L’inflammasome NLRP3, un complexe protéique pro-inflammatoire, joue un rôle essentiel dans le processus pathologique de l’athérosclérose et du diabète de type 2 (DT2). Il est responsable de la maturation de la pro-IL-1β et de la pro-IL-18 respectivement en IL-1β et IL-18 biologiquement actives. L’objectif de cette étude consiste à identifier et caractériser un inhibiteur spécifique de l’inflammasome NLRP3 qui pourrait contribuer à limiter l’évolution des plaques d’athérome et l’installation du DT2. Au cours de cette thèse, nous avons isolé l’Arglabine d’une plante, Artemisia glabella, connue pour ses vertus anti-tumorales. L’effet de l’Arglabine a été étudié au niveau des macrophages et des cellules β-pancréatiques, et chez des souris ApoE2.Ki et ApoE2.Ki/NLRP3-/- placées sous régime High Fat Diet (HFD). Les résultats in vitro montrent que l’Arglabine réduit, d’une façon dose-dépendante, l’activité de l’inflammasome NLRP3 et inhibe l’expression des protéines Nlrp3, IL-1β et caspase-1. Elle induit l’autophagie en augmentant significativement l’expression de la LC3-II au niveau des macrophages murins en culture. L’injection intra-péritonéale de deux doses journalières d’Arglabine (2.5 ng/g de m.c.) à des souris ApoE2.Ki placées sous régime HFD, normalise le profil lipidique et réduit l’oxydation des LDL au niveau du plasma des souris. Elle réduit le nombre des monocytes pro-inflammatoires (Ly-6Chigh) et augmente le nombre des monocytes anti-inflammatoires (Ly-6Clow). Au niveau des lésions artérielles, l’Arglabine oriente les macrophages présents vers un phénotype anti-inflammatoire M2. L’ensemble de ces résultats montre un rôle athéroprotecteur de l’Arglabine : elle réduit la surface des lésions artérielles au niveau du sinus aortique ainsi qu’au niveau de la totalité de l’aorte des souris ApoE2.Ki placées sous régime athérogène. De plus, le traitement par l’Arglabine normalise le profil glycémique et insulinémique des souris ApoE2.Ki. Elle réduit également l’activité de la caspase 3 au niveau des îlots de Langerhans et augmente de manière dose-dépendante l’expression de la protéine Bcl-2 au niveau des cellules β-pancréatiques. Par ailleurs, nous avons montré une augmentation de l’expression de protéines impliquées dans l’autophagie telles que la Becline 1 et la LC3-II sous l’effet de l’Arglabine. Ainsi, l’Arglabine réduit non seulement l’activité de l’inflammasome NLRP3 mais améliore aussi la survie des cellules β-pancréatiques. L’Arglabine constitue donc une molécule très prometteuse dans le traitement des maladies cardiovasculaires et le DT2. / The NLRP3 inflammasome activity is abnormally elevated in many human inflammatory diseases, including cardiovascular and metabolic diseases such as atherosclerosis and type 2 diabetes mellitus (T2DM) respectively. Therefore, there is considerable interest in the identification of effective therapeutics that selectively inhibit the NLRP3 inflammasome pathway. In this study, we have identified Arglabin as a potential small molecule inhibitor that targets the NLRP3 inflammasome activity in cell culture and in an animal model, the ApoE2.Ki mice fed a high-fat Western-type diet (HFD). Arglabin, a plant sesquiterpene lactone, has been used extensively as an herbal remedy that proved effective in treating cancer of the liver, lungs and breast at early stages. Arglabin inhibited, in a concentration-dependent manner, IL-1β and IL-18 production in lipopolysaccharide and cholesterol crystal-activated cultured mouse peritoneal macrophages. In addition, Arglabin activated autophagy as evidenced by the increase in LC3-II protein. Intraperitoneal injection of Arglabin (2.5 ng/g body weight twice daily for 13 weeks) into female ApoE2.Ki mice fed a HFD resulted in a decreased IL-1β plasma level and reduced plasma levels of total cholesterol and triglycerides. Treatment of ApoE2.Ki mice fed a HFD with Arglabin significantly reduced the plasma concentration of anti-oxLDL antibodies. Moreover, Arglabin oriented the proinflammatory M1 macrophages into the anti-inflammatory M2 phenotype in spleen and arterial