Cellular response to inflammatory cytokines involves concerted changes in gene expression. Cytokines, such as IL-6 and TNF-α, regulate gene expression through multiple intracellular signaling pathways. The activation of transcription factors is one of the important mechanisms through which these cytokines regulate gene expression, and NF-κB is a key transcription factor that is activated by TNF-α during inflammation. In this study, we have utilized green fluorescent protein reporter cells along with fluorescence microscopy, image analysis and mechanistic modeling to determine the activation dynamics of NF-κB in H35 rat hepatoma cells upon TNF-α stimulation. NF-κB reporter cells were monitored for induction of GFP expression for 24 hours following continuous stimulation with 2.5ng/mL, 10ng/mL and 25ng/mL TNF-α. As expected, TNF-α addition resulted in a significant increase in fluorescence. Relative fluorescence profiles were generated from the fluorescence intensity data, and indicated that fluorescence increases up to 24 hours after an initial delay of approximately four hours. The fluorescence data was also used to develop a model describing significant events leading to NF-κB activation and GFP expression. In addition, a model describing regulatable expression of GFP upon stable integration into the genome was also developed. Comparing these two models led to the construction of a third model depicting NF-κB activation dynamics. Simulation of the model representing NF-κB activation dynamics yielded an NF- κB activation profile, which demonstrated that in the presence of constant TNF-α stimulation, there is an approximate 90 minute hour time delay followed by a rapid increase in nuclear NF-κB, that reaches a steady state value at approximately two hours. This study establishes a method to derive NF-κB activation from reporter cell fluorescence data, and can be used to infer dynamics of activation of other transcription factors using GFP reporter cell lines.

Notre travail a permis tout d’abord d’investiguer les effets des nucléotides extracellulaires sur les cellules dendritiques (DCs) qui sont des cellules présentatrices d’antigènes capables d’initier et de réguler la réponse immunitaire. Afin d’avoir une vue globale de l’action des nucléotides extracellulaires sur les DCs, un profil d’expression génique de l’ATPgS – dérivé stable de l’ATP - a été réalisé par microarray dans les cellules dendritiques dérivées de monocytes (MoDCs). Notre groupe a préalablement montré que malgré que l’ATP est considéré comme un signal de danger, il confère des propriétés immunosuppressives aux DCs (Marteau et al, 2005). Nous nous sommes focalisés sur des régulations géniques pouvant être mises en relation avec un action anti-inflammatoire de l’ATP. Nous avons ainsi démontré que l’ATP était capable d’inhiber la sécrétion des chimiokines MCP-1 et MIP- 1a initiée par l’action du LPS, ce qui a pour conséquence de diminuer la capacité des DCs à recruter des monocytes ou d’autres DCs. Ce travail a fait l’objet d’une publication en tant que premier auteur (Horckmans et al, 2005). Un grand nombre d’autres gènes régulés liés à la réponse immune et à l’inflammation a été identifiée dans le profil microarray de l’ATPgS. Nous avions notamment pu identifier une augmentation de la sécrétion de VEGF-A en réponse à l’ATP, amplifiée en présence de LPS. Cette régulation est extrêmement intéressante au vu de l’action immunosuppressive du VEGF sur les DCs. Par ailleurs, cette régulation pourrait constituer un lien entre les DCs et l’angiogénese. Ce travail a fait l’objet d’une publication en tant que premier co-auteur dans la revue Journal of Immunology (Bles et al, 2007). En conclusion, nos données nous ont ainsi permis de montrer que les nucléotides adényliques peuvent avoir par leur action sur les cellules dendritiques une action anti-inflammatoire voire pro-angiogénique, en inhibant le recrutement leucocytaire et une action immunosuppressive en stimulant la sécrétion de VEGF.

