Dissertations / Theses on the topic 'Macrophages'
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1
Svensson, Ulf. "Macrophage activation by bacteria signalling to prostaglandin and cytokine responses /." Lund : Dept. of Medical & Physiological Chemistry, Lund University, 1994. http://books.google.com/books?id=sAhrAAAAMAAJ.
2
Higuera, González Laura 1993. "Novel transcription regulators of tissue macrophages and alternative macrophage polarization." Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2021. http://hdl.handle.net/10803/672702.
Abstract:
Macrophages play crucial roles in the defense of the organism against a wide range of pathogens. Macrophages can rapidly adapt to perturbations in the microenvironment due to the existence of a network of transcription factors that modulates their responses. While transcription factors that regulate macrophage identity have been widely described in the past decades, the role of transcription regulators that fine-tune tissue macrophage responses in homeostasis and infection is starting to be elucidated.
Our group has previously identified transcription regulators of pro-inflammatory macrophage responses, and in the present work we have explored the function of novel transcription mechanisms that participate in the regulation of the homeostatic distribution of tissue macrophages and in anti-inflammatory macrophage responses. We have studied ontogenically different macrophage populations inhabiting different tissues and have characterized their transcription regulation. We have also compared the anti-inflammatory response of tissue macrophages and identified a specific transcriptional control of anti-inflammatory gene expression that depends on their ontogeny.Los macrófagos juegan un papel muy importante en la defensa del organismo frente a una amplia variedad de patógenos. Los macrófagos se adaptan rápidamente a las perturbaciones en el microambiente gracias a que existe una compleja red de factores de transcripción que modulan sus respuestas. En los últimos años se han identificado factores de transcripción que regulan la identidad de los macrófagos, sin embargo, apenas se está comenzando a conocer la importancia de otros factores de transcripción que permiten adaptar la respuesta de los macrófagos, tanto en condiciones homeostáticas como frente a infecciones. Anteriormente nuestro grupo identificó reguladores transcripcionales de las respuestas pro-inflamatorias de los macrófagos, y en este trabajo hemos explorado la función de nuevos mecanismos reguladores que participan en la regulación de la distribución de los macrófagos en homeostasis, así como en las respuestas anti-inflamatorias de los macrófagos. Hemos estudiado poblaciones de macrófagos con diferentes ontogenias que habitan dentro de los tejidos y hemos caracterizado su regulación transcripcional. Además, hemos comparado la respuesta anti-inflamatoria de los diferentes macrófagos tisulares y así hemos identificado que existe un mecanismo transcripcional específico que controla la expresión de genes anti-inflamatorios según el origen del macrófago.
3
Tabata, Yasuhiko. "Macrophage phagocytosis of polymer microspheres and antitumor activation of macrophages." Kyoto University, 1987. http://hdl.handle.net/2433/74704.
4
Raborn, Erinn Shenee. "Cannabinoid Modulation of Chemotaxis of Macrophages and Macrophage-like Cells." VCU Scholars Compass, 2007. http://hdl.handle.net/10156/1333.
5
Grand-Perret, Thierry A. R. "Induction d'une activité anti-tumorale chez les macrophages péritonéaux murins." Paris 11, 1986. http://www.theses.fr/1986PA112301.
6
Bouchareychas, Laura. "Implication des phagocytes mononuclées dans l'évolution de la plaque d'athérosclérose et relation avec l'homéostasie du cholestérol et des lipoprotéines." Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066282/document.
Abstract:
L'athérosclérose est un processus physiopathologique chronique impliqué dans la majorité des maladies cardio-vasculaires. Le développement des lésions d'athérosclérose est caractérisé par l'accumulation de lipides extra et intracellulaires dans la paroi artérielle à l'origine d'une forte réponse inflammatoire impliquant notamment les macrophages. Les macrophages sont considérés comme des acteurs clés dans le développement des plaques d'athérosclérose. En effet, de par leur capacité à métaboliser le cholestérol (captation, stockage, efflux), à réguler l'inflammation et à phagocyter les cellules apoptotiques, ils exercent des fonctions pro et/ou anti-athèrogènes qui peuvent être modulées à des fins thérapeutiques. Dans cette perspective, nous avons évalué le pouvoir thérapeutique des " macrophages protégés de l'apoptose " sur la progression des lésions d'athérosclérose constituées. Nous avons démontré que l'augmentation de la survie des macrophages permet de ralentir la progression des lésions, de stabiliser les lésions et de diminuer la cholestérolémie. Ces effets athéro-protecteurs sont attribués à l'augmentation des cellules de Kupffer et des monocytes Ly-6Clow en partie par leur capacité à produire de l'apolipoprotéine E. Nous montrons également que les cellules de Kupffer participent à la clairance des lipoprotéines pro-athérogènes. L'augmentation du pool d'apoE ainsi que l'augmentation des cellules de Kupffer permettent de diminuer la cholestérolémie et ainsi de diminuer la progression des lésionsAtherosclerosis represents a chronic pathophysiological process implicated in the majority of cardiovascular diseases. The development of atherosclerotic lesions is characterized by an accumulation of extra and intracellular lipids in the arterial wall at the origin of a strong inflammatory response involving macrophages.Macrophages are considered key actors in the development of atherosclerotic plaques. Indeed, because of their ability to metabolize cholesterol (capture, storage, efflux), to regulate inflammation and to phagocyte apoptotic cells, they exert pro and/or anti-atherogenic functions that may be modulated therapeutically. In this context, we evaluated the therapeutic potential of macrophages protected against apoptosis, on the progression of established atherosclerotic lesions.We have demonstrated that increased macrophage survival can slow down the progression of established lesions, stabilize lesion and reduce cholesterol levels. These athero-protective effects are attributed to the increase in Kupffer cells and Ly-6Clow monocytes partly due to their ability to produce apolipoprotein E. We also show that Kupffer cells are involved in the clearance of pro-atherogenic lipoproteins. The increase in ApoE pool and in Kupffer cells reduces cholesterol levels and thus lesion progression
7
Di, Maggio Paula. "Dietary lipids and inflammation : chylomicron remnants suppress pro-inflammatory pathways and activate antioxidant defence mechanisms in human macrophages." Thesis, Royal Veterinary College (University of London), 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.618287.
8
Georges, George Tharwat. "Novel Characteristics of Murine Bone Marrow-Derived Macrophages and Human Macrophage-Like Cells." VCU Scholars Compass, 2006. http://scholarscompass.vcu.edu/etd/932.
Abstract:
These studies provide evidence for novel properties of macrophages derived from bone marrow stem cells. In study 1, treatment of activated mouse bone marrow-derived macrophages (BMM) with either catecholamine synthesis inhibitors (α-methyl-para-tyrosine and fusaric acid) or the β2 adrenergic receptor antagonist ICI 118,551 demonstrated that BMM produce catecholamines. The catecholamines modulated macrophage cytokine production through autocrine actions on adrenergic receptors. In study II, undifferentiated human bone marrow cells were incubated in 30% mouse L929 fibroblast conditioned medium and generated adherent cells within three days. The cells were clearly identifiable as macrophages based on surface proteins and phagocytic activity but produced only low levels of the cytokines tumor necrosis factor-α and interleukin-lβ. Cytokine production did not increase in response to the bacterial endotoxin lipopolysaccharide (LPS). Generation of these macrophage-like cells was not repeatable with other samples of human bone marrow, but the cells continue to proliferate in cell culture and will be investigated further in future studies.
9
Awomoyi, Agnes Abiola Oluwatoyin. "Genetics of susceptibility to tuberculosis." Thesis, Open University, 2000. http://oro.open.ac.uk/58012/.
Abstract:
Convincing evidence that activated macrophages play a critical role in control of mycobacterial diseases has been clearly established from animal and in-vitro studies. Macrophages produce a variety of molecules upon appropriate stimulation, which act in concert towards eventual killing of bacteria. People with SUb-optimal macrophage activation are more susceptible to infection with intracellular pathogens. My project aims to answer two questions relating to genetic regulation of macrophage activation in tuberculosis: do macrophage genes regulate microbial-induced responses and do macrophage genes influence susceptibility to tuberculosis? A whole blood assay was used to investigate IFN-y responsiveness in healthy individuals and those who develop tuberculosis in The Gambia. Cytokine responses to lipopolysaccaride (LPS), Lipoarabinomanan (LAM) and the enhancing effect of IFN-y on these stimulants were measured. LPS induced IL-l 0 levels was higher in recovered TB cases than in controls (p=0.02). LPS and LAM induced cytokines were highly correlated (p<0.0001) similarly, levels of IL-IB and TNF were highly correlated (P
10
Suñer, Navarro Clara. "CPEB4 function in macrophages." Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/663483.
Abstract:
As innate immune cells, macrophages sense endogenous and exogenous danger signals and respond orchestrating inflammatory processes. For the rapid induction and efficient resolution of inflammatory responses, macrophages induce the expression of pro- inflammatory and anti-inflammatory mediators, which cross-regulate each other through feedback loops. This process requires tightly controlled gene expression at multiple levels. Recently, the regulation of mRNA deadenylation has emerged as a key regulator of the strength and, critically, the duration of transient inflammatory responses.
Cytoplasmic Polyadenylation Element Binding (CPEB1-4) family of RNA-binding proteins target mRNAs containing Cytoplasmic Polyadenylation Elements (CPEs) in their 3’UTR. CPEBs orchestrate the assembly of two types of ribonucleoprotein complexes (mRNPs) which can repress or stimulate the translation of target mRNAs by modulating the length of poly(A) tail. Several inflammatory mediators harbour CPEs in their 3’UTRs and are potential CPEB targets. Thus, we hypothesized that CPEBs could be an additional checkpoint to control inflammatory responses.
