The focus of this thesis is the role of dengue virus in the induction of chemokines. Dengue virus (DENV) occurs as four distinct serotypes, called DENV 1,2,3,and 4. Symptomatic DENV infection ranges from a self limited febrile illness, dengue fever (DF), to a more severe disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). DHF is characterized by increased capillary permeability resulting in decreased plasma volume, which may be accompanied by hemorrhagic manifestations. Many factors including T cell cross reactivity, viral burden, antibody dependent enhancement and induction of chemokines and cytokines have been reported in DHF and may play a role in the pathogenesis of DENV infection. Cytokines have been shown to modulate endothelial cell permeability [1-3]. Recent studies have shown that DENV-infected endothelial cells secrete the chemokine, interleukin (IL)-8 in vitro [4]. In addition, the permeability of an endothelial cell monolayer was found to be increased by interleukin-8 (IL-8) in vitro[5]. This thesis examines the effects of DEN2V infection on the induction of chemokines, and specifically, which DEN2V viral protein(s) are involved in the induction of IL-8. The chemokine induction profile following DEN2V infection was initially assessed in various cell lines that may represent potential targets in vivo, including monocytes, liver cells and endothelial cells. We hypothesized that distinct profiles of chemokine secretion can be induced by DEN2V infection of various cell types in vitro. We found RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) and IL-8 were induced in two of the five cell lines. DEN2V infection of primary monocyte-derived dendritic cells induced RANTES and IL-8 along with macrophage inflammatory protein-1α (MIP-1α), MIP-1β and monocyte chemoattractant protein-1 (MCP-1) but at an earlier time post infection than in the cell lines. These results showed that DEN2V infection induces distinct chemokine profiles in many cell types. In addition, monocytic-derived DCs can secrete chemokines upon infection with DEN2V. Characterization of the signaling pathways induced by DEN2V revealed that DEN2V induction of chemokines in human embryonic kidney (HEK293A) cells is mainly through the nuclear factor kappaB (NFκB) pathway, as previously reported for endothelial cells and 293T cells [4,6]. Alternatively, the liver cell line (HepG2) activated mainly activator protein (AP)-l. In addition, DENV infection can induce the activation of the interferon-stimulated response element (ISRE) driven promoter. IL-8 has been shown to have multiple effects on the immune system ranging from recruiting cells to the site of infection to countering the antiviral effects of type I interferon (IFN) [7,8]. Previous reports have shown that viral proteins can induce chemokines such as seen with IL-8 induction with the nonstructural protein 5A (NS5A) and core proteins from hepatitis C virus [9,10]. We hypothesized that protein(s) from DENV could induce chemokine production. The expression of DENV proteins was analyzed for effects on IL-8 and RANTES production in HEK293A cells. The effects of viral replication on IL-8 and RANTES induction were also analyzed using a DENV replicon that contains genes for the capsid protein and the nonstructural proteins. Transfection of plasmids expressing NS5 or the DEN2V replicon induced the expression and secretion of IL-8 but not RANTES. We attributed the lack of RANTES induction to the inability of NS5 or the DEN2V replicon to induce transcription from the ISRE driven promoter. We also found that NS5 and the DEN2V replicon induced IL-8 mainly through the CCAAT/enhancer binding protein (c/EBP) and AP-1 pathways. The profile of transcription factor activation is different from what was seen with DENV infection of HEK293A cells and suggests that the transient expression of the NS5 protein and the replication and/or translation of the DEN2V genome use different pathways than viral infection to induce IL-8. In addition, we found that the expression of prM-E, known to produce virus-like particles, could induce IL-8 secretion and activate transcription from the IL-8 promoter. As with the expression of NS5, RANTES was not induced. Analysis of the transcription factors involved in IL-8 induction using luciferase reporter constructs indicated that expression of prM-E induced transcription of IL-8 through NFκB, AP-1 and c/EBP, similar to what was seen with DEN2V infection of HEK293A cells. These results suggest that production of virions or virus-like particles induce IL-8 but that another mechanism in the viral life cycle is responsible for the induction of RANTES expression by DEN2V infection. We were also interested in the effects of drugs that have been used previously to inhibit cytokine or chemokine production on chemokine induction during DEN2V infection. We hypothesized that pharmacological inhibitors of cytokines will inhibit secretion of chemokines in DEN2V infected cells. We found that the pharmacological inhibitors SB203580 and rolipram enhanced chemokine production in a DEN2V infected liver cell line (HepG2), whereas dexamethasone had the same effect in a kidney epithelial cell line (HEK293A). We conclude that drugs that inhibit signaling pathways involved in cytokine production in other experimental systems can have variable effects on chemokine induction in different cell types during DEN2V infection. The data generated in this thesis extend our understanding of how DEN2V manipulates the host cell during viral infection to produce chemokines and perhaps enhance viral propagation and dissemination through the induction of IL-8. In addition, this study provides insight into the variable effects pharmacological drug treatment may have on disease progression during DENV infection. These results increase our understanding of DENV pathogenesis and may be helpful in finding better strategies for treatment and prevention.

