1 and CXCL2); interleukins (IL): -17, -6, -10 and INF-y were measured in lung using Enzyme Linked ImmunoSorbent Assay (ELISA). We observed a change in the mechanics of the respiratory system with increased tissue resistance (Gtis) and tissue elastance (Htis) in CS/LPS group. In addition, in CS/Sal and CS/LPS groups we observed increase of Lm (subpleural), and in CS/LPS group we observed the increase in Lm in peribronchial spaces. CS exposure and LPS challenge induced an increase in neutrophils, macrophages, CD4+ and CD8+ T cells. CS/LPS challenge association intensified this response and lung parenchyma damage. Treg density cells, IL-17 and IL-6 levels was increased in both LPS groups, while IL-10 level was increased only in Control/LPS group. The increase of Stat3, -5, PhosphoStat3, -5 positive cells corroborates with higher values for IL-17 and Treg cells. Although the CS/LPS challenge association induced both Th17 and Treg cells differentiation, there is no increase for IL-10 expression, suggesting that a failure in IL-10 production play a pivotal role in the inflammatory process exacerbation

Les thérapies anticancéreuses ont apporté un gain largement reconnu en matière de réduction de la charge tumorale, de survie des patients et d’amélioration de leur qualité de vie, dans un certain nombre de cancers. Hélas, ces thérapies exercent un effet immunosuppresseur en détruisant les effecteurs ou en bloquant l’activité de certains facteurs biologiques impliqués dans le recrutement des acteurs du système immunitaire. D’autre part, plusieurs travaux ont permis de démontrer que ces traitements pouvaient avoir un effet contraire en générant ou en favorisant l’induction d’une réponse immunitaire anti-tumorale, soit par effet direct sur le recrutement et l’activation des effecteurs de l’immunité, soit en potentialisant les interactions cellulaires par des mécanismes biologiques. Ces derniers faisant intervenir les cytokines, la stimulation des TLR, l’augmentation des interactions entre cellules du SI; ce qui permet de passer d’une anergie immunologique vers un véritable système d’éradication des cellules cancéreuses.Dans notre laboratoire, nous avons essayé d’évaluer l’implication du système immunitaire dans la réponse thérapeutique induite par des agents cytotoxiques conventionnels. Ici, nous décrivons les effets d’un inhibiteur de cyclines kinases multi-cibles « CDKi PHA-793 887 » testé dans un essai de phase I mené sur deux sites en Europe. C’est le constat inattendu que 6 des 15 patients, traités par ce médicament (PHA-793887) ont développé de graves infections bactériennes et virales et que 6 d’entre eux ont présenté la réactivation du virus de l’herpès qui nous a conduit à étudier ces effets sur le système immunitaire et en particulier sur le dialogue entre cellules dendritiques (CD) et cellules natural killer (NK). Ce travail met en évidence que ce médicament inhibe le signalling des récepteurs toll-like (TLR) réduisant par conséquent l’interaction CD/NK in vitro. Enfin la stimulation des cellules des patients sous traitement démontre une réduction importante de ce signalling ex-vivo. Ainsi, cet effet immunosuppresseur inattendu a permis une réactivation virale chez 40% des patients. La deuxième partie de ce travail, concerne les effets du cyclophosphamide (CTX) utilisé à faible dose. L’injection d'une faible dose chez la souris ou d’un dosage métronomique chez l'homme, promeut la différenciation des cellules lymphocytaires vers Th17 (sécrétant de l’interleukine-17 (IL-17)) et Th1 (sécrétant de l’interféron-γ (IFN)). Ceux-ci ont été retrouvés dans le sang et dans des ascites carcinomateuses de patients. Ainsi, le CTX pourrait participer à la génération de réponses anti-tumorale via la différenciation Th 17 comme cela fut suggéré par de récentes études précliniques montrant l’existence d’une corrélation étroite entre le taux des lymphocytes Th17 infiltrant la tumeur et la destruction tumorale. / Cancer therapies have made a gain widespread recognition in the reduction of tumor burden, patient survival and improved quality of life in a number of cancers. Unfortunately, these therapies exert an immunosuppressive effect by killing effectors or blocking the activity of certain biological factors involved in recruiting of the immune system. On the other hand, several studies have shown that these treatments could have the opposite effect by generating or promoting the induction of antitumor immune response, either by direct effect on the recruitment and activation of effectors immunity, either by potentiating cellular interactions by biological mechanisms. The latter involving cytokines, TLR stimulation, increased interactions between cells of the IS; which toggles between immunological anergy to a real system to eradicate cancer cells. In our laboratory, we tried to evaluate the involvement of the immune system in the therapeutic response induced by conventional cytotoxic agents. Here, we describe the effects of an inhibitor of cyclin kinases multi-target "CDKIs PHA-793887" tested in a phase I trial conducted at two sites in Europe. This unexpected finding is that 6 of 15 patients treated with this drug (PHA-793887) developed severe bacterial and viral infections and six of them showed reactivation of