We find that CPEB4 is a novel player in macrophage response to LPS. Upon LPS treatment, CPEB4 is upregulated and its polyadenylation function is activated, a process mediated by the MAPK p38α and ERK1/2 and two AU Rich Element Binding Proteins (ARE-BPs). Interestingly, the pattern of CPEB4 expression and activity suggests that it participates in late LPS-response, when the resolution of inflammation occurs. Indeed, myeloid-specific Cpeb4KO mice display increased sensitivity to LPS-induced endotoxic shock. We identify CPEB4 target mRNAs by RNA-Immunoprecipitation and Sequencing (RIP-Seq), uncovering that CPEB4 regulates the expression of negative regulators of MAPK signalling, thus creating the negative feedback loop needed the resolution of inflammation. Moreover, we also describe how the interplay between CPEB4, HuR and TTP defines mRNA behaviour during the different temporal windows of inflammatory responses.Como células del sistema inmune innato, los macrófagos detectan señales de peligro endógenas y exógenas y responden desencadenando procesos inflamatorios. Estas respuestas inflamatorias tienen que ser inducidas rápidamente pero a su vez, deben ser eficientemente resueltas. Para ello, los macrófagos inducen la expresión de mediadores pro- y anti- inflamatorios que controlan la expresión unos de otros mediante complejos circuitos regulatorios. Estos procesos requieren un estricto control de la expresión génica a distintos niveles. En los últimos años, se ha descrito que la regulación de los mRNAs por deadenilación es un elemento crucial para regular intensidad y sobretodo la duración de las respuestas inflamatorias. La família de proteínas de unión al RNA CPEBs (Cytoplasmic Polyadenylation Element Binding, CPEB1-4), unen mRNAs que contienen CPEs (Cytoplasmic Polyadenylation Elements) en su 3’UTR. Las CPEBs pueden reclutar dos tipos de complejos en los mRNAs que unen. Estos complejos modulan la longitud de la cola poly(A) y, por tanto, pueden reprimir o estimular su traducción. Los mRNAs de múltiples mediadores inflamatorios y son susceptibles de ser regulados por las CPEBs ya que contienen CPEs en sus 3’UTRs. Por tanto, las CPEBs podrían ser un nuevo mecanismo regulador del desarrollo de las respuestas inflamatorias. En este trabajo hemos descubierto que CPEB4 participa en la respuesta de los macrófagos frente a LPS. El tratamiento con LPS provoca un incremento en los niveles de CPEB4 y promueve que su función sea de polyadenylación. Este proceso es mediado por las MAPK p38α y ERK1/2 y dos proteínas que regulan mRNAs mediante la unión a AREs. El patrón de expresión de CPEB4 sugiere que esta proteína participa en la fase tardía de la respuesta a LPS, cuándo la respuesta inflamatoria es resuelta. Apoyando esta hipótesis, ratones KO para CPEB4 en las células mieloides son más sensibles al shock séptico inducido por LPS. Identificando los mRNAs que CPEB4 regula en este contexto, hemos descrito que CPEB4 regula la expresión de inhibidores de la señalización de la vía MAPK. De este modo, CPEB4 es necesaria para la resolución de la inflamación en respuesta a LPS. Además, hemos descrito como la regulación de mRNAs por CPEB4, HuR y TTP define diferentes patrones temporales de expresión durante el desarrollo de respuestas inflamatorias.
11
Alvarez-Hernandez, J. "Iron metabolism in macrophages." Thesis, University of Glasgow, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.375442.
12
Ng, Gilbert Sai Ho. "Inflammatory mechanosensing in macrophages." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709113.
13
Kotter, Mark Reinhard. "Macrophages and CNS remyelination." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614264.
14
Meng, Luxi. "Adipocytes, Macrophages and Inflammation." Thesis, The University of Sydney, 2013. http://hdl.handle.net/2123/10183.
Abstract:
While there is a very strong correlation between obesity and the development of insulin resistance and Type II diabetes, the molecular mechanisms involved are still unknown. However, one clue is that in both human subjects and animal models, obese adipose tissue is infiltrated with M1 macrophages and this creates a systemic low-grade, chronic inflammatory state that could be responsible for the negative consequences of obesity. A better understanding of the relationship between the adipocytes and macrophages under the inflammatory milieu may inform intervention strategies. In this study, the cross-talk between adipocytes and macrophages were investigated under inflammation at a transcriptional level. The gene expression of the adipocyte, 3T3-L1, was monitored in response to the pro-inflammatory secretions from an activated macrophage, RAW 264.7 cells. It was found that following exposure to this pro-inflammatory cocktail, adipocytes were able to respond to inflammatory stimuli in much the same way as immune cells, and displayed transient and drastic transcriptional regulation. Most excitingly, we showed that adipocytes exhibit a transcriptional memory effect, with the expression of many being quite different upon rechallenge with a second stimulus when compared to naïve cells. Microarray analysis revealed that this “memory” was demonstrated in a diverse group of sequences that were both up and down-regulated, including inflammatory and adipocyte-specific function associated sequences. Results suggested that the mechanism(s) behind this memory effect were both transcriptionally and post-transcriptionally regulated. Overall, this thesis shows that the inter-cellular communication between adipocytes and macrophages is strongly dependent on the environmental and temporal context of the adipocytes. This has far reaching implications for the study of the aetiology of the inflammatory state in obesity and, ultimately, the relationship between overweight and disease.
15
Chanez, Pascal. "Hétérogéneité morphologique et fonctionnelle des macrophages des voies aériennes de l'asthmatique." Montpellier 1, 1994. http://www.theses.fr/1994MON1T038.
16
Gui, Philippe. "Caractérisation de la migration trans-tissulaire des macrophages." Toulouse 3, 2014. http://thesesups.ups-tlse.fr/2612/.
Abstract:
L'infiltration de macrophages dans les tumeurs est associée à un mauvais pronostic. Le contrôle de leur migration trans-tissulaire représente donc un enjeu thérapeutique important. Ma thèse a consisté à identifier les mécanismes impliqués dans cette migration. Grâce à des approches d'observation directe du comportement migratoire des cellules dans des tissus vivants (microscopie intravitale et explants tissulaires ex vivo), je montre que les macrophages in vivo adoptent un mode de migration distinct selon le tissu. Dans un fibrosarcome (tissu dense), ils ont une migration de type mésenchymateux (dépendante des protéases), tandis que dans le derme sain adjacent, ils ont une migration de type amiboïde (indépendante des protéases). De plus, j'ai identifié une protéine, p27kip1, impliquée dans la migration mésenchymateuse. Ainsi, en montrant que la migration mésenchymateuse des macrophages existe in vivo, notamment dans les tumeurs, elle pourra devenir une cible thérapeutique prometteuseThe infiltration of macrophages inside tumors is associated with a poor prognosis. Therefore, the specific control of their trans-tissular migration represents an important therapeutic challenge. My thesis has consisted in identifying the mechanisms involved in this migration. Using approaches allowing the observation of the migration behavior of cells directly inside living tissues (intravital microscopy and ex vivo tissue explants), I show that macrophages adopt a distinct migration mode in vivo depending on the tissue. In a fibrosarcoma (dense tissue), they use a mesenchymal-like migration (protease-dependent), whereas in the healthy surrounding derma, they use an amoeboid-like migration (protease-independent). Moreover, I identified a protein, p27kip1, involved in mesenchymal migration. In conclusion, by showing that the mesenchymal migration of macrophages exists in vivo, particularly in tumors, it could become a promising therapeutic target
17
Alhanghari, Mofeda Abdussalam. "The Anti-Apoptotic Effect of HSV-1 ON Murine Macrophages: RAW 246.7Murine Macrophage Cell Line." Wright State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1472747895.
18
Manriquez, Rojas Valeria. "Role of the innate immune response in vascular damage caused by Neisseria meningitidis infection Vascular colonization by Neisseria meningitidis triggers a delayed and inefficient neutrophil response Intermittent pili-mediated forces fluidize Neisseria meningitidis aggregates promoting vascular colonization Adhesion to nanofibers drives cell membrane remodeling through 1D wetting." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCB076.
Abstract:
Neisseria meningitidis est un diplocoque à Gram négatif responsable de méningite et de choc septique. Alors que la méningite est la forme d'infection la plus fréquente, la septicémie fulminante est responsable de 90% de la mortalité imputable à N. meningitidis. La septicémie méningococcique est caractérisée par une éruption purpurique due à des lésions vasculaires. Les observations au niveau histologique révèlent des méningocoques associés aux cellules endothéliales, des thromboses, des hémorragies périvasculaires et des infiltrations de cellules inflammatoires. Les mécanismes conduisant à ces lésions vasculaires ainsi que les raisons pour lesquelles le système immunitaire inné est incapable de contrôler l'infection avant l'atteinte de ce stade pathologique sont inconnus. Dans ce travail de doctorat, nous abordons ces questions en utilisant un modèle murin humanisée par xénogreffe de peau humaine chez des animaux immunodéficients. Nous rapportons que la prolifération bactérienne dans les capillaires est rapide et mène à l'occlusion des vaisseaux en moins de 3 heures post-infection. Dans ce contexte, les macrophages périvasculaires jouent un rôle de sentinelles car ils phagocytent efficacement les bactéries intraluminales adhérentes, aux stades précoces de l'infection et sont essentiels pour recruter les neutrophiles au site d'infection. L'imagerie intravitale et les expériences de déplétion des neutrophiles indiquent que ceux-ci jouent un rôle important dans la destruction des bactéries adhérentes par un processus de migration inverse c'est à dire de l'extérieur vers l'intérieur des vaisseaux et, par conséquent, diminuent les dommages vasculaires induits par les bactéries. L'analyse de la cinétique de recrutement des neutrophiles montre que ceux-ci atteignent un pic de recrutement entre 16h et 24h post-infection chez la souris infectée par voie intravasculaire, comme c'est le cas lors d'une infection naturelle alors que cela ne prend seulement 3h lorsque les bactéries sont injectées par voie intradermique. Ces résultats montrent que la détection intraluminale des bactéries par les macrophages périvasculaires conduit finalement au recrutement des neutrophiles et au contrôle des lésions vasculaires, mais cette réponse dépendante des macrophages périvasculaires est initiée trop tard pour être pleinement efficaceNeisseria meningitidis is a gram-negative diplococcus responsible for meningitis and septic shock. While meningitidis is the most frequent form of infection, fulminant septicemia is responsible for 90% of the mortality imputable to N. meningitidis. Meningococcal sepsis is characterized by a purpuric rash due to vascular damages. Observations at the histological level reveal meningococci associated to endothelial cells, thrombosis, perivascular hemorrhage and inflammatory cells infiltrates. The mechanisms leading to this vascular damage and the reasons for which the innate immune system is unable to control the infection before reaching this pathological stage are unknown. In this doctoral work, we address these questions using a humanized skin xenograft mouse model of Neisseria meningitidis infection. We report that bacterial proliferation inside capillaries is rapid leading to vessel occlusion in less than 3 hours post-infection. In this context, perivascular macrophages play a role of sentinels as they efficiently phagocytose adhering intraluminal bacteria at early stages of infection and are essential to recruit neutrophils to the site of infection. Intravital imaging and neutrophils depletion experiments indicate that neutrophils play an important role in killing adherent bacterial through a reverse migration process and as a consequence decrease the vascular damages induced by the bacteria. Interestingly, detailed analysis of the kinetics of neutrophil recruitment show that while neutrophil numbers reach a peak between 16h and 24h post-infection in mice challenged by the intravascular route as during the natural infection, this takes only 3h when bacteria are injected intra-dermally. These results show that intraluminal detection of bacteria by perivascular macrophages eventually leads to neutrophil recruitment and vascular damage control but this perivascular macrophage-dependent response is initiated too late to be fully efficient
19
Jones, Hilary Francis. "Helicobacter pylori survival in macrophages /." Title page and abstract only, 2004. http://web4.library.adelaide.edu.au/theses/09SBT/09sbtj762.pdf.
