Recentemente, a expressão gênica dos receptores TLR foi encontrada em diversos tipos de células tumorais. A sua participação na biologia do câncer é controversa já que foram descritas ações pró e anti-tumorais após a ativação de sua sinalização. Na Leucemia Mielóide Crônica (LMC) nada se tem demonstrado. BCR-ABL é uma oncoproteína quimérica cujo sítio tirosina quinasa constitutivamente ativado promove inúmeras vias de sinalizações que desencadeia a transformação celular. Este trabalho se inicia com a hipótese de existir uma relação entre o oncogene BCR-ABL e a expressão dos receptores TLRs. Nós verificamos em células murinas TonB210.1 com expressão de BCR-ABL induzível por doxiciclina que Tlr1 e Tlr2 tem sua expressão gênica relativa aumentada na presença da oncoproteína. A regulação positiva de Tlr1 é dependente da ação tirosina quinasa de BCR-ABL. Também mostramos que as vias p38 e JNK estão reprimindo a expressão de Tlr1 induzida por BCR-ABL enquanto que a via ERK é utilizada pelo BCR-ABL para promovê-la. Por outro lado, observamos que a ligação de TLR1/TLR2 com seu agonista sintético Pam3CSK4 em células TonB210.1 BCR-ABL positivas induz um aumento da produção de IL-6 e leva ao aumento da resistência a morte quando induzida pelas drogas Ara-C e VP16. Em conclusão, estes resultados indicam que BCR-ABL esta regulando a expressão gênica de alguns TLRs. Por tanto esses dados contribuem para a compreensão sobre o comportamento de células tumorais BCR-ABL positivas em um contexto de infecção e por conseqüência, dão margem ao estudo de novos alvos de fator de risco para a LMC. / Recently, the gene expression of TLR receptors have been described in several kinds of tumour cells. Its participation in cancer biology is controversial because roles were already been described in pro and anti-tumoral activities after their signaling activation. In Chronic Myeloid Leukemia (CML) there are no published data. BCR-ABL is a quimeric protein and its tyrosine-kinase site is activated constitutively. Thus, many signaling pathways are activated and several cell processes are altered thereby resulting in cellular transformation. This work has started with the hypothesis that a putative relationship between the oncogene BCR-ABL and the expression of TLR receptors could exists. We verified in murine cells TonB210.1 BCR-ABL expression inducible by doxycicline that Tlr1 and Tlr2 have their relative gene expression up-regulated in the presence of the oncoprotein. Therefore the Tlr1 regulation is dependent of BCR-ABL tyrosine kinase action. Using MAPK inhibitors we showed that p38 and JNK pathways are suppressing the TLR1 induction by BCR-ABL while ERK pathway is used by the oncoprotein for promote it. On the other hand, we observed in TonB210.1 BCR-ABL positive cells that the binding of TLR1/TLR2 heterodimer to their synthetic agonist Pam3CSK4 induced an increased production of IL-6 and when these cells were induced by Ara-C and VP-16 drugs the apoptosis resistance increased. In conclusion, these results indicate that the oncoprotein regulates the gene expression of some TLRs. Therefore, this fact gives us data about the behavior of BCR-ABL positive tumor cells in the context of infection and in consequence the study of new risk factor targets for CML.

Dans le travail portant sur l'immunorégulation de la réponse immunitaire dans le modèle expérimental d'asthme, nous avons montré que les basophiles étaient activés par des ARN double brin poly(A:U) et que cette stimulation exacerbait les réponses d'asthme. Nous avons ensuite étudié la modulation de l'asthme en utilisant des agonistes de la voie TLR/IL1R. Les résultats obtenus ont montré que le R848, agoniste de TLR7 qui induit les réponses immunitaires antivirales de type Th1, et l'IL-33 qui favorise les réponses Th2, activaient les cellules NKT dont la production rapide et modulée des cytokines pourrait avoir un effet modulateur dans l'asthme. Nous avons montré que les cellules NKT avaient une fonction régulatrice sur le développement et l'activité des cellules Th17. Enfin, nous avons décrit les effets protecteurs et suppresseurs induits par le R848 dans le modèle d'asthme et montré que ces effets suppresseurs étaient dépendants des cellules T régulatrices et du TGF-β.

