Coxiella burnetii é a bactéria intracelular causadora da Febre Q, capaz de subverter funções celulares e evadir o reconhecimento do sistema imune da célula hospedeira permitindo o estabelecimento do seu nicho replicativo nas células-alvo: macrófagos e monócitos. É um patógeno altamente virulento, sendo necessário poucos organismos para desencadear a doença, e é considerado como um potencial agente de bioterrorismo da categoria B. Entre os danos econômicos causados por C. burnetii destaca-se a infecção de animais de gado, seu reservatório natural, pois causa aborto espontâneo dos filhotes e, consequentemente, prejuízo aos produtores. Seres humanos também adquirem a infecção, por inalação de partículas contaminadas. Hospedeiros imunocompetentes são capazes de restringir a infecção por C. burnetii apesar dos diversos mecanismos de evasão da resposta imune do hospedeiro, como interação com vias de sinalização celular e utilização de efetores bacterianos. No entanto, em hospedeiros não competentes a infecção pode evoluir para casos de Febre Q crônica e levá-los à morte. Modelos murinos são frequentemente utilizados para entender as interações patógeno-hospedeiro nas infecções por essa bactéria. A diferença na suscetibilidade de macrófagos de diferentes linhagens murinas e de diferentes tipos celulares à infecção por C. burnetii ainda é pouco conhecida. No entanto, sabe-se que C. burnetii fase II sucumbe a macrófagos derivados da medula óssea (BMDMs) de camundongos C57BL/6, enquanto células das linhagens BALB/c e A/J são suscetíveis à infecção. Nesse contexto, considerando a relevância biomédica de C. burnetii, faz-se necessário a determinação de um modelo relevante para melhor compreender as relações patógeno-hospedeiro nas infecções por essa bactéria. Neste trabalho, caracterizamos um novo modelo de estudo com macrófagos primários que permite avaliar a infecção com C. burnetii fase II in vitro, além de elucidar os mecanismos relacionados à suscetibilidade das células à bactéria. Por meio de quantificação do DNA genômico bacteriano por qPCR verificamos que macrófagos alveolares (AMs) murinos são altamente suscetíveis à replicação da bactéria, mesmo no fundo gênico restritivo C57BL/6. Caracterizamos a replicação da bactéria em AMs por microscopia de fluorescência e microscopia eletrônica de transmissão e demonstramos que essa replicação ocorre dentro dos vacúolos parasitóforos típicos. Pela análise de expressão gênica por RT-PCR efenotipagem de marcadores de superfície por FACS identificamos que a alta suscetibilidade dos AMs se deve a uma polarização dessas células para um padrão M2. Por fim, validamos a relevância desse modelo pela análise da suscetibilidade de AMs murinos deficientes para NOS2, IFN-? e IL-4 como prova de princípio. Verificamos que a suscetibilidade de AMs é comparável à de células Vero e células THP-1, modelos celulares imortalizados descritos como altamente suscetíveis à replicação de C. burnetii, e que AMs podem ser utilizados para estudos utilizando células derivadas de camundongos com fundo gênico C57BL/6. Dessa forma, nosso trabalho caracterizou os AMs murinos como um relevante modelo celular primário altamente suscetível para o estudo das interações patógeno-hospedeiro frente à infecção in vitro por C. burnetii fase II, além de elucidar os mecanismos por trás dessa suscetiblidade / Coxiella burnetii is the intracellular bacterium that causes Q fever, capable of subverting cellular functions and evading recognition by the host cell immune system, thus allowing the establishment of its replicative niche in its the target cells: macrophages and monocytes. It\'as a highly virulent pathogen, with few organisms being sufficient to cause the disease, and considered a potential class B bioterrorism agent. Among the economic damages caused by C. burnetii are infection of cattle animals, its natural reservoir, that leads to spontaneous abortion of the offspring and financial loss to the producers. Human beings also acquire the infection, through inhalation of contaminated air particles. Immunocompetent hosts are able to restrict infection by C. burnetii despite the several evasion mechanisms from the host immune system, which includes interaction with cell signaling pathways and utilization of bacterial