Trichophyton rubrum, importante agente de dermatofitoses, é um fungo queratinofílico, capaz de parasitar tecidos como pele e unha. É o principal responsável pelas dermatofitoses crônicas e refratárias ao tratamento e como é uma espécie antropofílica encontra-se muito bem adaptado ao parasitismo humano. Por tratar-se de uma micose cutânea, torna-se necessário o estudo dos fenômenos que ocorrem durante o encontro deste fungo com uma das principais células do sistema imunológico que primeiramente reconhecem o antígeno. Assim sendo, o objetivo deste trabalho é estudar a interação de T.rubrum com macrófagos, para aumentar o conhecimento dos mecanismos envolvidos na resposta imunológica nesta importante patologia. Para isso, foram realizados ensaios de fagocitose de conídios de T.rubrum, seguidos da análise da expressão de moléculas de superfície celular, dosagem de citocinas e viabilidade de macrófagos. Verificamos que o exoantígeno de T.rubrum provocou diminuição da fagocitose de conídios e partículas de zymosan pelos macrófagos. Entretanto, o exoantígeno não interferiu na expressão de moléculas de superfície celular e não foi capaz de estimular os macrófagos a secretar TNF-α, IL-12, IL-10 e óxido nítrico. Já os conídios fagocitados por macrófagos, provocaram diminuição significativa na expressão de suas moléculas de superfície, tais como MHC classe II, CD80 e CD54. Após fagocitose de conídios, os macrófagos foram capazes de secretar uma grande quantidade de TNF-α e IL-10 e após 8 horas de cultivo, os conídios internalizados iniciaram processo de formação de hifa, provocando lise e a conseqüente morte destas células. Por estes achados e pelos estudos prévios já realizados com o T.rubrum, pensamos que a persistência desta infecção fúngica possa estar relacionada com a ação inibitória do fungo sobre os macrófagos, levando à cronicidade observada nestas lesões. / Trichophyton rubrum is the most common pathogen causing dermatophytosis, accounting for approximately 80% of the reported cases of onychomycosis. Since 90% of the chronic dermatophyte infections are caused by T. rubrum, it is likely that this pathogen must have evolved mechanisms that evade or suppress cell-mediated immunity. Several reports have highlighted the participation of phagocytes in the immune defense against fungi; however, few studies have addressed the role of these cells in dermatophytosis. In this study, we investigated the interactions of resident and peritoneal macrophages with T. rubrum. We show here that the interaction of T. rubrum conidia with resident macrophages results in the production of TNF-α and IL-10 but not IL-12 and nitric oxide. Infected macrophages down-regulated the expression of co-stimulatory molecules (CD80 and CD54). We also show that phagocytosis of T. rubrum conidia is inhibited by the addition of fungal exoantigens or mannan. Cytotoxicity assays indicated that after 8 h of conidia ingestion macrophage viability decreased drastically. Electron microscopy revealed that the ingested conidia grow and differentiate into hyphae inside macrophages leading to rupture of the macrophage membrane.

Background: COPD is associated with an increased lung macrophage burden. Whilst lung macrophages may self-renew, recruitment of peripheral blood monocytes from the systemic circulation is considered to represent their principal means of replenishment. Through modulating expression of monocytic chemokines CCL2/CCL3 and their respective receptors (CCR2/CCR1+CCR5), IL-6 could play a key role in facilitating the recruitment of monocytes to the lungs of COPD patients. COPD is associated with enhanced pulmonary and systemic IL-6 levels; concentrations of the soluble IL-6 receptor sIL-6R may be an important determinant of IL-6 signalling in COPD. Trans-signalling through sIL-6R, IL-6 may facilitate recruitment of monocytes in COPD by influencing chemokine and chemokine receptor expression. Aims: 1) To compare levels of IL-6, sIL-6R, CCL2 and CCL3 in the plasma and sputum of COPD and controls. 2) To examine of the effects of IL-6 stimulation on monocyte chemokine receptor gene expression (CCR1, CCR2 and CCR5). 3) To compare subtypes (CD14++CD16-, CD14+CD16+, CD14-CD16++) and chemokine receptor expression (CCR1, CCR2, CCR5) of monocytes in COPD (paired stable & exacerbating) and controls. 4) To compare the migratory ability of monocytes from COPD and controls. 