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Conventional cancer therapies such as chemotherapy and radiation often lead to severe side effects since they are unable to specifically target the tumor. Additionally, they do not guarantee the prevention of metastasis or recurrence. Recent developments on small-molecule inhibitors, such as kinase inhibitors that target cellular pathways characteristically upregulated in cancer cells, show promise. However, significant challenges such as tolerance and mutations causing drug resistance need to be overcome. Immunotherapy, wherein the host's immune system is leveraged to recognize and target cancer cells, is a better alternative that shows reduced toxicity. Macrophages are an attractive target for immunotherapy seeing as they constitute 50% of the infiltrating leukocytes in the tumor microenvironment. Their plastic nature allows them to be modulated from pro-tumor to anti-tumor phenotype. Although, it does not work for everyone, necessitating a need to monitor response to medication at earlier time points. In this thesis, I have designed an HDL mimicking nanoparticle system to target tumor associated M2 macrophages through the SRB1 receptor. The nanoparticle was optimized for better stability, better loading of the targeting peptide, and the drug as well. It was used to deliver a CSF1R inhibitor drug to successfully repolarize pro-tumor M2 macrophages to anti-tumor M1 phenotype. In addition to that, it was also used to deliver an Arginase-responsive probe that only fluoresces when engulfed by arginase-producing M2 macrophages, differentiating them from arginase non-producing M1 phenotype. Through this study, the SRB1 receptor was successfully targeted to effectively deliver small molecules. This can be used to target and modulate tumor-associated macrophages.

A functional immune system is a potential barrier to tumor growth and progression. Cancer is caused, in part, by the loss of immune surveillance leading to the inability of the immune system to destroy the cancer cells. Macrophages (Mfs) are essential cellular components of the immune system; they influence immune responses in diverse and fundamental ways. As a consequence, Mfs present targets for tumors to evade, thereby enhancing tumor survival and growth. An interaction between CD40 on Mfs and CD40L on T cells is required for cell-mediated inflammatory responses. The CD40/CD40L interaction is bi-directional; suppressed expression of either protein by the tumor will prevent activation of both Mfs and T cells. We showed that tumor growth suppresses T-cell CD40L expression. Decreased CD40L expression disrupted Mf activation pathways, leading to impaired production of immunostimulatory cytokines, interleukin (IL)-12 and IL-18 by tumor-bearing host (TBH) Mfs. Disruption of CD40L expression, via dysregulation of IL-12 and IL-18 production, impeded T-cell interferon (IFN)-g production, which in turn exacerbated Mf dysfunction. We showed that IFN-g induced interferon consensus sequence binding protein (ICSBP) expression is impaired in TBH Mfs due to tumor cell-derived TGF-b and, to a lesser extent, IL-10. ICSBP induces CD40L, IL-12, and IL-18 expression. Disruption of the CD40/CD40L interaction via lowered CD40L expression generates an immunosuppressive loop that may be a strategy for tumor survival and growth. This was demonstrated by impaired cytotoxicity; via impaired tumor necrosis factor (TNF)-a and nitric oxide (NO) production by TBH Mfs against Meth-KDE tumor cells. Collectively, these studies show that multiple antitumor mechanisms could be enhanced by restoration of CD40L expression. / Master of Science

Titre de l'écran-titre (visionné le 29 février 2016). / L'asthme allergique est le résultat d'altérations de plusieurs mécanismes immunorégulateurs. Le maintien de l'homéostasie est assuré principalement par les cellules de première défense, comme les macrophages alvéolaires (MA). Pour empêcher les réponses inflammatoires exagérées, ces cellules expriment des molécules anti-inflammatoires, comme le CD200. Cette molécule est exprimée plus fortement à la surface des MA de rats naïfs suite à l'exposition à un allergène. Le CD200 agit sur les cellules myéloïdes, notamment les mastocytes qui jouent un rôle important dans l'asthme allergique. En somme, l'objectif principal de cette étude est de caractériser l'effet immunomodulateur de l'expression du CD200 à la surface des MA sur les fonctions des mastocytes. Une série d'expériences in vitro de coculture mastocytes-MA permet de conclure que le relâchement de médiateurs préformés, leucotriènes et cytokines produites de novo sont modulés par le CD200 des MA. Ces résultats suggèrent que le CD200 est une cible thérapeutique intéressante dans l'asthme. / Allergic asthma may be caused by alterations of many immunoregulatory mechanisms. Alveolar macrophages (AM) are central regulators of pulmonary immune responses. They express anti-inflammatory molecules, such as CD200, to avoid exaggerated immune responses. CD200 is upregulated on AM following allergen challenge in naïve rats. CD200 interacts with its receptor only expressed on myeloid cells, including mast cells that are central effector cells in allergic asthma. Thus, we investigated modulation of mast cell functions by AM CD200. Antigen-stimulated mast cells were investigated in vitro using co-culture and mast cell degranulation, lipid mediator production, and cytokine release were measured. Results suggest that inhibition of antigen-stimulated mast cells by CD200 expression on AM has an important role in asthma regulation.

