Au cours de ce travail de thèse intitulé " Identification chimique de métabolites secondaires de certains microorganismes, évaluation de leurs effets dans les domaines pharmaceutiques et agronomiques ", nous nous sommes intéressés à l'étude des effets des conditions de culture sur la production des composés organiques volatils microbiens à partir de deux souches bactériennes co-existantes dans le sol Français : Burkholderia sp. et Bacillus megaterium. A partir des différents extraits préparés, plus que cent composés ont été identifiés, comprenant les dicétopipérazines, les alcools, les composés soufrés, les esters et les acides carboxyliques, par le biais de plusieurs techniques chimiques, analytiques et spectroscopiques. Les résultats obtenus ont montré que les conditions de culture sont les pricipales responsables de la production des différentes familles chimiques des volatiles. Nous avons identifiés des composés qui sont rapportés pour la première fois à partir des bactéries tel que: la N-butylbenzènesulfonamide, triacontane, le proponaoate de 3- (3,5-di-tert-butyl-4-hydroxyphényl), (E) -5-chloro-3-(hydroxyimino) indoline-2-one et 1,3,5-triméthyl-2-octadecylcyclohexane. Sur le plan biologique, on a montré que les résultats obtenus sont fortement influencés par les conditions de culture utilisées pour cultiver les bactéries testées. En parallèle à cette investigation, nous avons montré que les extraits de Burkholderia sp. sont dotés d'un très important potentiel allélopathique. Enfin, une série des analogues de dicétopipérazines a été préparée et évaluée pour leurs activités anti-xanthine oxydase, anti a-amylase et anti 5-lipoxygénase ainsi que pour leurs activités cytotoxiques contre les lignées cellulaires suivantes ; OVCAR, MCF7 et HCT116. Un certain nombre de ces dérivés de dicétopiperazine ont montré des activités anti a-amylase et cytotoxique importantes. / In this thesis entitled " Chemical identification of secondary metabolites from microorganism, evaluation of their effects on pharmaceutical and agronomic fields ", we are interested in studying the effect of culture conditions on the production of microbial volatiles organic compounds by two bacteria that inhabit French soil which are: Burkholderia sp. and Bacillus megaterium. From different prepared extracts, more than one hundred compounds were identified, including diketopiperazine, alcohols, sulfur containing compounds, esters and carboxylic acids, by means of several chemical, analytical and spectroscopic techniques. Results showed that culture conditions of different bacteria are the mainly responsible of production of different blend of volatiles. Many identified compounds including N-butylbenzenesulfonamide, triacontane, octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate, (E)-5-chloro-3-(hydroxyimino)indolin-2-one and 1,3,5-trimethyl-2-octadecylcyclohexane are reported for the first time from bacteria.Biologically, we have shown that obtained results are greatly influenced by the cultures conditions used in cultivation of tested bacteria. In addition to that, we have shown that Burkholderia sp. extracts possessed a very good allelopathic potential. Finally, a series new protested deketopiperazine derivatives have been prepared and evaluated in vitro against xanthine oxidase, a-amylase and 5-lipoxygenase enzymes, OVCAR, MCF7 and HCT116 cancer cell lines. Some of these molecules have been shown to be potent inhibitors of a-amylase and different cancer cell lines.

Durante o repasto sanguíneo, os mosquitos hematófagos inoculam os componentes de seu coquetel salivar na pele do hospedeiro podendo causar reações inflamatórias. Existem vários trabalhos que descrevem o infiltrado celular após a picada de mosquito utilizando abordagens clássicas de histologia. No entanto, são escassos os trabalhos que avaliem esse infiltrado celular de maneira quantitativa/qualitativa com técnicas mais modernas. Adicionalmente, a literatura científica carece de modelos simples para o estudo das moléculas envolvidas na migração celular induzida pelos componentes salivares de mosquitos. Diante disto, em um primeiro momento estudamos o infiltrado celular na pele da orelha decorrente da picada do mosquito Aedes aegypti em animais sensibilizados ou não com o extrato da glândula salivar (EGS) destes mosquitos utilizando a técnica de citometria de fluxo. Observamos que enquanto o edema em animais não sensibilizados alcança um pico 6 horas após a exposição aos mosquitos, o mesmo só acontece após 24 horas nos animais sensibilizados, persistindo ainda até 72 horas. A fenotipagem das células imunes presentes no tecido por citometria de fluxo mostrou a complexidade de populações celulares presentes na reação inflamatória à picadas de mosquito, destacando-se o infiltrado de linfócitos B, neutrófilos e eosinófilos que ocorreram em ambos os grupos, porém de maneira mais intensa após 24 horas no grupo sensibilizado. Dentre essas populações, os eosinófilos foram as células que proporcionalmente mais aumentaram em relação àquelas encontradas na orelha de animais naïve. Em uma segunda abordagem, desenvolvemos um modelo de inflamação eosinofílica na cavidade peritoneal de camundongos inoculados com o EGS de A. aegypti. Utilizando esse modelo, fomos capazes de mostrar que a migração de eosinófilos se deve a atividade dos componentes salivares do mosquito e não a uma possível contaminação por endotoxina presentes na preparação. Além disso, a desnaturação das proteínas do EGS aboliu sua capacidade de induzir migração de eosinófilos, reforçando a importância da integridade das moléculas salivares nesse processo. Finalmente, mostramos que a migração de eosinófilos nesse