lesions. Consequently, a marked reduction in atherosclerotic lesions was observed in the median areas in the sinus and whole aorta. In comparison to vehicle-treated mice, Arglabin reduced plasma levels of glucose and insulin. Immunohistochemical analysis revealed the presence of active caspase 3 in Langerhans islets of ApoE2.Ki mice fed a HFD that was significantly inhibited by Arglabin treatment. Moreover, Arglabin reduced susceptibility to apoptosis in cultured INS-1 cells by increasing concentration-dependently Bcl-2 levels, which led to concomitantly decreased Bax/Bcl-2 ratio. In cultured INS-1 cells, Arglabin increased the expression of the autophagic markers Becline 1 and LC3-II in a concentration-dependent manner. Consequently, our results indicate survival-promoting properties of the Arglabin molecule in pancreatic β-cells.In conclusion, our findings demonstrate that Arglabin may represent a promising new drug to treat atherosclerosis and T2DM.

Le Sepsis et le choc septique, associés à une inflammation systémique sévère et incontrôlée, sont les principales causes de mortalité dans les unités de soins intensifs. Les macrophages jouent un rôle central dans ces pathologies. Ils participent à l'initiation et à la régulation de l'inflammation. Lors d'une infection bactérienne, ils reconnaissent le LPS de la paroi bactérienne par l’intermédiaire du TLR4 ce qui déclenche l’activation des MAPK, des facteurs de transcription NF-kB et AP1 et, in fine, la production des cytokines pro-inflammatoires dont le TNF et l'IL6. L’expression de la protéine GILZ dans les macrophages limite, in vitro, la production d'IL6 et de TNF en réponse au LPS. Cet effet est attribué à une inactivation de NF-kB. D’autre part, l'expression de GILZ décroît dans les macrophages humains et murins après une stimulation par du LPS.Compte tenu des effets régulateurs de GILZ dans les macrophages, les objectifs de notre étude sont 1) de déterminer si l’expression de GILZ est altérée dans les monocytes/macrophages (M/M) au cours du Sepsis, 2) de déterminer si une modulation de l’expression de GILZ dans les M/M est suffisante pour influencer l’inflammation systémique, et 3) d’identifier le mécanisme d'action de GILZ dans les M/M humains.Nous avons mesuré l’expression de GILZ dans les M/M de patients atteints de choc septique ou de syndrome de détresse respiratoire aiguë, et dans un modèle murin d’endotoxémie. Nous avons observé une diminution significative de GILZ dans ces contextes pathologiques chez l’homme et la souris. L'impact de cette altération a été exploré dans des souris transgéniques uniques dont les macrophages surexpriment GILZ de façon non régulable. Nous avons confirmé que la surexpression de GILZ limite la production de TNF et favorise celle de l'IL-10 dans les macrophages stimulés in vitro par du LPS. Nous avons ensuite étudié la réponse inflammatoire et la survie de ces souris dans un modèle d’endotoxémie et de choc septique, et montré que cette surexpression de GILZ restreinte aux macrophages limite la production sérique de cytokines pro-inflammatoires et, par conséquent, l'inflammation systémique en améliorant significativement la survie des souris. Ces résultats mettent en évidence les conséquences, au niveau systémique, de la régulation des macrophages par GILZ.Dans l’optique d’élucider les mécanismes impliqués dans la régulation des macrophages par GILZ, nous avons confirmé que GILZ inhibe NF-kB dans les macrophages humains sans toutefois retrouver l’interaction directe décrite entre GILZ murin et la sous-unité p65 de NF-kB.Ce résultat nous a conforté dans la nécessité de caractériser l’interactome de GILZ dans les macrophages humains. Deux approches complémentaires ont été utilisées. La première est un criblage pan-génomique des interactants de GILZ humain par la technique du double-hybride. La seconde méthode consiste en une purification d'affinité en tandem (TAP-TAG) de la protéine GILZ et de ses interactants, suivie d'une identification de ces protéines par spectrométrie de masse. Ce complexe a été isolé à partir d'extraits nucléaires