Le macrophage constitue l’un des mécanismes de défense cellulaire contre les pathogènes. Le rôle principal du macrophage est l’initiation ainsi que la résolution de l’inflammation lors d’infection. Notre laboratoire s’intéresse au CD200, une molécule anti-inflammatoire présente sur les macrophages. Certaines études ont montré que le CD200 est impliqué dans la résolution de l’inflammation. Notre hypothèse était qu’une absence de CD200 augmente les fonctions inflammatoires des macrophages dérivés de la moelle osseuse (BMDMs) ce qui pourrait être causé par une modulation de la différenciation des BMDMs. Pour ce faire, nous avons utilisé des BMDMs différenciés avec un facteur de stimulation des colonies de granulocytes et de macrophages (GM-CSF) pendant 9 jours à partir de moelle osseuse de rats Sprague Dawley déficients pour le CD200 (CD200 KO) (générés par notre équipe). L’impact de l’absence du CD200 sur la différenciation et l’activation des BMDMs par stimulation au lipopolyssaccharide (LPS) a été étudié. L’expression de molécules présentatrices d’antigènes a été mesurée par cytométrie en flux, la sécrétion de cytokines par ELISA et la phagocytose par essai fluorométrique avec Pseudomonas aeruginosa. Au cours de la différenciation, les BMDMs augmentent l’expression de CD200 et CD200R. L’absence du CD200 altère l’expression des molécules associées à la présentation d’antigènes (MHCII, CD80 et CD86) lors de la différentiation. Par contre, l’absence du CD200 n’affecte pas la phagocytose. De plus, l’absence du CD200 diminue la sécrétion de cytokines suite à une stimulation au LPS. Ces résultats sont associés à une diminution de la surexpression du Toll-like receptor 4(TLR-4), le récepteur du LPS. En conclusion, l’absence de CD200 module la différenciation des BMDMs in vitro et,contrairement à notre hypothèse, l’absence du CD200 sur les BMDMs limite l’expression des molécules présentatrices d’antigènes et la sécrétion de cytokines. Les mécanismes de réponse aux pathogènes que l’absence du CD200 affecte sont encore inconnus, mais pourraient impliquer la voie du TLR-4. / The first line of cell defense against pathogens is the macrophages. The main function of this cell is to be able to maintain immune homeostasis. The CD200 is an anti-inflammatory molecule expressed by on many cell types, including macrophages. Some studies have shown that the CD200-CD200R pathway is involved in the resolution of inflammation. Our hypothesis was that the absence of CD200 on bone marrow derived macrophages (BMDMs) will change their differentiation pattern and increase their inflammatory functions. BMDMs were differentiated from Sprague Dawley rat’s bone marrow with GM-CSF for 9 days, and the expression of CD200 and CD200R was measured by flow cytometry. The inflammatory response of BMDMs from wild type and CD200 knock out rats (generated by our team) was measured after LPS stimulation. The expression of molecules associated with antigen presentation was quantified by flow cytometry. Cytokine secretion was measured by ELISA and phagocytosis activity was measured by a fluorescence assay with Pseudomonas aeruginosa. BMDMs increased CD200 and CD200R expression during the differentiation process. The absence of CD200 altered the expression of antigen presentation molecules (MHCII, CD80 and CD86) during the differentiation, but did not modulate the phagocytosis function of BMDMs. In addition, CD200 KO BMDMs secreted lower levels of proinflammatory and anti-inflammatory cytokines following LPS exposition. These results are associated with a reduction of TLR-4 overexpression, the LPS receptor. In conclusion, CD200 deficiency modulates the differentiation process of BMDMs and limits the expression of markers associated with pathogen-presentation and cytokines secretion. The implication of CD200 absence in pathogens response mechanism is still unknow but could include markers from the TLR-4 pathway.