modelo é dependente de IL-5 e de produtos da 5-lipoxigenase. Em conjunto, esses dados contribuem para a caracterização da resposta inflamatória do hospedeiro aos componentes salivares do mosquito A. aegypti e para o entendimento das moléculas envolvidas na reação eosinofílica presente em indivíduos sensibilizados. / During their blood feeding, hematophagous mosquitoes inoculate the components of their salivary cocktail in the host skin causing inflammatory reactions. Several works describe the cellular infiltrate following the mosquito bites using classical histological approaches. However, only few studies evaluate the cellular infiltrate in a quantitative/qualitative manner employing recent techniques. Additionally, the scientific literature lacks simple models to study the molecules involved in cell migration induced by the salivary components of mosquitoes. In view of this facts, we first studied the cellular infiltrate in the skin of the ear following Aedes aegypti mosquito bites in animals sensitized or not with the salivary gland extract (SGE) of these mosquitoes, using the flow cytometry technique. We observed that, while the swelling reached a peak at 6 hours after exposure to mosquitoes in non-sensitized animals, the same occurred only after 24 hours in sensitized animals, and the edema still persisted at 72 hours. Immune cell phenotyping of the tissue by flow cytometry showed the complexity of cell populations present in the inflammatory reaction to mosquito bites, especially the infiltration of B lymphocytes, neutrophils and eosinophils that occurred in both groups, but more intensely after 24 hours in the sensitized group. Among these populations, eosinophils were the cells that most increased proportionally compared those found in the ear of naïve animals. In a second approach, we have developed a model of eosinophilic inflammation in the peritoneal cavity of mice inoculated with the A. aegypti SGE. Using this model, we were able to show that the migration of eosinophils was due to the activity of the mosquito salivary components and not to a possible endotoxin contamination of the preparation. Moreover, the denaturation of SGE proteins abolished its ability to induce eosinophil migration, underscoring the importance of the integrity of salivary molecules in this process. Finally, we showed that the eosinophil migration in this model is dependent on IL-5 and 5-lipoxygenase products. Together, these data contribute to the characterization of the host inflammatory response to salivary components of A. aegypti mosquitoes and to the understanding of the molecules involved in this eosinophilic reaction in sensitized individuals.

Durante a resposta inflamatória, diversos mediadores são liberados localmente com o objetivo de estimular a resposta imune celular e humoral. Por meio da ação das enzimas ciclooxigenases e lipoxigenases ocorrerão modificações estruturais na cadeia do ácido araquidônico levando a síntese de prostaglandinas ou leucotrienos e lipoxinas, respectivamente. Tais mediadores são responsáveis pela regulação da expressão dos genes RANK, RANKL e OPG, moduladores da osteoclastogênese. Dessa maneira, o objetivo deste estudo foi avaliar a expressão do RNA mensageiro (RNAm) para as enzimas envolvidas no metabolismo do ácido araquidônico, ciclooxigenase-2 (COX-2) e 5- lipoxigenase (5-LO), e para os mediadores da osteoclastogênese (RANK, RANKL e OPG) no tecido ósseo, após inoculação de lipopolissacarídeo bacteriano (LPS) nos canais radiculares de molares de camundongos. Posteriormente foi investigado o efeito do bloqueio farmacológico da via COX-2 induzida pelo LPS, na expressão de mediadores da osteoclastogênese e de genes envolvidos no metabolismo ósseo. Foram utilizados 144 camundongos C57BL/6, com 6 semanas de idade, pesando de 18 a 20 gramas, nos quais os canais radiculares dos primeiros molares foram inoculados com uma solução contendo lipopolissacarídeo bacteriano de E. coli (0,1, 1,0 e 10mg/ml). Decorridos os períodos experimentais de 7, 14, 21 e 28 dias, os animais foram submetidos à eutanásia e os blocos contendo dente e osso foram removidos para extração do RNA total. Em seguida, foi realizada a avaliação da expressão gênica por meio de transcrição reversa e reação da polimerase em cadeia em tempo real (qRT-PCR). A análise global da expressão de RNAm para proteínas envolvidas no metabolismo ósseo foi realizada por meio de um ensaio de PCR Array (Osteogenesis RT² Profiler PCR Array). Os valores de expressão relativa de cada RNAm, para cada grupo, foram comparados por meio da análise de variância (ANOVA) de duas vias seguido pelo pós-teste de Bonferroni ou por ANOVA de uma via seguido pelo pós-teste de Dunnett (&alpha = 0,05). A inoculação de LPS nos canais radiculares de molares de camundongos foi capaz de induzir a expressão dos genes PTGS2 e ALOX5, responsáveis pela codificação das enzimas COX-2 e 5-LO, envolvidas no metabolismo do ácido araquidônico, concomitantemente à modulação da expressão dos genes TNFRSF11A, TNFSF11 e TNFRSF11B, responsáveis pela codificação dos moduladores da osteoclastogênese RANK, RANKL e OPG, respectivamente. A administração de Indometacina, um inibidor não seletivo de COX-2, inibiu a expressão de RNAm para RANK e RANKL e estimulou a expressão de OPG durante os períodos iniciais de resposta à inoculação de LPS nos canais radiculares. A inibição da via COX-2 de metabolismo do ácido araquidônico nos períodos iniciais de resposta à inoculação de LPS nos canais radiculares modulou diferencialmente a expressão de genes envolvidos no catabolismo e anabolismo ósseo, indicando possíveis papéis para os mediadores derivados no ácido araquidônico na