O endométrio é a mucosa que reveste o útero. A receptividade uterina é definida como um estado em que o endométrio se encontra receptivo à implantação do blastocisto. E, a preparação do endométrio para a implantação não é somente uma questão de estimulação hormonal adequada, depende da interação entre o blastocisto e o endométrio. Esta interação envolve uma complexa sequência de eventos de sinalização e uma variedade de moléculas. As concentrações de interleucina-8 (IL-8) e interleucina-1β (IL-1β) estão correlacionadas com o processo de implantação. Em humanos, a taxa de insucesso desse processo é alta e ocasionada por diversos fatores. A síndrome dos ovários policísticos (SOP) é um distúrbio endócrino-ginecológico que afeta de 6 a 8 % das mulheres em idade reprodutiva, e se caracteriza, principalmente, por anovulação crônica e hiperandrogenismo, estando diretamente relacionada à infertilidade feminina. Apesar da incerteza sobre a causa primária da SOP, há relatos sobre a importância da hiperinsulinemia na sua promoção. O objetivo deste trabalho foi estabelecer um modelo de hiperinsulinemia e hiperandrogenismo em células estromais endometriais in vitro, simulando características de SOP; identificar o melhor gene normalizador para estudos de expressão gênica em amostras das células em cultivo; avaliar o efeito da metformina sobre a proliferação celular e expressão gênica da IL-8 e IL-1β no modelo proposto. O tecido endometrial foi obtido de pacientes submetidas a histerectomia. A cultura primária das células estromais foi padronizada e as células foram divididas em sete grupos de tratamento: estradiol (G1); estradiol e progesterona (G2); estradiol, progesterona e insulina (G3); estradiol, progesterona e diidrotestosterona (G4); estradiol, progesterona e metformina (G5); estradiol, progesterona, insulina e diidrotestosterona (G6); estradiol, progesterona, insulina, diidrotestosterona e metformina (G7). Foi realizada análise de imunocitoquímica para vimentina para confirmação do cultivo com células estromais. Para avaliar a viabilidade e proliferação celular ao longo do tempo foi utilizado o ensaio de MTT em dois tempos diferentes de cultivo. As extrações de RNA foram realizadas e o cDNA obtido das amostras foi utilizado para a amplificação do mRNA de cinco genes candidatos a normalizadores e para avaliar a expressão dos genes da IL-8 e IL-1β através de PCR em tempo real.O estabelecimento da cultura de células estromais foi confirmado através da coloração positiva para a proteína vimentina. As células mantiveram-se viáveis durante todo o período de cultivo, apresentando aumento significativo na proliferação celular no tempo 8 em relação ao tempo 4 em todos os grupos. O grupo G7 (tratado durante 48 horas com metformina) apresentou uma menor taxa de proliferação em relação aos grupos G2, G3 e G6. Para análise de expressão gênica nestas células, o gene que mostrou os melhores parâmetros de estabilidade de expressão no modelo celular proposto foi o gene HPRT1. Observamos uma maior expressão do gene da IL-8 no grupo G5 tratado durante 48 horas em relação ao mesmo grupo tratado durante o período de 24 horas. Verificou-se maior expressão do gene da IL-1β no grupo G5 quando comparado a todos os outros grupos no período de 48 horas de tratamento com metformina. Entretanto, o grupo G7, também tratado com metformina, não apresentou diferença estatística em relação ao tempo de tratamento em nenhum dos genes estudados. Esses resultados demonstram que o modelo de hiperinsulinemia e hiperandrogenismo em cultura de células estromais endometriais é viável. Neste modelo, em que foram testados cinco genes em relação à sua estabilidade de expressão, o gene HPRT1 apresentou uma boa estabilidade, ao contrário de outros genes frequentemente utilizados como genes de referência. O tratamento com metformina apresentou um efeito antiproliferativo nas células do grupo hiperinsulinêmico e hiperandrogênico. No período de 48 horas aumentou a expressão do gene da IL-1β no grupo tratado somente com o medicamento. Sugerindo uma ação inibitória da insulina sobre a expressão dos genes da IL-8 e IL-1β no grupo hiperinsulinêmico e hiperandrogênico. Mais estudos são necessários para melhor entendimento do efeito da metformina nos fatores envolvidos durante a implantação. / The endometrium is the mucosa lining the uterus. The uterine receptivity is defined as a condition in which the endometrium is receptive to implantation of the blastocyst. The preparation of the endometrium for implantation is not only a matter of proper hormonal stimulation, it depends on the interaction between the blastocyst and the endometrium. This interaction involves a complex sequence of events and a variety of signaling molecules. The concentrations of interleukin-8 (IL-8) and interleukin-1β (IL-1β) are correlated to the implantation process. In humans, the failure rate of this process is high and caused by several factors. The polycystic ovary syndrome (PCOS) is an endocrine-gynecological disorder that affects from 6 to 8 % of women of reproductive age. It is characterized mainly by chronic anovulation and hyperandrogenism and directly related to female infertility. Despite the uncertainty about the primary cause of PCOS, there are reports about the importance of hyperinsulinemia in promoting it. The aim of this study was to establish a model of hyperinsulinemia and hyperandrogenism in endometrial stromal cells in vitro, simulating features of PCOS; to identify