Résumé : La voie des kynurénines est l’une des trois voies de biosynthèse du NAD, qu’elle produit à partir de la dégradation du plus rare des acides aminés essentiels, le tryptophane. Cette voie catabolique est initiée par trois enzymes distinctes, dont l’indoléamine 2,3-dioxygénase 1 (IDO-1). L’IDO-1 est la seule des trois enzymes dont l’expression est induite par des stimuli pro-inflammatoires tels que l’IFN-γ, le TNF-α ainsi que des produits bactériens et viraux. De plus, son expression est induite de manière particulièrement importante au cours de la maturation des cellules dendritiques (DC) qui jouent un rôle clé, tant dans les réponses immunes innées qu’adaptatives. Par conséquent, outre la production d’un cofacteur essentiel du métabolisme cellulaire, le catabolisme du tryptophane participe également à la régulation de la réponse immune. En effet, la fonction d’agent antimicrobien fut la première attribuée à l’IDO-1 parce qu’en réponse aux stimuli inflammatoires, celle-ci dégrade le tryptophane et limite la prolifération d’organismes pathogènes auxotrophes pour cet acide aminé. Ensuite, l’inhibition de l’activité catalytique de l’IDO-1 par une approche pharmacologique permit de mettre en évidence la contribution de cette enzyme au phénomène de tolérance maternelle pour les fœtus semi-allogéniques qu’elle porte. Enfin, sa participation à la tolérance périphérique fut proposée étant donné sa capacité à induire le développement de Treg ou à leur servir de mécanisme effecteur. Cependant, ces deux dernières fonctions ne sont pas très claires puisque les souris invalidées pour le gène de l’IDO-1 (IDO-1-/-) ne présentent ni défaut reproducteur, ni signe d’auto-immunité spontanée. Comme deux publications récentes suggéraient que l’inhibition pharmacologique de l’IDO-1 affecte la maturation des moDC humaines, nous avons effectué une analyse détaillée du compartiment immunitaire des souris IDO-1-/-, avec une attention toute particulière pour les DC. Au début, nous avons également constaté un important défaut de la maturation des BMDC IDO-1-/- générées in vitro en présence de GM-CSF. Cependant, le défaut se révéla extrêmement dépendant des conditions de culture, puisqu’un changement de substrat de culture ou de facteur de croissance suffit à restaurer une maturation normale de ces cellules. De même, l’analyse de la maturation des DC spléniques démontra de manière claire que l’IDO-1 n’est certainement pas essentielle à la maturation des DC in vivo. Nous avons ensuite montré que l’expression fonctionnelle d’IDO-1 protège les cellules et potentiellement les souris qui l’expriment lorsqu’elles sont soumises à des stress oxydatifs, suggérant que l’IDO-1 puisse consommer les anions O2- afin d’assurer son activité catalytique. Contre toute attente, nous avons ensuite constaté que les souris IDO-1-/- survivent plus longtemps aux infections par la forme pléiomorphe du parasite Trypanosoma brucei brucei. Bien que la levée de l’inhibition de la lymphoprolifération chez les souris IDO-1-/- soit l’explication la plus évidente de l’augmentation de leur survie, nous suggérons plutôt que c’est la perte de la fonction antioxydante de l’IDO-1-/- qui leur confère cette résistance. En conclusion, l’IDO-1 ne semble pas jouer un rôle important dans la différenciation et maturation des cellules dendritiques. Nos observations préliminiares indiquent cependant que cette enzyme pourrait jouer un rôle anti-oxydant, et protége donc les cellules dendritiques d’un stress oxydant potentiellement causé lors des réponses innées anti-microbiennes.

Short-chain fatty acids (SCFAs) are anaerobic bacterial metabolites. Cystic fibrosis (CF) lung disease is a condition caused by mutations in the cystic fibrosis transmembrane conductange regulator (CFTR) gene and is characterized by persistent lung inflammation and bacterial colonization. We measured the concentrations of SCFAs in sputum of patients with CF and tested the effect of these compounds on bacterial growth. Furthermore we found that SCFAs can influence the inflammatory protein expression and cytokine release in airway epithelial cells. SCFAs differentially alter cytokine release in CF bronchial epithelial cells (CFBE) compared to CFBE expressing wild-type CFTR. We also studied the effect of SCFAs in an acute lung injury model in BALB/cJ mice and found that intratracheally administered SCFAs can affect the inflammatory environment of the airways in vivo. We conclude that SCFAs may be important in the airways and that further investigation is warranted to understand their effects on inflammation and infection.