regulação do metabolismo ósseo. Estes resultados sugerem alvos terapêuticos importantes para intervenção precoce em doenças inflamatórias, como lesões periapicais para evitar a reabsorção do tecido ósseo. / During an inflammatory response, several mediators are locally released in order to stimulate cellular and humoral immune response. Through the action of cyclooxygenase and lipoxygenase enzymes structural changes occur in the arachidonic acid chain leading to synthesis of prostaglandins or leukotrienes and lipoxins, respectively. Such mediators are responsible for the regulation of RANK, RANKL and OPG gene expression, osteoclastogenesis modulators. Thus, the objective of this study was to evaluate the expression of messenger RNA (mRNA) for the enzymes involved in arachidonic acid metabolism, cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), and the osteoclastogenesis mediators (RANK, RANKL and OPG) in bone tissue after injection of bacterial lipopolysaccharide (LPS) in murine dental root canals. Then, COX-2 pathway was pharmacologically blocked for investigation of expression of osteoclastogenesis mediators and genes involved in bone metabolism. We used 144 C57BL/6 mice, 6 weeks-old, weighing 18-20 grams, which had the first molars root canals inoculated with a solution containing LPS from E. coli (0.1, 1.0 and 10 mg/ml). After 7, 14, 21 and 28 days the animals were euthanized and the tooth-and-bone blocks were removed for total RNA extraction. Subsequently, the evaluation of gene expression was performed by reverse transcription and polymerase chain reaction in real time (qRT-PCR). Global analysis of mRNA expression for proteins involved in bone metabolism was performed using PCR arrays (Osteogenesis RT² Profiler PCR Array). The values for relative expression of each mRNA for each group were compared using two-way analysis of variance (ANOVA) followed by Bonferroni post-test or one-way ANOVA followed by Dunnett\'s test (α=0.05). The injection of LPS into the root canals was induced expression of genes PTGS2 and ALOX5, responsible for encoding COX-2 and 5-LO enzymes, involved in the metabolism of arachidonic acid, simultaneously to the modulation of gene expression of TNFRSF11A, TNFSF11 and TNFRSF11B, responsible for encoding the osteoclastogenesis modulators RANK, RANKL and OPG, respectively. Administration of Indomethacin, a non-selective inhibitor of COX-2, inhibited the expression of mRNA for RANK and RANKL and stimulated the expression of OPG during the initial response to the root canals contamination with LPS. Inhibition of the COX-2 pathway from arachidonic acid metabolism in the initial periods of response to LPS injection into the root canals differentially modulated the expression of genes involved in bone catabolism and anabolism, indicating possible roles for mediators derived from arachidonic acid in the regulation of bone metabolism. These results suggest important therapeutic targets for early intervention in inflammatory diseases such as apical periodontitis to avoid resorption of bone tissue.

Durante a resposta inflamatória, diversos mediadores são liberados localmente com o objetivo de estimular a resposta imune celular e humoral. Por meio da ação das enzimas ciclooxigenases e lipoxigenases ocorrerão modificações estruturais na cadeia do ácido araquidônico levando a síntese de prostaglandinas ou leucotrienos e lipoxinas, respectivamente. Tais mediadores são responsáveis pela regulação da expressão dos genes RANK, RANKL e OPG, moduladores da osteoclastogênese. Dessa maneira, o objetivo deste estudo foi avaliar a expressão do RNA mensageiro (RNAm) para as enzimas envolvidas no metabolismo do ácido araquidônico, ciclooxigenase-2 (COX-2) e 5- lipoxigenase (5-LO), e para os mediadores da osteoclastogênese (RANK, RANKL e OPG) no tecido ósseo, após inoculação de lipopolissacarídeo bacteriano (LPS) nos canais radiculares de molares de camundongos. Posteriormente foi investigado o efeito do bloqueio farmacológico da via COX-2 induzida pelo LPS, na expressão de mediadores da osteoclastogênese e de genes envolvidos no metabolismo ósseo. Foram utilizados 144 camundongos C57BL/6, com 6 semanas de idade, pesando de 18 a 20 gramas, nos quais os canais radiculares dos primeiros molares foram inoculados com uma solução contendo lipopolissacarídeo bacteriano de E. coli (0,1, 1,0 e 10mg/ml). Decorridos os períodos experimentais de 7, 14, 21 e 28 dias, os animais foram submetidos à eutanásia e os blocos contendo dente e osso foram removidos para extração do RNA total. Em seguida, foi realizada a avaliação da expressão gênica por meio de transcrição reversa e reação da polimerase em cadeia em tempo real (qRT-PCR). A análise global da expressão de RNAm para proteínas envolvidas no metabolismo ósseo foi realizada por meio de um ensaio de PCR Array (Osteogenesis RT² Profiler PCR Array). Os valores de expressão relativa de cada RNAm, para cada grupo, foram comparados por meio da análise de variância (ANOVA) de duas vias seguido pelo pós-teste de Bonferroni ou por ANOVA de uma via seguido pelo pós-teste de Dunnett (&alpha = 0,05). A inoculação de LPS nos canais radiculares de molares de camundongos foi capaz de induzir a expressão dos genes PTGS2 e ALOX5, responsáveis pela codificação das enzimas COX-2 e 5-LO, envolvidas no metabolismo do ácido araquidônico, concomitantemente à modulação da expressão dos genes TNFRSF11A, TNFSF11 e TNFRSF11B, responsáveis pela codificação dos moduladores da osteoclastogênese RANK, RANKL e OPG, respectivamente. A administração de Indometacina, um inibidor não seletivo de COX-2, inibiu a expressão de RNAm para RANK e RANKL e estimulou a expressão de OPG durante os