[Truncated abstract] The last few decades have witnessed an increase in the prevalence, morbidity and economic burden associated with asthma and allergic disease. This rising incidence cannot be completely explained by changes in genetic factors or by improvements in diagnostic procedures. Environmental factors, particularly those associated with a westernised lifestyle, are considered to be involved in this increase. In the late 1980’s Strachan was the first to link environmental factors with allergic disease, this theory became to be known as the ‘hygiene hypothesis’. This hypothesis links the “cleaner” more “healthy ” environment we now live in, with an increased risk of developing allergic disease. This effect is highlighted by studies linking farm and animal exposure (rich in microbial compounds) during early life with a decrease in allergic disease. Since then numerous studies have been undertaken to ascertain the factors present in the microbe rich environment, which elicit this protective effect. Many studies have revolved around endotoxin, however microbial components (mainly from Gram-positive bacteria) which signal through Toll-like receptor 2 (TLR2), have also shown that they can alter the allergic immune response. In mice models TLR2 has been shown to both exacerbate and inhibit allergic disease. The above research highlights the need for further studies into the effect of TLR2 ligands, and to define the mechanisms by which they exert their effects in human allergic disease. These mechanisms will be relevant to understanding the pathogenesis of allergy, but also might provide novel ways to treat allergy. The aims of the study outlined in this thesis were to determine whether in vitro exposure to TLR2 ligands could modify the established immune response to house dust mite allergen (HDM), and to examine the mechanisms by which this occurs. ... The addition of glucocorticoids to LTA enhanced the ability of this TLR2 ligand to inhibit IL-5 and IL-13 production by HDM-activated blood mononuclear cells. In conclusion, this study shows that TLR2 ligands have the ability to inhibit the Th2 response to mite allergen in previously sensitized individuals by an as yet unknown mechanism. However the findings described herein do provide an impetus for future studies designed to uncover novel mechanisms by which allergic responses can be ameliorated, and may open new treatment modalities.

Made available in DSpace on 2015-07-22T20:49:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-28 / Objetivo: Verificar o efeito inflamatório do exercício agudo exaustivo sobre diferentes depósitos de tecido adiposo branco (mesentérico e retroperitoneal), assim como, sobre músculos esqueléticos (sóleo: fibra muscular tipo I; EDL: fibra muscular tipo II). Métodos: os animais (ratos Wistars) foram submetidos a uma sessão exaustiva de exercício, após período de adaptação. Os depósitos de tecido adiposo branco, retroperitoneal e mesentérico, como, os músculos EDL e sóleo, foram retirados logo após o sacrifício e analisados para a expressão gênica (PCR-real time) e protéica (ELISA) da citocinas IL-6, IL-10, TNF-α, IL-2 e IL-4. O sacrifício foi realizado logo após a sessão de exercício, duas e seis horas após, e o grupo controle não foi submetido à sessão exaustiva de exercício. Por Western Blotting foi analisado o conteúdo de TLR-4, e sua via de sinalização TRAF6 e MYD88. Resultados: As citocinas IL-6, IL-10 e TNF-α foram encontradas elevadas em todos os tecidos e grupos dos ratos exercitados. No entanto, a razão IL-10/TNF-α foi maior no músculo e encontrou-se diminuída no tecido adiposo. O TLR-4, assim como sua via (TRAF6 e MYD88), foi aumentada nos depósitos de tecido adiposo branco de ratos submetidos ao exercício exaustivo, sem alteração no EDL e sóleo. Já as citocinas IL-2 e IL-4 tiveram suas concentrações alteradas no sóleo e EDL, mas não foram moduladas no tecido adiposo pelo protocolo de exercício exaustivo. Conclusão: O exercício exaustivo apresentou um aumento da resposta antiinflamatória no músculo esquelético, pelo aumento da razão IL-10/TNF-α. Já o tecido adiposo observado (mesentérico e retroperitoneal) apresentou um aumento da resposta inflamatória, evidenciada pela diminuição da razão IL-10/TNF-α e pelo aumento da expressão protéica do TLR-4. Além disso, a IL-4 foi aumentada 6 horas após o protocolo de exercício no EDL, concomitante com o aumento do MyoD. Esse aumento pode levar a um aumento da hiperplasia muscular. / Aim: to verify the inflammatory effects of exhaustive exercise on adipose tissue (mesenteric and retroperitoneal), and skeletal muscle (sóleus: fiber type I, EDL: Fyber type II). Methods: Wistar rats were randomly assigned to four groups: group control and group exercised until exhaustion, these rats were sacrificed in three different times, after the exhaustion, two hours after and six hours after exhaustive exercise. Cytokines IL-2, IL-4, TNF-α, IL-6 and IL-10 were analyzed by mRNA (PCR Real Time) and protein (ELISA) expression. TLR-4 and your inflammatory via (TRAF6, MYD88), were analyzed by Western Blotting. Results: IL-6, IL-10 and TNF-α were elevated in all tissues and groups compared with control group. However, IL-10/TNF-α ratio was higher in skeletal muscle, and opposite response was found in adipose tissue. TLR-4 and your via was elevated in adipose tissue, but not altered in skeletal muscle after exhaustive exercise. The concentrations of IL-2 and IL-4 were altered in skeletal muscle, but were not modulated in adipose tissue, after exercise protocol. Conclusion: exhaustive exercise showed one increased in anti-inflammatory response in skeletal muscle, by increased of IL-10/TNF-α ratio. On the other hand, adipose tissue showed increased in inflammatory response, evidenced by decreased in IL-10/TNF-α ratio and increased in TLR-4 content. Moreover, the IL-4 was increased in EDL, concomitant with raised in Myod. This results could showed one increased in hyperplasia in skeletal muscle. / TEDE / BV UNIFESP: Teses e dissertações