the best housekeeping gene for gene expression studies in the cultured cells; to evaluate the effects of metformin on cell proliferation, as well as IL-8 and IL-1β gene expression in the proposed culture model. The human endometrial tissue was obtained from patients undergoing hysterectomy. The primary culture of stromal cells was standardized and divided into seven treatment groups: estradiol (G1); estradiol and progesterone (G2); estradiol, progesterone and insulin (G3); estradiol, progesterone and dihydrotestosterone (G4); estradiol, progesterone and metformin (G5); estradiol, progesterone, insulin and dihydrotestosterone (G6); estradiol, progesterone, insulin, dihydrotestosterone and metformin (G7). Immunocytochemistry analysis for vimentin were performed. Cell viability and proliferation were evaluated by MTT assay at two days in different times of cultivation. RNA extractions were performed and the cDNA obtained from primary culture was used to amplify five candidates to housekeeping genes mRNA and to evaluate IL-8 and IL1β expression by real time PCR. The stromal culture cell establishment was confirmed by positive staining for vimentin. The cells remained viable throughout the cultivation period, with significant cell proliferation increase at day 8 compared to day 4 in all groups. The G7 group (metformin treated for 48 hours) showed lower proliferation rate than G2, G3 and G6 groups. For gene expression analysis in these cells, the gene showing the best parameters of stability of expression was HPRT1. Increased gene expression of IL-8 was observed in G5 group treated for 48 hours compared to the same group during 24 hours. Similarly, the G5 group showed higher IL-1β gene expression when compared to all other groups treated with metformin for 48 hours. However, the G7 group, also metformin treated, did not show statistically significant difference in treatment time of any studied genes. These results suggest that model of hyperinsulinemia and hyperandrogenism in endometrial stromal cells is viable and provides a good cell sampling to molecular analysis under different experimental conditions. In this model, several genes were tested for expression stability. HPRT1 presented the best values, unlike others classical housekeeping genes. The metformin treatment showed an antiproliferative effect on cells in hyperinsulinemic and hyperandrogenic group and at 48 hours increased IL-1β gene expression in the treated group with the drug alone. It suggests an inhibitory action of insulin on these genes expression in the hyperinsulinemic and hyperandrogenic group. More studies are needed to better understand the effect of metformin on the factors involved during implantation.

O endométrio é a mucosa que reveste o útero. A receptividade uterina é definida como um estado em que o endométrio se encontra receptivo à implantação do blastocisto. E, a preparação do endométrio para a implantação não é somente uma questão de estimulação hormonal adequada, depende da interação entre o blastocisto e o endométrio. Esta interação envolve uma complexa sequência de eventos de sinalização e uma variedade de moléculas. As concentrações de interleucina-8 (IL-8) e interleucina-1β (IL-1β) estão correlacionadas com o processo de implantação. Em humanos, a taxa de insucesso desse processo é alta e ocasionada por diversos fatores. A síndrome dos ovários policísticos (SOP) é um distúrbio endócrino-ginecológico que afeta de 6 a 8 % das mulheres em idade reprodutiva, e se caracteriza, principalmente, por anovulação crônica e hiperandrogenismo, estando diretamente relacionada à infertilidade feminina. Apesar da incerteza sobre a causa primária da SOP, há relatos sobre a importância da hiperinsulinemia na sua promoção. O objetivo deste trabalho foi estabelecer um modelo de hiperinsulinemia e hiperandrogenismo em células estromais endometriais in vitro, simulando características de SOP; identificar o melhor gene normalizador para estudos de expressão gênica em amostras das células em cultivo; avaliar o efeito da metformina sobre a proliferação celular e expressão gênica da IL-8 e IL-1β no modelo proposto. O tecido endometrial foi obtido de pacientes submetidas a histerectomia. A cultura primária das células estromais foi padronizada e as células foram divididas em sete grupos de tratamento: estradiol (G1); estradiol e progesterona (G2); estradiol, progesterona e insulina (G3); estradiol, progesterona e diidrotestosterona (G4); estradiol, progesterona e metformina (G5); estradiol, progesterona, insulina e diidrotestosterona (G6); estradiol, progesterona, insulina, diidrotestosterona e metformina (G7). Foi realizada análise de imunocitoquímica para vimentina para confirmação do cultivo com células estromais. Para avaliar a viabilidade e proliferação celular ao longo do tempo foi utilizado o ensaio de MTT em dois tempos diferentes de cultivo. As extrações de RNA foram realizadas e o cDNA obtido das amostras foi utilizado para a amplificação do mRNA de cinco genes candidatos a normalizadores e para avaliar a expressão dos genes da IL-8 e IL-1β através de PCR em tempo real.O estabelecimento da cultura de células estromais foi confirmado através da coloração positiva para a proteína vimentina. As células mantiveram-se viáveis durante todo o período de cultivo, apresentando aumento significativo na proliferação celular no tempo 8 em relação ao tempo 4 em todos os grupos. O grupo G7 (tratado durante 48 horas com metformina) apresentou