períodos iniciais de resposta à inoculação de LPS nos canais radiculares. A inibição da via COX-2 de metabolismo do ácido araquidônico nos períodos iniciais de resposta à inoculação de LPS nos canais radiculares modulou diferencialmente a expressão de genes envolvidos no catabolismo e anabolismo ósseo, indicando possíveis papéis para os mediadores derivados no ácido araquidônico na regulação do metabolismo ósseo. Estes resultados sugerem alvos terapêuticos importantes para intervenção precoce em doenças inflamatórias, como lesões periapicais para evitar a reabsorção do tecido ósseo. / During an inflammatory response, several mediators are locally released in order to stimulate cellular and humoral immune response. Through the action of cyclooxygenase and lipoxygenase enzymes structural changes occur in the arachidonic acid chain leading to synthesis of prostaglandins or leukotrienes and lipoxins, respectively. Such mediators are responsible for the regulation of RANK, RANKL and OPG gene expression, osteoclastogenesis modulators. Thus, the objective of this study was to evaluate the expression of messenger RNA (mRNA) for the enzymes involved in arachidonic acid metabolism, cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), and the osteoclastogenesis mediators (RANK, RANKL and OPG) in bone tissue after injection of bacterial lipopolysaccharide (LPS) in murine dental root canals. Then, COX-2 pathway was pharmacologically blocked for investigation of expression of osteoclastogenesis mediators and genes involved in bone metabolism. We used 144 C57BL/6 mice, 6 weeks-old, weighing 18-20 grams, which had the first molars root canals inoculated with a solution containing LPS from E. coli (0.1, 1.0 and 10 mg/ml). After 7, 14, 21 and 28 days the animals were euthanized and the tooth-and-bone blocks were removed for total RNA extraction. Subsequently, the evaluation of gene expression was performed by reverse transcription and polymerase chain reaction in real time (qRT-PCR). Global analysis of mRNA expression for proteins involved in bone metabolism was performed using PCR arrays (Osteogenesis RT² Profiler PCR Array). The values for relative expression of each mRNA for each group were compared using two-way analysis of variance (ANOVA) followed by Bonferroni post-test or one-way ANOVA followed by Dunnett\'s test (α=0.05). The injection of LPS into the root canals was induced expression of genes PTGS2 and ALOX5, responsible for encoding COX-2 and 5-LO enzymes, involved in the metabolism of arachidonic acid, simultaneously to the modulation of gene expression of TNFRSF11A, TNFSF11 and TNFRSF11B, responsible for encoding the osteoclastogenesis modulators RANK, RANKL and OPG, respectively. Administration of Indomethacin, a non-selective inhibitor of COX-2, inhibited the expression of mRNA for RANK and RANKL and stimulated the expression of OPG during the initial response to the root canals contamination with LPS. Inhibition of the COX-2 pathway from arachidonic acid metabolism in the initial periods of response to LPS injection into the root canals differentially modulated the expression of genes involved in bone catabolism and anabolism, indicating possible roles for mediators derived from arachidonic acid in the regulation of bone metabolism. These results suggest important therapeutic targets for early intervention in inflammatory diseases such as apical periodontitis to avoid resorption of bone tissue.

Chronic myeloid leukemia (CML) is a disease characterized by the expansion of granulocytic cells. The BCR-ABL tyrosine kinase inhibitor imatinib, the frontline treatment for Ph+ leukemias, can induce complete hematologic and cytogenetic response in most chronic phase CML patients. Despite the remarkable initial clinic effects, it is now recognized that imatinib will unlikely cure patients because a small cell population containing leukemic stem cells (LSCs) with self-renewal capacity is insensitive to tyrosine kinase inhibitors. In Chapter I, I briefly review the BCR-ABL kinase and its related signaling pathways. BCR-ABL kinase activates several signaling pathways including MAPK, STAT, and JNK/SAPK. BCR-ABL also mediates kinase-independent pathways through SRC family kinases. I will also discuss pathways involving β-catenin, hedgehog, FoxO and Alox5 are critical to the regulation of self-renewal and differentiation in LSC of CML. As detailed in Chapter II, I describe our work evaluating the effects of omacetaxine, a novel CML drug inducing cell apoptosis by inhibition of protein synthesis, on self-renewal and differentiation of LSCs and BCR-ABL-induced CML and acute lymphoblastic leukemia (B-ALL) in mice. We found that treatment with omacetaxine decreased the number of LSCs and prolonged the survival of mice with CML or B-ALL. In chapter III, I describe that Alox5 is an essential gene in the function of LSCs and CML development. We show evidence that Alox5 affects differentiation, cell division, and survival of long-term LSCs. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly and prolonged survival. In chapter IV, I present evidence of our work showing a further dissection the Alox5 pathway by comparing the gene expression profiles of wild type and Alox5-/- LSCs. We show that Msr1 deletion causes acceleration of CML development. We also show that Msr1 affects CML development by regulating the PI3K-AKT pathway and β-catenin. Taken together, these results demonstrate that some pathways including Alox5 and Msr1 play an important role in regulating the self-renewal and differentiation of LSC. More efforts should be put into developing the novel strategies that may effectively target LSCs and thus cure CML.

