Orientador: Carlos Rossa Júnior / Resumo: Os macrófagos representam uma das principais pontes entre imunidade inata e adaptativa e estão presentes em grande número tanto no estroma circundando tumores sólidos quanto no interior da massa tumoral (TAMs, tumor-associated macrophages), onde podem representar até 50% da massa da lesão. Nos carcinomas espinocelulares de cabeça e pescoço (HNSCC), TAMs apresentam predominantemente o perfil M2 (pró-tumoral), e a quantidade de TAMs é inversamente relacionada ao prognóstico. A efetividade do tratamento não-cirúrgico (quimio/radioterapia ou imunoterapia) dos tumores sólidos é diretamente relacionada à indução de morte das células neoplásicas. No processo de reparo, as células apoptóticas são removidas por fagocitose por outros tipos celulares, num processo denominado eferocitose. Embora seja um processo importante para o reparo e homeostasia tecidual, a eferocitose pode afetar o fenótipo dos macrófagos, favorecendo a polarização para o perfil M2, associado à progressão de HNSCC. A galanina é um peptídeo de 29 aminoácidos amplamente distribuída no organismo e com amplo espectro de efeitos biológicos. A expressão constitutiva de galanina por células de OSCC está associada à maior agressividade do tumor e tem sido estudado como marcador prognóstico de agressividade do tumor e também como um possível alvo terapêutico de HNSCC. O objetivo do presente trabalho é determinar a influência dos produtos secretados pelas células de carcinoma espinocelular de cabeça e pescoço, bem como da ga... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Macrophages are a major link between innate and adaptive immunity and are present both in the surrounding stroma and within various solid tumors (when they are denominated tumor-associated macrophages, TAMs), where these cells may represent up to 50% of the tumor mass. In head and neck squamous cell carcinomas (HNSCC), TAMs are predominantly of the M2 phenotype and the prognosis is inversely correlated with their abundance. The effectiveness of non-surgical treatment of solid tumors (chemotherapy/radiotherapy) is directly related to the induction of neoplasic cell death. In the repair process, apoptotic/dead cells must be removed by phagocytosis by other cell types (particularly macrophages), in a process called efferocytosis. Although this is a critical step in the repair and reestablishment of tissue homeostasis, efferocytosis itself may affect the phenotype of macrophages, skewing the cells towards an M2 phenotype, which is deemed as a 'pro-tumoral' phenotype. Galanin is a 29 aminoacid peptide of 29 produced by various cell types and with a broad spectrum of biological effects. The constitutive expression of galanin by head and neck cancer cells is associated with the increased of tumor aggressiveness and has been proposed as a biomarker of tumor aggressiveness and also as a possible therapeutic target in HNSCC. Thus, the goal of the present study was to determine the role of secreted products from head and neck cancer cells, and of galanin independently, on macrophage phe... (Complete abstract click electronic access below) / Mestre

Galectin-3 is a β-galactoside binding lectin expressed in tumour cells and macrophages and has been associated with increased malignancy in a variety of cancers. Previous work has shown that galectin-3 is an important regulator of macrophage function, promoting an alternative (M2) phenotype which potentiates chronic inflammation and fibrosis. Tumour associated macrophages (TAMs) adopt an M2 phenotype and are thought to promote tumour growth by down regulating T cell effector function and promoting angiogenesis. This project examines the hypothesis that host galectin-3 promotes lung cancer growth and spread. In order to test this hypothesis, Lewis Lung Carcinoma tumour growth and metastasis was investigated in strain matched mice either expressing or deficient in galectin-3. The Lewis Lung Carcinoma cell line (LLC1) is a spontaneous lung carcinoma line, derived from C57BL/6 mice, which readily forms tumours when transplanted. Furthermore, LLC1 cells were stably transfected with a Luciferase expressing vector in order to assist detection of tumour growth and metastasis in vivo. An orthotopic model of LLC1 growth suggested that galectin-3-/- animals do not support lung carcinoma growth and spread. This finding was confirmed by a subcutaneous model of cancer growth, where it was found that wild type animals display a higher proportion of macrophages expressing a prototypic M2 marker around tumour sites compared to galectin-3-/- animals. M2-promoting cytokine transcripts were also reduced in galectin-3-/- mice. Additionally, tumours of wild type mice were more invasive