uma menor taxa de proliferação em relação aos grupos G2, G3 e G6. Para análise de expressão gênica nestas células, o gene que mostrou os melhores parâmetros de estabilidade de expressão no modelo celular proposto foi o gene HPRT1. Observamos uma maior expressão do gene da IL-8 no grupo G5 tratado durante 48 horas em relação ao mesmo grupo tratado durante o período de 24 horas. Verificou-se maior expressão do gene da IL-1β no grupo G5 quando comparado a todos os outros grupos no período de 48 horas de tratamento com metformina. Entretanto, o grupo G7, também tratado com metformina, não apresentou diferença estatística em relação ao tempo de tratamento em nenhum dos genes estudados. Esses resultados demonstram que o modelo de hiperinsulinemia e hiperandrogenismo em cultura de células estromais endometriais é viável. Neste modelo, em que foram testados cinco genes em relação à sua estabilidade de expressão, o gene HPRT1 apresentou uma boa estabilidade, ao contrário de outros genes frequentemente utilizados como genes de referência. O tratamento com metformina apresentou um efeito antiproliferativo nas células do grupo hiperinsulinêmico e hiperandrogênico. No período de 48 horas aumentou a expressão do gene da IL-1β no grupo tratado somente com o medicamento. Sugerindo uma ação inibitória da insulina sobre a expressão dos genes da IL-8 e IL-1β no grupo hiperinsulinêmico e hiperandrogênico. Mais estudos são necessários para melhor entendimento do efeito da metformina nos fatores envolvidos durante a implantação. / The endometrium is the mucosa lining the uterus. The uterine receptivity is defined as a condition in which the endometrium is receptive to implantation of the blastocyst. The preparation of the endometrium for implantation is not only a matter of proper hormonal stimulation, it depends on the interaction between the blastocyst and the endometrium. This interaction involves a complex sequence of events and a variety of signaling molecules. The concentrations of interleukin-8 (IL-8) and interleukin-1β (IL-1β) are correlated to the implantation process. In humans, the failure rate of this process is high and caused by several factors. The polycystic ovary syndrome (PCOS) is an endocrine-gynecological disorder that affects from 6 to 8 % of women of reproductive age. It is characterized mainly by chronic anovulation and hyperandrogenism and directly related to female infertility. Despite the uncertainty about the primary cause of PCOS, there are reports about the importance of hyperinsulinemia in promoting it. The aim of this study was to establish a model of hyperinsulinemia and hyperandrogenism in endometrial stromal cells in vitro, simulating features of PCOS; to identify the best housekeeping gene for gene expression studies in the cultured cells; to evaluate the effects of metformin on cell proliferation, as well as IL-8 and IL-1β gene expression in the proposed culture model. The human endometrial tissue was obtained from patients undergoing hysterectomy. The primary culture of stromal cells was standardized and divided into seven treatment groups: estradiol (G1); estradiol and progesterone (G2); estradiol, progesterone and insulin (G3); estradiol, progesterone and dihydrotestosterone (G4); estradiol, progesterone and metformin (G5); estradiol, progesterone, insulin and dihydrotestosterone (G6); estradiol, progesterone, insulin, dihydrotestosterone and metformin (G7). Immunocytochemistry analysis for vimentin were performed. Cell viability and proliferation were evaluated by MTT assay at two days in different times of cultivation. RNA extractions were performed and the cDNA obtained from primary culture was used to amplify five candidates to housekeeping genes mRNA and to evaluate IL-8 and IL1β expression by real time PCR. The stromal culture cell establishment was confirmed by positive staining for vimentin. The cells remained viable throughout the cultivation period, with significant cell proliferation increase at day 8 compared to day 4 in all groups. The G7 group (metformin treated for 48 hours) showed lower proliferation rate than G2, G3 and G6 groups. For gene expression analysis in these cells, the gene showing the best parameters of stability of expression was HPRT1. Increased gene expression of IL-8 was observed in G5 group treated for 48 hours compared to the same group during 24 hours. Similarly, the G5 group showed higher IL-1β gene expression when compared to all other groups treated with metformin for 48 hours. However, the G7 group, also metformin treated, did not show statistically significant difference in treatment time of any studied genes. These results suggest that model of hyperinsulinemia and hyperandrogenism in endometrial stromal cells is viable and provides a good cell sampling to molecular analysis under different experimental conditions. In this model, several genes were tested for expression stability. HPRT1 presented the best values, unlike others classical housekeeping genes. The metformin treatment showed an antiproliferative effect on cells in hyperinsulinemic and hyperandrogenic group and at 48 hours increased IL-1β gene expression in the treated group with the drug alone. It suggests an inhibitory action of insulin on these genes expression in the hyperinsulinemic and hyperandrogenic group. More studies are needed to better understand the effect of metformin on the factors involved during implantation.

No description available.

No description available.