It has been reported that opioid receptor activation mimics ischemic preconditioning, which may protect the heart from the development of infarction. Toll-like receptor 4 (TLR-4) during infarction stimulates cytokine production leading to inflammation and injury of the heart tissue. Our aim was to study the effect of morphine in vitro on the viability and oxidative state of H9c2 cells (rat cardiomyoblasts) and the role of TLR-4 during oxidative stress. Our experiments showed that pretreatment with morphine before tert-butylhydroperoxide (t-BHP)-, 2,2'-bipyridyl (BP)- and lipopolysaccharide (LPS)-induced oxidative stess had protective effect on the viability of H9c2 cells and markedly reduced the production of reactive oxygen species (ROS). The protective effect of morphine was diminished after naloxone treatment, which confirms the role of opioid receptors in preconditioning. TLR-4 inhibition by TAK-242 pretreatment and silencing TLR-4 by RNA interference resulted in a partial increase in cell viability but significant attenuation of ROS production after t-BHP and BP treatment. The action of LPS was reduced in response to TLR-4 silencing. Interestingly, naloxone pretreatment and suppression of TLR-4 markedly alleviated oxidative stress and resulted in a significant improvement of cell viability. We...

The kinetics of tumor growth and progression are governed by the interaction between tumor cells, the non-malignant stroma and both innate and adaptive immune cell lineages. Innate immunity has a critical role in the control of tumor cell growth and metastasis. The microenvironment of many tumors is populated with innate immune cells, including regulatory natural killer (NK) cells and dendritic cells (DCs), tumor associated macrophages, and myeloid derived suppressor cells, that suppress normal immune function. Much of our understanding of interactions between tumors and the innate immune system is based on experimental studies performed in mouse “syngenic” models. However, there is clear need for a mechanistic understanding of the human innate immune system within the tumor microenvironment. The goal of my thesis is to characterize the interactions between human innate immune cells and tumors and to define specific pathways and cell lineages that are targets for immune modulation. A central focus of my thesis is the use of cutting-edge humanized mouse models based on the immunodeficient NOD-scid IL2Rgnull (NSG) mouse strain to study human immuno-oncology. In the first section of my thesis I describe studies that evaluate the influence of inflammatory stimuli on innate immune control of tumors. Agents that induce inflammation have been used since the 18th century for the treatment of cancer. The inflammation induced by agents such as toll-like receptor (TLR) agonists is thought to stimulate tumor-specific immunity in patients and augment control of tumor burden. While NSG mice lack murine adaptive immunity (T and B cells), these mice maintain a residual murine innate immune system that responds to TLR agonists. Here I describe a novel NSG mouse strain lacking TLR4 that fails to respond to lipopolysaccharide (LPS). NSG-Tlr4null mice support human immune system engraftment and enables the study of human specific responses to TLR4 agonists. My data demonstrate that specific stimulation of TLR4 activates human innate immune system and promotes regression of human patient derived xenograft (PDX) tumors. In the second section of my thesis I describe the development of an NSG mouse strain that constitutively expresses human Interleukin 15 (IL15) and supports the development of functional human NK cells. Using humanized NSG-IL15 transgenic mice (NSG-Tg(Hu-IL15), my data clearly demonstrate a critical role for human NK cells in limiting growth of a PDX melanoma. In the third section of my thesis I describe, the use of the bone marrow/liver/thymus (BLT) humanized mouse model to study the interactions between the human immune system and PDX melanoma and to evaluate the response of the melanoma to immunotherapy modalities. My results collectively suggest that mice engrafted with human immune systems and bearing human tumors can be harnessed as translational models, which are critically needed as tools to study tumor immunotherapy. These humanized mouse models are an ideal translational tool to advance our understanding of human immuno-oncology and for development and testing of novel immune therapies for the treatment of malignancies.