20
Brown, Heidi Catherine. "Macrophages and the nervous system." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320118.
21
Mukhopadhyay, Subhankar. "Innate immune activation of macrophages." Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414236.
22
Newsholme, Philip. "Studies on metabolism in macrophages." Thesis, University of Oxford, 1987. http://ora.ox.ac.uk/objects/uuid:3a4fa0f1-03dd-47f1-beff-a48cbd2b14c1.
Abstract:
A general metabolic profile of macrophages was established by measurement of maximum catalytic activities of enzymes in energy-producing pathways and rates of utilisation of glucose, glutamine, fatty acids and ketone bodies under various conditions. It was found that glucose, glutamine and fatty acids can be used to satisfy the energy requirement of the cell. Although a significant proportion of utilised glutamine or fatty acid was converted to C02 by the macrophage, most glucose was not oxidised and was converted, almost stoichiometrically, to lactate. Utilised fatty acids were not only oxidised by the macrophage, but were incorporated into cellular lipid (mainly triacylglycerol and phospholipid). The triacylglycerol rich macrophage was shown to be able to release fatty acids into the culture medium. The importance of glutamine in macrophages was indicated from the high activity of phosphate-dependent glutaminase. Glutamine is probably metabolised by the following enzymes in macrophages: phosphate-dependent glutaminase, aspartate aminotransferase (or other amino acid aminotransferases), oxoglutarate dehydrogenase followed by enzymes of the TCA cycle and metabolism of oxaloacetate by phosphoenolpyruvate carboxykinase. Pyruvate derived via this pathway may be metabolised via pyruvate dehydrogenase or pyruvate carboxylase. A study of the sub-cellular distribution of some of these enzymes suggested that phosphate-dependent glutaminase has a cytosolic as well as a mitochondrial localisation. Further characterisation suggested that the non-mitochondrial activity could be associated with the plasma membrane. To the author's knowledge, this is the first report of a non-mitochondrial localisation for phosphate-dependent glutaminase. Glutaminase was shown to be activated by phosphate and inhibited by glutamate and 2-oxoglutarate. Significant inhibition of glutaminase occurred only at high concentrations of these compounds. Glucose and glutamine were utilised at very high rates by the macrophage, but were not fully oxidised even though the cells were incubated in aerobic conditions. The significance of these high rates of utilisation to the macrophage is discussed.
23
McNally, Oonagh Rose. "Monokine secretion by tumouricidal macrophages." Thesis, University of Ulster, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359349.
24
Guo, Manman. "Phagosome proteomes in activated macrophages." Thesis, University of Dundee, 2015. https://discovery.dundee.ac.uk/en/studentTheses/c5a94702-2164-4acc-9763-bdfcaf1229dc.
Abstract:
Macrophages play key roles in innate and adaptive immune systems not only in the response to pathogens but also in tissue homeostasis. They are extremely plastic and recognize external stimuli such as cytokines with substantial changes to the proteome and molecular functions. One major macrophage function altered by cytokine activation is phagocytosis and phagosome maturation, through which macrophages engulf foreign material such as microbes or apoptotic cells to form phagosomes which then fuse with endosome and finally lysosomes, where the particles are finally degraded. My project aims at investigating the phagosome functions regulated in activated macrophages and further exploring the mechanism by which alternative activation regulates phagosome biogenesis. First of all, a comparison of phagosome proteomes of BMDMs and RAW 264.7 cells was performed, suggesting that there are significant differences for a large number of proteins including important receptors such as mannose receptor 1 and Siglec-1. Moreover, BMDM phagosomes mature considerably faster when validated using fluorogenic phagosome function assays. For the main goal of my project, I have performed a thorough proteomics analysis of the phagosome proteomes of non-activated (RestingMΦ), alternative-activated (IL4 treated, AAMΦ), classical-activated (LPS and IFNγ treated, CAMΦ) and reprogrammed (IL4 activated then LPS and IFNγ treated, ReMΦ) BMDMs. Results indicate that alternative activation leads to phagosomal recruitment of proteins in favour of apoptotic cell clearance, enhanced fusion with lysosomes as well as with parts of the endoplasmic reticulum (ER). Both proteomics and phagosome function assays showed that the phagosome maturation is enhanced in AAMΦ and reduced in CAMΦ and ReMΦ. As side projects, I have also compared phagosome proteomes in IL4 treated, IL13 treated and IL10 treated BMDMs and analysed cellular total proteomes of AAMΦ and RestingMΦ. Furthermore, proteomic data suggest the specific recruitment of TAK1/MKK7/JNK signalling to the phagosomes in AAMΦ, which was confirmed by immunoblotting and fluorescence microscopy. I uncovered that K63 polyubiquitylation of phagosomal proteins is enhanced in AAMΦ, which is responsible for the translocation of TAK1 complex. In AAMΦ, 55 K63 polyubiquitylation sites on 33 phagosomal proteins were identified, including macrophage scavenger receptor 1 (MSR1/SRA). This receptor was further found to be specifically polyubiquitylated on phagosomes upon alternative activation, and MSR1 activation leads to enhanced JNK activation in AAMΦ. Finally, three hypotheses of the function of JNK pathway on phagosomes were described. Firstly, proteomics reveals a reduction of ER and lipid metabolic proteins to phagosomes by the inhibition of JNK, suggesting that TAK1/MKK7/JNK signalling might regulate phagosomal lipid handling. Secondly, JNK might phosphorylate the lipid-activated transcription factor, PPARγ, to regulate macrophage gene expression in lipid metabolism. Finally, loss of MSR1 impairs oxLDL induced JNK activation and M2-to-M1 shift of macrophages, indicating that MSR1/JNK cascade mediates phenotypic shift of AAMΦ upon lipid laden. In conclusion, the work in this thesis provides comprehensive characterisation of phagosomal and cellular proteomes in activated BMDMs. Moreover, TAK1/MKK7/JNK signalling was found for the first time to be specially recruited to phagosomes by K63 polyubiquitylation, and 55 novel K63 polyubiquitylation sites on 33 phagosomal proteins were identified, including MSR1. We hypothesise that JNK signalling might regulate lipid metabolism in AAMΦ.
25
Jubb, Alasdair. "Effects of glucocorticoids in macrophages." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/19521.
Abstract:
Glucocorticoids (GC) are powerful metabolic hormones with anti-inflammatory actions. Despite major side effects they remain widely prescribed therapies. GC regulates gene expression through an intracellular receptor (GR), which is a ligand activated transcription factor. Macrophages are innate immune cells and major targets of GC. Traditionally repression of pro-inflammatory genes in the context of an inflammatory stimulus has been considered the primary mode of action of GC in macrophages. The work described in this thesis has demonstrated that GC act primarily as inducers of gene expression in primary macrophages from both mouse and man, but the set of induced genes is very different between the two species. Chromatin immunoprecipitation and sequencing (ChIP-seq) in each species using anti-GR antibodies revealed candidate enhancers in the vicinity of inducible genes that were generally not shared between mouse and man. The differences in binding were correlated with DNA sequence changes at the enhancer sites between the two species, that caused gain or loss of predicted GR receptor-binding motifs. The mechanism of action of GC was investigated by imaging several different target loci using fluorescence in situ hybridisation in macrophage nuclei. Chromatin at specific GC responsive loci was found to decondense within minutes of exposure of macrophages to the ligand. The apparent decondensation was effect was maintained for at least 24 hours and was not prevented by inhibitors of transcription. The general principles of the GC response were shared between species. However the divergence found underlines the caution that must be used when translating specific findings from mouse to man. Additionally, the data support a role for GR driven changes to chromatin structure in gene regulation in macrophages.
26
Swee, Mei Hua. "Effects of Lysolecithin on Macrophages." Thesis, North Texas State University, 1988. https://digital.library.unt.edu/ark:/67531/metadc798400/.
Abstract:
The effect of lysolecithin on the macrophage was studied using five macrophage function assays. The results of indicate that lyso lecithin is a macrophage activating agent which causes enhanced cell spreading, increased phagocytosis of sheep erythrocytes, heightened membrane activity in the presence of damaged autologous red blood cells, chemotaxis, and vigorous phagocytosis and intracellular killing of Staphylococcus albus.
27
Zlatanova, Ivana. "Macrophages, inflammation et réparation cardiaque." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB047/document.