ou cytoplasmiques de cellules humaines différenciées en macrophages et génétiquement modifiées afin d’exprimer la protéine GILZ flanquée des deux étiquettes nécessaires à sa purification. Ces deux approches ont mis en évidence des interactions nouvelles entre GILZ et des protéines clés de la signalisation du TLR4 dans les macrophages humains ainsi qu'un rôle probable de GILZ comme facteur régulateur de la transcription.Ces résultats montrent que la régulation de la réponse anti-inflammatoire des macrophages par GILZ a un impact sur l’inflammation systémique in vivo et améliore la survie dans un modèle de choc septique sévère. De plus, ces travaux identifient pour la première fois les partenaires cytoplasmiques et nucléaires de GILZ dans les macrophages humains et devraient permettre dans le futur, une meilleure compréhension de cette protéine. / Sepsis and septic shock, associated with a severe and uncontrolled systemic inflammation, are the main causes of death in intensive care units. Macrophages play a central role in these pathologies. They are involved in the initiation and regulation of inflammation. They recognize LPS from the bacterial cell wall via TLR4, which triggers the activation of MAPK signaling pathway and transcription factors such as NF-KB and AP1 and ultimately, the production of pro-inflammatory cytokines including TNF and IL6. The expression of the protein GILZ in macrophages limits in vitro the production of IL6 and TNF in response to LPS. This effect is attributed to inactivation of NF-kB. Moreover, GILZ expression decreases in human and mouse macrophages exposed to LPS.Given the regulatory effects of GILZ in macrophages, the objectives of our study were 1) to determine whether GILZ expression is down-regulated in monocytes / macrophages (M/M) in the sepsis, 2) to determine whether the modulation of GILZ expression in M/M is sufficient to influence systemic inflammation, and 3) to identify GILZ mechanism of action in human M/M.GILZ expression was measured in the M/M of patients with septic shock or acute respiratory distress syndrome, and in a murine model of endotoxemia. We observed a significant reduced expression of GILZ in these pathological contexts in human and mice. The impact of this alteration was explored in unique transgenic mouse model in which macrophages stably overexpress GILZ (CD68-GILZ).We confirmed that GILZ overexpression limits TNF production and promotes IL-10 production in in vitro LPS-stimulated macrophages. We further studied the inflammatory response and survival of these mice in models of endotoxemia and septic shock. We showed that GILZ overexpression restricted to macrophages, limits serum pro-inflammatory cytokines production, therefore decreases systemic inflammation and significantly improves mice survival. These results highlight the effects of macrophage polarization by GILZ at a systemic level.This result confirmed the need to characterize GILZ interactome in human macrophages. Two complementary approaches have been used. The first one consists of a pan-genomic double hybrid screening of human GILZ partners. The second method consists of a tandem affinity purification (TAP-TAG) of GILZ protein and its associated partners, followed by the identification of these partners by mass spectrometry. Analyses have been performed independently on nuclear and cytoplasmic extracts from human macrophage cells, genetically engineered to express GILZ protein with the two tags required for purification. This dual approach led us to identify new direct and indirect interactions between GILZ and other key proteins of TLR4 signaling pathway in human macrophages and highlight a likely role of GILZ as a transcription regulatory factor.These results confirm the anti-inflammatory role of GILZ on systemic inflammation and enhancement of lifetime in murine models of endotoxemia and septic shock. Furthermore, this work identifies for the first time the cytoplasmic and nuclear GILZ partners in human macrophages and would allow in the future, a better understanding of GILZ mechanism of action.