and presented more mature blood vessels compared to galectin-3-/- mice. To specifically address the role of recruited cells on tumour growth, metastasis and the inflammation profile around tumour sites, in relation to galectin-3 expression, bone marrow cells (BMCs) were transplanted from wild type to galectin-3-/- mice and vice versa. It was shown that galectin-3 positive BMCs restore the wild type phenotype of tumour growth in galectin-3-/- mice, while galectin-3 deficient BMCs impair tumour growth in wild type animals. Furthermore, macrophage ablation experiments demonstrated incapacity for tumour establishment in the absence of macrophages. A series of experiments investigating reported inhibition of galectin-3 by modified citrus pectin (MCP) via competitive inhibition did not provide conclusive results. MCP had no effect in vivo, but was able to inhibit LLC1 cell growth in vitro. Most importantly though, results were inconclusive as to whether galectin-3 binds MCP. Some ligand displacement was seen, but direct binding of the molecules could not be shown. In general, the results obtained demonstrate a strong pro-tumoural effect of galectin-3 on growth, tissue invasion and metastasis of LLC1 tumours via an increased proportion of Ym1-expressing macrophages around tumour sites. It was shown that macrophages are key cells for tumour initiation and that BMC phenotype in relation to galectin-3 expression determines the phenotype of tumour development in subcutaneous and orthotopic LLC1 models. Therefore, galectin-3 has a strong regulatory effect on tumour phenotype and could present a key target in the management of lung carcinomas.

Cardiovascular disease (CVD) is highly prevalent in chronic kidney disease (CKD) patients. Whilst this can in part be explained by the high incidence of traditional CVD risk factors such as hypertension and diabetes evident in CKD patients, recent focus has been on non-traditional risk factors and their role in CVD progression. These include endothelial dysfunction, arterial stiffness, inflammation and oxidative stress. The potent vasoconstrictor endothelin-1 (ET-1) has been implicated in the pathogenesis of CKD and the CVD associated with it. Further understanding of the mechanisms by which it contributes to CKD and CVD pathogenesis, specifically its interactions with non-traditional risk factors are still required. Additionally, the potential applications of ET antagonists in renal disease have not been fully explored. This thesis aims to investigate the role of ET-1 in the development of renal disease and the associated inflammation, hypertension and vascular dysfunction through a series of in vitro, in vivo and clinical studies. I have demonstrated using in vitro techniques that murine macrophages (Mϕ) express both endothelin A (ETA) and endothelin B (ETB) receptors but that ET-1 does not elicit either a classical pro-inflammatory or alternative anti-inflammatory phenotype in Mϕ. I was however, able to show that M display chemokinesis towards ET-1 and M ETB receptors provide a novel clearance mechanism for ET-1 through receptor mediated dynamin-dependent endocytosis In an in vivo study I investigated whether ET-1 mediates the progressive renal injury after renal ischaemia reperfusion injury (IRI) that leads to the development of CKD. I demonstrated that endothelin A receptor antagonism provided long term beneficial effects reducing blood pressure and preventing progressive kidney injury, inflammation, and the development of fibrosis resulting from an episode of acute kidney injury (AKI). Similar benefits were observed with calcium channel blockade, suggesting hypertension may mediate some of the long term effects of renal IRI and anti-hypertensive treatments could prevent the development of CKD after AKI. Finally, in a clinical study I showed for the first time that CKD patients lack the diurnal variation in arterial stiffness that is seen in matched subjects without CKD. Alteration in the circadian variation of the ET-1 system may contribute to this. In summary, my studies have furthered our understanding of the role of ET-1 in CKD progression and the cardiovascular risk associated with it. Mϕ were shown to express both ET receptors and a novel mechanism of ET-1 clearance was observed in Mϕ. Using an in vivo model of AKI I was able to identify ETA receptor antagonism as a novel therapeutic agent in preventing the development of CKD caused by AKI where data are limited. Finally, alterations in the circadian rhythm of the cardiovascular system is emerging as an important factor in disease pathogenesis. Here the diurnal variation in arterial stiffness was described for the first time in a group of CKD patients and matched controls.