5) To compare numbers of marginated CX3CR1+ monocytes in the pulmonary microvasculature and proliferation status (Ki67 positivity) of alveolar macrophages in COPD and controls. Methods: 1) MSD soluble marker analysis was performed on plasma and sputum supernatant. 2) Monocytes underwent stimulation with IL-6 and sIL-6R; chemokine receptor expression was determined by quantitative PCR. 3) Flow cytometry was performed on whole blood to determine monocyte subtype and chemokine receptor expression. 4) Monocyte migration towards sputum supernatant was assessed using a transwell system incorporating fluorescence based detection of DNA from migrated cells. 5) Immunofluorescence and immunohistochemistry was performed on lung tissue (obtained from patients undergoing surgical resection of lung carcinoma) to identify marginated (CX3CR1+CD14+, CX3CR1+CD16+) monocytes and proliferating alveolar macrophages (Ki67) respectively. Results and Conclusion: Levels of sIL-6R were increased in the lungs and systemic circulation of COPD patients implying potential for enhanced IL-6 trans-signalling: monocytes cultured in the presence of IL-6+sIL-6R upregulated expression of the CCR5 gene. A greater proportion of circulating COPD CD14++CD16- and CD14+CD16+ monocytes were demonstrated to express CCR5 compared to controls indicating that CCR5 ligands may have an important influence over monocyte migration in COPD. Levels of CCR5 ligand CCL3 were significantly elevated in COPD sputum supernatant; IL-6 levels were positively associated with CCL3 indicating that IL-6 trans-signalling may mediate lung chemokine expression. Nevertheless, COPD monocytes demonstrated impaired migration towards sputum supernatant and reduced margination to pulmonary microvessels. Despite this, the number of alveolar macrophages in COPD was increased; however this was not likely to be related to self-replication owing to low alveolar macrophage Ki67 expression.

L'insuffisance cardiaque (IC) est une pathologie évolutive avec une forte morbi-mortalité suggérant que les traitements actuels ne modifient que peu les mécanismes physiopathologiques responsables de la maladie. L'inflammation et la réponse immune inappropriée sont récemment apparues comme de nouveaux acteurs de la progression de l'insuffisance cardiaque (IC). La réponse inflammatoire lors d'un stress tissulaire semble initiée par la néo-synthèse ou la libération de protéines normalement non présentes ou non accessibles dans le tissu. Ces molécules, appelées " damage associated molecular pattern " ou DAMPs, sont des ligands des récepteurs Toll like capable d'induire l'expression de cytokines et de chimiokines pro-inflammatoires. La ténascine-C (TNC), une protéine présente dans la matrice extracellulaire lors d'état inflammatoire est considérée comme une DAMP car elle est capable d'induire l'expression de cytokines pro-inflammatoires via TLR4. Bien que l'expression de la TNC soit augmentée lors de pathologies cardiaques, son rôle exact dans les processus inflammatoires au cours d'une surcharge de pression cardiaque est encore peu défini. Nous avons montré que la TNC est exprimée dans le cœur hypertrophié où elle se concentre en foyers associés à un fort infiltrat monocytaire. L'utilisation de chimères hématopoïétiques a permis de montrer que la TNC est exprimée exclusivement par les cellules stromales du compartiment cardiaque et notamment les fibroblastes et non les cellules inflammatoires. Grâce à l'utilisation de souris dont le gène codant pour la TNC a été invalidé (TNCKO), nous avons mesuré l'impact de l'absence de TNC sur la fonction et le remodelage cardiaques après constriction de l'aorte transverse (TAC). L'analyse des paramètres échocardiographiques a montré une meilleure fonction contractile cardiaque chez les souris TNCKO par rapport aux souris WT après TAC. De plus la réponse inflammatoire cardiaque induite par la constriction est fortement atténuée chez les souris TNCKO par rapport aux WT. En effet, l'expression cardiaque des cytokines et des chimiokines pro-inflammatoires ainsi que l'infiltration monocytaire sont prévenues chez les souris TNCKO après TAC. Le développement d'un lentivecteur recombinant pour la TNC (LV-TNC), a permis de montrer que cette protéine stimule, in vitro, l'expression de cytokines et de chimiokines pro-inflammatoires (CCL2, CCL5) par les fibroblastes cardiaques. De plus, le microenvironement produit par la TNC induit un effet attractant sur les monocytes in vitro et in vivo et favorise la polarisation des