No description available.

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) is the leading cause of infectious disease-related death worldwide. The critical role of adaptive immunity in anti-TB host defence has been firmly established; thus, current efforts in developing novel vaccination strategies against TB are primarily focused on generating protective adaptive immunity at the infection site, the lungs. Innate immunity has not been a target for vaccination strategies against TB due to the belief that innate immune cells cannot exhibit memory-like characteristics which are known to be central to the long-lasting immunity created by vaccines. Also, the importance of innate immunity in anti-TB immunity has been overlooked. However, over 25% of individuals that are heavily exposed to M.tb clear infection without any detectable conventional T cell immune responses, suggesting a crucial role for innate immune cells in bacterial clearance. Interestingly, the early protection in these individuals is associated with their Bacillus Calmette-Guerin (BCG) vaccination status. Epidemiological studies have shown that BCG is capable of providing protection against numerous infections unrelated to TB in an innate-immune dependent manner. Such observations suggest that the innate immune system exhibits memory-like characteristics, capable of remembering the exposure to the vaccine and thereby responding in an augmented manner to future systemic infections. Nonetheless, it still remains unknown whether parenteral BCG immunization modulates the innate immune cells in the lung and airways, and if so, what role the trained innate immune cells play in early protection against pulmonary TB. Using a subcutaneous BCG immunization and pulmonary TB challenge murine model, we show that early protection against M.tb is independent of adaptive responses in the BCG immunized host. Our data suggest that enhanced early protection is mediated by the BCG-trained memory alveolar macrophages that we have shown to be functionally, phenotypically, metabolically, and transcriptionally altered following immunization. These novel findings suggest a significant anti-TB immune role for the innate immune memory established in the lung following parenteral BCG immunization and have important implications for the development of novel vaccination strategies against TB. / Thesis / Master of Science (MSc) / Pulmonary tuberculosis (TB) is a disease of the lung and is now one of the leading causes of human mortality worldwide. For more than eight decades, parenterally administered Bacillus Calmette–Guérin (BCG) vaccine has been globally used as the only approved vaccine against TB. Recently, it has also been observed that BCG vaccination provides protection against other diseases unrelated to TB and reduces childhood mortality in many developing countries where it is routinely administered to children shortly after birth. The mechanisms underlying the off-target protective effects of BCG vaccine remains largely under-investigated. In this project, we investigated how BCG vaccination enhances the immune system responses against TB and other unrelated infectious diseases. A better understanding of how the BCG vaccination modulates our immune system will provide us with the knowledge that will be useful in the development of more effective vaccination strategies against infectious diseases.

CSF-1R (colony-stimulating factor 1 receptor) est un récepteur transmembranaire à activité tyrosine kinase exprimé à la surface des monocytes, des macrophages et de leurs progéniteurs. Son ligand, CSF-1, oriente les cellules souches hématopoïétiques vers le lignage myéloïde et permet la différenciation des monocytes en macrophages. Une localisation nucléaire de CSF-1R a été décrite dans certaines lignées tumorales, dans des tumeurs mammaires primitives et dans les macrophages murins. Dans le noyau de ces cellules, CSF-1R régulerait la phosphorylation de protéines nucléaires et l'expression de gènes de la prolifération. Nous avons identifié une localisation nucléaire de CSF-1R dans les monocytes primaires humains par différentes approches et différents anticorps. La forme nucléaire de CSF-1R correspond à la protéine entière monomérique qui est transportée depuis la membrane plasmique vers le noyau, de manière