Abstract:
L’inflammation joue un rôle crucial dans les processus de réparation du muscle cardiaque consécutifs à un infarctus du myocarde (IM). En particulier, les monocytes (MO) et les macrophages (Mf) assument une fonction majeure dans la régulation de la taille d’infarctus et de la fibrose du coeur infarci. Dans un premier travail, nous avons analysé le rôle coordonné de la myeloidepithelial- reproductive protein tyrosine kinase (Mertk) et du milk fat globule epidermal growth factor (Mfge8) dans la propension des MO et des Mf à éliminer les débris cellulaires. Nous avons notamment émis l'hypothèse que ces médiateurs pourraient jouer un rôle clé dans la synchronisation de l’efférocytose dans le tissu cardiaque infarci. Les souris dépourvues de Mertk et Mfge8 présentaient un phénotype cardiaque défectueux par rapport aux souris contrôles. Fait intéressant, au 3ème jour après l’IM, le niveau de protéine cardiaque d’un facteur pro-angiogénique majeur, le VEGF-A, était plus faible chez les souris chimères Mertk-/ -Mfge8-/-par rapport aux contrôles. À partir de ces données, nous avons postulé qu'une altération du taux de la protéine VEGF-A pourrait être liée à une réduction de la capacité des MO/Mf cardiaques à libérer ces facteurs et donc à participer au remodelage bénéfique du coeur infarci. De fait, après un IM, la suppression du VEGF-A dans les cellules myéloïdes réduit la fonction cardiaque. Ces effets étaient associés à une taille d'infarctus supérieure et à un nombre limité de capillaires dans les coeurs infarcis des souris présentant une invalidation conditionnelle du VEGF-A dans les cellules myéloïdes. Ainsi, le VEGF-A dérivé des cellules myéloïdes joue un rôle essentiel dans la régulation de la fonction cardiaque, probablement grâce à l'activation du processus angiogénique dans la zone ischémique. Dans un deuxième travail, nous nous sommes intéressés au rôle de l’hepcidine dans les fonctions réparatrices des MO et des Mf. L'hepcidine est une hormone qui régule activement le trafic du fer. De façon inattendue, la déficience complète en hepcidine restaure considérablement la fonction cardiaque après un IM. L'hepcidine est exprimée par les Mf cardiaques. En outre, les souris ayant une moelle déficiente en hepcidine (Hamp -/-) ou présentant une invalidation conditionnelle dans les cellules de la lignée myéloide (LysMCre +/Hampf/f) ont une fonction cardiaque fortement améliorée. Cet effet a été associé à une réduction de la taille de l'infarctus, de la fibrose et à une stimulation imprévue du renouvellement des cardiomyocytes. De même, dans un modèle de régénération cardiaque induite par résection apicale chez des souris nouveaux nés, nous avons révélé que les macrophages déficients en hepcidine favorisent la prolifération des cardiomyocytes. L'impact des Mf déficients en hepcidine repose sur l'altération de leur métabolisme du fer et sur la libération d'IL-4 et d'IL-13. D’ailleurs, la suppression génétique de l'IL-4 et de l'IL-13 dans les Mf dépourvus d'hepcidine annihile leur effet bénéfique sur la fonction et la réparation cardiaque. Ainsi, l'hepcidine commande la réparation et la régénération cardiaque induite par les Mf par une voie liée à l'IL-4 / IL-13. Ces travaux ont permis de démontrer l’importance des Mf dans la revascularisation et la régénération post-ischémique et de souligner l’efficacité potentielle d’un traitement basé sur la modulation de l’activité des cellules immunitaires dans les pathologies ischémiques cardiaquesNo abstract
28
Adler, Heiko. "Fetal bovine bone marrow-derived macrophages : a model for studying basic aspects of macrophage biology and pathogen-macrophage interaction in cattle /." [S.l.] : [s.n.], 1994. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
29
Laval-Gilly, Philippe Ferard Jean-François. "Analyse de la mobilité des macrophages pour le développement d'un biocapteur atmosphérique." [S.l.] : [s.n.], 2000. ftp://ftp.scd.univ-metz.fr/pub/Theses/2000/Laval_Gilly.SMZ0052.pdf.
30
Willems, Jorine Joanna Lamberta Paulina. "Apoptosis-driven activation of macrophages by starry-sky B-cell lymphoma." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/18738.
Abstract:
In high-grade ‘starry-sky’ non-Hodgkin’s lymphoma (NHL), particularly Burkitt’s lymphoma (BL), large numbers of apoptotic tumour cells are engulfed by infiltrating tumour-associated macrophages (TAM). In situ studies suggest that in starry-sky TAM in a xenograft model of BL various tumour-promoting, trophic, angiogenic, tissue remodelling, and anti-inflammatory pathways are activated. Furthermore, apoptotic cells have been shown to activate expression of tumour-promoting matrix metalloproteinases in macrophages. This work investigates the hypothesis that apoptotic cells or factors released from apoptotic cells induce additional aspects of the starry-sky TAM signature, which serve to promote tumour growth. Macrophages at different stages of maturation, cultured in vitro in the presence of large numbers of apoptotic cells, were shown to differ in phenotype, giving credibility to the hypothesis. Less mature mouse bone marrow-derived macrophages (BMDM) were better at migrating towards apoptotic cells, whereas mature BMDM were better at phagocytosing them. Lactoferrin, which is released from cells undergoing apoptosis and inhibits the migration of neutrophils, was selected as a candidate mediator in the activation of macrophages by apoptotic cells. Lactoferrin was shown to bind viable human and murine monocytes and macrophages, however only high concentrations, which are unlikely to be physiologically or clinically relevant, were found to affect expression of starry-sky TAM genes or reduce classically-activated macrophage cytotoxicity. The direct effect of apoptotic cells on macrophage activation was assessed. Mature BMDM were not used for these studies as their development in vitro in a highly apoptotic environment was judged likely to bias their activation state toward that of TAM, therefore macrophages were first classically-activated with IFN-γ and LPS. This reduced the expression of many starry-sky TAM genes, including several genes associated with responses to apoptotic cells. However, classical activation did not inhibit apoptotic cell engulfment, but rather enhanced it. Co-culture with apoptotic cells, but not viable cells, increased the gene expression of Gas6, Mrc1, Cd36, Timp2, and Pparg, and the latter was dependent on direct interaction with macrophages rather than factors released from apoptotic cells. Furthermore, classically-activated macrophages were found to induce apoptosis in lymphoma cells, and although pre-co-culture of the macrophages with apoptotic cells did not reduce their ability to induce apoptosis, it enhanced tumour cell growth. Macrophage deficiency of IL-4Rα or galectin-3 did not affect classically-activated macrophage responses to apoptotic cells. However, classical activation of galectin-3 deficient macrophages appeared to restore the decreased ability of galectin-3 deficient, untreated macrophages to phagocytose apoptotic cells. Because of the unique new method of laser-capture microdissection by which starry-sky TAM signatures were established, direct comparisons with expression databases of tissue and in vitro cultured macrophages were not possible, but indirect comparisons suggest starry-sky TAM activation reflects the activation phenotype of a mixture of tissue macrophages. Furthermore, it highlighted phagocytosis as one of the most important pathways activated by starry-sky TAM. Together the results presented here suggest apoptotic lymphoma cells can shape TAM activation signatures in starry-sky NHL, even when macrophages are pre-activated to induce apoptosis in lymphoma cells. This is important when considering the consequences of anti-cancer therapies that induce apoptosis or aim to redirect phagocyte activation.
31
Leclercq, Yves. "Interactions transferrines macrophages : rôle des glycannes dans la reconnaissance par les récepteurs membranaires du macrophage péritonéal de souris." Lille 1, 1987. http://www.theses.fr/1987LIL10151.
32
Merlin, Johanna. "Étude de l’influence de la glutaminolyse des macrophages dans les maladies cardio-métaboliques." Thesis, Université Côte d'Azur, 2020. http://theses.univ-cotedazur.fr/2020COAZ6016.
Abstract:
Les maladies chroniques inflammatoires telles que l’obésité ou l’athérosclérose constituent un problème majeur de santé publique dans les pays occidentaux. Il est désormais bien établi que les cellules immunitaires, et en particulier les macrophages, jouent un rôle essentiel dans l’initiation et la progression de ces maladies métaboliques. En effet, les macrophages résidents au sein des tissus contrôlent leur homéostasie comme par exemple l’expansion du tissu adipeux viscéral ou la thermogenèse du tissu adipeux brun. Cependant, les mécanismes restent mal compris. L’immuno-métabolisme est un nouveau domaine de recherche qui illustre l’adaptabilité des macrophages à leur environnement nutritionnel pour le maintien de leurs fonctions. Nous nous sommes ainsi intéressés au rôle de la glutamine dans l’homéostasie des macrophages et son impact sur l’obésité et l’athérosclérose. Pour cela, nous avons génétiquement supprimé l’enzyme limitante hydrolysant la glutamine en glutamate, appelée glutaminase 1 (Gls1), spécifiquement au sein des cellules myéloïdes. Dans notre première étude, nos données démontrent que l’absence de Gls1 dans les cellules myéloïdes conduit à une intolérance au glucose sous régime riche en graisses, associée à une diminution des niveaux de norépinephrine dans le tissu adipeux brun conduisant alors à un défaut de thermogenèse. Nos résultats mettent en évidence une diminution de l’adhésion des macrophages de la moelle épinière aux neurones glutamatergiques et diminuant ainsi l’activation de ces derniers. Notre étude démontre donc le rôle de la glutaminolyse des macrophages dans le contrôle du tonus sympathique des tissus adipeux thermogéniques. Dans un second temps nous avons étudié l’impact la glutaminolyse des cellules myéloïdes sur le développement de l’athérosclérose. L’invalidation de la glutaminolyse des cellules myéloïdes entraîne une augmentation de la surface de la plaque athéromateuse. En particulier, nous avons pu observer une augmentation de la nécrose des plaques suggérant une nouvelle fonction de la glutaminolyse des macrophages. Nous avons également pu valider cette association dans des plaques athéromateuses humaines. Bien que la déficience en Gls1 dans les macrophages n’altère pas leur survie, nous avons pu mettre en évidence un rôle clé de cette voie dans le processus d’efférocytose. L’analyse des mécanismes en aval a révélé une altération de la polarisation alternative des macrophages chez ces souris associée à une reprogrammation du métabolisme mitochondrial. La modulation de ces voies conduit à une baisse d’activité de Rac1 expliquant ainsi le défaut d’efférocytose. Ainsi, notre seconde étude identifie la glutaminolyse des cellules myéloïdes comme un acteur essentiel dans le développement des maladies cardiovasculairesChronic inflammatory diseases such as obesity or atherosclerosis are a major public health concern in the western countries. It is now well established that immune cells, and in particular macrophages, play a critical role in the initiation and progression of cardiometabolic diseases. Indeed, tissue resident macrophages control tissue homeostasis such as visceral adipose tissue expansion and brown adipose tissue thermogenesis. However, the underlying mechanisms remain unknown. Immuno-metabolism is a new research area that illustrates macrophage adaptability to their nutritional environment for their function maintenance. We therefore looked at the role of glutamine in macrophage homeostasis and its impact on obesity and atherosclerosis. Therefore, we genetically abolished the limiting enzyme hydrolyzing glutamine to glutamate, called glutaminase 1 (Gls1), specifically in myeloid cells.In our first study, our data demonstrate that Gls1 deficiency in myeloid cells leads to glucose intolerance on a high-fat diet. This is associated with a decrease in norepinephrine levels in brown adipose tissue leading to defective thermogenesis. Our results highlight a decrease in spinal cord macrophage adhesion to glutamatergic neurons leading therefore to a decrease in neuron activation. Thus, our study demonstrates the role of macrophage glutaminolysis in controlling the sympathetic tone of thermogenic adipose tissue.Secondly, we studied the impact of myeloid cell glutaminolysis on atherosclerosis development. Glutaminolysis invalidation in myeloid cells leads to an increase atherosclerotic plaque area. In particular, we observed an increase in plaque necrosis suggesting a new function for macrophage glutaminolysis. We also validated this association in human atheromatous plaques. Although Gls1 deficiency in macrophages does not affect their survival, we showed a key role of this pathway in efferocytosis. Further analyses of the downstream mechanisms revealed an alteration in macrophage alternative polarization associated with mitochondrial metabolism reprogramming. Modulation of these pathways leads to a drop in Rac1 activity, thus explaining the efferocytosis defect. Therefore, our second study identifies myeloid cell glutaminolysis as an essential actor in atherosclerosis development
33
Naiken, Tanesha. "Étude de la reprogrammation métabolique dans les macrophages polarisés." Thesis, Nice, 2014. http://www.theses.fr/2014NICE4039.