the herpes virus that has led us to study these effects on the immune system and in particular on the dialogue between dendritic (DCs) and natural killer (NK) cells. This work shows that this drug inhibits the signaling of toll-like receptor (TLR) thereby reducing the interaction DC / NK in vitro. Finally, stimulation of the cells of treated patients demonstrated a significant reduction of this signaling ex vivo. Thus, this immunosuppressive effect has an unexpected viral reactivation in 40% of patients. The second part of this work concerns the effects of metronomic dose of cyclophosphamide (CTX). The injection of a low dose in mice or metronomic dosing in humans, markedly promotes the differentiation of CD4+ T helper 17 (Th17) cells that can be recovered in both blood and tumor beds. However, CTX does not convert regulatory T cells into Th17 cells and promotes cell differentiation into Th17 lymphocytes (secreting interleukin-17 (IL-17)) and Th1 (secreting interferon-γ (IFN)). These were found in blood and in ascites carcinoma patients. Thus, CTX may participate in the generation of antitumor responses through Th 17 differentiation as was suggested by recent preclinical studies showing the existence of a correlation between the rate of Th17 lymphocytes infiltrating the tumor and tumor destruction.

Experiências vivenciadas durante o período pré-natal são determinantes para a saúde do feto. A ocorrência de infecções maternas e a consequente ativação do sistema imune da mãe ocasionam uma série de alterações estruturais e funcionais no cérebro da prole, podendo predispor o indivíduo a transtornos psiquiátricos na vida pós-natal, como esquizofrenia e autismo. No entanto, estudos que investigam as alterações imunes na prole ainda são escassos na literatura. Dessa forma, o objetivo do presente estudo foi avaliar, na prole, o impacto da ativação imune materna sobre a atividade imune periférica, a resposta imune-inflamatória no sistema nervoso central (SNC), e sobre o desenvolvimento da encefalomielite autoimune experimental (EAE), o modelo murino de Esclerose Múltipla. Camundongos fêmeas prenhes receberam uma administração de salina ou lipopolissacarídeo (LPS) ao final da gestação (dia gestacional 17) e, quando adulta, a prole foi submetida a 3 experimentos principais, analisando: (1) produção de citocinas, atividade de células da periferia e desenvolvimento da hipersensibilidade do tipo tardia; (2) produção de mediadores inflamatórios por células residentes do SNC e; (3) desenvolvimento dos sintomas clínicos e da resposta imune no decorrer da EAE. Nossos resultados mostraram que a ativação imune materna provocou na prole alterações imunes periféricas, como aumento da produção de Interleucina(IL)- 12 e exacerbação da resposta de hipersensibilidade do tipo tardia; potencialização da produção das citocinas IL-1β e IL-6 em cultura primária de células residentes do SNC e; piora na severidade dos sintomas clínicos causados pela EAE, que coincide com aumento do infiltrado de linfócitos e macrófagos no SNC e ativação imuneinflamatória das células da glia. Tomados em seu conjunto, os dados do presente trabalho sugerem que condições inflamatórias durante a gestação, particularmente durante o final da gestação, podem predispor o feto a distúrbios autoimunes e neurodegenerativos na vida adulta. / Prenatal period experiences are crucial for the fetal health. The occurrence of maternal infections and subsequent maternal immune system activation cause a number of structural and functional changes in the brain of the offspring that may predispose individuals to psychiatric disorders in post-natal life, such as schizophrenia and autism. However, studies investigating offspring´s immune alterations are still scarce in the literature. The aim of this study was to evaluate, in mice offspring taken from LPS-treated dams, the impact of maternal immune activation on peripheral immune cell activity, central nervous system (CNS) inflammatory response, and development of experimental autoimmune encephalomyelitis (EAE), the murine model of multiple sclerosis. Pregnant female mice received a dose of either saline or lipopolysaccharide (LPS) during late gestation (gestational day 17), and offspring were used in three experiments to analyze: (1) cytokine production and activity by peripheral immune cells and development of delayed type hypersensitivity, (2) production of inflammatory mediators by resident CNS cells and, (3) development of clinical symptoms and immune response during the course of EAE. Our results showed that maternal immune activation resulted in immune alterations in the offspring, such as increased peripheral production of interleukin (IL) -12 and exacerbated response of delayedtype hypersensitivity; enhancement of IL-1β and IL-6 productions in primary CNS resident cells culture and; increased severity of EAE clinical symptoms, which is positively correlated with the increased lymphocytes and macrophages infiltration within the CNS and also with the immune-inflammatory activation of glial cells. Taken together, the data from this study suggest that inflammatory conditions during pregnancy, especially during the late pregnancy, may predispose the fetus to autoimmune and neurodegenerative disorders in adulthood.