rétrograde, après activation par son ligand et avec celui-ci. L'utilisation d'inhibiteurs de l'activité kinase de CSF-1R diminue la quantité de récepteur dans le noyau. En revanche le blocage des mécanismes d'export nucléaire dépendant de CRM1 par la leptomycine B conduit à l'accumulation de la protéine dans ce compartiment. Dans les monocytes, CSF-1R est localisé sur la chromatine, dans les régions intergéniques et introniques et colocalise avec la marque H3K4me1 présente au niveau des enhancers activés. CSF-1R est situé à proximité de gènes régulant la morphogénèse, le développement du système nerveux, l'ossification et la différenciation cellulaire. Le récepteur est présent sur le promoteur du gène PU.1, facteur de transcription clé dans la différenciation myéloïde et la génération des monocytes, ainsi que sur des gènes impliqués dans la différenciation, la polarisation, la survie et les fonctions des macrophages. Au niveau de la chromatine, CSF-1R interagit avec des facteurs de transcription comme EGR1 sur lequel il exerce un effet co-répresseur. Cette localisation nucléaire de CSF-1R est conservée lorsque les monocytes se différencient en macrophages en réponse à CSF-1. CSF-1R nucléaire est alors relocalisé vers les régions promotrices et exoniques où il colocalise avec la marque H3K4me3. Il est présent à proximité de gènes régulant la vascularisation, la phagocytose, le métabolisme, la réponse au stress et à l'hypoxie. Il interagit avec les facteurs de transcription ELK1 et YY1, et joue un rôle de co-activateur. Lorsque les monocytes sont différenciés en macrophages par une autre cytokine, le GM-CSF, CSF-1R reste dans le noyau des cellules mais sa localisation sur la chromatine et ses interacteurs diffèrent de ceux des monocytes et des macrophages générés par CSF-1, démontrant un régulation différentielle de CSF-1R nucléaire selon le stade de différenciation et les signaux environnementaux. Dans des monocytes de patients atteints de leucémie myélomonocytaire chronique, l’expression, la localisation sur l’ADN et les interacteurs de CSF-1R sont modifiés, indiquant une dérégulation des fonctions nucléaires du récepteur en condition pathologique. CSF-1R est donc localisé dans le noyau des monocytes et des macrophages où il exerce un rôle de régulation de l'expression des gènes dont PU.1. Des résultats préliminaires suggèrent une localisation nucléaire du récepteur dans certaines populations de progéniteurs myéloïdes où il pourrait participer à la regulation de la différenciation. De nombreux inhibiteurs de CSF-1R sont en développement afin de cibler les macrophages infiltrant les tumeurs. Nos résultats démontrent que certains inhibiteurs ont la capacité de cibler la forme membranaire et la forme nucléaire du récepteur et donc d'inhiber l'ensemble des activités de CSF-1R dans les cellules, renforçant l'activité potentielle de ces traitements. / CSF-1R (colony-stimulating factor 1 receptor) is a transmembrane receptor with a tyrosine kinase activity. It is expressed at the cell surface of monocytes, macrophages and their progenitors. Its ligand, CSF-1, has an instructive role on hematopoietic stem cells to direct their differentiation into the myeloid lineage. CSF-1R is also able to differentiate monocytes into macrophages. A nuclear location was described for CSF-1R in cancer cell lines, primary breast tumors and murine macrophages. In the cell nucleus, CSF-1R was suggested to regulate nuclear protein phosphorylation and gene expression. We demonstrate that a small part of CSF-1R is in the nucleus of primary human monocytes, using different antibodies and technical approaches. Nuclear CSF-1R corresponds to full length monomeric receptor. After activation by its ligand, CSF-1R is translocated form cell surface to the nucleus through a retrograde transport, together with CSF-1. Kinase activity inhibitors impaired this process while inhibitors of CRM1-dependant nuclear export (leptomycin B) can revert this effect. In monocytes, CSF-1R is localized on chromatin, mainly on intergenic and intronic regions. It colocalizes with H3K4me1 mark which signs active enhancers. The receptor is present around genes involved in morphogenesis, nervous system development, ossification and cell differentiation. CSF-1R is also located on PU.1 promoter, which is a master transcription