Escherichia coli enteropatogênica (EPEC) é um dos principais agentes etiológicos da diarreia infantil tanto em países desenvolvidos como em países em desenvolvimento. Esta bactéria possui dois fatores de virulência comprovadamente envolvidos na patogênese, intimina e bundle-forming pilus (BFP). Este patotipo está dividido em EPEC típica e EPEC atípica, ambos apresentam uma ilha de patogenicidade cromossomal denominada locus of enterocyte effacement (região LEE) onde está localizado o gene eae (E. coli attachment effacement), que codifica a intimina, uma proteína de membrana externa que medeia a adesão íntima da bactéria ao enterócito. Diferente da EPEC típica, as cepas de EPEC atípica não possuem o plasmídeo EAF (EPEC adherence factor) no qual encontra-se o operon bfp (bundle forming pilus) constituído por 14 genes incluindo bfpA o qual codifica a bundlina (BfpA), principal subunidade da fímbria Bfp, que possibilita a agregação bacteriana. Na infecção por EPEC ocorre grave disfunção da barreira epitelial, e uma das conseqüências é a inflamação. Na literatura, é bem descrito a interação entre as proteínas efetoras de EPEC com as células epiteliais e os processos iniciais da interação bactéria à célula hospedeira. Entretanto, poucos são os estudos que analisam a produção de citocinas em infecções por EPEC ou suas moléculas efetoras com relação a ativação de macrófagos, fundamentais para o controle do processo inflamatório e geração da resposta imune durante esta infecção. A análise das citocinas produzidas constitui uma parte importante da resposta imune e representa a tentativa do hospedeiro em lidar com um determinado microrganismo. Em função disto analisou-se o papel da intimina e do BfpA na capacidade de ativar a resposta inata mediada por macrófagos in vitro, onde avaliou-se a produção de citocinas pró-inflamatórias (TNF-α, IL-1, IL-6 e IL-12), citocina antiinflamatória (IL-10) e quimiocina (MCP-1). Os resultados demonstraram que as proteínas recombinantes intimina e BfpA são potentes ativadores de macrófagos, de forma dose dependente, produzindo TNF-α, IL-12 e IL-6, IL-10 e MCP-1, mas não IL-1β. Neste estudo não foi observado efeito sinérgico na produção de citocinas pró-inflamatórias ao associar intimina e BfpA, entretanto em dose mais elevada potenciou a produção de IL-10, um mediador antiinflamatório. O efeito imune obtido foi atribuído majoritariamente a estas proteínas uma vez que o tratamento destas com polimixina B não alterou a produção de TNF-α. Conclui-se que intimina e BfpA são potentes ativadores de macrófagos durante a resposta inata podendo colaborar para o controle do processo inflamatório durante a infecção por EPEC. / Enteropathogenic Escherichia coli (EPEC) is a common cause of childhood diarrhea in developed countries as well as developing countries. This bacterium has two proven virulence factors involved in pathogenesis, intimin and bundle-forming pilus (BFP). This pathotype EPEC is divided into typical and atypical EPEC, both having a chromosomal pathogenicity island called locus of enterocyte effacernent (LEE region) which contains the gene eae (E. coli attachment effacement). eae encodes intimin, an outer membrane protein that mediates the intimate adherence of bacteria to the enterocyte. Unlike typical EPEC, atypical EPEC strains do not possess the plasmid EAF (EPEC adherence factor) which is in the operon bfp (bundle forming pilus) consisting of 14 genes including bfpA, which encodes bundlin (BfpA), the main subunit of BFP allowing bacterial aggregation. EPEC infection occurs in severe dysfunction of the epithelial barrier, and one consequence is inflammation. In the literature, the interaction between effector proteins of EPEC and epithelial cells and the