A doença do enxerto contra o hospedeiro (DECH) é uma das maiores causas de mortalidade e morbidade pós-transplante de células-tronco hematopoiéticas. A DECH, em sua manifestação crônica (DECHc), ainda não tem sua fisiopatologia totalmente esclarecida; entretanto, o envolvimento do sistema imunológico, por meio de respostas imunes inatas e adaptativas é bem estabelecido na literatura. A DECHc afeta múltiplos órgãos, incluindo as glândulas salivares, o que tem, como causa imediata, a xerostomia. Essas alterações são largamente desconhecidas e sub-relatadas. Assim, esse estudo indagou se o sintoma de xerostomia na DECHc é decorrente de alterações morfológicas e funcionais das glândulas salivares. Para responder essa pergunta analisamos de forma qualitativa e por meio da morfometria, espécimes de biopsias de glândulas salivares labiais de pacientes com DECHc e com sintoma de xerostomia. Foram utilizados como controles, espécimes de biopsias de glândulas salivares de pacientes diagnosticados com Síndrome de Sjögren (SSp) (que é um modelo clássico de xerostomia) e de pacientes com líquen plano oral (LPO) (cujas manifestações clínicas orais podem se assemelhar às da DECHc) e queixa de xerostomia. Também foram analisadas, por meio de ensaio multiplex, as citocinas relacionadas à resposta imune Th17 (importante via imunológica na patogenia da DECHc) na saliva de 21 pacientes de DECHc, 27 de SSp, 10 de LPO e 15 voluntários saudáveis. Os principais achados morfológicos nas glândulas salivares dos pacientes de DECHc foram a extensa fibrose, fibroplasia periductal, atrofia ductal e acinar; alterações vasculares representadas pela congestão e formação de trombos hialinos; e infiltrado inflamatório intersticial difuso de aspecto leve a moderado. As glândulas salivares de SSp, entretanto, apresentaram um infiltrado inflamatório na forma de focos de linfócitos ao redor dos ductos excretores de intensidade moderada a severa; os ductos excretores apresentaram-se atróficos, ectásicos, exibindo metaplasia oncocítica e fibroplasia periductal; as alterações vasculares, por sua vez, se apresentaram em maior proporção na forma de vasculite. No LPO as alterações teciduais foram menos intensas. Quando analisadas as concentrações das citocinas, na DECHc, foram encontradas maiores concentrações de IL-17A, IL-4, IL-17F e IL-10, em relação aos grupos controles, essas citocinas estão envolvidas em mecanismos prófibróticos, o que permitiu a correlação dessa expressão aos eventos escleróticos nas glândulas salivares dos pacientes de DECHc. Entre elas, a IL-17F apresentou uma tendência de aumento em relação a proporção da área de fibrose nas glândulas salivares destes pacientes. A CD40L, que também esteve presente em maior concentração nos pacientes de DECHc, é uma molécula de ligação capaz de amplificar a resposta imunológica no mecanismo de rejeição do enxerto no hospedeiro, além de regular o mecanismo de apoptose e ativar o endotélio para a formação de trombos. As citocinas IL-31, IL-23 e IL-22, também apresentaram relevância na saliva dos pacientes de DECHc, sendo participantes do mecanismo das alterações liquenóides no LPO. Através das análises comparativas foi possível correlacionar a presença de citocinas envolvidas na resposta Th17 com as alterações glandulares e consequente xerostomia nos pacientes de DECHc. / The graft versus host disease (GVHD) is one of the biggest causes of mortality and morbidity after hematopoietic stem cells transplantation. The pathophysiology in the chronical manifestation of the disease (cGVHD), is not entirely elucidated, however the involvement of the immunological system, by means of the innate and adaptive responses are depicted in the literature concerning the disease development. The cGVHD affects multiple organs, including the salivary glands, leading to xerostomia. These alterations are under reported and mostly unknown. Therefore, this study investigated if the symptom of xerostomia in cGVHD is triggered by functional e morphological changes in minor salivary glands. To answer this inquiry specimens of biopsied labial salivary glands from patients of cGVHD and xerostomia were analyzed qualitatively and through morphometry. Specimens of biopsied salivary glands from patients with Sjögren\'s Syndrome (SS) (which is a classic model