factor involved in myeloid and monocyte differentiation. CSF- 1R is also present on genes implicated in macrophage functions, differentiation, polarization and survival. At the chromatin level, CSF-1R interacts with different transcription factors like EGR1 and exerts a co-repressive role to decrease or limit gene expression. CSF-1R nuclear localization persists in macrophages generated by exposure of monocytes to CSF-1. It entails CSF-1R relocalization on promoter-TSS and exonic regions where it colocalizes with H3K4me3 mark. The receptor is close to genes regulating vascularization, phagocytosis, metabolism, stress and hypoxia responses. CSF-1R interacts with ELK1 and YY1 to promote macrophage functions. When monocytes are differentiated into macrophages with GM-CSF, CSF-1R also remains in the nucleus. However, its chromatin localization and interactions change compared to monocytes and CSF-1 differentiated macrophages. This indicates that nuclear CSF-1R is differentially regulated, depending on the cytokine that triggers cell differentiation. In monocytes from chronic myelomonocytic leukemia, CSF-1R expression, chromatin localization and interactors are modified, indicating a deregulated CSF-1R nuclear function under pathological state. Altogether, we showed that CSF-1R is localized in the nucleus of human monocytes and macrophages where it regulates gene expression including PU.1. Preliminary results suggest CSF-1R nuclear location in myeloid progenitor subsets where the receptor could directly regulate the expression of myeloid differentiation genes. Targeting CSF-1R is currently tested as a therapeutic strategy to impair tumor infiltrating macrophages. Our results show that CSF-1R inhibitors are able to target both membrane and nuclear forms and thus to inhibit all CSF-1R activities in the cells, enhancing the potential therapeutic effects of these molecules.

Ischaemia/Reperfusion Injury (IRI) is the most common cause of acute kidney injury- a devastating clinical problem lacking any specific treatments to promote renal recovery. Macrophages (Mφ) are pleiotropic cells of the innate immune system, with roles spanning host defence, cytotoxicity, clearance of apoptotic cells and promotion of tissue repair. Mφ are also known to be important mediators of renal injury in other experimental models of renal disease including transplantation, obstruction and glomerulonephritis. This work sought to examine the role of Mφ in mediating renal IRI. Conditional renal Mφ and monocyte depletion prior to experimental IRI was achieved by administering diphtheria toxin to the CD11b-DTR transgenic animal. This had no impact on either renal function or structural injury. In contrast liposomal clodronate mediated Mφ depletion provided functional and structural protection from injury. Administration of exogenous apoptotic cells also protected renal function if delivered 24h prior to IRI. Immunodeficient SCID mice exhibited a protected injury phenotype after IRI, however derived no additional protection from the administration of either liposomal clodronate or i.v. apoptotic cells. These findings suggest that the protective phenotype must involve either lymphocyte populations or circulating antibody. Preliminary work demonstrates that SCID mice lack IgM natural antibody which deposits in the kidney in the first 30 minutes after IRI. It was also demonstrated that apoptotic cells bind IgM natural antibody present within the circulation. The potential for the key antioxidant enzyme Heme oxygenase-1 (HO-1) to protect renal function was also examined in aged mice using hemearginate (HA) - a potent HO-1 inducer. Echoing epidemiological studies in humans aged mice had increased susceptibility to IRI, whilst failing to induce medullary HO-1. The main site of medullary HO-1 induction by HA was in medullary Mφ, and the protective phenotype was abolished by Mφ ablation, implicating Mφ as the key mediators of HA induced protection in renal IRI. Final studies employed adenoviral transduction to overexpress HO-1 within bone marrow derived Mφ, leading to a modified phenotype with increased IL- 10 and phagocytosis, and reduced TNFα and NO production. When these were introduced in vivo after IRI renal function was improved, potentially due to accelerated clearance of renal platelet deposition.