L’arthrose (OA) est une maladie dégénérative et multifactorielle caractérisée par une destruction de cartilage, une formation d’ostéophytes et une inflammation au niveau de la membrane synoviale. Le 4-hydroxynonénal (HNE), un produit final de la peroxydation lipidique, a été identifié récemment comme un facteur catabolique et un médiateur inflammatoire dans le cartilage arthrosique humain. Notre projet vise à étudier l’effet du HNE sur la régulation de la prostaglandine E2 synthase-1 microsomale (mPGES-1) et de la protéine activante 5-lipoxygénase (FLAP)/5-lipoxygénase (5-LOX) dans les chondrocytes arthrosiques humains. Lorsque les cellules sont traitées une seule fois avec 10 µM HNE, les résultats de Western blot et de PCR en temps réel montrent que l’expression de la cyclooxygénase-2 (COX-2) et de la mPGES-1 augmente de manière significative et atteint respectivement le maximum après 8 et 16 heures d’incubation puis diminue graduellement. Cependant, lorsque les cellules sont traitées plusieurs fois avec 10 µM HNE à 2 heures d’intervalle, l’expression de la COX-2 et de la mPGES-1 augmente en fonction du temps sans subir une baisse après 24 heures d’incubation. Le HNE induit l’activité du promoteur de la mPGES-1 via l’activation du facteur de transcription Egr-1. L’investigation de la 2ème voie du métabolisme de l’acide arachidonique, à savoir 5-LOX/FLAP, montre que le HNE induit l’expression de FLAP après 24 heures de stimulation et celle de 5-LOX seulement après 48 heures. Ceci semble survenir à l’étape de transcription au cours de laquelle HNE induit l’expression de l’ARNm et l’activité du promoteur du gène 5-LOX. Nous avons démontré aussi que le niveau de leukotriène B4 (LTB4) augmente et suit le même profil que celui de la 5-LOX. L’étude des mécanismes moléculaires susceptibles d’être impliqués dans la régulation de la 5-LOX/FLAP par le HNE montre que ce dernier stimule leur expression via l’action de prostaglandine E2 (PGE2) et du facteur de croissance transformant-beta 1 (TGF-β1). En conclusion, notre étude démontre que le HNE induit à court-terme d’incubation la voie de COX-2/mPGES-1 puis par la suite stimule celle de FLAP/5-LOX à long-terme d’incubation dans les chondrocytes arthrosiques humains. Ces résultats suggèrent que la mPGES-1 et 5-LOX/FLAP sont des potentielles cibles thérapeutiques intéressantes pour contrôler la production de PGE2 et LTB4 dans OA. / 4-hydroxynonenal (HNE), a lipid peroxidation end-product, is produced abundantly in osteoarthritic (OA) articular tissues. Recently, we reported that HNE-induced cyclooxygenase-2 (COX-2) decreased gradually in human OA chondrocytes after 8 h of incubation. This study aimed to investigate whether COX-2 down-regulation is attributed to HNE depletion and is responsible for the switch from COX-2 to 5-lipoxygenase-activating protein (FLAP)/5-lipoxygenase (5-LOX). Treatment of chondrocytes with 10 µM HNE induced prostaglandin E2 (PGE2) release as well as COX-2 and microsomal prostaglandin E2 synthase-1 (mPGES-1) expression at the protein and mRNA levels, with a plateau reached at 8-16 h of incubation, followed by a subsequent decline. However, 8 repeated treatments with 10 µM HNE prevented the reduction of COX-2 and mPGES-1 expression. We demonstrated that HNE induced mPGES-1 promoter activity mainly through transcription factor Egr-1 activation. On the other hand, when COX-2 expression decreased, leukotriene B4 (LTB4) level rose after a long period of stimulation (48 and 72 h). At the mRNA level, HNE induced FLAP and 5-LOX expression after 24 and 48 h of stimulation, respectively. The addition of a nonspecific COX-2 inhibitor (naproxen) to cultured chondrocytes revealed that FLAP and 5-LOX regulation by HNE required PGE2 production. Furthermore, our data showed that 10 µM HNE significantly induced transforming growth factor-beta 1 (TGF-β1) production. The addition of anti-TGF-β antibody to culture medium reduced HNE-induced 5-LOX/FLAP expression by 40%, indicating the involvement of a TGF-β1-dependent mechanism. Our data demonstrate that the shunt to the FLAP/5-LOX pathway in HNE-induced human OA chondrocytes is attributed to COX-2 inhibition, probably due to HNE depletion. PGE2 and TGF-β1 are suggested to be involved in this regulation. Further experiments are in progress to determine other molecular mechanisms underlying this switch in OA chondrocytes.