effectors. However, in non-competent hosts the infection can evolve to chronic Q Fever and lead to death. Murine models are frequently used to understand the host-pathogen interactions during infections by this bacterium. The difference in the susceptibility among macrophages from different murine strains, as well as different cell types, still poorly known. However, it is known that C. burnetii phase II succumbs to bone-marrow derived macrophages (BMDMs) from C57BL/6 mice, whereas cells from BALB/c and A/J strains are susceptible to infection. In this context, considering the biomedical relevance of C. burnetii, it\'s necessary to establish a relevant study model to better understand the host-pathogen relations in infections by C. burnetii. In this work, we characterized a new study model with primary macrophages to assess the infection by C. burnetii phase II in vitro and elucidated the mechanisms underlying the susceptibility to C. burnetii. By using qPCR for quantification of bacterial genomic DNA, we saw that murine alveolar macrophages (AMs) are highly susceptible to C. burnetii replication, even in the usually restrictive C57BL/6 backgound. We characterized the bacterium replication in murine AMs by florescence microscopy and transmission electron microscopy, showing that this replication occurred inside the typical C. burnetii-containing vacuole. We also identified, through the gene expression by RT-PCR and the phenotypic profile of surface markers by FACS, that the increased susceptibility of murine AMs were due to a polarization of these cells into a M2 profile. To finish, we validated the relevance of thismodel through the analysis of the susceptibility of murine AMs deficient to NOS2, IFN-? and IL-4 as a proof of principle. We verified that the susceptibility of AMs is comparable to Vero cells and THP-1 cells, immortalized cell models described as highly susceptible to C. burnetii replication, and that AMs can be used to studies using cells derived from mice with the C57BL/6 background. In this way, our work characterized murine AMs as a relevant primary cell model highly susceptible to studies among the host-pathogen interactions in infections with C. burnetii phase II in vitro, and we also elucidate the mechanisms underlying this susceptibility

No description available.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A doença pulmonar obstrutiva crônica (DPOC) causa a redução da capacidade respiratória e seu desenvolvimento é associado à fumaça de cigarro. O cigarro possui mais de 4800 substâncias tóxicas e causa a morte de seis milhões de pessoas por ano no mundo. Estudos buscam meios de reverter os males causados pela fumaça de cigarro. A própolis (P) é um produto produzido por abelhas que possui várias propriedades. O objetivo deste trabalho foi avaliar os efeitos antioxidantes da P em macrófagos murinos e na inflamação pulmonar aguda induzida pela fumaça de cigarro (CS) em camundongos. A análise dos compostos fitoquímicos do extrato alcóolico da P (EAP) foi feita por cromatografia gasosa acoplada à espectrometria de massa (GC-MS). Células da linhagem RAW 264.7 foram tratadas em diversas concentrações de P durante 24 horas. Após tratamento, as seguintes análises foram realizadas: polifenóis totais; viabilidade celular (MTT); potencial redutor (DPPH); espécies reativas de oxigênio totais (ROS) e de malondialdeído (MDA). Trinta camundongos C57BL/6 foram divididos em 3 grupos (n=10/grupo): Controle+P, CS e CS+P. Ambos os grupos CS foram expostos a 6 cigarros/dia durante 5 dias. O grupo CS foi tratado com veículo. O pulmão e o lavado broncoalveolar (BAL) foram coletados para análise histológica e contagem diferencial de células. As análises para mieloperoxidase (MPO), superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx), glutationa reduzida (GSH) e oxidada (GSSG), MDA, nitrito e western blotting para TNF-alfa foram realizadas. A análise fitoquímica do EAP mostrou a presença dos ácidos hidrocinâmicos e coumárico, a artepilina C e a drupanina. Foi observado o aumento concentração-dependente dos níveis de polifenóis totais (p<0,001), do MTT (p<0,001) e do DPPH (p<0,001), e o inverso com o MDA (p<0,001). Os níveis de ROS diminuem nas concentrações de 15,6 e 31,2 mg/mL (p<0,05, ambos). A histologia pulmonar do grupo