Abstract:
Les cellules tumorales requièrent un approvisionnement continu de nutriments pour produire de l’énergie. Cependant le micro-environnement autour de la tumeur en fournit une quantité insuffisante. Nous avons donc émis l'hypothèse qu'il existe une symbiose nutritionnelle entre les cellules stromales et les cellules tumorales, qui contribue à la tumorigenèse et, nous nous sommes concentrés sur les macrophages. Typiquement, ils sont classés comme classiques (M1-like) ou alternatifs (M2-like) et dans le stroma, les macrophages ont tendance à avoir un phénotype M2-like. Concernant leur profil métabolique, les macrophages M1-like ont un métabolisme glycolytique. Toutefois, les caractéristiques métaboliques des macrophages M2-like ne sont toujours pas clairement définies. Dans nos travaux, en utilisant la lignée cellulaire murine de macrophage, les RAW 264.7, nous avons confirmé que les macrophages M1-like sont exclusivement glycolytiques alors que les M2-like ont plutôt un profil oxydatif. Nous avons démontré qu’il existe une certaine plasticité métabolique des cellules M2-likes car elles sont capables de basculer vers la glycolyse quand la respiration mitochondriale est inhibée. De plus, un blocage de la glycolyse n'a révélé aucune adaptation métabolique des macrophages M1-like mais influe sur les cellules M2, en réduisant leur capacité respiratoire. Finalement, nous avons observé que la polarisation fonctionnelle des macrophages M1-like pourrait être affectée par des changements dans le métabolisme cellulaire. Ces données suggèrent que le métabolisme est un facteur déterminant du phénotype fonctionnel des macrophagesTumor cells require a constant supply of nutrients and yet, their tumor microenvironment supplies insufficient amount of nutrients. We therefore hypothesize that it exists a nutritional symbiosis between stromal cells and tumor cells which contribute to tumorigenesis. Of these stromal cells, we focused on macrophages. Typically, they are classified as inflammatory (M1-like) or alternative (M2-like) and within the stroma, macrophages tend to exhibit an M2-like phenotype. Concerning their metabolic profile, M1-like macrophages have been described to exhibit a glycolytic metabolism. However, the metabolic features of M2-like macrophages remain pretty unclear. In our work, using the mouse monocyte macrophage cell line RAW 264.7, we have confirmed that M1-like cells are exclusively glycolytic whereas M2-like macrophages appear as mainly oxidative. We were able to demonstrate some metabolic plasticity for M2 cells that shift to glycolysis when mitochondrial respiration is inhibited. The targeting of glycolysis through the blockade of downstream lactic acid export catalyzed by monocarboxylate transporters, did not reveal any metabolic adaptation of M1-like macrophages but impacted on M2 cells, reducing their respiratory rate. By manipulating metabolic features of M1- and M2-like macrophages (i.e. glycolysis vs oxidative phosphorylation), we also investigated whether metabolic pathways themselves could impact on macrophage polarization phenotype. We observed that functional M1 polarization of macrophages could be affected by changes in cell metabolism. Taken together, these data suggest that metabolism is a crucial determinant of macrophage functional phenotype
34
Rivier, Agnès. "Activation des phagocytes mononucléés chez les sujets normaux et les sujets asthmatiques/ Agnès Rivier." Montpellier 1, 1994. http://www.theses.fr/1994MON1T020.
35
Marie-Anaïs, Florence. "Mécanismes de formation et de fermeture des phagosomes dans les macrophages." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCB072/document.
Abstract:
La phagocytose est un mécanisme cellulaire essentiel de l’organisme. Elle joue un rôle à la fois dans le maintien de l’homéostasie tissulaire mais également dans le système immunitaire. Ce processus, réalisé par des cellules phagocytaires, telles que les cellules dendritiques, les polymorphonucléaires neutrophiles ou les macrophages, permet l’ingestion et l’élimination quotidienne de particules de grandes tailles (>0,5 µm) : bactéries, champignons ou débris cellulaires. Il est induit par de nombreux récepteurs phagocytaires tels que les récepteurs aux fragments cristallisables des immunoglobulines (FcR) et les récepteurs au complément (CR3). Ceux-ci induisent des cascades de signalisation différentes mais aboutissant, toutes deux, à un remodelage du cytosquelette d’actine et de la membrane plasmique. Il y alors formation d’une coupe phagocytaire entourant et enfermant la particule à internaliser dans un compartiment clos appelé phagosome. Alors que de nombreuses études ont permis de disséquer l’organisation des coupes phagocytaires induites par les FcR, le mécanisme de fermeture des phagosomes n’était pas élucidé. Par ailleurs, les mécanismes moléculaires impliqués dans la formation des phagosomes suite à l’engagement des CR3 sont moins bien décrits. Au cours de ce travail, nous avons analysé le rôle de la dynamine 2, une GTPase impliquée dans les mécanismes de fission des vésicules d’endocytose, au cours de la formation et de la fermeture des phagosomes. Nous avons utilisé un système expérimental original utilisant la microscopie à ondes évanescentes pour montrer, que la dynamine 2 est recrutée avec l’actine dans les coupes phagocytaires en formation et qu’elle s’accumule au site de fermeture des phagosomes dans des macrophages vivants. L’inhibition de son activité GTPase induit une inhibition de l’efficacité de phagocytose et un défaut de la dynamique de l’actine lors de l’extension des coupes phagocytaires. De façon surprenante, la dépolymérisation de l’actine conduit à un défaut de recrutement de la dynamine 2 au site de la phagocytose mettant en évidence une régulation croisée entre la dynamine 2 et l’actine. Enfin cette étude a montré que la dynamine 2 joue un rôle critique dans la scission du phagosome. Dans un second temps, nous avons initié l’étude des mécanismes impliqués dans la régulation de l’activité du récepteur au complément CR3. L’activation de ce récepteur phagocytaire, qui fait partie de la famille des intégrines, requiert un ancrage à l’actine nécessaire à la signalisation vers la polymérisation d’actine et à la formation des coupes phagocytaires. L’ensemble de ces résultats contribue à une meilleure connaissance des mécanismes moléculaires fins impliqués dans la phagocytosePhagocytosis is an important cellular mechanism. It plays a role in both the maintenance of tissue homeostasis and in the immune system. This process, performed by phagocytic cells, including dendritic cells, polymorphonuclear neutrophils or macrophages, enables daily ingestion and elimination of large particles (> 0.5 microns) e.g. bacteria, fungi or cellular debris. It is induced by many phagocytic receptors such as the receptors for crystallizable fragments of immunoglobulins (FcR) and complement receptor (CR3). These receptors induce different signaling cascades but ultimately lead to a remodelling of the actin cytoskeleton and the plasma membrane. Next there is the formation of a phagocytic cup which surrounds and encloses the ingested particle in a closed compartment called the phagosome. While many studies have dissected the phagocytic cup organization induced by the FcR, the mechanism of phagosome closure was not understood. Furthermore, the molecular mechanisms involved in phagosome formation following CR3 engagement are less well described. In this work, we analyzed the role of dynamin 2, a GTPase involved in fission mechanisms of endocytosis vesicles, and in the formation and closure of phagosomes. We used an original experimental system using the total internal reflection fluorescence microscopy (TIRFM) to show that dynamin 2 is recruited with actin during phagocytic cup formation and accumulates at the site of phagosome closure in living macrophages. The inhibition of its GTPase activity induced an inhibition of phagocytosis and a defect in actin dynamics during pseudopod extension. Surprisingly, the depolymerization of actin lead to a defective recruitment of dynamin 2 at the phagocytic site showing there is a cross-regulation between dynamin 2 and actin. Finally, this study showed that dynamin 2 plays a critical role in the scission of the phagosome. Secondly, we initiated the study of the mechanisms involved in regulating the activity of the complement receptor CR3. Enabling this phagocytic receptor, part of the integrin family, requires anchoring actin which is necessary for signaling to the actin polymerization and the formation of phagocytic cups. All these results contribute to a better understanding of the molecular mechanisms involved in phagocytosis purposes
36
Pasquier, Benoit. "Dualité fonctionnelle du récepteur aux fragments constants des Immunoglobulines A : RFcαI ou CD89." Paris 5, 2004. http://www.theses.fr/2004PA05N032.