Short-chain fatty acids (SCFAs) are anaerobic bacterial metabolites. Cystic fibrosis (CF) lung disease is a condition caused by mutations in the cystic fibrosis transmembrane conductange regulator (CFTR) gene and is characterized by persistent lung inflammation and bacterial colonization. We measured the concentrations of SCFAs in sputum of patients with CF and tested the effect of these compounds on bacterial growth. Furthermore we found that SCFAs can influence the inflammatory protein expression and cytokine release in airway epithelial cells. SCFAs differentially alter cytokine release in CF bronchial epithelial cells (CFBE) compared to CFBE expressing wild-type CFTR. We also studied the effect of SCFAs in an acute lung injury model in BALB/cJ mice and found that intratracheally administered SCFAs can affect the inflammatory environment of the airways in vivo. We conclude that SCFAs may be important in the airways and that further investigation is warranted to understand their effects on inflammation and infection.

Chronic inflammation in response to Aß peptide deposits is a pathological hallmark of Alzheimer's disease (AD). The inflammatory environment includes populations of reactive and proliferating microglia and astrocytes and perturbed vasculature. However, the association between activated glial cells and cerebrovascular dysfunction remain largely unknown. This study has used Aß1-42 intrahippocampal injection as an animal model of inflamed AD brain to characterize mechanisms of glial-vasculature responses as a basis for chronic inflammation. Preliminary findings suggested Aß1-42-injected brain demonstrated vascular remodeling including evidence for formation of new blood vessels (angiogenesis). This result led to study of the effects of the anti-angiogenic/anti-inflammatory compound, thalidomide on activated glial cells and perturbations in the vasculature in an Aß1-42 peptide-injected rat model. First, Aß1-42 injection was found to cause perturbations in vasculature including new blood vessel formation and increased BBB leakiness. Second, thalidomide decreased the vascular perturbations and the glial reactivity and conferred neuroprotection. Overall, these results suggest that altered cerebral vasculature is integral to the overall inflammatory response induced by peptide. Experiments then examined the level of parenchymal plasma proteins in brain tissue from AD and nondemented (ND) individuals. AD, but not ND, brain tissue demonstrated high levels of fibrinogen immunoreactivity (ir). Aß1_42 injection into the rat hippocampus increased the level of parenchymal fibrinogen, which was reduced by treatment with the defibrinogenating agent, ancrod. In addition, ancrod also attenuated microglial activation and prevented neuronal injury. Overall, these results demonstrate that extravasation of blood protein and a leaky BBB are important in promoting and amplifying inflammatory responses and causing neuronal damage in inflamed AD brain. Microglial chemotactic responses to VEGF (vascular endothelial growth factor) receptor Flt-1 were next studied. Treatment with a monoclonal antibody to Flt-1 (anti-Flt-1 Ab) in the peptide-injected hippocampus diminished microglial reactivity and provided neuroprotection. Secondly, anti-Flt-1 Ab inhibited the AI3142-induced migration of human microglia. These results suggest critical functional roles for Flt-1 in mediating microglial chemotaxis and inflammatory responses in AD brain. The overall conclusion from my work is that AP deposits induce microglial reactivity which subsequently causes vascular remodeling resulting in an amplified inflammatory microenvironment which is damaging to bystander neurons.