Parasitic helminths infect millions of people and animals worldwide. A key feature of their lifecycle is the longevity of survival within a single host, which is often attributed to the ability of the parasite to divert or modulate the immune response against it. The excretory-secretory (ES) products released by helminths are of interest as the mediators of such immunomodulation. Heligmosomoides polygyrus is an excellent model of gastrointestinal (GI) helminth infection in rodents, and has been used here to investigate several aspects of the immune response, and the manipulation of these, in mice. Firstly, the roles of B cells and antibodies in infection with H. polygyrus and towards the adult ES (HES) were investigated. Using several B cell-deficient mouse strains, a minimal effect on immunity to primary infection with H. polygyrus was demonstrated. However, primary infection serum binds to a select set of highly immunodominant components of the complex protein mixture of HES, which were identified as venom allergen-like proteins (VALs). Utilising four strains of mice that vary in their resistance phenotype to H. polygyrus, several aspects of immunity towards the worm were investigated. Increased levels of markers of alternatively activated macrophages, which are a key component of the granulomatous inflammatory response around invading H. polygyrus larvae, were found in the most resistant strains, SJL and BALB/c. Depletion of macrophages, by administration of clodronate, severely disrupted the granuloma and parasite clearance. Numbers of innate lymphoid cells and the subsequent Th2 response, specificity range and titre of antibody, and activation of regulatory T cells all correlate with a resistant phenotype. A deficiency in the cytokine macrophage migration inhibitory factor (MIF) renders a resistant BALB/c mouse completely susceptible to infections with H. polygyrus, and Nippostronygylus brasiliensis, an acute model of GI helminth infection. This is accompanied by a failure to induce both ILCs and an early myeloid-derived cell population upon infection. The influx of alternatively activated macrophages around larvae in the mucosa of the small intestine is delayed in MIF-/- mice, although all immunological parameters are comparable to wild-type by day 14 post-infection. The susceptible phenotype of MIF-/- mice can be replicated using a chemical inhibitor of MIF in BALB/c mice. Finally, the previously documented transforming growth factor-β (TGF-β) activity of HES was dissected out further using two methods of fractionation. Distinct fractions with TGF-β activity were subjected to mass spectrometry to identify protein components that could be potential candidates for this activity.

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.

Macrophages are sentinels present in most tissues of the body, where they recognise and respond to biological dangers. Recognition and uptake of particles is mediated through phagocytic receptors which upon activation induce appropriate responses. These responses need to be tightly regulated in order to destroy pathogens but prevent uncontrolled inflammation. Phagocytosis is an evolutionarily conserved process required for host defence and homeostasis. During phagocytosis, particles are recognised by cell surface receptors that trigger rearrangement of the actin cytoskeleton and internalization of the bound particle into a de novo, membranous organelle known as the phagosome. Regulation of phagocytosis and phagosome maturation can be achieved through changes in transcription/translation and differential recruitment of proteins but also through their non-translational modifications. Here I explored the role of ubiquitylation in the phagosome biogenesis of Interferon-gamma (IFN-ɣ) activated macrophages. Ubiquitylation is a diverse, reversible post-translational modification which is not only involved in protein degradation but also in vesicle trafficking and immune signalling. My data shows that phagosomes are enriched in polyubiquitylation, which is further enhanced by IFN-ɣ. I applied a targeted AQUA peptide approach by which we quantified ubiquitin chain linkage peptides from phagosome samples by PRM. This data shows that all chain linkages apart from M1/linear chains are present on phagosomes. Furthermore, IFN-ɣ activation enhanced K11, K48 and K63 chains significantly. In order to identify the molecular function of this polyubiquitylation, I characterized the ubiquitinome of phagosomes of IFN-γ activated macrophages and can demonstrate that ubiquitylation is preferentially attached to proteins involved in vesicle trafficking, thereby delaying fusion with late endosomes and lysosomes. I demonstrated that most ubiquitin chains are on the cytoplasmic site of the phagosome enabling an interaction of ubiquitin chains with cytosolic proteins such as Rab7. Rab7 a major regulator of vesicle trafficking could be shown to be ubiquitylated on phagosomes. I further showed that phagosomal recruitment of the E3 ligase RNF115 is enhanced upon IFN-γ stimulation and RNF115 is responsible for most of the increase of K63 polyubiquitylation of phagosomal proteins. Knock-down of RNF115 promotes phagosome maturation and induces an increased pro-inflammatory response to Toll-like receptor (TLR) agonists, indicating that RNF115 is a negative regulator of vesicular trafficking to the lysosome and disruption of this pathway induces pro-inflammatory responses in macrophages. In conclusion, this is the first study showing unbiasedly that ubiquitylation plays an important role in vesicle trafficking to the lysosome.