Abstract:
Les IgA sériques sont décrites comme ayant des propriétés anti-inflammatoires, alors que les complexes immuns à IgA induisent des réponses inflammatoires. Nous avons identifié le récepteur aux IgA, le RFcα comme le médiateur de ces deux fonctions, activatrice et inhibitrice. Les IgA en l'absence d'antigène, ou le fragment Fab d'un Acm anti-RFcαI inhibe la réponse de récepteurs hétérologues, alors que l'agrégation du RFαI par des complexes à IgA médie l'activation cellulaire. Nous avons identifié le motif ITAM de la chaîne γ comme étant impliqué dans ces deux fonctions. L'absence d'agrégation du RFcαI induit une phosphorylation partielle de la chaine γ, permet le recrutement de la phosphatase SHP-1 au RFcαI et induit la déphosphorylation des protéines Syk, LAT et ERKSerum IgA is considered a housekeeper of the immune system with anti-inflammatory functions whereas IgA-immune complexes mediate inflammation. Here, we identify FcαRI or CD89 as the molecular device that determines the nature of IgA responses In the absence of sustained aggregation, receptor targeting by IgA or anti-FcαRI Fab inhibits activatory responses of heterologous receptors. The inhibitory mechanism involves recruitment of the tyrosine phosphatase SHP-1 to FcαRI, thereby affecting Syk, LAT and ERK phosphorylation. Conversely, sustained aggregation of FcαRI by multimeric ligands stimulates cell activation. Both signals require the FcαRγ-ITAM motif. Anti-FcαRI fab treatment suppresses manifestations of allergic asthma in FcαRI transgenic mice
37
Chapple, Cheryl Christine. "The characterisation of macrophage functions in untreated adult periodontitis." Thesis, The University of Sydney, 2000. http://hdl.handle.net/2123/4467.
Abstract:
Macrophages play a critical role in many chronic inflammatory diseases. The pleiotrophic functional capacity of these cells includes phagocytosis and killing of opsonised microorganisms, antigen presentation to T cells, cytotoxicity and the secretion of potent angiogenic growth factors, of central importance in the maintenance or restoration of tissue homeostasis. Although the pathology of the chronic inflammatory disease, periodontitis, has been studied extensively there is a paucity of data relating to the role of macrophages. Recent studies of the untreated advanced periodontitis lesion have revealed extensive vascular pathology, including alteration in blood vessel morphology with thickening of the basement membrane, the accumulation of fragments of vascular basement membrane, persistence of foci of degenerate plasma cells and a restricted capacity to develop reparative granulation tissue. In relation to these pathological features, the distribution and functional status of macrophages assumes prime importance. Macrophage populations in untreated advanced periodontitis biopsies were compared with those in biopsies of clinically healthy, minimally inflamed, gingival tissue. The immunohistochemical investigation used high specificity monoclonal antibodies including a pan-macrophage marker and probes for acute inflammatory, resident histiocytic, and reparative macrophage phenotypes. Results indicated that advanced periodontits and minimally inflamed tissues displayed similar distribution patterns and numbers for the macrophage phenotypic markers. Regionally-specific differences in the populations occurred, however. Further studies investigated macrophage expression of the functional activation markers MHC class II for antigen presentation, and acid phosphatise (AP) and tartrate-resistant acid phosphatise (TRAP) for lysosomal enzyme activity. In the advanced periodontitis lesion there was little evidence of macrophage activation for the expression of HLA-DR and TRAP, although strong expression of HLA-DR was observed in association with blood vessels. Macrophages expressing AP showed a distinct regional distribution; however this was not associated with foci of degenerate plasma cells. These data support the hypothesis that macrophages do not express appropriate activation for key functions in the unrelated lesion of periodontitis. It is postulated that the apparent failure of recruitment and activation of macrophages may in part be both a cause and a consequence of the unusual pathological features of this disease. Although periodontisis is a chronic inflammatory disease of the highly vascularised supporting tissues of the teeth, little is known about the vascular changes in untreated advanced periodontitis. Observations of vascular features of the pathology were investigated and defined, forming the basis for a study of two angiogenic growth factors in periodontitis. In the connective tissue subjacent to the altered epithelium lining the periodontal pocket, there was a significant increase in the numerical density of vascular profiles, primarily accounted for by vessels ≥25μm in diameter. In addition, vascular basement membranes were thickened and there was regional accumulation of non-vascular basement membrane remnants. The co-localisation of type IV collagen and laminin and the discrete nature of these structures was confirmed, using 3-D reconstruction. The distribution of two major angiogenic growth factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), in untreated periodontitis was studied using immunohistochemistry. Basic fibroblast growth factor was not expressed by macrophages. Although bFGF was consistently associated with blood vessels, there was no regional variation in its distribution. In contrast, there was marked regional variation in the intensity of immunostaining for VEGF, with significantly reduced staining of the pocket epithelium. Few macrophages of the Ber-MAC3 phenotype expressed VEGF, as determined by dual immunofluorescence and confocal microscopy. It is considered that the changes in the vascularity of the periodontal connective tissues in untreated advanced periodontitis are, in part, a consequence of altered expression of angiogenic activity by the epithelium and limited expression of angiogenic growth factors by macrophages. Macrophage anergy and vascular disruption observed in the described studies indicate that tissue defence, maintenance and repair are compromised in the untreated lesion of advanced periodontitis.
38
Chapple, Cheryl Christine. "The characterisation of macrophage functions in untreated adult periodontitis." University of Sydney, 2000. http://hdl.handle.net/2123/4467.
Abstract:
Doctor of PhilosophyMacrophages play a critical role in many chronic inflammatory diseases. The pleiotrophic functional capacity of these cells includes phagocytosis and killing of opsonised microorganisms, antigen presentation to T cells, cytotoxicity and the secretion of potent angiogenic growth factors, of central importance in the maintenance or restoration of tissue homeostasis. Although the pathology of the chronic inflammatory disease, periodontitis, has been studied extensively there is a paucity of data relating to the role of macrophages. Recent studies of the untreated advanced periodontitis lesion have revealed extensive vascular pathology, including alteration in blood vessel morphology with thickening of the basement membrane, the accumulation of fragments of vascular basement membrane, persistence of foci of degenerate plasma cells and a restricted capacity to develop reparative granulation tissue. In relation to these pathological features, the distribution and functional status of macrophages assumes prime importance. Macrophage populations in untreated advanced periodontitis biopsies were compared with those in biopsies of clinically healthy, minimally inflamed, gingival tissue. The immunohistochemical investigation used high specificity monoclonal antibodies including a pan-macrophage marker and probes for acute inflammatory, resident histiocytic, and reparative macrophage phenotypes. Results indicated that advanced periodontits and minimally inflamed tissues displayed similar distribution patterns and numbers for the macrophage phenotypic markers. Regionally-specific differences in the populations occurred, however. Further studies investigated macrophage expression of the functional activation markers MHC class II for antigen presentation, and acid phosphatise (AP) and tartrate-resistant acid phosphatise (TRAP) for lysosomal enzyme activity. In the advanced periodontitis lesion there was little evidence of macrophage activation for the expression of HLA-DR and TRAP, although strong expression of HLA-DR was observed in association with blood vessels. Macrophages expressing AP showed a distinct regional distribution; however this was not associated with foci of degenerate plasma cells. These data support the hypothesis that macrophages do not express appropriate activation for key functions in the unrelated lesion of periodontitis. It is postulated that the apparent failure of recruitment and activation of macrophages may in part be both a cause and a consequence of the unusual pathological features of this disease. Although periodontisis is a chronic inflammatory disease of the highly vascularised supporting tissues of the teeth, little is known about the vascular changes in untreated advanced periodontitis. Observations of vascular features of the pathology were investigated and defined, forming the basis for a study of two angiogenic growth factors in periodontitis. In the connective tissue subjacent to the altered epithelium lining the periodontal pocket, there was a significant increase in the numerical density of vascular profiles, primarily accounted for by vessels ≥25μm in diameter. In addition, vascular basement membranes were thickened and there was regional accumulation of non-vascular basement membrane remnants. The co-localisation of type IV collagen and laminin and the discrete nature of these structures was confirmed, using 3-D reconstruction. The distribution of two major angiogenic growth factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), in untreated periodontitis was studied using immunohistochemistry. Basic fibroblast growth factor was not expressed by macrophages. Although bFGF was consistently associated with blood vessels, there was no regional variation in its distribution. In contrast, there was marked regional variation in the intensity of immunostaining for VEGF, with significantly reduced staining of the pocket epithelium. Few macrophages of the Ber-MAC3 phenotype expressed VEGF, as determined by dual immunofluorescence and confocal microscopy. It is considered that the changes in the vascularity of the periodontal connective tissues in untreated advanced periodontitis are, in part, a consequence of altered expression of angiogenic activity by the epithelium and limited expression of angiogenic growth factors by macrophages. Macrophage anergy and vascular disruption observed in the described studies indicate that tissue defence, maintenance and repair are compromised in the untreated lesion of advanced periodontitis.
39
Warby, Tammra. "Interactions between granulocyte-macrophage colony-stimulating factor and human monocyte-derived macrophages following infection with HIV-1 /." [St. Lucia, Qld.], 2006. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe19318.pdf.
40
Finney-Hayward, Tricia Kate. "Monocyte-derived macrophages as a lung macrophage model in chronic obstructive pulmonary disease : characterisation and functional output." Thesis, Imperial College London, 2005. http://hdl.handle.net/10044/1/8330.
41
Walter, Michaela Roylene Valerie. "Macrophages in bovine footrot, the effect of Porphyromonas levii on macrophage function and pro-inflammatory cytokine production." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/MQ65143.pdf.