By 2030 major depression is predicted to be the leading cause of disease burden in the world; as such, it is critical to understand factors that contribute to the development of depression. The social signal transduction theory of depression hypothesizes that adversity and social threat upregulate pro-inflammatory biomarkers leading to depression. The current study examined whether pro-inflammatory biomarkers (interleukin-6, interleukin-8, c-reactive protein, and tumor necrosis factor alpha) mediate the association between various types of childhood maltreatment (physical abuse, sexual abuse, emotional abuse, physical neglect, emotional neglect) and adult depression symptoms in a sample of 740 adults (372 female; Mage= 51.6 years, SD = 13.6) who provided retrospective report of childhood maltreatment as part of the Midlife in the United States (MIDUS) Refresher Biomarker study. Additionally, it explored whether these relations differ for males versus females. A series of linear regression analyses were run in SPSS; separate models were run for each form of childhood maltreatment and for interleukin-6, interleukin-8, c-reactive protein, and tumor necrosis factor-alpha. The results showed that childhood maltreatment is a robust predictor of adulthood depression; however, this association did not differ between biological sexes. In addition, only interleukin-6 was shown to partially mediate the association between childhood maltreatment and adulthood depression. These findings highlight the need to explore the use of interleukin-6 to screen for depression in youth. / M.S. / By 2030 major depression is predicted to be the leading cause of disease burden in the world; as such, it is critical to understand factors that contribute to the development of depression. It has been hypothesized that adversity and social threat activate pro-inflammatory biomarkers, which are proteins that can detect inflammation in the body, leading to depression. The current study examined whether several pro-inflammatory biomarkers explain the association between several types of childhood maltreatment (physical abuse, sexual abuse, emotional abuse, physical neglect, emotional neglect) and adult depression symptoms in a sample of 740 adults (372 female; Mage= 51.6 years, SD = 13.6) who provided report of past experiences of childhood maltreatment. Additionally, it explored whether these relations differ for males versus females. The results showed that childhood maltreatment is a robust predictor of adulthood depression for males and females. Of the inflammatory biomarkers examined, only interleukin-6 was shown to partially explain the association between childhood maltreatment and adulthood depression symptoms. These findings highlight the need to explore the use of interleukin-6 to screen for depression in youth.

Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease, characterized by progressive and largely irreversible airflow limitation due to alveolar destruction (emphysema), small airway narrowing, and chronic bronchitis. It is one of the leading causes of morbidity and mortality worldwide and in the UK, it may affect approximately 1.5 per cent of the population; and up to one in eight emergency admissions may be due to COPD,corresponding to over one million bed days, with some 24160 people in the UK dying as a result of COPD in 2005 (Burden of Lung Disease 2nd Edition,British Thoracic Society 2006). Most cases of COPD are triggered by chronic inhalation of cigarette smoke.However, some people do not suffer from COPD even if they smoke for many years. COPD cannot be cured, and patients usually live with poor life quality. Treatments include giving up smoking, medication and oxygen therapy. Genetic factors contribute to the development of COPD. In Northern Europe,Z-AT homozygotes (342Glu Lys) develop emphysema in their third or forth decade. One explanation is AT deficiency because they form inactive polymers. However, this cannot explain why bronchoalveolar lavage fluid (BALF) from Z-AT homozygotes with emphysema contains more neutrophils than BALF from individuals with emphysema and normal AT (M-AT). Inhaling pollutants which include smoking (cigarettes, pipes, cigars, etc.) and other fumes such as those found in many industrial work environments probably also plays a role in an individual’s development of COPD. Previously, it has been shown that the polymeric conformer of AT is present in BALF from Z-AT homozygotes and that it is a chemoattractant for neutrophils in vitro (Parmar JS, 2002). These findings have been confirmed by others (Mulgrew AT, 2004). However, it is unknown where the polymers form and if 4 they are chemotactic in vivo. My colleague Dr Carl Atkison† showed that polymers of Z 1-AT are present in the alveolar wall of Z-AT homozygotes with emphysema, which accounts for 20% of the total AT from lung homogenates.These Z-AT individuals also have an excess of neutrophils in the alveolar wall compared with M-AT homozygotes. Furthermore, neutrophils and polymeric AT co-localize in the alveolar wall (Mahadeva R, 2005). To investigate whether there was a direct relationship between polymers of Z-AT and the excess neutrophils, polymers of AT were instilled into the lungs of wild-type mice (Mahadeva R, 2005). This produced a significant increase in neutrophil influx into the lungs compared with instillation of the native protein.Examination of the time course demonstrated that the influx of neutrophils was closely linked to the presence of polymeric AT. The mechanism of neutrophil recruitment in this mouse model was subsequently shown to be a direct chemotactic effect rather than stimulation of IL-8 homologues or