Cellular response to inflammatory cytokines involves concerted changes in gene expression. Cytokines, such as IL-6 and TNF-α, regulate gene expression through multiple intracellular signaling pathways. The activation of transcription factors is one of the important mechanisms through which these cytokines regulate gene expression, and NF-κB is a key transcription factor that is activated by TNF-α during inflammation. In this study, we have utilized green fluorescent protein reporter cells along with fluorescence microscopy, image analysis and mechanistic modeling to determine the activation dynamics of NF-κB in H35 rat hepatoma cells upon TNF-α stimulation. NF-κB reporter cells were monitored for induction of GFP expression for 24 hours following continuous stimulation with 2.5ng/mL, 10ng/mL and 25ng/mL TNF-α. As expected, TNF-α addition resulted in a significant increase in fluorescence. Relative fluorescence profiles were generated from the fluorescence intensity data, and indicated that fluorescence increases up to 24 hours after an initial delay of approximately four hours. The fluorescence data was also used to develop a model describing significant events leading to NF-κB activation and GFP expression. In addition, a model describing regulatable expression of GFP upon stable integration into the genome was also developed. Comparing these two models led to the construction of a third model depicting NF-κB activation dynamics. Simulation of the model representing NF-κB activation dynamics yielded an NF- κB activation profile, which demonstrated that in the presence of constant TNF-α stimulation, there is an approximate 90 minute hour time delay followed by a rapid increase in nuclear NF-κB, that reaches a steady state value at approximately two hours. This study establishes a method to derive NF-κB activation from reporter cell fluorescence data, and can be used to infer dynamics of activation of other transcription factors using GFP reporter cell lines.

Lemnalol (8-isopropyl-5-methyl-4-methylene-decahydro-1, 5-cyclo-naphthalen- 3-ol) is a natural compound isolated from the marine soft coral Lemnalia cervicorni. In the present study, we focused to determine whether lemnalol has in vivo antinociceptive and anti-inflammatory effects on carrageenan-induced rat paw edema and the hyperalgesia model using the thermal hyperalgesia test, paw edema assay, and histological examination. Furthermore, we also explored the possible cellular mechanisms of lemnalol on carrageenan-induced inflammatory responses by immunohistochemistry. The present results show that both systemic and central administration of lemnalol significantly inhibits carrageenan-evoked thermal hyperalgesia behavior. Moreover, intramuscular injection of lemnalol also significantly decreases paw edema and improves the inflammatory status of the tissues with reduced neutrophil infiltration. From behavioral and immunohistological observations, we found that lemnalol exerts its anti-inflammatory and analgesic effects on carrageenan-induced inflammatory responses; the cellular mechanisms of these effects involved the inhibition of elevated inflammatory mediators, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), vascular endothelial cell growth factor (VEGF), tumor necrosis factor-£\ (TNF-£\), and deletion of phosphatase and tensin homologues on chromosome 10 (PTEN). Since 1962, the carrageenan-induced rat model of inflammation has been widely used as an animal model to investigate inflammatory mechanisms and anti-inflammatory activity of drugs and has been fully characterized. However, the use of rats as an animal model has certain limitations such as those associated with breeding, economy, efficiency, and higher dosage. Recently, many studies have demonstrated zebrafish (Danio rerio) to be a valuable animal model for the investigation of bioactive compounds and molecular mechanisms of various diseases. In the present study, we attempted to establish an inflammatory animal model in zebrafish. Our research results showed that intraperitoneal (i.p.) administration of carrageenan significantly increased body edema and caused death of zebrafish resulting in a decrease in the cumulative survival rate. These effects were significantly inhibited by i.p. administration of methylprednisolone. Immunohistochemical observations revealed i.p. carrageenan-induced upregulation of iNOS, heat shock protein 25 (Hsp25), heat shock protein 90 (Hsp90), and PTEN; however, heat shock protein 70 (Hsp70) was observed to be downregulated. The present study provides a new in vivo inflammatory model for the screening of small volumes of drugs or compounds.

Thèse d'exercice : Pharmacie : Université de Nantes : 2003. / Bibliogr. f. 170-182 [120 réf.].

Thèse d'exercice : Médecine. Médecine générale : Université de Nantes : 2005. / Bibliogr. f. 97-102 [48 réf.].