42
Graham, Susan. "Studies on the activation of rainbow trout (Salmo gairdneri) macrophages and the characterization of a macrophage activating factor." Thesis, University of Aberdeen, 1989. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU498113.
Abstract:
Rainbow trout macrophages were stimulated with PMA to produce 02- and H2O2 as detected by the reduction of nitroblue tetrazolium (NBT) and the oxidation of phenol red respectively. Addition of DDC or nitroprusside, inhibitors of superoxide dismutase (SOD) increased O2-levels and decreased H2O2 levels, whereas addition of exogenous SOD had the reverse effect. Such data are indicative of a respiratory burst pathway in teleost macrophages comparable with that of mammals. Respiratory burst activity, acid phosphatase activity and RNA synthesis in rainbow trout macrophages which have been stimulated in vitro with the mitogen Concanavalin A (Con A) or in vivo by injection of formalin-fixed Aeromonas salmonicida in Freund's incomplete adjuvant (FIA) was analysed. With Con A, in vitro stimulated head kidney (HK) or elicited macrophages had increased O2-production and RNA synthesis but no significant increases in H2O2 or acid phosphatase activity after 72h post-stimulation with Con A. In contrast, all functions were increased in in vivo stimulated macrophages compared with FIA-elicited peritoneal macrophages. In a bactericidal assay, Con A stimulated macrophages did not show an increase in killing of an avirulent strain of A. salmonicida (004) above control levels whereas in vivo stimulated macrophages not only displayed increased killing of the avirulent strain of bacteria but also acquired the ability to kill a virulent strain (048). Thus, Con A stimulated macrophages only possessed some of the features of activation whereas in vivo stimulated macrophages were activated as defined by the increased bactericidal activity. Peritoneal washes obtained in the collection of activated macrophages were able to increase NBT reduction in normal HK macrophages suggesting the presence of a soluble activating factor. Lymphokine (LK)-containing supernatants produced using either HK or blood derived leucocytes, by pulsing with 10ug/ml Con and 5ng/ml PMA, were able to increase O2- and H2O2 production, to enhance the killing of an avirulent strain of A. Salmonicida and conferred the ability to kill a virulent strain of A. salmonicida. The LK present in these supernatants was therefore designated a macrophage activating factor (MAF). The use of potential second signals to enhance the killing of bacteria by LK-treated macrophages, met with limited success. Only A. salmonicida (strain 004) LPS was able to produce a small increase in killing above LK-treatment. The MAF produced in this study was tested for antiviral/interferon (IFN) activity. The results showed that the supernatants did contain IFN activity. Attempts to semi-purify the MAF from antiviral activity showed the two activities to co-purify, indicating that both activities may be due to the same molecular species. The retention time of the MAF/IFN, coupled with the results of SDS-PAGE analysis showed the molecular weight of the moiety to be approximately 19K daltons. Both activities were sensitive to low pH (pH 2), high temperature (60oC) and trypsin, providing further evidence that the MAF and IFN activity produced in these studies may be due to the same molecular species, possibly akin to IFN- of higher vertebrates.
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Mossadegh, Rashti Noushin. "Ontogeny of testicular macrophages, the guardians of fertility." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0141/document.
Abstract:
Les macrophages sont des cellules de l’immunité innée et sont localisés dans la majorité des organes du corps, présentant des fonctions spécifiques dépendant de leur lieu de résidence.Les macrophages d’origine embryonnaire sont la source majeure des macrophages tissulaires et sont capables de se maintenir à long terme dans la plupart des organes adultes.Cependant, il reste certains organes comme le testicule, où l’origine des macrophages n’est pas clairement déterminée. Le testicule est considéré comme un organe immuno-privilégié et a cette nécessité de protéger de tous contacts les spermatozoïdes des cellules immunitaires, qui pourraient induire une auto-immunité.Les macrophages testiculaires (tMφ) contribuent à maintenir ce statut d’organe immuno-privilégié en produisant des cytokines immunosuppressives. Pour ces raisons, les tMφ peuvent être considérés comme des “ gardiens de la fertilité”. Dans les testicules adultes, deux différentes populations de macrophages, nommées interstitielles et péritubulaires, ont été identifiées en se basant sur leurs morphologies et localisations distinctes, mais leur origine et leur mode de développement et de maintenance restent encore inconnus. En combinant des méthodes de traçage cellulaire et la mise au point d’un modèle de transfert adoptif dans des souriceaux, j’ai démontré que les macrophages d’origine embryonnaire contribuaient exclusivement à la population de tMφ interstitielle dès la naissance et que les tMφ péritubulaires proviennent exclusivement de la moelle osseuse. Après avoir caractérisé les tMφ, mes prochaines investigations se porteront sur l’étude des fonctions de chacune de ces deux populationsMacrophages are innate immune cells residing in most of the organs of the body and ensure proper organ function. Traditionally, it has been known that macrophages can be derived from HSC progenitors in the bone-marrow (BM), but technology using fate-mapping tools has revealed that macrophages can already be generated from embryonic progenitors. Embryo-derived macrophages are a major source of tissue-resident macrophages and can self-maintain during adulthood. The origin of resident macrophages in the testis, however, so far has not been well studied.Importantly, the testis is considered as an immune-privileged organ by protecting the highly immunogenic spermatozoa sequestrated in the seminiferous tubules from the entrance of immune cells. In the adult testis, macrophages participate in the creation of an immune suppressive microenvironment preventing auto-immune attack. Therefore, testicular macrophages tMφ could be considered as the guardians of fertility. Recently,two different macrophage populations have been identified in the adult testis, called interstitial and peritubular, based on their distinct localization and morphology,but their developmental origin and homeostatic maintenance were unknown.Combining the genetic lineage tracing and the neonatal adoptive transfer model, I could demonstrate that the embryo-derived macrophages give rise exclusively to interstitial tMφ. Peritubular tMφ, however, only emerge postnatally from BM-derived progenitors. .My findings provide framework for future investigations into the distinct functions of these two tMφ populations in establishment of immune-privilege as well as the support of spermatogenesis and male hormone production
44
Stephens, Janet. "A study of biochemical and morphological aspects of macrophage function in experimental murine Nocardia asteroides and Nocardia brasiliensis infections." Doctoral thesis, University of Cape Town, 1987. http://hdl.handle.net/11427/27213.
Abstract:
It is submitted in this thesis that the degree of activation or inhibition of macrophage function may differ in N. asteroides and N. brasiliensis infections with respect to release of plasminogen activator and of lysozyme The pattern of secretion of plasminogen activator and lysozyme in N. asteroides infections appears to differ in N. brasiliensis infection; and there is possibly a difference in the amount of lysozyme released by 2 day N. asteroides-activated macrophages and 2 day N. brasiliensis -activated macrophages. Strains of Nocardia organism did not influence macrophage morphology or ultrastructure. The study also shows the biochemical characteristics of plasminogen activator and lysozyme release, but not macrophage morphology and ultrastructure, are modified in the first 21 days of experimental Nocardia infections. There are three apparent mechanisms by which virulent strains of N. asteroides manage to survive within macrophages: (i) an ability to inhibit phagosome-lysozome fusion: (ii) alteration in the intraphagosomal pH: and (iii) alteration in the activity of the lysozomal enzyme acid-phosphatase. This study attempted to elucidate further the mechanisms enabling Nocardia organisms to persist and grow within macrophages. Reduced lysozyme release reflects diminished functional status of the macrophages of mice inoculated with N. asteroides or N. brasiliensis at certain times during infection. Reduced intracellular lysozyme levels have been linked with defects in bactericidal function. Such a reduction in intracellular and consequently extracellular levels of lysozyme might explain the capacity of Nocardia to survive intracellularly and to proliferate in the macrophage host.
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Mastora, Chrysoula. "Genetically Modified Macrophages and Renal Regeneration." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/129675.
Abstract:
After ischemic damage in kidney, alternatively activated or M2 macrophages are key players in resolving inflammation and participating in kidney repair. Obtaining this particular phenotype is a process controlled by deregulation of a series of genes.
Modulation of those genes could result in controlling macrophages phenotype and functions, aiming new approaches in resolving ARF.
The first aim of this study was to identify genes that are related to M2 macrophages that are involved in tissue repair. By functional genomics we have identified the expression of 14 genes crucial for the induction of the endogenous reparative capacity of the kidney that are modulated by the presence of macrophages.
We also demonstrated that modulation of Ivns1abp gene can modulate renal repair. More concrete we show that over-expression of the Ivns1abp gene can enhance macrophages regenerative capacity and switch their phenotype towards M2, provoking modulation of their inflammatory state and increasing its resistance against inflammation inputs. We also demonstrated that silencing of the Ivns1abp genet to macrophages leads them to acquire a M1 phenotype and decrease their reparative and phagocytosis capacity.
As a last step, we define pathways that regulate the transcription of the Ivns1abp gene. We show that Ivns1abp gene expression in macrophages is regulated by the c-myc transcription factor and modulated by the presence of cytokines determining cell fate.Después de la lesión isquémica en el riñón, los macrófagos activados alternativamente o M2 no solo participan en la reparación del daño sino son factores clave en la resolución de la inflamación. La obtención de este fenotipo particular es un proceso controlado por la desregulación de una serie de genes. Modulando estos genes, se podría controlar el fenotipo y las funciones de los macrófagos, con el objetivo de nuevos enfoques en el tratamiento de FRA. El primer objetivo de este estudio fue identificar los genes que están implicados en la reparación de tejidos y que están relacionados con los macrófagos M2. Por genómica funcional hemos identificado la expresión de 14 genes cruciales para la inducción de la capacidad regeneradora endógena del riñón y modulados por la presencia de macrófagos. A continuación hemos demostrado que la modulación del gen Ivns1abp puede modular la reparación renal. En concreto hemos demostrado que la sobre-expresión del gen Ivns1abp en macrófagos puede mejorar su capacidad regenerativa y cambiar su fenotipo hacia M2, provocando la modulación de su estado inflamatorio y aumento de su resistencia frente la inflamación. Se demostró además que el silenciamiento del gen Ivns1abp en macrófagos disminuye su capacidad reparadora y fagocítica transformando su fenotipo a M1. Como ultimo, hemos definido las vías que regulan la transcripción del gen Ivns1abp. Hemos demostrado que la expresión del gen Ivns1abp en los macrófagos está regulada por el factor del trascripción c-myc y modulada por la presencia de citoquinas, determinando el destino de la célula.