L’arthrose (OA) est une maladie dégénérative et multifactorielle caractérisée par une destruction de cartilage, une formation d’ostéophytes et une inflammation au niveau de la membrane synoviale. Le 4-hydroxynonénal (HNE), un produit final de la peroxydation lipidique, a été identifié récemment comme un facteur catabolique et un médiateur inflammatoire dans le cartilage arthrosique humain. Notre projet vise à étudier l’effet du HNE sur la régulation de la prostaglandine E2 synthase-1 microsomale (mPGES-1) et de la protéine activante 5-lipoxygénase (FLAP)/5-lipoxygénase (5-LOX) dans les chondrocytes arthrosiques humains. Lorsque les cellules sont traitées une seule fois avec 10 µM HNE, les résultats de Western blot et de PCR en temps réel montrent que l’expression de la cyclooxygénase-2 (COX-2) et de la mPGES-1 augmente de manière significative et atteint respectivement le maximum après 8 et 16 heures d’incubation puis diminue graduellement. Cependant, lorsque les cellules sont traitées plusieurs fois avec 10 µM HNE à 2 heures d’intervalle, l’expression de la COX-2 et de la mPGES-1 augmente en fonction du temps sans subir une baisse après 24 heures d’incubation. Le HNE induit l’activité du promoteur de la mPGES-1 via l’activation du facteur de transcription Egr-1. L’investigation de la 2ème voie du métabolisme de l’acide arachidonique, à savoir 5-LOX/FLAP, montre que le HNE induit l’expression de FLAP après 24 heures de stimulation et celle de 5-LOX seulement après 48 heures. Ceci semble survenir à l’étape de transcription au cours de laquelle HNE induit l’expression de l’ARNm et l’activité du promoteur du gène 5-LOX. Nous avons démontré aussi que le niveau de leukotriène B4 (LTB4) augmente et suit le même profil que celui de la 5-LOX. L’étude des mécanismes moléculaires susceptibles d’être impliqués dans la régulation de la 5-LOX/FLAP par le HNE montre que ce dernier stimule leur expression via l’action de prostaglandine E2 (PGE2) et du facteur de croissance transformant-beta 1 (TGF-β1). En conclusion, notre étude démontre que le HNE induit à court-terme d’incubation la voie de COX-2/mPGES-1 puis par la suite stimule celle de FLAP/5-LOX à long-terme d’incubation dans les chondrocytes arthrosiques humains. Ces résultats suggèrent que la mPGES-1 et 5-LOX/FLAP sont des potentielles cibles thérapeutiques intéressantes pour contrôler la production de PGE2 et LTB4 dans OA. / 4-hydroxynonenal (HNE), a lipid peroxidation end-product, is produced abundantly in osteoarthritic (OA) articular tissues. Recently, we reported that HNE-induced cyclooxygenase-2 (COX-2) decreased gradually in human OA chondrocytes after 8 h of incubation. This study aimed to investigate whether COX-2 down-regulation is attributed to HNE depletion and is responsible for the switch from COX-2 to 5-lipoxygenase-activating protein (FLAP)/5-lipoxygenase (5-LOX). Treatment of chondrocytes with 10 µM HNE induced prostaglandin E2 (PGE2) release as well as COX-2 and microsomal prostaglandin E2 synthase-1 (mPGES-1) expression at the protein and mRNA levels, with a plateau reached at 8-16 h of incubation, followed by a subsequent decline. However, 8 repeated treatments with 10 µM HNE prevented the reduction of COX-2 and mPGES-1 expression. We demonstrated that HNE induced mPGES-1 promoter activity mainly through transcription factor Egr-1 activation. On the other hand, when COX-2 expression decreased, leukotriene B4 (LTB4) level rose after a long period of stimulation (48 and 72 h). At the mRNA level, HNE induced FLAP and 5-LOX expression after 24 and 48 h of stimulation, respectively. The addition of a nonspecific COX-2 inhibitor (naproxen) to cultured chondrocytes revealed that FLAP and 5-LOX regulation by HNE required PGE2 production. Furthermore, our data showed that 10 µM HNE significantly induced transforming growth factor-beta 1 (TGF-β1) production. The addition of anti-TGF-β antibody to culture medium reduced HNE-induced 5-LOX/FLAP expression by 40%, indicating the involvement of a TGF-β1-dependent mechanism. Our data demonstrate that the shunt to the FLAP/5-LOX pathway in HNE-induced human OA chondrocytes is attributed to COX-2 inhibition, probably due to HNE depletion. PGE2 and TGF-β1 are suggested to be involved in this regulation. Further experiments are in progress to determine other molecular mechanisms underlying this switch in OA chondrocytes.

Contexte - La prévalence de la maladie de Crohn (MC), une maladie inflammatoire chronique du tube digestif, chez les enfants canadiens se situe parmi les plus élevées au monde. Les interactions entre les réponses immunes innées et acquises aux microbes de l'hôte pourraient être à la base de la transition de l’inflammation physiologique à une inflammation pathologique. Le leucotriène B4 (LTB4) est un modulateur clé de l'inflammation et a été associé à la MC. Nous avons postulé que les principaux gènes impliqués dans la voie métabolique du LTB4 pourrait conférer une susceptibilité accrue à l'apparition précoce de la MC. Dans cette étude, nous avons exploré les associations potentielles entre les variantes de l'ADN des gènes ALOX5 et CYP4F2 et la survenue précoce de la MC. Nous avons également examiné si les gènes sélectionnés montraient des effets parent-d'origine, influençaient les phénotypes cliniques de la MC et s'il existait des interactions gène-gène qui modifieraient la susceptibilité à développer la MC chez l’enfant. Méthodes – Dans