The mammalian immune system is capable of detecting and responding to different infectious conditions with specificity at the adaptive level, however whether this ability extends to individual cells of the innate immune system is unclear. The hypothesis of this thesis is that macrophages, as individual cells, can distinguish between productive and non-productive virus infections and respond differently at the gene expression and secreted protein level. To test the hypothesis, mouse bone marrow derived macrophages (BMDMs) were infected in parallel with either a productive (live) and non-productive (attenuated) strain of murine cytomegalovirus (MCMV) and profiled temporally using a range of techniques. Both productive and non-productive MCMV infection resulted in strong type I IFN induction in BMDMs, however induction was significantly more rapid in response to productive infection. In addition, chemoattractant and pro-inflammatory cytokines TNFα, IL-6, RANTES, MIG and MIP-2 were secreted to significantly higher levels in response to productive MCMV infection, and curtailed in response to non-productive MCMV infection. Furthermore, genome-wide microarray profiling revealed a number of co-expressed gene networks regulated differentially in response to the two conditions. This consisted of macrophage gene networks targeted for modulation by de novo MCMV proteins, and late macrophage response genes regulated specifically in response to productive MCMV infection. To further explore the mechanisms of transcriptional regulation during macrophage antiviral response, BMDMs from mice lacking either the type I IFN receptor (Ifnar1) or the IFNβ (Ifnb1) gene were profiled using a similar approach. The resulting genome-wide transcriptional data provided a unique insight into the relationship between type I IFN regulation and the macrophage transcriptome in response to MCMV infection. Overall, the study utilizes a combination of genetic mutants from both host and pathogen to investigate mechanisms of virus detection and host transcriptional regulation during the innate immune response to MCMV infection in macrophages.

The successful resolution of inflammation requires removal of neutrophils from the inflammatory site to prevent release of histotoxic contents that may potentiate inflammatory processes and promote progression to a chronic state associated with impaired repair mechanisms and/or autoimmune responses. Macrophages are “professional” phagocytes required for rapid and efficient clearance of apoptotic neutrophils. Macrophage phagocytic capacity can be critically regulated by a number of environmental factors, including cytokines, bacterial products, and glucocorticoids. We have hypothesised that modulation of macrophage phagocytic capacity may represent an effective strategy for promoting resolution of inflammation in diseases where clearance of neutrophils may be impaired or inefficient. The aim of this thesis was to investigate the molecular mechanisms underlying glucocorticoid-augmentation of macrophage phagocytosis. We have demonstrated that long-term exposure of human peripheral blood monocytes to the synthetic glucocorticoid dexamethasone dramatically increases phagocytic capacity for “early” membrane-intact apoptotic neutrophils. Increased phagocytic potential was associated with a “switch” from a serum-independent to a serum-dependent apoptotic cell recognition mechanism. We initially employed an “add back” approach to rule out several well-defined opsonins in apoptotic neutrophil clearance, including immune complexes, IgG, complement proteins, pentraxin-3, fibronectin, annexin I, and platelet-derived factors. Using a multi-step purification scheme involving anion exchange and gel filtration chromatography, we purified a high molecular weight fraction that contained the prophagocytic activity of serum and analysis by mass spectrometry identified C4-binding protein as a candidate protein. C4-binding protein circulates in human plasma bound predominately in a >570kDa complex with protein S and the presence of protein S in high molecular weight fractions was confirmed by immunoblotting. We found that protein S was equivalent to unfractionated serum in its ability to enhance phagocytosis of apoptotic neutrophils by dexamethasone-treated monocyte-derived macrophages (Dex-MDMo) and that immunodepletion of protein S resulted in loss of prophagocytic activity. Protein S was found to opsonise apoptotic neutrophils in a calcium-dependent manner and enhanced phagocytic potential by Dex-MDMo through stimulation of Mer tyrosine kinase (Mertk), a receptor that is upregulated on the surface of Dex-MDMo compared to untreated MDMo. The studies presented in this thesis have provided novel insight into the underlying molecular mechanisms required for high capacity clearance of apoptotic neutrophils by macrophages following treatment with glucocorticoids and may form the foundations for further studies investigating glucocorticoid action for development of safer and more selective therapies.