46
Patel, Dilipkumar. "Cholesterol metabolism in monocyte-derived macrophages." Thesis, Imperial College London, 1990. http://hdl.handle.net/10044/1/46492.
47
Cessford, Erin Lauren. "Mechanistic Insights into Necroptosis of Macrophages." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31771.
Abstract:
Cell death is an imperative mechanism for the development, homeostasis and survival of an organism. Various forms of cell death have been documented and recent reports indicate that the mode of cell death elicited can have a profound influence on the development and perpetuation of inflammation. Apoptosis is the predominant, programmed pathway of cell death, which ensures physiological elimination of unwanted cells. On the other hand, another cell death pathway described as programmed necrosis (necroptosis), has recently been revealed. The induction of necroptosis and its impact in host biology is not clear. Herein I have evaluated the mechanisms of necroptosis in macrophages, an important cell type of the immune system. My experiments indicate that type I interferon (IFN-I) signaling through transcription factors STAT1, STAT2 and IRF9, collectively described as the ISGF3 complex, is indispensable for necroptosis of macrophages. Furthermore, my results indicate that IFN-I signaling promotes the sustained phosphorylation of receptor interacting protein kinase 3 (Rip3), a key protein required for the execution of necroptosis. My findings also reveal that dynamin-dependent endocytosis following IFNβ stimulation and caspase inhibition is necessary for the induction of necroptosis. The results presented in this thesis provide new insights into the molecular mechanisms of necroptosis and therefore contribute to a deeper understanding of multiple inflammatory pathologies.
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Nyberg, Lindborg Kristina. "Phagolysosomal pH measurements in alveolar macrophages /." Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4512-8/.
49
Nho, Boram. "Characterization of elastolytic cathepsins in macrophages." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/40665.
Abstract:
Atherosclerosis is characterized by a thickening of the arterial wall and loss of its elasticity. The elasticity of the arterial wall is impaired when the extracellular matrix undergoes extensive proteolytic remodeling. Cathepsins are papain-like cysteine proteases that are known to have elastolytic/fibrinolytic activities. They are highly expressed in macrophages present in plaque areas of diseased blood vessels and are thought to contribute to the tissue remodeling. Using cathepsin deficient macrophages and various protease inhibitors, the elastolytic activities of cathepsins B, K, L, and S were quantitatively determined. Up to 60% of the total elastase activity of macrophages was attributed to cathepsin activities. Deficiencies in single cathepsins appeared to be compensated by other cathepsins. The capability and potency of cathepsins B, K, L, and V to hydrolyze fibrin was also determined.
The exact quantification of individual cathepsin activities with the help of inhibitors or enzyme deficiencies in biological samples is difficult due to compensatory effects. Thus, specific substrates could be a viable alternative. Commercially available cathepsin activity assay kits that exploit fluorogenic peptidyl substrates are widely used to measure individual cathepsin activities in biological samples. However, substrates marketed as cathepsin K, L and S specific were found to be only marginally specific or completely non-specific, and were hydrolyzed by various other cathepsins. Furthermore, the presence of highly potent endogenous inhibitors in biological samples and the lack of specificity of the substrates skew the measurements towards cathepsin B which is relatively resistant to endogenous inhibitors. Thus, data obtained using commercial substrate kits are to be interpreted with great caution.
50
Platt, Andrew M. "Intestinal macrophages in health and inflammation." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/690/.
Abstract:
The healthy large intestinal mucosa contains a vast pool of macrophages (mφ) that are close to the local bacterial flora and have several unique phenotypic and functional properties compared with other mφ populations. Although human colonic mφ retain some of the hallmark functions of mφ, such as the ability to phagocytose particulate material and exert bactericidal activity, they are unable to produce pro-inflammatory mediators. Thus it has been suggested that intestinal mφ are functionally adapted to the microbe-rich, immunostimulatory environment of the gut, where strong inflammatory responses to harmless commensal bacteria would lead to continuous inflammation and ultimately tissue pathology. Indeed, there is mounting evidence that mφ play an essential role in maintaining homeostasis and epithelial renewal in the normal intestine. In contrast, mφ from the intestine of patients with inflammatory bowel disease (IBD) differ markedly from those present physiologically, exhibiting heightened inflammatory and bactericidal activities, and contributing to the tissue damage. How these differing properties of colonic mφ are controlled and how this potentially dangerous population is kept quiescent under physiological conditions are important questions. Most existing information comes from either simple, observational studies of human tissue, or from work on cell culture systems which aim to reproduce the unusual phenotype of resident intestinal mφ in vitro. Importantly analogous experiments of resident and inflammatory mφ have not been carried out in murine systems, where it would be possible to characterise the cells fully and explore their origin, function and role in inflammatory processes. Therefore, the aims of this thesis were first to characterise mφ in the resting murine colon both functionally and phenotypically, focussing particularly on their expression of toll-like receptors (TLR) and responsiveness to TLR ligation and on their population dynamics in vivo. By comparing colonic mφ with other tissue mφ populations, I hoped to gain an understanding of how resident gut mφ might have adapted to their local environment. In the second part of my work, I examined how the properties of colonic mφ altered in inflammation, employing a well-established experimental model of colitis, with the aim of determining how resident and inflammatory mφ might relate to each other. Lastly, I explored the effects of the ES-62 parasite product, known to have potent anti-inflammatory effects on mφ, on experimental colitis in vivo. Experiments detailing my initial characterisation of the myeloid cells expressing the F4/80 mφ marker in the colon of normal mice are described in Chapter 3. These revealed that the F4/80+ population in the gut is extremely heterogeneous compared with other mφ populations in the body. Virtually all in vitro-differentiated BM mφ (BMM) and mφ from the resting peritoneum (PEC mφ) exhibited the conventional F4/80+CD11b+CD11c- phenotype of classical mφ and upregulated costimulatory molecules in response to TLR ligation. In stark contrast, the colon contained three F4/80+ subsets, one F4/80+CD11b+CD11cint, one F4/80+CD11b+CD11c- and a smaller population of F4/80+CD11b-CD11c- cells. None of these subsets expressed co-stimulatory molecules, even after LPS stimulation, but unlike other mφ, the majority of colonic mφ expressed high levels of class II MHC without stimulation. BMM and PEC mφ also produced several pro-inflammatory cytokines and chemokines following stimulation, whereas colonic mφ showed no mediator production under these conditions. Nevertheless, colonic mφ did retain avid endocytic and phagocytic activities, indicating that colonic mφ may engulf bacteria without initiating inflammation. In Chapter 4, I explored the unresponsiveness of resting colonic mφ to microbial stimuli in more detail and found that the TLR refractoriness is associated with reduced expression of TLR2, 3, 4 and 9. Apart from a small proportion of mφ that retained TLR2 expression, TLR expression was downregulated both at the protein level and to some extent also at the mRNA level; TLRs were not re-expressed following ex vivo culture of purified mφ. This global downregulation of TLRs could not be reproduced in BMM by treatment with TLR ligands, and was also present in colonic mφ taken from mice unable to signal via TLR2 or TLR4, suggesting it was not simply a form of endotoxin “tolerance”. However, the mechanism seemed to involve IL-10, as colonic mφ from IL-10-deficient animals displayed a heightened level of TLR expression and responsiveness, even prior to the onset of intestinal inflammation. In Chapter 5, I examined the phenotype and function of mφ during the experimental colitis induced by feeding dextran sodium sulphate (DSS). During inflammation, the absolute number of F4/80+ mφ increased 6-fold, the majority of which now expressed TLR, CD11b and low levels of CD11c. This new population of colitic mφ also expressed class II MHC, low levels of co-stimulatory molecules and produced large amounts of TNFα. In Chapter 6, I went on to examine the population dynamics of colonic mφ under resting conditions and during inflammation, showing that the overall turnover rate of the total mφ population was increased during colitis, as assessed by uptake of BrdU in vivo. The increased turnover was mainly due to the TLR-expressing, TNFα+ population of mφ and more detailed analysis showed that the small number of these cells present in resting colon had identical turnover rates to those found in colitis. In contrast, the TLR negative mφ had much lower turnover rates in resting and inflamed colon, suggesting that the TLR+ and TLR- subsets may represent distinct mφ populations with different population dynamics, and that during intestinal inflammation, the TLR+ subset may display a preferential recruitment into the gut. Indeed, proliferation in situ was minimal, indicating that the recently divided, TLR-expressing mφ proliferated outside the intestine before being recruited into the gut. My subsequent experiments suggested that this recruitment may involve the CCR2 chemokine receptor, which was expressed at high levels specifically by the TLR+ subset of mφ both in resting and inflamed colon. Finally in Chapter 7, I treated colitic mice with ES-62, a phosphorylcholine (PC)-containing glycoprotein secreted by the filarial nematode, Acanthocheilonema viteae, which has been shown to modulate pro-inflammatory cytokine production by mφ in vitro. ES-62 treatment had no significant effect on weight loss or pro-inflammatory cytokine production in the colon of mice with DSS colitis, although it slightly delayed the onset of the clinical signs of disease. Thus further studies of ES-62 as a modulator of mφ-dependent intestinal inflammation may be warranted. Taken together, my data suggest that under resting conditions, intestinal mφ are heterogeneous and adapt to their microenvironment by being non-inflammatory via active downregulation of TLR expression and function, which may be partly dependent on IL-10. During inflammation, large numbers of TLR expressing, fully responsive mφ appear, probably via CCR2-dependent recruitment of recently divided blood-derived monocytes. Interestingly, small numbers of these TLR-expressing, rapidly turning over mφ are also present in normal colon and my data suggest that these pro-inflammatory mφ may be quite distinct from the more sessile mφ which are the dominant “resident” population in normal gut. A delicate balance between these two mφ populations must ensure homeostasis and appropriate responses to inflammation.