macrophages vers un phénotype pro-inflammatoire. En plus de son effet sur le chimiotactisme monocytaire, nous avons observé que la TNC modifie également la clairance des macrophages du tissu cardiaque hypertrophié. Le suivi des monocytes/macrophages après marquage par des billes fluorescentes nous a permis de montrer que leur migration vers les ganglions lymphatiques drainants le cœur est augmentée chez les souris KO après TAC. En effet la TNC semble inhiber l'expression de CCR7 par les macrophages cardiaques, récepteur majeur impliqué dans le processus de drainage des cellules immunes dans les vaisseaux lymphatiques. En conclusion, la TNC aggrave le remodelage cardiaque et la transition vers l'IC dans un modèle murin de surcharge de pression. Elle entraine un état inflammatoire en induisant l'expression de gènes pro-inflammatoires par les fibroblastes cardiaques favorisant ainsi l'infiltration monocytaire dans le tissu. La TNC maintient l'inflammation en polarisant les macrophages vers un phénotype pro-inflammatoire. Enfin, la TNC inhibe le drainage des macrophages cardiaques vers les ganglions lymphatiques en diminuant l'expression de CCR7. Cette accumulation de cellules inflammatoires dans le tissu cardiaque induite par la TNC semble favoriser la dysfonction contractile et pourrait participer à l'apparition de l'IC. / Sustained pressure overload leads to myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Several studies suggest a potential role of inflammation induced by the Danger Associated Molecular Patterns (DAMPs) in the development of heart failure. These molecules are able to induce pro-inflammatory cytokines synthesis through Toll like receptors (TLRs). Tenascin-C (TNC), an extracellular matrix glycoprotein considered as DAMP is slightly present in adult tissue but highly expressed in various cardiac diseases. We investigated the prospective role of TNC on inflammation in a cardiac remodeling induced by pressure overload. An experimental model of pressure overload (transverse aortic constriction or TAC) was established in WT and knockout mice for TNC (TNCKO). TNC was greatly expressed after transverse aortic constriction in cardiac tissue of WT animals. TNCKO mice displayed no ventricular dilatation and a preserve cardiac function compared with wild type animals after TAC. In absence of TNC, mice showed a blunted cardiac inflammatory response and a decreased myocardial remodeling. Bone marrow transplantation from wild-type mice into TNCKO animals and vice versa indicated that TNC is exclusively synthesized by cardiac cells and not by hematopoietic cells. Furthermore, immunostaining experiments showed that TNC was found in areas where infiltration of monocytes/macrophages was abundant. To better understand the role of TNC, cardiac fibroblasts were transduced by a lentivector encoding for GFP (LV-GFP) or TNC (LV-TNC). TNC production by fibroblasts stimulated their expression of pro-inflammatory cytokines and chemokines (CCL2, CCL5). In vitro chemotaxis assays showed that conditioned medium from LV-TNC transduced cardiac fibroblasts exhibited chemoattractant properties for bone marrow derived monocytes. Moreover, TNC production by cardiac fibroblasts increased macrophages phagocytic activity, pro-inflammatory markers expression and nitrite release suggesting a pro-inflammatory polarization of macrophages. The administration of a recombinant TNC lentivector in the myocardium was sufficient to induce monocyte infiltration, promote cytokine and chemokine expression and a contractile dysfunction. Finally, TNC did not modify macrophage apoptosis and proliferation but hindered cardiac macrophage clearance by reducing their migration into lymph nodes. In presence of TNC, expression of CCR7 on monocyte/macrophage was down-regulated. CCR7 has been described as the major receptor controlling immune cells migration to lymph nodes. In vivo tracking of monocytes/macrophages by the injection of fluorescents phagocytic beads confirmed a higher number of bead-positive macrophages in lymph nodes of TNCKO mice than in WT mice after TAC. Our results identified cardiac TNC as a monocyte chemoattractant factor increasing chemokines synthesis, decreasing emigration signals and thus promoting the maintenance of monocyte/macrophage in pathological heart. By promoting chronic inflammation, TNC accentuated cardiac dilation and deleterious outcome of cardiac remodeling and function during pressure overload.