Controle+P foi similar ao do CS+P e foi observado um influxo de macrófagos e neutrófilos no grupo CS (p<0,01 e p<0,001, respectivamente). Os níveis de MPO foram aumentados no grupo CS (526,534,72 mU/mg ptn, p<0,01), mas houve uma redução no grupo CS+P (385,127,64 mU/mg ptn, p<0,05) comparável ao Controle+P (13412,99 mU/mg ptn, p<0,001), o mesmo aconteceu com as enzimas antioxidantes: SOD (Controle+P: 523,529,6 U/mg ptn; CS: 523,529,6 U/mg ptn, p<0,001; CS+P: 246,815,69 U/mg ptn, p<0,001); CAT (Controle+P: 37,383,39 U/mg ptn; CS: 92,686,24 U/mg ptn, p<0,001; CS+P: 59,844,55 U/mg ptn, p<0,05); GPx (Controle+P: 2,230,17 (M/min/mg ptn) x 10-1; CS: 4,510,31 (M/min/mg ptn) x 10-1, p<0,001; CS+P: 2,640,19 (M/min/mg ptn) x 10-1, p<0,05). O inverso ocorreu com a relação GSH/GSSG (Controle+P: 1,0880,17; CS: 0,7360,07, p<0,05; CS+P: 1,2580,10, p<0,05). Os níveis de MDA (Controle+P: 0,2660,05 nMol/mg ptn; CS: 0,940,076 nMol/mg ptn, p<0,001; CS+P: 0,4980,06 nMol/mg ptn, p<0,01) e de nitrito (Controle+P: 50,014,19 Mol/mg ptn; CS: 108,77,73 Mol/mg ptn, p<0,001; CS+P: 58,843,42 nMol/mg ptn, p<0,01) estavam aumentados no CS que em outros grupos. A expressão de TNF-&#945; foi observada no grupo CS. O tratamento da P apresentou ação anti-inflamatória e antioxidante em macrófagos e em camundongos expostos à fumaça de cigarro, possivelmente pela ação dos polifenóis presentes nela / Chronic obstructive pulmonary disease (DPOC) causes a respiratory capacity reduction and its development is be associated with cigarette smoke. Cigarette has more than 4800 toxic substances in your composition and it causes death of six million people in the world. Studies had been made to find methods to change cigarette-induced health problems. Propolis (P) has been reported as a natural product with several properties. Here, we used two types of experiments, in vitro and in vivo, to study the potential antioxidant use of P. Phytochemical analyze was made to evaluate which compounds are within P. Murine macrophages, RAW 264.7 cell line, are exposed to different concentrations of propolis and following analyses were made: total polyphenols levels; cell viability (MTT); reduction potential (DPPH); total reactive oxygen species levels (ROS) and malondialdehyde (MDA). Thirty C57BL6 mice were divided into 3 groups: Control+P, CS and CS+P. Both CS groups were exposed to 6 cigarettes per day for 5 days. CS group was treated with vehicle. Lung and bronchoalveolar lavage (BAL) were collected for histological analysis and differential cell counts. Analysis for mieloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), oxidized glutathione (GSSG), MDA, nitrite and western blotting for TNF-alpha were performed. Phytochemical analyses of P showed which possesses four main compounds: hydrocinnamic and coumaric acids; artepillin C and drupanin. P showed an increase in total polyphenols (p<0.001), cell viability (p<0.001) and DPPH levels (p<0.001) in concentration-dependent manner, different from MDA (p<0.001) which was inverse pattern. P decreased ROS levels in 15.6 and 31.2 mg/mL groups (p<0.05). Lung histology of Control+P group was similar to CS and CS+P groups, however significant inflammatory cell influx was observed in CS group. Propolis administration reduced significantly macrophages and neutrophils compared with CS group (p<0.01 e p<0.001, respectively). MPO levels was increased in CS (526.534.72 mU/mg ptn, p<0.01), but was shown to be reduced in CS+P (385.127.64 mU/mg ptn, p<0.05) compared with Control+P (13412.99 mU/mg ptn, p<0.001), similar pattern was found in antioxidant enzymes: SOD (Controle+P: 523.529.6 U/mg ptn; CS: 523.529.6 U/mg ptn, p<0.001; CS+P: 246.815.69 U/mg ptn, p<0.001), CAT (Controle+P: 37.383.39 U/mg ptn; CS: 92.686.24 U/mg ptn, p<0.001; CS+P: 59.844.55 U/mg ptn, p<0.05) and GPx (Controle+P: 2.230.17 (M/min/mg ptn) x 10-1; CS: 4.510.31 (M/min/mg ptn) x 10-1, p<0.001; CS+P: 2.640.19 (M/min/mg ptn) x 10-1, p<0.05) and different from GSH/GSSG ratio (Controle+P: 1.0880.17; CS: 0.7360.07, p<0.05; CS+P: 1.2580.10, p<0.05). MDA (Controle+P: 0.2660.05 nMol/mg ptn; CS: 0.940.076 nMol/mg ptn, p<0.001; CS+P: 0.4980.06 nMol/mg ptn, p<0.01) and nitrite levels (Controle+P: 50.014.19 Mol/mg ptn; CS: 108.77.73 Mol/mg ptn, p<0.001; CS+P: 58.843.42 nMol/mg ptn, p<0.01) increased in CS group when compared with other groups. TNF-&#945; expression was observed in CS only. P administration showed an antioxidant action in murine macrophages and reduced cigarette smoke-induced lung inflammation and oxidative stress, probably, by action of its phytochemical compounds actions