Pneumocystis carinii is an opportunistic fungal pathogen that causes lifethreatening pneumonia in immunocompromised individuals. Infants appear to be particularly susceptible to Pneumocystis (PC) pulmonary infections. The higher incidence of PC as well as other pulmonary infections among infants is likely due to an immature immune system. The neonatal lung environment is deficient immunologically in preterm as well as term infants (1, 2). Decreased phagocytic capacity of macrophages in newborns may increase the risk of infection from inhaled pathogens (1, 2). We have previously demonstrated that there is approximately a 3-week delay in the clearance of PC organisms from pup mouse lungs compared to adults. Herein, we demonstrate that there is also a 1-week delay in the infiltration of AMs in pup compared to adult PC-infected mice. We go on to show that there is a delay in pup versus adult lung macrophage phenotypic expression and cytokine production in response to PC organisms. We demonstrated that pup AMs are competent to produce cytokine in response to LPS and that stimulation with zymosan generates cytokine production in pup AMs that is comparable to adult cytokine production. These data indicate that pup lung macrophages are specifically poorly responsive to PC organisms and likely require exogenous stimulation to mount a significant immune response and expedite clearance of the organism. We go on to show that heat-killed Escheriae coli improves cytokine response, cellular infiltration and reduces organism burden in PC-infected pup mice. The clinically relevant cytokine, GM-CSF, has been used to improve the clearance of several pulmonary infections, including PC in adult animal models. We show that monotherapy with GM-CSF is insufficient to improve PC clearance in pup mice; however, when combined with TMP/SMX it improves PC clearance and maintains a reduced PC burden following discontinuation of therapy. Furthermore, we have shown that GM-CSF improves the ability of human infant lung macrophages to phagocytose PC organsms without generating an increased inflammatory response. These data suggest that combination therapy with TMP/SMX and GM-CSF may be a viable treatment option for infants failing or intolerant to standard therapy.

Matrix metalloproteinase-2 (MMP-2, gelatinase A, type IV collagenase) is a member of a family of zinc-dependent metalloendopeptidases that functions in the degradation of elastin, collagens, and other components of extracellular matrix (ECM). Both secretion and activation of MMP-2 are elevated in human atherosclerotic lesions and abdominal aortic aneurysms (AAA). In this dissertation project, we sought to test the hypothesis that MMP-2 plays a critical role in both atherosclerosis and AAA. We also sought to determine the detailed mechanism. We first examined the atherosclerosis and AngII-induced AAAs development in MMP-2-/- x apolipoprotein (apoE)-/- mice in vivo. It was surprising that MMP-2 deficiency did not reduce the incidence of AngII-induced AAAs or the size of atherosclerosis in apoE-/- mice. However, the cellular and ECM content of atherosclerotic plaques were modified in MMP-2-/- x apoE-/- mice as compared to MMP-2+/+ x apoE-/- control mice. To explain the apparent paradox between this result and the hypothesis, we investigated the morphological characteristics of the aortic wall of MMP-2-/- mice. We detected an enhanced MMP-9 level in the aortic wall of MMP-2-/- x apoE-/- mice compared with MMP-2+/+ x apoE-/- mice. Interestingly, we also observed more branching of the elastin fibers in aortic wall of MMP-2-/- mice as compared with aorta of wild type mice. We also examined the behavior of macrophages from MMP-2-/- mice. Reduced adhesion, migration, and expression of integrin beta 3 were detected in MMP-2 deficient macrophages compared with wild type macrophages. Lastly, we examined whether MMP-2 deficiency in bone marrow-derived cells may influence AAAs and atherosclerosis using bone marrow transplantation technique. There was a significant reduction of both atherosclerosis development and AAAs formation in mice that were reconstituted MMP-2-/- bone marrow cells. In conclusion, the findings in this dissertation suggest that MMP-2 might play an important role in atherosclerosis and aneurysm through influencing inflammatory cell infiltration.