Macrophages, phagocytic cells of the immune system involved in host defence, homeostasis and development, are controlled and influenced by a variety of growth factors. In mammals, the colony stimulating factor 1 (CSF1) is a secreted cytokine that controls macrophages survival, proliferation and differentiation. It acts through the CSF1 receptor (CSF1R), a transmembrane receptor tyrosine kinase, expressed mainly in mononuclear phagocytes. Mammalian CSF1R is found exclusively at the surface of the mononuclear phagocytes and their progenitors. CSF1R-/- knockout mice display more severe phenotypes than the CSF1-deficient mice, thus suggesting the existence of another CSF1R ligand. Indeed, recent studies have shown that interleukin 34 (IL34) also binds to and activates CSF1R and regulates monocyte viability in vitro. While the exact role of this protein is yet to be fully elucidated, studies in mammals thus far implied its involvement in embryogenesis and development. CSF1R system is highly conserved within vertebrates and has been identified in variety of mammals. Chicken has been used extensively as a model for vertebrate development and to identify fundamental biological processes. Previous studies by colleagues in the lab demonstrated that the CSF1R system is conserved in the chicken, where it controls the generation of monocytes and tissue macrophages. This thesis provides a thorough evolutionary and structural analysis to fully demonstrate the similarities and differences between avian and mammalian CSF1R systems. The primary objective of this thesis was the comparative functional and structural analyses of the three proteins in birds and mammals, using evolutionary and experimental approaches. Here the presence of CSF1, CSF1R and IL34 genes and protein products is identified in a number of evolutionary diverse birds, indicating that the system is well maintained within the group. Avian genes were cloned and sequenced or otherwise extracted from different databases, and the mammalian sequences were gathered from available online sources. Whilst the gene regulation and the differential expression of the mammalian CSF1R, CSF1 and IL34 are reasonably well understood, they have not been extensively studied in birds. Preliminary comparison between these two groups provided in this thesis suggests a number of similar patterns are involved in regulation of avian CSF1R system. The mammalian CSF1/CSF1R and IL34/CSF1R ligand:receptor peptide interface has been previously resolved and was used to model similar structures in the chicken. The models were then utilised to determine which amino acids are involved in receptor binding in birds. The apparent lack of cross-species reactivity between the chicken CSF1 and zebra finch CSF1R provided a basis for an experimental validation of the in silico binding site predictions. Altogether the structural modelling, evolutionary analysis and experimental confirmation provided sufficient proof for the location of avian CSF1/CSF1R interface. Finally, an extensive bioinformatics analysis has been performed on both the coding DNA and the protein structures of the CSF1R system. The results uniformly showed that IL34 remains under purifying selection in both groups. CSF1 is diverse amongst most mammalian species, while avian CSF1 is only positively selected along particular lineages. This implies the rapid evolution of mammalian CSF1, probably in response to the selection pressure from pathogens. Contrasting situation is found in the CSF1R. Whilst mammalian CSF1R remains positively selected only along particular branches, avian CSF1R presents a number of pervasively positively selected sites, found mostly in the extracellular domains of the receptor. That suggests that in birds it is the receptor, not CSF1, which remains under strong selective pressure. These indicates that birds employ a unique way of competing in the hostpathogen arms race, suggesting the existence of yet unknown pathogen-encoded protein interacting with the avian receptor.

COPD is a condition characterised by chronic inflammation in which macrophages arethought to play a central role. Corticosteroids are the most widely used antiinflammatoryagents to treat COPD. There is a subset of inflammatory mediatorswhich are corticosteroid insensitive and so there is a need for novel anti-inflammatorytherapies to treat COPD. One such target is the liver X receptor (LXR), a cholesterolsensing nuclear hormone receptor with anti-inflammatory properties. Before investigating the anti-inflammatory potential of LXR, I aimed to validate thealveolar macrophage in vitro culture model. I investigated the effect of differentculture times on unstimulated and stimulated cytokine release, dexamethasoneinhibition of cytokine release, and transcription factor phosphorylation in alveolarmacrophages from COPD patients and controls. I found that freshly isolatedmacrophages release higher levels of cytokines, are less responsive to dexamethasoneand have increased levels of phosphorylated p38 MAPK.I next investigated LXR gene and protein expression levels in alveolar macrophages andwhole lung tissue from COPD patients and controls, the effect of LXR activation on thesuppression of inflammatory mediators from LPS stimulated COPD alveolarmacrophages, and the effect of LXR activation on the induction of genes associatedwith alternative macrophage polarisation. The levels of LXR mRNA were significantlyincreased in whole lung tissue extracts in COPD patients and smokers compared tonever smokers. The expression of LXR protein was significantly increased in smallairway epithelium and alveolar epithelium in COPD patients compared to controls. Nodifferences in LXR mRNA and protein levels were observed in alveolar macrophagesbetween patient groups. The LXR agonist GW3965 significantly induced the expressionof the LXR dependent genes ABCA1 and ABCG1 in alveolar macrophage cultures. InLPS stimulated alveolar macrophages, GW3965 suppressed the production of CXCL10and CCL5, whilst stimulating IL-10 production. GW3965 did not significantly suppressthe production of TNFα, IL-1β, or CXCL8. The major finding is that LXR activation hasanti-inflammatory effects on CXC10, CCL5 and IL-10 production from alveolarmacrophages. Finally, I investigated whether dysfunctional lipid homeostasis is a feature of COPDalveolar macrophages. I found that alveolar macrophages from current smokers withand without COPD had increased levels of neutral lipids compared to never smokersand ex-smokers with and without COPD. Dysfunctional lipid homeostasis may play arole in COPD.