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A doença pulmonar obstrutiva crônica (DPOC) causa a redução da capacidade respiratória e seu desenvolvimento é associado à fumaça de cigarro. O cigarro possui mais de 4800 substâncias tóxicas e causa a morte de seis milhões de pessoas por ano no mundo. Estudos buscam meios de reverter os males causados pela fumaça de cigarro. A própolis (P) é um produto produzido por abelhas que possui várias propriedades. O objetivo deste trabalho foi avaliar os efeitos antioxidantes da P em macrófagos murinos e na inflamação pulmonar aguda induzida pela fumaça de cigarro (CS) em camundongos. A análise dos compostos fitoquímicos do extrato alcóolico da P (EAP) foi feita por cromatografia gasosa acoplada à espectrometria de massa (GC-MS). Células da linhagem RAW 264.7 foram tratadas em diversas concentrações de P durante 24 horas. Após tratamento, as seguintes análises foram realizadas: polifenóis totais; viabilidade celular (MTT); potencial redutor (DPPH); espécies reativas de oxigênio totais (ROS) e de malondialdeído (MDA). Trinta camundongos C57BL/6 foram divididos em 3 grupos (n=10/grupo): Controle+P, CS e CS+P. Ambos os grupos CS foram expostos a 6 cigarros/dia durante 5 dias. O grupo CS foi tratado com veículo. O pulmão e o lavado broncoalveolar (BAL) foram coletados para análise histológica e contagem diferencial de células. As análises para mieloperoxidase (MPO), superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx), glutationa reduzida (GSH) e oxidada (GSSG), MDA, nitrito e western blotting para TNF-alfa foram realizadas. A análise fitoquímica do EAP mostrou a presença dos ácidos hidrocinâmicos e coumárico, a artepilina C e a drupanina. Foi observado o aumento concentração-dependente dos níveis de polifenóis totais (p<0,001), do MTT (p<0,001) e do DPPH (p<0,001), e o inverso com o MDA (p<0,001). Os níveis de ROS diminuem nas concentrações de 15,6 e 31,2 mg/mL (p<0,05, ambos). A histologia pulmonar do grupo Controle+P foi similar ao do CS+P e foi observado um influxo de macrófagos e neutrófilos no grupo CS (p<0,01 e p<0,001, respectivamente). Os níveis de MPO foram aumentados no grupo CS (526,534,72 mU/mg ptn, p<0,01), mas houve uma redução no grupo CS+P (385,127,64 mU/mg ptn, p<0,05) comparável ao Controle+P (13412,99 mU/mg ptn, p<0,001), o mesmo aconteceu com as enzimas antioxidantes: SOD (Controle+P: 523,529,6 U/mg ptn; CS: 523,529,6 U/mg ptn, p<0,001; CS+P: 246,815,69 U/mg ptn, p<0,001); CAT (Controle+P: 37,383,39 U/mg ptn; CS: 92,686,24 U/mg ptn, p<0,001; CS+P: 59,844,55 U/mg ptn, p<0,05); GPx (Controle+P: 2,230,17 (M/min/mg ptn) x 10-1; CS: 4,510,31 (M/min/mg ptn) x 10-1, p<0,001; CS+P: 2,640,19 (M/min/mg ptn) x 10-1, p<0,05). O inverso ocorreu com a relação GSH/GSSG (Controle+P: 1,0880,17; CS: 0,7360,07, p<0,05; CS+P: 1,2580,10, p<0,05). Os níveis de MDA (Controle+P: 0,2660,05 nMol/mg ptn; CS: 0,940,076 nMol/mg ptn, p<0,001; CS+P: 0,4980,06 nMol/mg ptn, p<0,01) e de nitrito (Controle+P: 50,014,19 Mol/mg ptn; CS: 108,77,73 Mol/mg ptn, p<0,001; CS+P: 58,843,42 nMol/mg ptn, p<0,01) estavam aumentados no CS que em outros grupos. A expressão de TNF-&#945; foi observada no grupo CS. O tratamento da P apresentou ação anti-inflamatória e antioxidante em macrófagos e em camundongos expostos à fumaça de cigarro, possivelmente pela ação dos polifenóis presentes nela / Chronic obstructive pulmonary disease (DPOC) causes a respiratory capacity reduction and its development is be associated with cigarette smoke. Cigarette has more than 4800 toxic substances in your composition and it causes death of six million people in the world. Studies had been made to find methods to change cigarette-induced health problems. Propolis (P) has been reported as a natural product with several properties. Here, we used two types of experiments, in vitro and in vivo, to study the potential antioxidant use of P. Phytochemical analyze was made to evaluate which compounds are within P. Murine