of xerostomia) and from patients with oral lichen planus (OLP) (whose clinical oral manifestations resemble the cGVHD lesions) were used as controls. Also, the cytokines related to the immunological response Th17 (important immune pathway in cGVHD pathophysiology) in the saliva of 21 cGVHD patients, 27 of SS, 10 patients of OLP and 165 healthy individuals were analyzed using the multiplex assay. The major morphological findings revealed on the salivary glands of cGVHD patients were the extensive fibrosis, periductal fibrosis, ductal and acinar atrophy. Congestion and hyaline thrombi formation were the most important vascular changes shown among these specimens. A diffuse interstitial inflammatory infiltrate was observed, with varied intensity. The SS salivary glands, however, portrayed a focal inflammatory infiltrate, with moderate to severe intensity around the excretory ducts. These ducts exhibited atrophy, ectasia, periductal fibrosis and oncocytic metaplasia. The main vascular change presented in these patients was the manifestation of vasculitis. The salivary glands from the OLP patients showed a lesser amount of alterations. The multiplex assay revealed a higher concentration of the cytokines IL-17A, IL-4, IL-17F and IL-10 in the cGVHD samples, when compared to the other groups. These cytokines are involved within the promotion of fibrosis, which endorsed the association of these secretions with the salivary glands sclerotic mechanisms. The secretion of CD40L was higher in cGVHD samples; this membrane protein is capable of amplifying the immunological response in graft rejection, besides the capacity to regulate apoptosis and activate the endothelium in thrombi formation. The cytokines IL-31, IL-23 and IL-22, also presented a higher concentration in cGHVD patients\' saliva, these secretions are actively involved in the mechanisms of lichenoid lesions in OLP, corroborating the perceived morphological changes. The comparative analysis of the morphological and salivary changes in cGVHD confirmed the correlation of Th17 immunological response within the minor salivary glands injuries and consequent xerostomia in these patients.

L’importance respective des lymphocytes T régulateurs naturels générés dans le thymus ou induits en périphérie dans la régulation immunitaire et la résolution de l’inflammation est désormais bien établie. Nous avons contribué à mettre en évidence une nouvelle voie d’induction de lymphocytes T régulateurs périphériques à partir de cellules T humaines CD4+CD25- naïves et mémoires. Nous avons montré que l’engagement de la molécule ubiquitaire transmembranaire CD47 sur la cellule T par un anticorps monoclonal ou par le peptide 4N1K (peptide dérivé du domaine carboxy-terminal de la thrombospondine-1 et spécifique du site de liaison à CD47) induisait des lymphocytes T CD4+ régulateurs exerçant une fonction suppressive sur les lymphocytes T effecteurs. Les propriétés suppressives induites par la thrombospondine-1 confortent les fonctions anti-inflammatoires de cette protéine de la matrice extracellulaire. L’inhibition exercée par les lymphocytes T régulateurs induits dépend du contact intercellulaire entre les cellules T régulatrices et leurs cibles, et est indépendante du TGF-. Nos résultats démontrent également le rôle de CD47 sur le lymphocyte T CD4+ dans la réponse immunitaire spécifique de l’antigène in vivo. En effet, les souris BALB/c déficientes pour CD47 présentent un biais de la sécrétion d’anticorps et de cytokines de type Th1, alors que les souris BALB/c sont décrites comme exprimant un profil de production de cytokines de type Th2. Nos travaux mettent en évidence le rôle de CD47 dans l’inhibition du développement d’une réponse cellulaire et humorale de type Th1 in vivo, confirmant de précédentes études in vitro réalisées avec des cellules T CD4+ humaines. Nous présentons également le rôle inhibiteur de l’engagement de CD28 in vitro sur la différenciation en cellules Th17 des lymphocytes T CD4+ naïfs isolés de souris BALB/c. Le mécanisme proposé est dépendant de la production de l’IL-2 et de l’IFN- et indépendant de la présence de lymphocytes T régulateurs. Notre étude du rôle de deux molécules transmembranaires CD47 et CD28 exprimées sur la cellule T CD4+, contribue à une meilleure connaissance des