Inflammatory airway disease (IAD) is a common performance limiting pulmonary disorder in young racehorses in training. Although the precise aetiopathogenesis is poorly understood, proposed mechanisms include opportunistic bacterial infections and/or suboptimal air-hygiene. Since alveolar macrophages (AMs) are the first line defence in the lungs of mammalian species, they may constitute an appropriate therapeutic target cell in the treatment and the prevention of opportunistic airway infections. This thesis aimed to investigate the basic biology of the equine AM. A series of experiments were conducted to investigate the function and phenotype of this cell and comparisons made with equine macrophages derived from other anatomical sites and macrophage datasets derived from other species. The lung environment is unique, and may direct a unique phenotype and function compared with macrophages derived from other sites. Macrophages were isolated from the lungs, peritoneal cavity and other regions of healthy horses. Excellent cell recovery was demonstrated and associated with good viability, RNA yield and a demonstrable response to several stimuli, both when fresh and following cryopreservation. AMs produced tumor necrosis factor alpha (TNFα) when stimulated with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly IC) and heat-killed Salmonella typhimurium and were actively phagocytic. By comparison, peritoneal macrophages (PMs) did not respond to these inducers and lacked phagocytic activity. In contrast to AMs, which showed high expression of the specific macrophage markers cluster of differentiation (CD) 14, CD163 and toll-like receptor 4 (TLR4), PMs lacked CD14. Moreover, gene expression analysis revealed an alternative macrophage activation for AMs, whereas PM showed a hybrid macrophage activation potentially attributed to the phenomenon of endotoxin tolerance. The response of equine AMs to LPS was analysed using microarrays. There was significant change in the expression of 240 genes. Those that were upregulated included well known inflammatory genes such as TNFα, IL1A and CXCL6. The pattern of response more closely resembled human and pig macrophages than mouse, including the LPS-induced expression of STAT4, IDO, IL7R genes and the failure to produce nitrite in response to LPS. These data suggest that the horse may represent a suitable animal model for human macrophage-associated lung inflammation, and conversely that data from humans may translate to horses. A final aim of this study was to investigate the effect of exercise on equine AM function. Therefore, AMs were isolated from bronchoalveolar lavage samples obtained from Standardbred racehorses at rest and during the training period and microarray analysis performed. Despite important limitations of the study, a few mechanisms at the molecular level were detected which may be involved in the development of either training-associated symptoms of, or susceptibility to IAD. Overall, this thesis aims to improve our understanding of equine macrophage biology and to provide useful information regarding the role of AMs in exercise-associated inflammation. Moreover, the findings presented here may help to inform future preventative pharmacological and/or managemental interventions for IAD.