macrophages, RAW 264.7 cell line, are exposed to different concentrations of propolis and following analyses were made: total polyphenols levels; cell viability (MTT); reduction potential (DPPH); total reactive oxygen species levels (ROS) and malondialdehyde (MDA). Thirty C57BL6 mice were divided into 3 groups: Control+P, CS and CS+P. Both CS groups were exposed to 6 cigarettes per day for 5 days. CS group was treated with vehicle. Lung and bronchoalveolar lavage (BAL) were collected for histological analysis and differential cell counts. Analysis for mieloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), oxidized glutathione (GSSG), MDA, nitrite and western blotting for TNF-alpha were performed. Phytochemical analyses of P showed which possesses four main compounds: hydrocinnamic and coumaric acids; artepillin C and drupanin. P showed an increase in total polyphenols (p<0.001), cell viability (p<0.001) and DPPH levels (p<0.001) in concentration-dependent manner, different from MDA (p<0.001) which was inverse pattern. P decreased ROS levels in 15.6 and 31.2 mg/mL groups (p<0.05). Lung histology of Control+P group was similar to CS and CS+P groups, however significant inflammatory cell influx was observed in CS group. Propolis administration reduced significantly macrophages and neutrophils compared with CS group (p<0.01 e p<0.001, respectively). MPO levels was increased in CS (526.534.72 mU/mg ptn, p<0.01), but was shown to be reduced in CS+P (385.127.64 mU/mg ptn, p<0.05) compared with Control+P (13412.99 mU/mg ptn, p<0.001), similar pattern was found in antioxidant enzymes: SOD (Controle+P: 523.529.6 U/mg ptn; CS: 523.529.6 U/mg ptn, p<0.001; CS+P: 246.815.69 U/mg ptn, p<0.001), CAT (Controle+P: 37.383.39 U/mg ptn; CS: 92.686.24 U/mg ptn, p<0.001; CS+P: 59.844.55 U/mg ptn, p<0.05) and GPx (Controle+P: 2.230.17 (M/min/mg ptn) x 10-1; CS: 4.510.31 (M/min/mg ptn) x 10-1, p<0.001; CS+P: 2.640.19 (M/min/mg ptn) x 10-1, p<0.05) and different from GSH/GSSG ratio (Controle+P: 1.0880.17; CS: 0.7360.07, p<0.05; CS+P: 1.2580.10, p<0.05). MDA (Controle+P: 0.2660.05 nMol/mg ptn; CS: 0.940.076 nMol/mg ptn, p<0.001; CS+P: 0.4980.06 nMol/mg ptn, p<0.01) and nitrite levels (Controle+P: 50.014.19 Mol/mg ptn; CS: 108.77.73 Mol/mg ptn, p<0.001; CS+P: 58.843.42 nMol/mg ptn, p<0.01) increased in CS group when compared with other groups. TNF-&#945; expression was observed in CS only. P administration showed an antioxidant action in murine macrophages and reduced cigarette smoke-induced lung inflammation and oxidative stress, probably, by action of its phytochemical compounds actions

Mycobacterium avium subsp. paratuberculosis (MAP) is a very slow growing bacterium that is the causative agent of Johne's disease (JD) in ruminants and has long been suggested to be associated with complications of Crohn's disease (CD) in humans. Although there is no direct evidence that MAP is the primary etiological agent for CD, most CD patients are found to have MAP in their intestinal tissues. The current control measures for JD in cattle, sheep, and goats have only been minimally effective, and there are only medications to treat the symptoms of mycobacterial infections associated with CD in humans. Along with not being able to cure MAP infections, there is no established laboratory animal model for testing therapeutics. When mice are infected with MAP they develop systemic infection and do not mimic disease observed in ruminants. J774A.1 murine macrophages typically have a very short lifespan of about 4-6 days, however MAP infected cell cultures can survive up to about 10 days. Using a modified protocol of Estrella et al. (2011), we have been able to establish a 45-60 day long-term MAP infected J774A.1 murine macrophage cell culture model. With the addition of retinoic acid (RA), vitamin D (VD), and phorbol myristate acetate (PMA) in combination in cell culture, we were able to screen novel therapeutics before embarking on in vivo testing in animals. This is a significant step forward in Crohn's and Johne's disease treatment research. We are not only able to test various drugs against specific strains of MAP to determine susceptibility, but we are also able to test a wide variety of drugs at the same time, with relatively minimal cost. We have evaluated the