initial processes of bacterial interaction with the host cell are well described. However, there are few studies that have examined cytokine production in EPEC infections or their effector molecules with respect to macrophage activation, essential for controlling inflammation and immune response during this infection. The production of cytokines is an important part of the immune response and represents the host\'s attempt to deal with a particular microorganism. Therefore, we examined in vitro the role of intimin and BfpA in the ability to activate the innate response mediated by macrophages, where we analyzed the production of the proinflammatory cytokines TNF-α, IL-1, IL-6 and IL-12, and the antiinflammatory cytokine IL-10 and chemokine MCP-1. The results show that recombinant intimin and BfpA are potent activators of macrophages in a dose-dependent manner, where the stimulated cells produce TNF-α, IL-12 and IL-6, IL-10 and MCP-1, but not IL-1β. In this study, no synergistic effect was observed in the production of proinflammatory cytokines by combining BfpA and intimin, although production of IL-10, an antiinflammatory mediator, was potentiated at a higher dose. The effect obtained was largely attributed to these proteins, as the treatment of proteins with polymyxin B did not alter the production of TNF-α. We conclude that intimin and BfpA are potent activators of macrophages during the innate response and may contribute to the control of inflammation during infection with EPEC.

Cheng, Siu Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 121-141). / Abstracts in English and Chinese. / Abstract (English) --- p.ii / (Chinese) --- p.V / Acknowledgements --- p.viii / List of Abbreviations --- p.x / Table of Contents --- p.xi / Chapter Chapter 1 --- Introduction and Literature Reviews --- p.1 / Chapter 1.1 --- Bacterial Infection in Neonates --- p.1 / Chapter 1.1.1 --- Overview of Bacterial Infection in Neonates --- p.1 / Chapter 1.1.2 --- Gram-Positive Bacteria and Infection in Newborns --- p.2 / Chapter 1.1.3 --- Lipoteichoic Acid - the Immunostumulatory Component of Gram-Positive Bacteria --- p.5 / Chapter Figure 1.1 --- Schematic Representation of Gram-Positive Bacterial Cell Wall --- p.8 / Chapter Figure 1.2 --- Structure of Lipoteichoic Acid Polymer --- p.9 / Chapter 1.2 --- The Immune System of the Neonate --- p.10 / Chapter 1.2.1 --- Overview of the Human Immune System --- p.10 / Chapter 1.2.2 --- Innate Immune System --- p.10 / Chapter 1.2.3 --- Adaptive immune system --- p.13 / Chapter 1.3 --- Macrophages --- p.15 / Chapter 1.3.1 --- Overview of Macrophages --- p.15 / Chapter 1.3.2 --- Functions of Macrophages --- p.16 / Chapter 1.3.3 --- Macrophages in Gram-Positive Bacterial Infection --- p.19 / Chapter 1.4 --- Natural Killer Cells --- p.21 / Chapter 1.4.1 --- Overview of Natural Killer Cells --- p.21 / Chapter 1.4.2 --- Activation of Natural Killer Cells --- p.23 / Chapter 1.4.3 --- Functions of Natural Killer Cells --- p.24 / Chapter 1.4.4 --- Activation Markers on Natural Killer Cell Surface --- p.27 / Chapter Figure 1.3 --- Schematic Diagram of CD 107a Expression in NK Cells During Degranulation --- p.30 / Chapter 1.5 --- Interactions Between Macrophages and Natural Killer Cells --- p.31 / Chapter 1.6 --- Mitogen-Activated Protein Kinases (MAPKs) and Infection --- p.32 / Chapter 1.7 --- ApolipoproteinA-1 (ApoA-1) and Infection --- p.34 / Chapter 1.8 --- Overall Objectives of the Study --- p.36 / Chapter Chapter 2 --- Materials and Methods --- p.37 / Chapter 2.1 --- Reagents Used --- p.37 / Chapter 2.2 --- Sampling Methods --- p.41 / Chapter 2.3 --- Mononuclear Cell Isolation --- p.41 / Chapter 2.4 --- Mononuclear Cell Cryopreservation --- p.42 / Chapter 2.5 --- Induction of Macrophage from MNC culture --- p.42 / Chapter 2.6 --- Enrichment of Natural Killer (NK) Cells from MNC --- p.43 / Chapter 2.7 --- Culture of NK Cells Using Conditioned Medium Collected From LTA-Stimulated Macrophages --- p.45 / Chapter 2.8 --- Western Blotting --- p.46 / Chapter 2.9 --- Cytokines in Culture Supernatant of Macrophages --- p.50 / Chapter 2.10 --- CD107a Assay --- p.51 / Chapter 2.11 --- CD69 Assay --- p.52 / Chapter 2.12 --- Statistical Analysis --- p.53 / Chapter Figure 2.1 --- A Representative Diagram to Show NK Cell Purity After Enrichment by Immuno-Magnetic Beads --- p.55 / Chapter Figure 2.2 --- A Diagram to Illustrate the Gating Method Used in CD 107a Assay --- p.56 / Chapter Figure 2.3 --- A Diagram to Illustrate the Gating Method Used in CD69 Assay --- p.58 / Chapter Chapter 3 --- Lipoteichoic Acid - Induced Inflammatory Responses of Cord Blood Macrophages in vitro --- p.59 / Chapter 3.1 --- LTA-Stimulated Secretion of Tumor Necrosis Factor-Alpha in Cord Blood Macrophages --- p.60 / Chapter 3.2 --- LTA-Stimulated Secretion of Interleukin-6 in Cord Blood Macrophages --- p.61 / Chapter 3.3 --- LTA-Stimulated Secretion of Interleukin-12 in Cord Blood Macrophages --- p.62 / Chapter 3.4 --- LTA-Stimulated Phosphorylation of P44/42 Mitogen-Activated Protein Kinase (ERK1/2) in Cord Blood Macrophages --- p.63 / Chapter 3.5 --- The Effect of Apolipoprotein A-l Pre-Treatment on LTA-Stimulated P44/42 Mitogen-Activated Protein Kinase (Erkl/2) Phosphorylation --- p.64 / Chapter 3.6 --- Discussion --- p.65 / Chapter Figure 3.1 --- LTA-Induced TNFa Secretion by Cord Blood Macrophages --- p.73 / Chapter Figure 3.2 --- LTA-Induced IL-6 Secretion by Cord Blood Macrophages --- p.74 / Chapter Figure 3.3 --- LTA-Induced IL-12 Secretion by Cord Blood Macrophages --- p.75 / Chapter Figure 3.4 --- Phosphorylation of P44/42 MAPK (ERK1/2) Stimulated by LTA in Cord Blood Macrophages --- p.76 / Chapter Figure 3.5 --- The Effect of ApolipoproteinA-1 on LTA-Stimulated P44/42 MAPK Phosphorylation --- p.78 / Chapter Chapter 4 --- Lipoteichoic Acid - Induced Inflammatory Responses of Natural Killer Cells in vitro --- p.80 / Chapter 4.1 --- Dose-Dependent Effect of IL-15 on CD69 Expression --- p.80 / Chapter 4.2 --- Effects of LTA on CD69 Surface Expression in NK Cells --- p.82 / Chapter 4.3 --- Effects of LTA on CD 107a Surface Expression in NK Cells --- p.84 / Chapter 4.4 --- Discussion --- p.87 / Chapter Figure 4.1 --- Dose-Dependent Effect of IL-15 on Cord Blood NK Cells CD69 Surface Expression --- p.93 / Chapter Figure 4.2 --- Effects of LTA and IL-15 on CD69 surface Expression in NK Cells --- p.95 / Chapter Figure 4.3 --- Effects of LTA and IL-15 on CD 107a surface Expression in NK Cells --- p.97 / Chapter Chapter 5 --- Effects of LTA-Primed Macrophage-Conditioned Medium on Autologous Natural Killer Cell Activation --- p.99 / Chapter 5.1 --- Effects of Macrophage-Conditioned Medium on Autologous NK Cell CD 107a Expression --- p.100 / Chapter 5.2 --- Effects of LTA-Stimulated Macrophages on Autologous NK cells CD69 Expression --- p.102 / Chapter 5.3 --- Discussion --- p.105 / Chapter Figure 5.1 --- Flow Chart of the Experiment Design --- p.110 / Chapter Figure 5.2 --- Effects of Macrophage-Conditioned Medium on Autologous NK CD 107a Expression --- p.111 / Chapter Figure 5.3 --- Effects of Macrophage-Conditioned Medium on Autologous NK CD69 Expression --- p.113 / Chapter Chapter 6 --- Conclusion and General Discussion --- p.115 / Chapter 6.1 --- Conclusion --- p.115 / Chapter 6.2 --- Potential Applications of the Findings --- p.117 / Chapter 6.3 --- Limitations --- p.118 / Chapter 6.4 --- Further Studies --- p.119 / References --- p.121