mécanismes impliqués dans la tolérance immunologique, la résolution de l’inflammation et la différenciation des cellules T "helper" CD4+. / Nowadays, the importance of natural regulatory T cells and adaptive regulatory T lymphocytes in immune regulation and resolution of inflammation are well established. We report a previously unknown pathway to generate adaptive regulatory T cells in the periphery from naive and memory human CD4+CD25- T cells. We show that the stimulation of the broadly expressed transmembrane proteins CD47 on T cells by a monoclonal antibody or by the 4NK1 peptide (carboxy-terminal peptide of thrombospondin-1 (TSP) specific of the binding site of CD47) induced regulatory T cells that exerted an inhibitory function on effector T cells. Our study on the suppressive proprieties of the TSP corroborates with reported anti-inflammatory activities of this extracellular matrix protein. The suppressive function of TSP induced regulatory T cells was contact-dependent and TGF--independent. Our data further demonstrate the role of CD47 expression on T cells in the antigenic-specific immune response in vivo. We report that the CD47-deficient BALB/c mice displayed a Th1-biased antibody and cytokine responses, instead of the Th2 cytokine profile observed in unmanipulated BALB/c mice. Our study outlines the role of CD47 as a self-control mechanism to negatively regulate type 1 cellular and humoral immune responses and most importantly confirm in vivo previous in vitro studies with human CD4+ T cells. We also report that soluble anti-CD28 monoclonal antibody suppressed in vitro differentiation of naïve CD4+ T cells isolated from BALB/c mice into IL-17-producing cells by mechanism that are IL-2 and IFN-γ-dependent but independent of the presence of regulatory T cells. Our studies highlight the suppressive function of two transmembrane molecules CD47 and CD28 expressed by CD4+ T cells in vitro and in vivo in human and mice. They thus may contribute to a better understanding of the mechanisms involved in the induction of immune tolerance, the resolution of inflammation and the differentiation of the T helper cells.

A doença do enxerto contra o hospedeiro (DECH) é uma das maiores causas de mortalidade e morbidade pós-transplante de células-tronco hematopoiéticas. A DECH, em sua manifestação crônica (DECHc), ainda não tem sua fisiopatologia totalmente esclarecida; entretanto, o envolvimento do sistema imunológico, por meio de respostas imunes inatas e adaptativas é bem estabelecido na literatura. A DECHc afeta múltiplos órgãos, incluindo as glândulas salivares, o que tem, como causa imediata, a xerostomia. Essas alterações são largamente desconhecidas e sub-relatadas. Assim, esse estudo indagou se o sintoma de xerostomia na DECHc é decorrente de alterações morfológicas e funcionais das glândulas salivares. Para responder essa pergunta analisamos de forma qualitativa e por meio da morfometria, espécimes de biopsias de glândulas salivares labiais de pacientes com DECHc e com sintoma de xerostomia. Foram utilizados como controles, espécimes de biopsias de glândulas salivares de pacientes diagnosticados com Síndrome de Sjögren (SSp) (que é um modelo clássico de xerostomia) e de pacientes com líquen plano oral (LPO) (cujas manifestações clínicas orais podem se assemelhar às da DECHc) e queixa de xerostomia. Também foram analisadas, por meio de ensaio multiplex, as citocinas relacionadas à resposta imune Th17 (importante via imunológica na patogenia da DECHc) na saliva de 21 pacientes de DECHc, 27 de SSp, 10 de LPO e 15 voluntários saudáveis. Os principais achados morfológicos nas glândulas salivares dos pacientes de DECHc foram a extensa fibrose, fibroplasia periductal, atrofia ductal e acinar; alterações vasculares representadas pela congestão e formação de trombos hialinos; e infiltrado inflamatório intersticial difuso de aspecto leve a moderado. As glândulas salivares de SSp, entretanto, apresentaram um infiltrado inflamatório na forma de focos de linfócitos ao redor dos ductos excretores de intensidade moderada a severa; os ductos excretores apresentaram-se atróficos, ectásicos, exibindo metaplasia oncocítica e fibroplasia periductal; as alterações vasculares, por sua vez, se apresentaram em maior proporção na forma de vasculite. No LPO as