other CXC chemokines. Oxidized AT (Ox-AT) promotes release of human monocyte chemoattractant protein-1 (MCP-1) and IL-8 from human lung type epithelial cells (A549) and normal human bronchial epithelial (NHBE) cells. Native, cleaved, polymeric AT and secretory leukoproteinase inhibitor (SLPI) and oxidized conformations of cleaved, polymeric AT and SLPI did not have any significant effect on MCP-1 and IL-8 secretion. These findings were supported by the fact that instillation of Ox-AT into murine lungs resulted in an increase in JE (mouse MCP-1) and increased macrophage numbers in the bronchoalveolar lavage fluid. The effect of Ox-AT was dependent on NF- B and activator protein-1 (AP-1)/JNK. These findings have important implications. They demonstrate that the oxidation of methionines in AT by oxidants released by cigarette smoke or inflammatory cells not only reduces the anti-elastase lung protection, but also converts AT into a proinflammatory stimulus. Ox-AT generated in the airway † My colleagues’ contributions are acknowledged in future text where appropriate by the following superscripts: (a) Dr Sam Alam, (b) Dr Jichun Wang, (c) Dr Carl Atkinson, (d) Dr Sabina Janciauskiene. 5 interacts directly with epithelial cells to release chemokines IL-8 and MCP-1,which in turn attracts macrophages and neutrophils into the airways. The release of oxidants by these inflammatory cells oxidizes AT, perpetuating the cycle, potentially contributing to the pathogenesis of COPD. Furthermore, this demonstrates that molecules such as oxidants, anti-proteinases, and chemokines, rather than acting independently, collectively interact to cause emphysema (Li Z, 2009). To investigate the molecular basis for the interaction between Z-AT and Ox-AT associated with cigarette smoking, female mice transgenic for normal (MAT)or Z-AT on CBA background were exposed to cigarette smoke (CS). Transgenic mice for Z-AT developed a significant increase in pulmonary polymers following acute CS exposure. Increased levels of neutrophils in CSZ lungs were tightly correlated with polymer concentrations. Oxidation of human plasma Z-AT by CS or -chlorosuccinimide greatly accelerated polymerization, which could be abrogated by antioxidants. The results showed that cigarette smoke accelerated polymerization of Z-AT by oxidative modification, which in so doing further reduced pulmonary defense and increased neutrophil influx into the lungs. These novel findings provided a molecular explanation for the striking observation of premature emphysema in ZZ homozygote smokers, and raised the prospect of anti-oxidant therapy in ZAT related COPD (Alam S, 2011).

Nous avons identifié l’expression du récepteur des angiopoïétines, le récepteur Tie2, à la surface des neutrophiles humains. De plus, nous avons démontré qu’Ang1 et Ang2 induisent des activités pro-inflammatoires sur les neutrophiles, comme l’adhésion aux cellules endothéliales (CEs) et la synthèse du facteur d’activation plaquettaire (PAF). Puisque le PAF augmente la viabilité des neutrophiles et que les angiopoïétines modulent la survie des CEs, nous avons voulu évaluer l’effet des angiopoïétines sur la survie des neutrophiles. Des neutrophiles humains ont été isolés à partir du sang de donneurs sains en accord avec le comité d’éthique de l’Institut de cardiologie de Montréal. La viabilité des neutrophiles a été mesurée par cytométrie en flux à l’aide de marqueurs d’apoptose et de nécrose. Un traitement avec des témoins positifs, soit l’interleukine 8 (IL-8; 25 nM) ou le PAF (100 nM), a augmenté la survie basale des neutrophiles de 34 et 27%, respectivement. De plus, un traitement avec Ang1 (1 pM – 10 nM) a augmenté la survie des neutrophiles jusqu’à 35%, alors qu’Ang2 n’a eu aucun effet. La combinaison de l’IL-8 ou du PAF avec Ang1 (10 nM) a eu un effet additif sur la viabilité des neutrophiles et a augmenté la survie de 56 et 60%, respectivement. Un prétraitement avec des anticorps bloquants contre l’IL-8 a permis d’inhiber l’activité anti-apoptotique de l’IL-8 et d’Ang1 de 92 et 80%, respectivement. Ainsi, notre étude est la première à démontrer la capacité d’Ang1 à prolonger la viabilité des neutrophiles, qui est principalement causée par la relâche d’IL-8. / We reported the expression of angiopoietin receptor Tie2 on the surface of human neutrophils. In addition, we reported that Ang1 and Ang2 are both capable to promote pro-inflammatory activities in neutrophils, namely their adhesion onto endothelial cells (ECs) and platelet-activating factor (PAF) synthesis. PAF is known to promote pro-survival activity on neutrophils and since both angiopoietins can modulate ECs viability, we addressed whether Ang1 and/or Ang2 could modulate neutrophil viability. Human neutrophils were isolated from blood of healthy volunteers in accordance with the guidelines of the Montreal Heart Institute’s ethical committee. Neutrophil viability was assessed by flow cytometry using apoptotic and necrotic markers. Treatment with anti-apoptotic mediators such as interleukin 8 (IL-8; 25 nM) and PAF (100 nM) increased neutrophil basal viability by 34 and 27%, respectively. In addition, treatment with Ang1 (1 pM – 10 nM) increased neutrophil viability by up to 35%, while Ang2 had no effect. Combination of IL-8 or PAF with Ang1 (10 nM) provided an additive effect on neutrophil viability and further increased viability by 56 and 60%, respectively. Pretreatment of the neutrophils with blocking anti-IL-8 antibodies inhibited the anti-apoptotic effect of IL-8 and Ang1 by 92 and 80%, respectively. In summary, our data are the first one to report Ang1 pro-survival activity on neutrophils, which is mainly driven through IL-8 release.