Guo, Cuixia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 75-85). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Abstract --- p.iii / 摘要 --- p.iv / Acknowledgment --- p.v / List of Tables --- p.vi / List of Figures --- p.vii / List of Abbreviations --- p.viii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction to immune system --- p.1 / Chapter 1.2 --- Immune effecter cells --- p.1 / Chapter 1.2.1 --- Macrophage --- p.1 / Chapter 1.2.2 --- Dendritic Cells (DCs) --- p.5 / Chapter 1.3 --- Immunomodulatory and antitumor activities of mushrooms --- p.8 / Chapter 1.3.1 --- Introduction to mushroom --- p.11 / Chapter 1.3.2 --- Mushroom polysaccharides --- p.11 / Chapter 1.3.3 --- Mushroom β-glucan --- p.14 / Chapter 1.4 --- The receptors for polysaccharides associated with immune effecter cells --- p.16 / Chapter 1.4.1 --- CR3 --- p.16 / Chapter 1.4.2 --- Dectin-1 --- p.18 / Chapter 1.4.3 --- TLR2 --- p.19 / Chapter 1.5 --- Nuclear factor-kappa B (NF-kB) activation --- p.19 / Chapter 1.6 --- Previous studies on mushroom sclerotium --- p.20 / Chapter 1.6.1 --- Pleurotus tuber-regium (PT) --- p.20 / Chapter 1.6.2 --- Polyporus rhinocerus (PR) --- p.21 / Chapter 1.7 --- Objectives --- p.21 / Chapter 2. --- Materials and Methods --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Mushroom sclerotia --- p.23 / Chapter 2.1.2 --- Animal --- p.23 / Chapter 2.1.3 --- Cell lines --- p.24 / Chapter 2.2 --- Methods --- p.24 / Chapter 2.2.1 --- Hot water extraction --- p.24 / Chapter 2.2.2 --- Measurement of monosaccharide profile --- p.25 / Chapter 2.2.2.1 --- Acid depolymerization --- p.25 / Chapter 2.2.2.2 --- Neutral sugar derivatization --- p.25 / Chapter 2.2.2.3 --- Gas chromatography (GC) --- p.26 / Chapter 2.2.3 --- Determination of molecular weight by size exclusion chromatography (SEC) --- p.27 / Chapter 2.2.4 --- Determination of total sugar by phenol-sulfuric acid method --- p.28 / Chapter 2.2.5 --- Determination of protein content by Lowry-Folin method --- p.28 / Chapter 2.2.6 --- Detection of endotoxin --- p.29 / Chapter 2.2.7 --- Immunomodulatory activities induced in RAW264.7 cell line and murine peritoneal macrophages (PMs) --- p.30 / Chapter 2.2.7.1 --- Isolation of murine peritoneal macrophages (PMs) --- p.30 / Chapter 2.2.7.2 --- Detection of cell surface antigens on RAW 264.7 cells and PMs --- p.30 / Chapter 2.2.7.3 --- Phagocytic uptake --- p.31 / Chapter 2.2.7.4 --- Reactive Oxygen Species (ROS) generation --- p.32 / Chapter 2.2.7.5 --- Nitric Oxide (NO) production --- p.32 / Chapter 2.2.7.6 --- Inducible Nitric Oxide Synthase (iNOS) expression --- p.32 / Chapter 2.2.7.6.1 --- Cell lysates preparation --- p.33 / Chapter 2.2.7.6.2 --- Determination of protein concentrations --- p.33 / Chapter 2.2.7.6.3 --- Western blot --- p.34 / Chapter 2.2.7.7 --- Tumor Necrosis Factor-alpha (TNF-α) production --- p.36 / Chapter 2.2.8 --- DC cell marker determination --- p.37 / Chapter 2.2.9 --- Nuclear factor kappa B (NF-kB) activation --- p.37 / Chapter 2.2.10 --- Determination of the expression of existing cell surface β-glucan receptors --- p.37 / Chapter 2.2.11 --- Statistical methods --- p.38 / Chapter 3. --- Results --- p.39 / Chapter 3.1 --- Yield and chemical composition of mushroom sclerotial polysaccharides --- p.39 / Chapter 3.2 --- Endotoxin examination --- p.41 / Chapter 3.3 --- Monosaccharide profiles of PTW and PRW by GC --- p.41 / Chapter 3.4 --- Molecular weight profile by size exclusion chromatography (SEC) --- p.43 / Chapter 3.5 --- Immunomodulatory activities induced in RAW264.7 cells and murine peritoneal macrophages (PMs) --- p.46 / Chapter 3.5.1 --- Detection of cell surface antigens on RAW 264.7 cells and PMs --- p.46 / Chapter 3.5.2 --- Phagocytic uptake --- p.49 / Chapter 3.5.3 --- ROS generation --- p.53 / Chapter 3.5.4 --- NO production --- p.56 / Chapter 3.5.5 --- iNOS expression --- p.59 / Chapter 3.5.6 --- TNF-α production --- p.60 / Chapter 3.5.7 --- Morphological changes of cells --- p.62 / Chapter 3.5.8 --- DC cell marker determination --- p.64 / Chapter 3.6 --- Receptors expression on RAW 264.7 cells and PMs --- p.66 / Chapter 3.7 --- NF-kB activation --- p.68 / Chapter 3.8 --- Discussion --- p.70 / Chapter 4. --- Conclusions and Future Works --- p.73 / Chapter 5. --- References --- p.75