efficacy of clarithromycin, azithromycin, isoniazid, amikacin, ethambutol, ciprofloxacin, levofloxacin, rifampicin, clofazimine, as well as a combination of clarithromycin, rifampicin, and clofazimine using our MAP infected macrophage cell culture model. We were able to determine the drugs' differential ability to kill intracellular MAP in the early stages of infection, versus chronic stages of infection, and against two different strains of MAP, 43015 and 19698 that affect humans and cattle respectively. The minimal inhibitory concentration (MIC) of each drug was determined as per NCCLS protocol in vitro, and the drugs were tested at the MIC value, along with one concentration above and below the MIC in our cell culture model. The antimicrobials were found to be effective at different stages of cell culture infection and in different strains of MAP. Some drugs were more effective at early stages of MAP infection, whereas others were more effective in chronic or latent stages of infections. It is important to note that although a drug may be effective at a certain stage of infection, it may not necessarily be effective against all strains of MAP. The most promising results were seen with a combination of clarithromycin, clofazimine, and rifampicin, which was effective at all stages of infection with both strains of MAP tested. This long term cell culture model will provide researchers with important screening tools for evaluating new therapeutics before embarking on costly in vivo testing, and allow the assessment of therapeutics at different stages of MAP infection but also against an array of intracellular pathogens. / Ph. D.

Dans cette étude, l'aptitude de trois extraits de Gui fermentés (VAF) issus de trois arbres hôtes, à induire ou moduler la sécrétion de cytokines, telles que l'IL-1β, l'IL-6 et le TNF-α, a été explorée à l'aide de deux modèles de macrophages murins. Des traitements prolongés par des concentrations cytotoxiques, mais non sub-cytotoxiques, de VAFs induisent la sécrétion d'IL-1β. Dans ces conditions, les concentrations sub-cytotoxiques de VAFs amplifient les sécrétions d'IL-1β induite après stimulations par le LPS puis l'ATP, ou par l'imiquimod. Par ailleurs, appliqués brièvement et à concentrations sub-cytotoxiques, les VAFs accélèrent la sécrétion d'IL-1β induite après stimulations par le LPS puis l'ATP. / In this study, the ability of fermented extracts from mistletoe grown on three host trees to induce or modulate the secretion of pro-inflammatory cytokines, like IL-1β, IL-6 and TNF-α has been explored. Applied for long times, cytotoxic, but not sub-cytotoxic concentrations of fermented mistletoe extracts induce the secretion of IL-1β. In these conditions sub-cytotoxic concentrations increase the IL-1β secretions induced either by LPS and ATP, or by imiquimod. When applied briefy at sub-cytotoxic concentrations, fermented mistletoe extracts can accelerate the secretion of IL-1β induced by LPS and ATP.

Dans cette étude, l'aptitude de trois extraits de Gui fermentés (VAF) issus de trois arbres hôtes, à induire ou moduler la sécrétion de cytokines, telles que l'IL-1β, l'IL-6 et le TNF-α, a été explorée à l'aide de deux modèles de macrophages murins. Des traitements prolongés par des concentrations cytotoxiques, mais non sub-cytotoxiques, de VAFs induisent la sécrétion d'IL-1β. Dans ces conditions, les concentrations sub-cytotoxiques de VAFs amplifient les sécrétions d'IL-1β induite après stimulations par le LPS puis l'ATP, ou par l'imiquimod. Par ailleurs, appliqués brièvement et à concentrations sub-cytotoxiques, les VAFs accélèrent la sécrétion d'IL-1β induite après stimulations par le LPS puis l'ATP. / In this study, the ability of fermented extracts from mistletoe grown on three host trees to induce or modulate the secretion of pro-inflammatory cytokines, like IL-1β, IL-6 and TNF-α has been explored. Applied for long times, cytotoxic, but not sub-cytotoxic concentrations of fermented mistletoe extracts induce the secretion of IL-1β. In these conditions sub-cytotoxic concentrations increase the IL-1β secretions induced either by LPS and ATP, or by imiquimod. When applied briefy at sub-cytotoxic concentrations, fermented mistletoe extracts can accelerate the secretion of IL-1β induced by LPS and ATP.