L’angiogenèse, caractérisée par la formation de nouveaux vaisseaux sanguins à partir de vaisseaux préexistants, est un processus accompagné par l’inflammation, impliquant la synthèse et la relâche de différents facteurs de croissance par les cellules inflammatoires. Parmi ces facteurs, seuls le vascular endothelial growth factor (VEGF) et les angiopoïétines (Ang1 et Ang2) peuvent participer à la régulation de l’inflammation et de l’angiogenèse. La famille des angiopoïétines comporte quatre membres, desquels l’Ang1 et l’Ang2 ont été les plus étudiés. Ces deux médiateurs inflammatoires sont capables d’activer le récepteur Tie2, dont l’expression a initialement été rapportée sur les cellules endothéliales (CE). Notre laboratoire a été le premier à démontrer l’expression de Tie2 à la surface des neutrophiles, ainsi que sa capacité, suite à son activation par l’Ang1 ou l’Ang2, à induire la synthèse du facteur d’activation plaquettaire (PAF), l’activation de la β2-intégrine, la migration des neutrophiles ainsi que leur adhésion aux CE. D’autres études ont montré que les CE emmagasinent et relâchent le VEGF et l’Ang2, tandis que les péricytes et les cellules musculaires lisses contiennent l’Ang1. Puisque les neutrophiles relâchent le VEGF et que les deux angiopoïétines ont la capacité d’activer Tie2 sur ces derniers, nous avons voulu déterminer si les neutrophiles contiennent l’Ang1 et/ou l’Ang2 et si elles peuvent être relâchées suite à une stimulation avec des agonistes proinflammatoires. Nous avons découvert que l’Ang1, mais pas l’Ang2 est présente dans les neutrophiles, et qu’elle est relâchée suite à une stimulation au phorbol myristate acetate (PMA). De plus, nous avons démontré que l’Ang1 est localisée au niveau du cytosol et que sa relâche est calcium-indépendante, contrairement au VEGF, qui est localisé dans les granules β et sa relâche est calcium-dépendante. Cette étude démontre pour la première fois l’expression et la localisation de l’Ang1 dans les neutrophiles. Une récente étude effectuée dans notre laboratoire a démontré que les angiopoïétines induisent la migration des neutrophiles en activant le récepteur Tie2 et la voie de la PI3K. De plus, les angiopoïétines ont potentialisé la migration induite par l’IL-8. Ainsi, nous avons émis l’hypothèse que l’Ang1 et/ou l’Ang2 seraient capables d’induire la relâche et/ou la synthèse de l’IL-8 par les neutrophiles. Nous avons démontré pour la première fois, la capacité de l’Ang1 à induire l’expression de l’ARNm, ainsi que la synthèse et la relâche d’IL-8 par les neutrophiles. Cependant, un traitement avec l’Ang2 seule ou en combinaison avec l’Ang1 n’a eu aucun effet sur les activités mentionnées ci-dessus. Nous avons aussi observé que la synthèse et la relâche d’IL-8 induite par l’Ang1 requièrent la transcription de l’ADN en ARNm, suivie par la stabilisation de ce dernier, qui ultimement induit la traduction de l’ARNm de l’IL-8 en sa protéine. Finalement, nous avons démontré que la stimulation des neutrophiles avec l’Ang1 induit ces activités en activant la voie de la p42/44 MAPK, tout en étant indépendantes de la p38 MAPK et la PI3K/Akt. Ces résultats sont en lien direct avec une récente étude dans laquelle nous avons observé que l’Ang1, mais pas l’Ang2 est capable d’augmenter la survie des neutrophiles via la relâche d’IL-8. / Angiogenesis is known as the formation of new blood vessels from pre-existent ones. This process is accompanied by inflammation, which involves the synthesis and release of numerous growth factors by inflammatory cells. Among the growth factors involved in these activities, only the vascular endothelial growth factor (VEGF) and the angiopoietins (Ang1 and Ang2) can modulate both the inflammatory and the angiogenic processes. The angiopoietins family has four fully characterized members, from which Ang1 and Ang2 have been the most extensively studied. Ang1 and Ang2 are both capable to activate the receptor Tie2, initially discovered on the endothelial cell (EC) surface. We were the first group to report the expression of Tie2 on neutrophils along with its activation by Ang1 and Ang2 which can enhance platelet-activating factor (PAF) synthesis, β2-integrin activation, neutrophil migration and adhesion onto EC. Other studies have shown that EC have endogenous stores of VEGF and Ang2, whereas pericytes and smooth muscle cells contain intracellular pools of Ang1. Since neutrophils can release VEGF and that both angiopoietins can activate Tie2 receptor, we wanted to assess if neutrophils contain Ang1 and/or Ang2, and if so, investigate their capacity to be released under inflammatory stimuli. We observed that Ang1, but not Ang2, is found in the neutrophils and that it can be only released upon phorbol myristate acetate (PMA) stimulation. Moreover, using the nitrogen sub-cellular fractionation technique, we demonstrated that Ang1 