La fibrose radio-induite (FRI) est une complication tardive de la radiothérapie souvent associée à une réponse inflammatoire chronique et à un infiltrat de macrophages. Aujourd’hui, les macrophages sont pressentis comme des médiateurs cellulaires important dans le processus de fibrose mais leur rôle n’a jamais été étudié dans le contexte de la FRI. Dans une précédente étude nous avions montré que l’irradiation (IR) induit une polarisation M1 des macrophages cardiaques après irradiation de souris ApoE-/- et est associée un score de fibrose élevé, ce qui suggérait que la polarisation des macrophages pourrait contribuer à la fibrogénèse radio-induite. Afin de valider cette hypothèse, nous avons cherché à caractériser le rôle des macrophages dans la FRI en utilisant un modèle classique de fibrose pulmonaire chez la souris C57Bl/6 induit après IR thoracique à 16Gy. Nous avons caractérisé les phénotypes et la fonction des macrophages alvéolaires (MA) et interstitiels (MI). Durant la phase précoce, les résultats montrent une déplétion des MA accompagnée de la sécrétion de CXCL1, MCP-1 et de MCSF. Cette déplétion est suivie d’une repopulation suite au recrutement et à la prolifération des monocytes/macrophages d’origine médullaire. La nouvelle population de MA présente une polarisation hybride accompagnée d’une augmentation simultanée de la sécrétion de cytokines Th1 et Th2. Durant la phase tardive les MI présentent une polarisation de type M2 accompagnée d’une diminution des cytokines Th1 et d’une augmentation de cytokines Th2 dans le lysat tissulaire. Nous avons ensuite cherché à caractériser la contribution des MA hybrides vs MI M2 dans le processus de fibrose. Nous avons montré que contrairement au MA hybrides, les MI M2 étaient capables d’induire l’activation des fibroblastes in vitro et l’expression de TGF-β1. De plus, la déplétion des MA hybrides avec une administration intranasale de clodronate exacerbe la FRI et induit l’augmentation de l’infiltrat de MI M2. Ensuite, nous nous somme interrogés à la contribution du processus de fibrose dans la polarisation des macrophages. Après 24h de coculture entre fibroblastes irradiés et macrophages pulmonaires non irradiés, une sécrétion de cytokines telles que M-CSF et TIMP-1 qui pourraient stimuler l’activation des fibroblastes est observée. De plus, l’inhibition de la FRI avec de la pravastatine montre que l’inhibition de la fibrose est accompagnée d’une augmentation des MI M1 et d’une diminution des MI M2 dans le poumon. En résumé, nos résultats montrent une contribution opposée des Macrophages Alvéolaires et des Macrophages Interstitiels dans le processus de fibrose radio-induite ainsi qu’une contribution du processus de fibrose dans le type d’activation des Macrophages interstitiels formant ainsi une boucle d’activation fibrogénique chronique. / Radiation-induced fibrosis (RIF) is a delayed complication of radiotherapy often associated with chronic inflammatory process and macrophage infiltration. Nowadays, macrophages are suggested to be important cellular contributors to fibrogenic process, but their implication in the context of RIF has never been investigated. In a previous study we have shown that irradiation (IR) induced the polarization of cardiac macrophages into M1 in ApoE-/- mice and was associated with a high fibrosis score in ApoE-/- mice, suggesting that macrophage polarization could drive tissue sensitivity to ionizing radiation. This observation prompted us to investigate the role of macrophages in RIF using a classical experimental model of lung fibrosis developed in C57Bl/6 mice after 16Gy thorax-IR. We profiled both alveolar macrophages (AM) and interstitial macrophages (IM). During the acute phase we found AM depletion associated with CXCL1, MCP-1 and M-CSF