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

Orientador: Ramon Kaneno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-04T01:09:50Z (GMT). No. of bitstreams: 1 Martins_PriscilaRaquel_M.pdf: 3935077 bytes, checksum: a342a73cd22f638b9ca5c0901503c580 (MD5) Previous issue date: 2004 / Resumo: A atividade imunomoduladora de cogumelos medicinais é atribuída principalmente às J}-glucanas. Neste estudo, avaliamos o efeito de peptidoglucanas extraídas do cogumelo Agaricus blazei (ATF) quanto à atividade candidacida, expressão de receptores de manose e produção de H2O2 e NO por macrófagos peritoneais murinos. Camundongos normais BALB/c receberam três inoculações intraperitoneais de solução salina (grupo controle) ou fração ATF (grupo ATF) e após 48 horas os macrófagos peritoneais foram coletados e ensaiados contra leveduras de Candida albicans. Nossos resultados indicam que o tratamento aumentou a atividade candidacida de macrófagos, produção de H2O2e expressão de receptores de manose, contudo, o tratamento não alterou a produção de NO. Nossos resultados sugerem que a fração ATF pode aumentar a resistência contra agentes infecciosos devido à estimulação da atividade microbicida de macrófagos / Abstract: Immunomodulatory activity of medicinal mushrooms is attributed to glucans. In the present study we avaluated the eifect of peptidoglycans of Agaricus blazei (ATF) on the candidacidal activity, the expression of mannose receptors (MR), production of H2O2 and NO by murine peritoneal macrophages. Normal BALB/c mice were i.p. treated with 3 inoculations of ATF (ATF group) or salt solution (control group) and after 48hr peritoneal macrophages were assayed against Candida albicans yest forms. Our results indicated that the treatment enhanced the candidacidal activity of peritoneal macrophages and increased the H2O2 production and MR expression. However ATF was not able to increase the spontaneous production of NO. The results suggest that ATF can enhance the host resistence against infectious agents due to the stimulation of the microbicidal activity of macrophages / Mestrado / Mestre em Farmacologia

The innate immune system is the first line of host defense against external exposures. During these initial encounters, antigen presenting cells - specifically monocytes and macrophages - modulate further inflammatory responses. Macrophages exist along a spectrum of phenotypic programs; on the inflammatory M1 end they enhance immune activity while on the anti-inflammatory M2 end they suppress further immune activation. Furthermore, within M2 macrophages there exist many subpopulations, namely M2a and M2d, each with specific roles during infection or exposure. We sought to compare the epigenetic profiles of these subpopulations of macrophages to determine key regulatory gene networks and factors that could be exploited for therapeutic benefit.While traditionally viewed as primitive and nonspecific, a growing body of clinical and experimental evidence argues the innate immune system develops memory as a result of previous exposures, allowing the innate system to respond with enhanced and broad immunological protection upon exposure to a secondary stimulus. This biological process of innate immunity has been termed trained immunity. Trained immunity shares many phenotypic and epigenetic characteristics with adaptive immune memory; however, one of the starkest distinctions is the propensity of trained immunity to develop against heterologous stimuli. Innate memory is not antigen specific, frequently protecting the host against unrelated organisms.

Extracellular components can be internalized by either receptor-mediated or fluid-phase endocytosis. Receptor-mediated endocytosis involves the internalization of receptor-ligand complexes into coated vesicles of about 0.1 μm in diameter. The average diameter of primary pinocytic vesicles has been calculated to be 0.24 - 0.28 μm. The discrepancy in size between coated vesicles and the average pinosome diameter can be explained if, in addition to coated vesicles, another endocytic process involving vesicles larger than 0.28 μm in diameter takes place. These two vesicle types could together produce an average diameter of 0.24 μm. This hypothesis suggests that coated vesicles cannot fully account for fluid-phase uptake. Hypertonic conditions can selectively inhibit receptor-mediated endocytosis, leaving fluid-phase uptake unaffected, again suggesting that an alternative to coated pit-mediated uptake exists. In this study we determined the volume-weighted average diameter of primary pinocytic vesicles under hypertonic conditions (0.52 osm) where receptor-mediated uptake of transferrin was selectively inhibited by 42%. Fluid-phase uptake of FITC-dextran was unaffected by 0.52 osm medium. The internalization rate of ³H-galactose-labelled plasma membrane was reduced from 2.6 %/min to 1.5 %/min. The decrease in the rate of membrane internalization, without a reduction in the rate of fluid uptake at hypertonicity, implied a reduced surface to volume ratio of the pinocytic vesicles formed under these conditions. This suggested an increase in the average diameter of primary pinocytic vesicles. Membrane internalization rates were calculated on the assumption that all labelled cell-surface constituents were internalized to the same relative extent, as has been shown previously for isotonic conditions. This assumption was also shown to hold true under isotonic conditions. The reduced rate of membrane internalization under hypertonic conditions was shown not to be due to the exclusion of any labelled protein species from internalized vesicles. The larger average vesicle size determined under conditions of selective reduction of coated vesicle formation (i.e. hypertonicity), demonstrates the existence of a population of larger pinosomes involved in a possible alternative mechanism to coated-pit-mediated endocytosis.