Padrões moleculares associados à patógenos (PAMPs) e citocinas pró-inflamatórias ativam o Eixo Imune-Pineal promovendo uma alternância temporária da síntese de melatonina da glândula pineal para células imuno-competentes. Esta regulação é mediada pela ativação do fator de transcrição factor nuclear kappa B (NF-&kappa;B), que de acordo com contexto fisiológico controla a transcrição do gene que codifica a enzima-chave na síntese de melatonina (arilalquilamina-N-acetiltransferase, AA-NAT), e altera a função da melatonina de cronobiótica a reguladora de respostas inflamatórias. Nosso objetivo foi investigar se a poluição do ar, um importante fator inflamatório ambiental é capaz de acionar o Eixo Imune-Pineal. Investigamos o efeito de partículas concentradas finas (<2,5 &mu;m) na síntese de melatonina noturna em ratos, e o efeito de partículas isoladas derivadas diesel (DEP) na síntese de melatonina por células RAW 264.7 e macrófagos pulmonares. A exposição ao PM aumentou a translocação nuclear de NF-&kappa;B e reduziu a expressão pineal de AA-NAT e a concentração de melatonina plasma. Em contraste, a DEP leva a uma translocação transitória de NF-&kappa;B em macrófagos resultando na expressão de AA-NAT e a síntese da melatonina. Nós confirmamos que a ativação de elementos kappa B no gene Aa-nat foi responsável por sua transcrição. Em conclusão, a poluição do ar, o que pode ser considerado um agente de sinalização de perigo, ativa o eixo imune-pineal, reduzindo a síntese da melatonina pineal noturno, e ativando a produção de melatonina por macrófagos. / Pathogen-associated molecular patterns (PAMPs) and pró-inflammatory cytokines activate the Immune-Pineal Axis promoting a temporary shift of melatonin synthesis from the pineal gland to immune-competent cells .This shift is mediated by the activation of the transcription factor nuclear factor kappa B (NF-&kappa;B ), which according to the cell milieu inhibits or induces the transcription of the gene that codifies the key enzyme in melatonin synthesis (arylalkylamine-N-acetyltransferase, AA-NAT), and alters melatonin function from chronobiotic to a regulator of inflammatory responses Our aim is to investigate whether air pollution, an important environmental danger-associated molecular pattern, triggers the immune-pineal axis. We investigated the effect of urban fine concentrated particulate matter (PM2.5, <2.5 mm) on the nocturnal melatonin synthesis in rats, and the effect of isolated derived diesel particles (DEP) on melatonin synthesis by RAW 264.7 cells, a model for studying macrophages. PM exposition increased nuclear translocation of NF-&kappa;B and reduced the pineal expression of AA-NAT and plasma melatonin concentration. In contrast, DEP leads to a transient translocation of NF-&kappa;B in RAW 274.6 cells resulting in the expression of AA-NAT and the synthesis of melatonin. We confirmed that the activation of &kappa;B elements in Aa-nat gene was responsible for its transcription. In conclusion, air pollution, which can be considered a danger-signalizing agent, activates the immune-pineal axis, reducing nocturnal pineal melatonin synthesis, and activating the production of melatonin by macrophages.

Thèse (Ph. D.)--Université Laval, 2005. / Titre de l'écran-titre (visionné le 28 mars 2007). Bibliogr.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.