No description available.

La réaction inflammatoire est un processus complexe et essentiel à la défense de l'hôte contre l'infection et le dommage tissulaire. Le déclenchement de la réponse inflammatoire s'effectue entre autre suite à l'activation de récepteurs appelés récepteurs ± toll-like ¿ par des molécules d'origine microbienne ou provenant des tissus endommagés. Le premier leucocyte circulant à atteindre le site inflammatoire est le neutrophile. Le recrutement du neutrophile de la circulation sanguine jusqu'au tissu est un processus finement régulé qui nécessite l'interaction du neutrophile avec 1'endothelium vasculaire. Les médiateurs lipidiques de l'inflammation, les leucotriènes et le facteur activateur des plaquettes, peuvent être produits par les neutrophiles et les cellules endothéliales et régulent plusieurs aspects indispensables au recrutement des neutrophiles soit leur adhésion, transmigration et chimiotaxie. Les travaux présentés dans cette thèse ont mené au développement d'un modèle de migration transendothéliale des neutrophiles humains in vitro permettant l'étude du rôle des médiateurs lipidiques dans la migration des neutrophiles. Les résultats obtenus au cours de ces expériences montrent que la présence d'une matrice extracellulaire dans le modèle de migration transendothéliale augmente l'impact du leucotriène B4 et du facteur activateur des plaquettes dans la migration des neutrophiles. L'utilisation de ce modèle de migration transendothéliale in vitro a également permis de démontrer que la production de novo du leucotriène B4 et du facteur activateur des plaquettes a un rôle clé dans la migration des neutrophiles induite par différents ligands des récepteurs ± toll-like ¿. En effet, l'inhibition de la biosynthèse ou le blocage des récepteurs du leucotriène B4 et du facteur activateur des plaquettes dans ce modèle réduit significativement (jusqu'à 90%) la migration transendothéliale des neutrophiles induite par les ligands des récepteurs ± tolllike ¿. Ce rôle majeur du leucotriène B4 et du PAF dans le recrutement des neutrophiles a également été confirmé in vivo dans le modèle de la poche d'air dorsale chez la souris. Les travaux présentés dans cette thèse contribuent donc significativement à l'amélioration des connaissances concernant les mécanismes régulant le recrutement des neutrophiles lors de la réponse inflammatoire.

No / The interleukin-2-deficient (IL-2¿/¿) mouse model of ulcerative colitis was used to test the hypothesis that colonic epithelial cells (CEC) directly respond to bacterial antigens and that alterations in Toll-like receptor (TLR)-mediated signaling may occur during the development of colitis. TLR expression and activation of TLR-mediated signaling pathways in primary CEC of healthy animals was compared with CEC in IL-2¿/¿ mice during the development of colitis. In healthy animals, CEC expressed functional TLR, and in response to the TLR4 ligand LPS, proliferated and secreted the cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1). However, the TLR-responsiveness of CEC in IL-2¿/¿ mice was different with decreased TLR4 responsiveness and augmented TLR2 responses that result in IL-6 and MCP-1 secretion. TLR signaling in CEC did not involve NF-B (p65) activation with the inhibitory p50 form of NF-B predominating in CEC in both the healthy and inflamed colon. Development of colitis was, however, associated with the activation of MAPK family members and upregulation of MyD88-independent signaling pathways characterized by increased caspase-1 activity and IL-18 production. These findings identify changes in TLR expression and signaling during the development of colitis that may contribute to changes in the host response to bacterial antigens seen in colitis.

Résumé confidentiel / Confidential abstract