is found in the cytosolic fraction and its release is calcium-independent, while VEGF is found in β-granules and its release is calcium-dependent. This study demonstrates for the first time the expression and release of Ang1 from the neutrophils and its localization in the cytosol. In one of our recent studies, we have shown that angiopoietins are capable to induce neutrophil migration through Tie2 activation and via the PI3K/Akt signalling pathway. Moreover, both angiopoietins were shown to potentiate IL-8-induced neutrophil migration. Thus, we sought to investigate the capacity of Ang1 and/or Ang2 to induce IL-8 synthesis and/or release from human neutrophils. We demonstrated for the first time, the capacity of Ang1 to induce IL-8 mRNA expression, along with its protein synthesis and release from the neutrophils. However, a treatment with Ang2, alone or in combination with Ang1, had no effect on these aforementioned activities. We also observed that Ang1-induced IL-8 protein synthesis and release requires the transcriptional mechanism from IL-8 DNA to mRNA, followed by the mRNA stabilization, which ultimately enhances its translation into IL-8 protein. Finally, we also observed that neutrophil stimulation with Ang1 enhances IL-8 mRNA expression, protein synthesis and release by activating the p42/44 MAPK signalling pathway, while being independent from p38 MAPK and PI3K/Akt. These results are in line with one of our recent studies, in which we observed that Ang1, but not Ang2, is capable to enhance neutrophil survival, by diminishing their apoptosis through the release of IL-8 by the neutrophils.

Les patients fibrose kystique (FK) souffrent de complications digestives qui incluent une inflammation intestinale modérée dont l’étiologie est méconnue. Les personnes atteintes de FK présentent également une malabsorption des vitamines liposolubles, telles la vitamine D. Or, la vitamine D possède des propriétés immunomodulatrices et anti-inflammatoires. Le présent projet vise à investiguer le rôle du CFTR, dont le gène est muté dans la FK, dans l’étiologie de cette inflammation intestinale et à étudier le potentiel anti-inflammatoire de la vitamine D sur celle-ci. Le CFTR a été invalidé génétiquement par la méthode des ARN interférents (shRNAi) et/ou inhibé pharmacologiquement par l’utilisation d’un antagoniste inhibiteur spécifique (CFTRinh-172). Un état inflammatoire a été induit par les cytokines pro-inflammatoires TNF-α et IL-1β. Afin d’évaluer le rôle anti-inflammatoire de la vitamine D, les cellules ont été pré-traitées avec la forme bioactive de la vitamine D, la 1,25(OH)2D3. La sécrétion et l’expression génique d’interleukine-8, ainsi que l’activation de la voie de signalisation p38MAPK et du facteur de transcription NFκB ont été évaluées. Pour explorer la voie par laquelle la vitamine D exerce ses actions anti-inflammatoires, les cellules ont été pré-incubées avec le BIRB796 pour inhiber la voie p38MAPK. Finalement l’expression génique du récepteur nucléaire de la vitamine D et des hydroxylases intestinales impliquées dans son métabolisme a été déterminée. Nos résultats suggèrent que le CFTR a un rôle dans l’étiologie de l’inflammation intestinale associée à la FK. De plus, la vitamine D semble moduler à la baisse la réponse inflammatoire de la cellule intestinale dont le CFTR a été génétiquement invalidé. / Cystic fibrosis (CF) patients suffer from various digestive complications including moderate intestinal inflammation which etiology is unknown. Individuals with CF also display malabsorption of fat-soluble vitamins; including vitamin D. Vitamin D is a hormone that has immunomodulatory and anti-inflammatory properties. This project aims to investigate the role of CFTR, the mutated gene in CF, in the etiology of CF-related intestinal inflammation and to study the anti-inflammatory potential of vitamin D at this level. CFTR was genetically depleted by means of short hairpin RNA interference (shRNAi) and/or pharmacologically inhibited by the use of a specific inhibitor (CFTRinh-172). An inflammatory state was induced by pro-inflammatory cytokines TNF-α and IL-1β. To evaluate the anti-inflammatory role of vitamin D, cells have been pre-treated with the bioactive form of vitamin D, 1,25(OH)2D3. The secretion and gene expression of interleukin-8, as well as the activation of the p38MAPK signaling pathway and the NFκB transcription factor were assessed. To explore how vitamin D exerts its anti-inflammatory actions, cells were incubated with the BIRB796 to inhibit the p38MAPK pathway. Finally, gene expression of the vitamin D nuclear receptor and the intestinal hydroxylases involved in vitamin D metabolism were assessed. Our results suggest that CFTR plays a role in the etiology of intestinal inflammation associated with CF and that vitamin D reduces the inflammatory responses of CFTR knockdown cells.