le cadre d’une étude de cas-parents et de cas-témoins, des cas confirmés, leurs parents et des contrôles ont été recrutés à partir de trois cliniques de gastro-entérologie à travers le Canada. Les associations entre les polymorphismes de remplacement d'un nucléotide simple (SNP) dans les gènes CYP4F2 et ALOX5 ont été examinées. Les associations allélique et génotypiques ont été examinées à partir d’une analyse du génotype conditionnel à la parenté (CPG) pour le résultats cas-parents et à l’aide de table de contingence et de régression logistique pour les données de cas-contrôles. Les interactions gène-gène ont été explorées à l'aide de méthodes de réduction multi-factorielles de dimensionnalité (MDR). Résultats – L’étude de cas-parents a été menée sur 160 trios. L’analyse CPG pour 14 tag-SNP (10 dans la CYP4F2 et 4 dans le gène ALOX5) a révélé la présence d’associations alléliques ou génotypique significatives entre 3 tag-SNP dans le gène CYP4F2 (rs1272, p = 0,04, rs3093158, p = 0.00003, et rs3093145, p = 0,02). Aucune association avec les SNPs de ALOX5 n’a pu être démontrée. L’analyse de l’haplotype de CYP4F2 a montré d'importantes associations avec la MC (test omnibus p = 0,035). Deux haplotypes (GAGTTCGTAA, p = 0,05; GGCCTCGTCG, p = 0,001) montraient des signes d'association avec la MC. Aucun effet parent-d'origine n’a été observé. Les tentatives de réplication pour trois SNPs du gene CYP4F2 dans l'étude cas-témoins comportant 225 cas de MC et 330 contrôles suggèrent l’association dans un de ceux-ci (rs3093158, valeur non-corrigée de p du test unilatéral = 0,03 ; valeur corrigée de p = 0.09). La combinaison des ces deux études a révélé des interactions significatives entre les gènes CYP4F2, ALOX et NOD2. Nous n’avons pu mettre en évidence aucune interaction gène-sexe, de même qu’aucun gène associé aux phénotypes cliniques de la MC n’a pu être identifié. Conclusions - Notre étude suggère que la CYP4F2, un membre clé de la voie métabolique LTB4 est un gène candidat potentiel pour MC. Nous avons également pu mettre en évidence que les interactions entre les gènes de l'immunité adaptative (CYP4F2 et ALOX5) et les gènes de l'immunité innée (NOD2) modifient les risques de MC chez les enfants. D'autres études sur des cohortes plus importantes sont nécessaires pour confirmer ces conclusions. / Background - The rates of Crohn’s disease (CD) a chronic inflammatory disease of the gastrointestinal tract, among Canadian children are the world’s highest. Interactions between the host microbial–innate-immune-responses are thought to underplay transition from physiological to pathological inflammation. Leukotriene B4 (LTB4) is a key modulator of inflammation and has been shown to be associated with CD. We postulated that key genes involved in the LTB4 metabolic pathway could confer susceptibility for early-onset CD. In this study we implemented a candidate gene approach to test for associations between DNA variants in the ALOX5 and CYP4F2 genes and early-onset of CD. We also explored whether the selected genes demonstrated parent-of-origin effects, influenced CD clinical phenotypes and whether there were gender-gene and gene-gene interactions that determined CD susceptibility. Methods – The study consisted of an exploratory phase (case-parent design) followed by a replication phase (case-control design). Confirmed cases, parents and controls were recruited from three tertiary gastroenterology clinics across Canada. Associations between tag-single nucleotide polymorphisms in the CYP4F2 and ALOX5 genes were examined. Allelic and/or genotype associations were examined using conditional on parental genotype (CPG) analysis for the case-parent data and contingency table and logistic regression for the case-control data. Gene-gene interactions were explored using multi-factor dimensionality reduction (MDR) methods. Results – The first phase of the study was based on 160 trios (case-parent design). CPG analysis for 14 tag-SNPs (i.e. 10 in the CYP4F2 and 4 in the ALOX5 gene, respectively) revealed significant allelic or genotypic associations between 3 tag-SNPs in the CYP4F2 gene (rs1272, p=0.04, rs3093158, p=0.00003, and rs3093145, p=0.02). No associations with ALOX5 tag-SNPs were evident. CYP4F2-haplotype analysis showed significant associations with CD (omnibus test p-value=0.035). Two specific haplotypes (GAGTTCGTAA, p=0.05; GGCCTCGTCG, p=0.001) showed evidence for association with CD. No parent-of-origin effects were observed. The second phase of the study retested the three CYP4F2 SNPs that showed association in the first stage and was based on 223 CD cases and 330 controls. Some indications of association with one SNP i.e. rs3093158 were present (genotypic uncorrected 1-sided p-value=0.03); however this genotype association did not withstand correction. Combining cases from the two phases of the study revealed significant interactions between the CYP4F2, ALOX and NOD2 genes. No gene-gender interactions were obvious nor were the study genes associated with specific clinical phenotypes of CD. Conclusions - Our study suggests that the CYP4F2, a key member of the LTB4 metabolic pathway is a potential candidate gene for CD. Furthermore there was evidence that interactions between adaptive immunity genes (CYP4F2 and ALOX5) and innate immunity genes (NOD2) genes modify risk for CD in children. Further studies on larger cohorts are required to confirm these findings.