alterações teciduais foram menos intensas. Quando analisadas as concentrações das citocinas, na DECHc, foram encontradas maiores concentrações de IL-17A, IL-4, IL-17F e IL-10, em relação aos grupos controles, essas citocinas estão envolvidas em mecanismos prófibróticos, o que permitiu a correlação dessa expressão aos eventos escleróticos nas glândulas salivares dos pacientes de DECHc. Entre elas, a IL-17F apresentou uma tendência de aumento em relação a proporção da área de fibrose nas glândulas salivares destes pacientes. A CD40L, que também esteve presente em maior concentração nos pacientes de DECHc, é uma molécula de ligação capaz de amplificar a resposta imunológica no mecanismo de rejeição do enxerto no hospedeiro, além de regular o mecanismo de apoptose e ativar o endotélio para a formação de trombos. As citocinas IL-31, IL-23 e IL-22, também apresentaram relevância na saliva dos pacientes de DECHc, sendo participantes do mecanismo das alterações liquenóides no LPO. Através das análises comparativas foi possível correlacionar a presença de citocinas envolvidas na resposta Th17 com as alterações glandulares e consequente xerostomia nos pacientes de DECHc. / The graft versus host disease (GVHD) is one of the biggest causes of mortality and morbidity after hematopoietic stem cells transplantation. The pathophysiology in the chronical manifestation of the disease (cGVHD), is not entirely elucidated, however the involvement of the immunological system, by means of the innate and adaptive responses are depicted in the literature concerning the disease development. The cGVHD affects multiple organs, including the salivary glands, leading to xerostomia. These alterations are under reported and mostly unknown. Therefore, this study investigated if the symptom of xerostomia in cGVHD is triggered by functional e morphological changes in minor salivary glands. To answer this inquiry specimens of biopsied labial salivary glands from patients of cGVHD and xerostomia were analyzed qualitatively and through morphometry. Specimens of biopsied salivary glands from patients with Sjögren\'s Syndrome (SS) (which is a classic model of xerostomia) and from patients with oral lichen planus (OLP) (whose clinical oral manifestations resemble the cGVHD lesions) were used as controls. Also, the cytokines related to the immunological response Th17 (important immune pathway in cGVHD pathophysiology) in the saliva of 21 cGVHD patients, 27 of SS, 10 patients of OLP and 165 healthy individuals were analyzed using the multiplex assay. The major morphological findings revealed on the salivary glands of cGVHD patients were the extensive fibrosis, periductal fibrosis, ductal and acinar atrophy. Congestion and hyaline thrombi formation were the most important vascular changes shown among these specimens. A diffuse interstitial inflammatory infiltrate was observed, with varied intensity. The SS salivary glands, however, portrayed a focal inflammatory infiltrate, with moderate to severe intensity around the excretory ducts. These ducts exhibited atrophy, ectasia, periductal fibrosis and oncocytic metaplasia. The main vascular change presented in these patients was the manifestation of vasculitis. The salivary glands from the OLP patients showed a lesser amount of alterations. The multiplex assay revealed a higher concentration of the cytokines IL-17A, IL-4, IL-17F and IL-10 in the cGVHD samples, when compared to the other groups. These cytokines are involved within the promotion of fibrosis, which endorsed the association of these secretions with the salivary glands sclerotic mechanisms. The secretion of CD40L was higher in cGVHD samples; this membrane protein is capable of amplifying the immunological response in graft rejection, besides the capacity to regulate apoptosis and activate the endothelium in thrombi formation. The cytokines IL-31, IL-23 and IL-22, also presented a higher concentration in cGHVD patients\' saliva, these secretions are actively involved in the mechanisms of lichenoid lesions in OLP, corroborating the perceived morphological changes. The comparative analysis of the morphological and salivary changes in cGVHD confirmed the correlation of Th17 immunological response within the minor salivary glands injuries and consequent xerostomia in these patients.