secretion, followed by a repopulation phase mediated by recruitment and proliferation of monocytes/macrophages from the bone marrow. Interestingly, the newly recruited AM exhibited a yet never described hybrid polarization (M1/M2), associated with the up-regulation of both Th1 and Th2 cytokines. At delayed times points, IM were M2-polarized and associated with downregulation of Th1 cytokines and upregulation of Th2 cytokines in tissue lysates. These results suggest a differential contribution of hybrid AM vs M2 IM to fibrogenesis. Interestingly, in contrast to activated hybrid AM, activated M2 IM were able to induce fibroblast activation in vitro mediated by an enhanced TGF-β1 expression. Therefore, specific depletion of hybrid AM using intranasal administration of clodrosome increased RIF score and enhanced M2 IM infiltration. We next evaluated if the fibrogenic process can in turn affect macrophage polarization. Interestingly, after coculture of irradiated fibroblast with non-irradiated pulmonary macrophages, secretion of cytokines such as M-CSF and TIMP-1, which can stimulate macrophage activation, was observed. Furthermore, RIF inhibition using pravastatin treatment showed that fibrosis inhibition was associated with a decrease in M2 IM accompanied by an increase in M1 IM, but had no effect on polarization of AM. These present study shows a dual and opposite contribution of alevolar versus intertitial macrophages in RIF and the contribution of the fibrogenic process to IM polarization, resulting thereby in a chronical fibrogenic loop.

Wound repair is a complex series of events that begins immediately after wounding, and can continue for a number of months to years. Various physiological and mechanical factors may impair the healing response, resulting in a chronic wound, characterised by a sustained inflammatory response. One of the main cells involved in both the inflammatory phase and proliferation phase of wound healing is the macrophage. There are thought to be different activation states which allow the macrophage to be involved in the two different phases of wound healing, namely the classically activated macrophage and the alternatively activated macrophage. Changes in the number of classically activated/alternatively activated macrophages in the wound is likely to have an effect on wound healing. Therefore a more thorough understanding of macrophage activation states during wound healing would broaden the understanding of the role of this cell in this process. The overall aim of this project was to investigate whether diabetic bone marrow progenitor cells or macrophages respond to activation stimuli differently in comparison to wild type cells. The hypothesis of this thesis is that diabetic macrophages will not respond to appropriate stimuli and thus alternative and classical macrophages will not behave 'appropriately', resulting in impaired healing. The results of this thesis indicate that impaired wound healing in the diabetic environment may be due to both the diabetic wound environment itself and intrinsic differences in diabetic macrophages. This work indicates that signals from the wound environment activates and influences macrophages, as these cells do not express activation markers until they enter the wound environment. However, the culture system devised in this study indicates that even before activation with signals they would receive in the wound environment, diabetic cells are more pro-inflammatory and have impaired migration. In addition, these macrophages respond differently to activation supporting the hypothesis that the macrophages are intrinsically different and that diabetic cells do not behave 'appropriately' which could contribute to impaired wound healing.