Chapter 1: Introductionn Staphylococci are gram positive rotund bacteria that grow in clusters; and hence get their name. The genus of Staphylococcus comprises of over 30 species of which S. epidermidis and S.aureus are significant in their interaction with humans and are known to cause diseases. S.aureus invades various soft tissues and causes a vast multitude of diseases spanning from simple boils and abscesses to osteomyelitis and endocarditis, which can become fatal upon the onset of bacteremia and toxic shock. S. aureus has also been established as one of the leading causes of nosocomial infections especially because of their multi-drug resistant traits and their ability to colonize prosthetic devices and catheters. The increasing incidence of the multi-drug resistant strains and the rising prevalence of community acquired S. aureus infections mandates a comprehensive understanding of the pathogen and its biology, its intracellular fate and the defense mechanisms in the host. Towards this end, we have attempted to delineate some aspects of the pathogen’s virulence and the host responses to them. S. aureus normally inhabits the skin and mucosal surfaces as a commensal. Upon the onset of permissive circumstances it turns into an opportunistic pathogen. Immuno-compromised conditions or breach of skin can serve as the portals of entry for the pathogen. Upon entry, the bacteria encounter macrophages as the first line of defense in the host. Macrophages appear at the site of infection and phagocytose the bacteria, subjecting the pathogen to phagolysosomal degradation which facilitates antigen presentation and pathogen clearance. As part of their immune evasion mechanism, various pathogens are known to adopt a multitude of strategies to subvert this fate and survive in the host cells. This dissertation work aims at gaining insight into the staphylococcus-macrophage interaction in the ongoing host-pathogen duel, to gain better understanding about the pathophysiology and etiology of the disease. Chapter 2: Intracellular Trafficking of Staphylococcus aureus in Macrophages Successful targeting of the pathogen necessitates a comprehensive understanding of its biology and physiology in its interactions with the host. With this objective we undertook a study to uncover the intracellular niche of S. aureus in RAW264.7 murine macrophage-like cells. Any invading pathogen once internalized by the macrophage is contained in a phagosome, which undergoes progressive acidification and maturation from the early endosome to late endosome and ultimately fuses with the phagolysosome, where where the invading pathogen is subject to degradation. Through exhaustive electron microscopy of the infected macrophages, we show that S. aureus is present as a single bacterium per vacuole through the entire period of infection. We have further monitored the intracellular trafficking of the bacteria in the macrophage through confocal studies with endosomal markers which serve as indicators of vesicle maturation. Soon after the onset of the infection, the bacteria were found to be present in the early endosome (EEA-1 positive vesicles) which gradually matured into LAMP1 positive, late endosomal vesicles. However, only a small fraction of the bacteria containing vesicles were found to fuse with the lysosomes, suggesting that the bacteria prevented phagolysosomal fusion. We further observed that the bacteria did not prevent the acidification of the vesicles they resided in, but only limited their fusion with the lysosome. Taken together, our studies delineating the intracellular niche of S. aureus in RAW macrophages revealed that the pathogen has successfully evolved immune evasion mechanisms to overcome its phagolysosomal relegation. Chapter 3: Staphylococcus aureus Succumbs to the Hepcidin in Murine Macrophage We have further attempted to study the intracellular fate of the bacteria in macrophages towards gaining greater insight into its biology. Our studies on the intracellular fate of S. aureus in RAW264.7 cells revealed a distinct biphasic fate of the bacteria. The pathogen was found to replicate initially and this proliferative phase was subsequently followed by a gradual fall in its numbers. Interestingly however, the pathogen is never found to be cleared from the system suggesting the presence of a residual infective pool in the macrophages. We thus explored the possible mechanisms which could attribute to this biphasic intracellular fate of the bacteria. Macrophages come armed with a rich repertoire of defense mechanisms to incapacitate the invading pathogens. They have in their arsenal, reactive oxygen (ROS) and nitrogen species (RNS) and many potent anti-microbial peptides, apart from the lysosomal machinery, to degrade the invading pathogen. Upon investigation, we find that the RAW macrophages do not mount a ROS/RNS response when infected with S. aureus. Induction of these responses in the macrophage by alternate means further reveals that the pathogen is recalcitrant to death by these oxidative/nitrosative bursts. Of the antimicrobial