Contexte - La prévalence de la maladie de Crohn (MC), une maladie inflammatoire chronique du tube digestif, chez les enfants canadiens se situe parmi les plus élevées au monde. Les interactions entre les réponses immunes innées et acquises aux microbes de l'hôte pourraient être à la base de la transition de l’inflammation physiologique à une inflammation pathologique. Le leucotriène B4 (LTB4) est un modulateur clé de l'inflammation et a été associé à la MC. Nous avons postulé que les principaux gènes impliqués dans la voie métabolique du LTB4 pourrait conférer une susceptibilité accrue à l'apparition précoce de la MC. Dans cette étude, nous avons exploré les associations potentielles entre les variantes de l'ADN des gènes ALOX5 et CYP4F2 et la survenue précoce de la MC. Nous avons également examiné si les gènes sélectionnés montraient des effets parent-d'origine, influençaient les phénotypes cliniques de la MC et s'il existait des interactions gène-gène qui modifieraient la susceptibilité à développer la MC chez l’enfant. Méthodes – Dans le cadre d’une étude de cas-parents et de cas-témoins, des cas confirmés, leurs parents et des contrôles ont été recrutés à partir de trois cliniques de gastro-entérologie à travers le Canada. Les associations entre les polymorphismes de remplacement d'un nucléotide simple (SNP) dans les gènes CYP4F2 et ALOX5 ont été examinées. Les associations allélique et génotypiques ont été examinées à partir d’une analyse du génotype conditionnel à la parenté (CPG) pour le résultats cas-parents et à l’aide de table de contingence et de régression logistique pour les données de cas-contrôles. Les interactions gène-gène ont été explorées à l'aide de méthodes de réduction multi-factorielles de dimensionnalité (MDR). Résultats – L’étude de cas-parents a été menée sur 160 trios. L’analyse CPG pour 14 tag-SNP (10 dans la CYP4F2 et 4 dans le gène ALOX5) a révélé la présence d’associations alléliques ou génotypique significatives entre 3 tag-SNP dans le gène CYP4F2 (rs1272, p = 0,04, rs3093158, p = 0.00003, et rs3093145, p = 0,02). Aucune association avec les SNPs de ALOX5 n’a pu être démontrée. L’analyse de l’haplotype de CYP4F2 a montré d'importantes associations avec la MC (test omnibus p = 0,035). Deux haplotypes (GAGTTCGTAA, p = 0,05; GGCCTCGTCG, p = 0,001) montraient des signes d'association avec la MC. Aucun effet parent-d'origine n’a été observé. Les tentatives de réplication pour trois SNPs du gene CYP4F2 dans l'étude cas-témoins comportant 225 cas de MC et 330 contrôles suggèrent l’association dans un de ceux-ci (rs3093158, valeur non-corrigée de p du test unilatéral = 0,03 ; valeur corrigée de p = 0.09). La combinaison des ces deux études a révélé des interactions significatives entre les gènes CYP4F2, ALOX et NOD2. Nous n’avons pu mettre en évidence aucune interaction gène-sexe, de même qu’aucun gène associé aux phénotypes cliniques de la MC n’a pu être identifié. Conclusions - Notre étude suggère que la CYP4F2, un membre clé de la voie métabolique LTB4 est un gène candidat potentiel pour MC. Nous avons également pu mettre en évidence que les interactions entre les gènes de l'immunité adaptative (CYP4F2 et ALOX5) et les gènes de l'immunité innée (NOD2) modifient les risques de MC chez les enfants. D'autres études sur des cohortes plus importantes sont nécessaires pour confirmer ces conclusions. / Background - The rates of Crohn’s disease (CD) a chronic inflammatory disease of the gastrointestinal tract, among Canadian children are the world’s highest. Interactions between the host microbial–innate-immune-responses are thought to underplay transition from physiological to pathological inflammation. Leukotriene B4 (LTB4) is a key modulator of inflammation and has been shown to be associated with CD. We postulated that key genes involved in the LTB4 metabolic pathway could confer susceptibility for early-onset CD. In this study we implemented a candidate gene approach to test for associations between DNA variants in the ALOX5 and CYP4F2 genes and early-onset of CD. We also explored whether the selected genes demonstrated parent-of-origin effects, influenced CD clinical phenotypes and whether there were gender-gene and gene-gene interactions that determined CD susceptibility. Methods – The study consisted of an exploratory phase (case-parent design) followed by a replication phase (case-control design). Confirmed cases, parents and controls were recruited from three tertiary gastroenterology clinics across Canada. Associations between tag-single nucleotide polymorphisms in the CYP4F2 and ALOX5 genes were examined. Allelic and/or genotype associations were examined using conditional on parental genotype (CPG) analysis for the case-parent data and contingency table and logistic regression for the case-control data. Gene-gene interactions were explored using multi-factor dimensionality reduction (MDR) methods. Results – The first phase of the study was based on 160 trios (case-parent design). CPG analysis for 14 tag-SNPs (i.e. 10 in the CYP4F2 and 4 in the ALOX5 gene, respectively) revealed significant allelic or genotypic associations between 3 tag-SNPs in the CYP4F2 gene (rs1272, p=0.04, rs3093158, p=0.00003, and rs3093145, p=0.02). No associations with ALOX5 tag-SNPs were evident. CYP4F2-haplotype analysis showed significant associations with CD (omnibus test p-value=0.035). Two specific haplotypes (GAGTTCGTAA, p=0.05; GGCCTCGTCG, p=0.001) showed evidence for association with CD. No parent-of-origin effects were observed. The second phase of the study retested the three CYP4F2 SNPs that showed association in the first stage and was based on 223 CD cases and 330 controls. Some indications of association with one SNP i.e. rs3093158 were present (genotypic uncorrected 1-sided p-value=0.03); however this genotype association did not withstand correction. Combining cases from the two phases of the study revealed significant interactions between the CYP4F2, ALOX and NOD2 genes. No gene-gender interactions were obvious nor were the study genes associated with specific clinical phenotypes of CD. Conclusions - Our study suggests that the CYP4F2, a key member of the LTB4 metabolic pathway is a potential candidate gene for CD. Furthermore there was evidence that interactions between adaptive immunity genes (CYP4F2 and ALOX5) and innate immunity genes (NOD2) genes modify risk for CD in children. Further studies on larger cohorts are required to confirm these findings.