peptides (AMPs) harbored by macrophages, we find that Hepcidin is up-regulated upon infection with S. aureus. Hepcidin is a peptide which is known to have a key regulatory role in iron homeostasis in addition to its potent antimicrobial functions. Since Hepcidin is known to be induced upon increased iron availability; we pre-treated the host cells with iron and monitored the effect of the same on bacterial fate. As expected, we observed that Hepcidin induction by pre-treatment with iron equips the macrophage to counter the pathogen better and thus leads to hastened and heightened clearance of the bacteria. This induction of hepcidin is significant at the mRNA and protein levels and is also corroborated by increased co-localisation of the bacteria with the anti-microbial peptide. Our studies thus identify hepcidin as a key line of the host defense towards countering the bacterial infection thus explaining the near complete bacterial clearance observed. Chapter 4: Global gene expression studies offering insight into potential immune evasion strategies of S.aureus in countering host offences. The interactions between host and the pathogen are multi-layered with the involvement of numerous players and many signaling cascades. In this light, we have attempted to get a holistic view of the host-pathogen interplay through microarray studies. These global profiling studies were aimed at identifying the important players in bacterial virulence and the macrophage response factors involved in countering the same in the context of S. aureus infection. The array was uniquely designed to incorporate both bacterial and host probes so as to facilitate parallel analysis of the host and pathogen gene expression profiles in the same sample. The expression profiling studies were carried out at three time points which represent the key phases of the bacterial infection viz. internalization, replication and clearance. A comprehensive analysis of the bacterial and host gene expression profiles under these phases provided insights into bacterial virulence and the host’s strategies to counter the same. We observe a large scale metabolic shut down in S. aureus subsequent to its internalization. We find the distinct up-regulation of a small subset of genes, majority of which are as yet uncharacterized. Amongst these were a few well-characterized virulence genes which remained active, representing the bacterial strategies to subvert the host immune response. The large scale down-regulation of gene expression can be possibly explained as the adaptation of the bacteria to the available metabolites and its submission to a quiescent phase of existence in the macrophage. In parallel, the host system exhibits the induction of TNF-α and up regulation of TLR2 and Nod2, which are typically triggered by a gram-positive infection. But simultaneously, we also observed a marked increase in the expression of anti-apoptotic and anti-inflammatory responses. This was re-iterated by a significant down-regulation in some of the pro-inflammatory, pro-apoptotic and antigen presentation involved genes and processes. We further find that the time course of the infection did not largely influence the gene expression kinetics. The macrophages were influenced and committed to a fate conducive for the bacteria fairly early in the infection regime. Thus, our studies of the expression profiles of the pathogen and the host under the different phases of the infection provide us with a comprehensive understanding the strategies of bacterial offense and host defenses thereby offering a window into this fascinating world of host-pathogen interactions. Chapter 5: Conclusion To summarize, we have attempted to study the intracellular fate of the S. aureus pathogen in macrophages. Our studies suggest that the bacterium attempts to evade clearance by the host immune system by actively preventing fusion with the lysosomal vesicles. We also find that despite these defenses, the pathogen appears to succumb to the host immune system as it is targeted by Hepcidin, an anti-microbial peptide. The lack of complete bacterial clearance under these conditions is however suggestive of an underlying strategy by the pathogen, possibly to maintain a chronic infective state in the host system. The microarray studies, in addition, shed light on the other possible immune evasion strategies that S.aureus might be employing to escape the host offences. The results are indicative of the bacteria influencing anti-apoptotic, anti-inflammatory and antigen presentation responses and thereby prolonging its survival in the macrophage. In conclusion, given the fact that the macrophages are itinerant cells with a long life span, the light thrown by our findings of the various immune evasion strategies that S.aureus is adopting; it suggests that the macrophages could serve as potential carriers which could account for the dissemination of the infection to new sites, which has perpetually been a major concern for any Staphylococcal infection.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal