Host defense against pathogenic organisms represents one of the most important and highly-conserved biological processes across the evolutionary timescale. The ability to detect, engulf and destroy particulates for either nutrition or host defense is conserved from single-celled protists to complex multicellular organisms. A central component of host defense is the recognition of invariant, conserved patterns on pathogenic organisms through the use of pattern recognition receptors (PRRs), such as macrophage receptor with collagenous structure (MARCO). MARCO modulates binding and phagocytosis of unopsonized microorganisms and particulates, tethers ligands to signalling complexes and enhances cellular adhesion. Current literature suggests these functions are mediated via the C-terminal scavenger receptor cysteine rich (SRCR) domain. The relative importance of this domain remains unclear, as other, closely-related scavenger receptors function independently of the SRCR domain via a shared lysine-rich motif. In chapter 3.1, we discovered and cloned a naturally-occurring transcript variant of MARCO which lacks the SRCR domain, termed MARCOII. We demonstrated that the SRCR domain is required for ligand binding and internalization and that MARCOII can form heteromeric complexes with MARCO and reduce receptor function. Furthermore, the SRCR domain enhanced TLR2/CD14-mediated pro-inflammatory responses to Streptococcus pneumoniae. Finally, it was demonstrated that the SRCR domain modulates MARCO-mediated cellular adhesion. In chapter 3.2, we used comparative phylogenetics to identify human specific mutations and residues undergoing positive selection in human MARCO. We demonstrated that humans possess a unique phenylalanine residue at position 282 that is polymorphic, with some humans encoding an ancestral serine residue. We also demonstrated that glutamine at position 452 is found in Denisovans, Neanderthals, and extant humans, but all other non-primate, terrestrial, and aquatic mammals possess an aspartic acid residue. We cloned the ancestral residues and loss-of-function mutations and demonstrated that these residues enhance ligand association and phagocytosis of Escherichia coli. / Thesis / Doctor of Philosophy (PhD) / Some of the most ancient mechanisms of host defense rely on invariant recognition of pathogens through the use of pattern recognition receptors, such as the macrophage receptor with collagenous structure (MARCO). MARCO plays an integral role to allow for specialized subsets of white blood cells to bind pathogens, activate signalling complexes and to bring pathogens inside the white blood cell for destruction. Current literature suggests the C-terminal Scavenger Receptor Cysteine Rich (SRCR) domain of MARCO is required for these processes. This remains under scrutiny, as other closely-related receptors have been shown to operate independently of the SRCR domain. Herein, we utilized a variant of MARCO which lacks the SRCR domain and patterns of evolution to confirm both that the SRCR domain is critical for receptor function and to discover novel sites within the human MARCO protein that play indirect, but important roles in receptor function.

The disease gonorrhea has plagued mankind at least as long as written records have been kept (Black and Sparling, 1985). N. gonorrhoeae is still an important cause of suffering, infertility, and occasional mortality despite the fact that treatment with antibiotics is relatively easy and highly effective, even with the recent increase in penicillin-resistant isolates (Jephcott, 1986). The continued existence of this public health problem is partly the result of a reservoir of asymptomatic carriers within the community who normally don't seek treatment and continue their usual sexual practices (Handsfield, 1983; Kavli et al., 1984). Asymptomatic carriers do not have the purulent discharge characteristic of gonococcal urethritis and cervicitis in which the neutrophil is such a prominent element. Since IgM is present in only trace amounts on genital mucosa (Schumacher, 1973), and this is the "naturally occurring" antibody against gonococci (Rich and Kasper, 1982); it is not unreasonable to assume that non-opsonic chemotaxis and non-opsonic phagocytosis by PMN may play important roles in initiating the inflammatory response and symptomatology seen with gonorrhea. Further, non-opsonic phagocytic killing may be important in eventually clearing gonococcal infection since the role of specific humoral immunity is limited by the ability of gonococcus to constantly vary its antigenic facade (Zak et al., 1984). I have found that three different gonococcal strains express certain outer membrane proteins of the protein II (P.II) family which stimulate neutrophil phagocytic killing and oxidative metabolism in a highly efficient, dose-dependent manner. Other P.IIs expressed by two of the strains are non-stimulatory. Since all P.IIs have very similar physicochemical properties, these results suggest that a specific receptor-ligand interaction occurs between the gonococcal P.II and some element of the neutrophil plasma membrane. The presence or absence of pili on the gonococcal surface has no apparent effect on the ability of certain P.IIs to stimulate neutrophils. Changes in gonococcal outer membrane protein I and lipopolysaccharide, which are thought to confer serum resistance, also have no apparent effect on P.II stimulation of human PMN. Therefore, gonococcal outer membrane P.II may be an important mediator in the inflammatory response to gonococcal infection. Once gonococci are phagocytized by human PMN killing occurs rapidly and there is no evidence of significant intracellular survival. Non-oxidative killing by human chronic granulomatous disease neutrophils is as effective as the killing seen with normal PMN. Extracellular killing of gonococci does not occur to any appreciable extent.

Despite the clinical importance of anaerobic bacteria in otitis media and the uncertainty regarding the proper treatment of the anaerobic focal infection, few experimental studies focused upon the role of these microorganisms in otitis media have been publis­hed. In the present investigation a guinea-pig model for the induc­tion of anaerobic monoinfections in the middle ear was described. Bacteroides fragilis and Propionibacterium acnes (4.0-10x10 colony forming units) injected via the tympanic membrane were capable of inducing clinical and histological otitis media with persistent seguele in the middle ear cavity. Bacteroides asacc-harolyticus, Peptostreptococcus micros and Peptost repto- coccus anaerobius failed to induce otitis media. B. fragilis otitis was accompanied by increased serum IgG and IgM antibody titres against the challenge organism, whereas P. acnes and P. anaerobius did not induce a humoral immune response. The results suggested true virulence of B. fragilis in guinea-pig middle ear monoinfections. Metronidazole was found to accelerate the elimination of B.fragilis from the middle ear. However, even high doses of metro­nidazole were nojt fully effective perhaps reflecting an incomplete anaerobiosis at the site of infection in some instances. At pre­sent, nitroimidazoles in chronic otitis media must be regarded as a possible alternative reguiring further study, particularly with regard to the dosage. In order to gain further knowledge of the interaction between poly­morphonuclear leukocytes and bacteria under anaerobic conditions an in vitro model was established. It was shown that P. acnes was readily phagocytosed with the aid of C3 activated either via the classical or alternative pathway and that killing of P. acnes was inefficient during anaerobiosis. The results suggest that P.acnes is maintained in the pus in chronic otitis media because it sur­vives phagocytosis. Finally, the interaction between the most common pathogen in acute purulent otitis media, Streptococcus pneumoniae, and human poly­morphonuclear leukocytes under anaerobic conditions was studied. Since purulent maxillary sinus effusion (and probably also purulent middle ear effusion), invariably has a pO^ approaching zero, such studies are highly relevant with regard to the host defence in sinuitis and perhaps also in otitis media. S. pneumoniae was killed by the phagocytes under anaerobic condi­tions although at a slower rate than in air. Degradation of pneumo­coccal teichoic acid, DNA and RNA took place after phagocytosis under aerobic as well as anaerobic conditions, whereas degradation of unsaturated cell membrane lipids took place only under aerobic conditions. Furthermore, the pneumococcal autolytic system did not participate in the killing or the degradation of the bacteria. / <p>Diss. Umeå, Umeå universitet, 1984, härtill 6 uppsatser</p> / digitalisering@umu

Rosetting is an adhesion property of malaria parasites whereby infected erythrocytes bind to two or more uninfected erythrocytes, forming a so-called rosette. Rosetting of Plasmodium falciparum is associated with disease severity and high parasitaemia in sub-Saharan Africa, although currently the function of rosetting remains unknown. An early IFNg response elicited from the innate immune system is associated with resolution of malaria infection in mice. Published data suggests that optimal IFNg production may require contact between peripheral blood mononuclear cells and P. falciparum infected erythrocytes. The first part of this thesis investigates the hypothesis that rosetting is an immune evasion strategy to hide infected erythrocytes from detection by innate immune cells. Across five laboratory strains of P. falciparum rosetting was not associated with differential IFNg production when parasites were grown in group O blood. Reappraisal of the data with respect to blood group for one strain found that rosetting significantly reduced the IFNg response to parasites grown in group A blood (P=0.022, Wilcoxon signed-rank test), where it is known that rosettes are bigger and stronger. This is consistent with the hypothesis that rosetting is an immune evasion strategy and the first study to find evidence for a function of rosetting. Further work is needed in order to generalise this finding. The cytokine response to P. falciparum varies between people and this variation may be indicative of disease progression. In mice infected with malaria it is also apparent that parasite strain can determine the cytokine response of the host. It is unclear whether P. falciparum strains vary in their ability to induce cytokines. The second part of this thesis investigates variation in cytokine induction between P. falciparum strains. Across four laboratory strains of P. falciparum, IFNg production was significantly dependent on parasite strain (F3,178= 48.49, P<0.001). Production of GM-CSF, IL-1b, IL-6, IL-10 and TNFa significantly correlated with production of IFNg (P<0.001, Pearson correlation) and followed the same strain-dependent pattern. The ratio of pro-inflammatory cytokines to IL-10 was also dependent on parasite strain. These data provide strong evidence for P. falciparum strain-dependent cytokine responses which may be an important determinant of disease outcome. Phagocytosis by splenic macrophages is proposed to be the principle mechanism of parasitaemia control in malaria infection. CD36 mediated phagocytosis may by an important mechanism of non-opsonic parasite clearance. The final part of this thesis investigates the hypothesis that rosetting is an immune evasion strategy of P. falciparum in order to evade phagocytic clearance, in particular that mediated by CD36. Overall the data obtained were inconsistent. Phagocytosis was significantly reduced in rosetting versus non-rosetting parasites in some strains (e.g. R29; P=0.048, paired T test), whereas others showed no effect (e.g. Muz12; P=0.228, paired T test) or increased versus non-rosetting parasites (e.g. HB3, P=0.004, paired T test). The relationship between CD36 binding and phagocytosis was also unclear, and anti-CD36 antibody did not effectively block phagocytosis, suggesting the involvement of alternative mechanisms. Further experiments are needed to clarify these observations. Data presented in this thesis are suggestive that rosetting in non-group O blood may be an immune evasion strategy with regard to IFNg production by innate immune cells, mechanistically linking rosetting with enhanced parasitaemia and disease severity. Furthermore, parasite strain significantly affects cytokine production and may be a determinant of disease outcome. This thesis demonstrates the importance of continued research into the effect of parasite virulence on the immune response, with particular emphasis on rosetting.

No description available.

Macrophages are key cells during inflammation and repair. Their activity is highly varied and requires precise regulation. The characterisation of cues coordinating macrophage functions has focussed on chemical and biological soluble mediators. Little is known about their responses to physical stimuli, in particular electric fields (EF) that are generated naturally in wounded tissue and infected tissue. Importantly, EFs are known to accelerate wound healing and limit infection but the mechanisms of this remain poorly understood. To address this gap in understanding, this study tested how key properties of human monocyte-derived macrophages are regulated by applied EFs equivalent to physiological EF strengths generated naturally. Using live-cell video microscopy, we show macrophage migration is directed anodally by EFs as low as 5 mV/mm and is EF-strength dependent, with effects peaking around 300 mV/mm. In contrast, monocytes, as macrophage-precursors, migrate in the opposite, cathodal direction. Strikingly, we show for the first time that EFs significantly enhance macrophage phagocytic uptake of a variety of targets, including carboxylate beads, apoptotic neutrophils and the nominal opportunist pathogen Candida albicans, all of which engage different classes of surface receptors. These EF-induced functional changes are accompanied by clustering of phagocytic receptors, enhanced PI3K and ERK activation, mobilization of intracellular calcium and actin polarization. EFs also selectively modulate cytokine production and augment effects of conventional polarising stimuli on cytokine secretion. Taken together, electrical signals have been identified as major contributors to the co-ordination and regulation of important human macrophage functions, including those essential for microbial clearance and healing. Our results open up a new area of research into effects of naturally occurring and clinically-applied EFs in conditions where macrophage activity is crucial.

Thesis advisor: Thomas N. Seyfried / Metastasis is the process by which cancer cells disseminate from the primary neoplasm and invade surrounding tissue and distant organs, and is the primary cause of morbidity and mortality for cancer patients. Most conventional cancer therapies are ineffective in managing tumor metastasis. This has been due in large part to the absence of in vivo metastatic models that represent the full spectrum of metastatic disease. Here I identify three new spontaneously arising tumors in the inbred VM mouse strain, which has a relatively high incidence of CNS tumors. Two of the tumors (VM-M2 and VM-M3) reliably expressed all of the major biological processes of metastasis to include local invasion, intravasation, immune system survival, extravasation, and secondary tumor formation involving liver, kidney, spleen, lung, and brain. Metastasis was assessed through visual organ inspection, histology, immunohistochemistry, and bioluminescence imaging. The metastatic VM tumor cells also expressed multiple properties of macrophages including morphological appearance, surface adhesion, phagocytosis, gene expression (CD11b, Iba1, F4/80, CD68, CD45, and CXCR4) and total lipid composition (glycosphingolipids and phospholipids). The third tumor (VM-NM1) grew rapidly and expressed properties of neural stem/progenitor cells, but was neither invasive nor metastatic. This thesis research also examined the influence of a genelinked up-regulation of the simple ganglioside GM3 in the metastatic VM-M3 tumor. Ganglioside GM3 has been shown to have anti-invasive effects through its ability to modulate integrins and matrix metalloproteases. Additionally, GM3 was previously shown to be elevated in resting macrophages when compared to activated macrophages. The bioluminescent VM-M3 cells (M3/Fluc) contain mostly GM2, GM1, and GD1a with undetectable levels of GM3. Additionally, the M3/Fluc cells express GalNAc-T, a key enzyme for the synthesis of complex gangliosides from GM3, the precursor used for complex ganglioside biosynthesis. Stable transduction of the M3/Fluc tumor with a lentiviral vector containing a cDNA sequence targeting the GalNAc-T gene (Fluc-TNG), resulted in a knock-down of GalNAc-T expression and an up-regulation of GM3 compared to the control (Fluc-csh) transduced M3/Fluc tumor cells. In vivo, the Fluc-TNG cells were significantly less invasive when implanted in the brain and less metastatic when implanted in the flank when compared to the control Fluc-csh tumors. My data indicate that spontaneous brain tumors can arise from different cell types in VM mice and that the ganglioside GM3 can inhibit invasion and metastasis in metastatic cancer cells with macrophage properties. The new VM tumor model will be useful for defining the biological processes of cancer metastasis and for evaluating potential therapies for tumor management. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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-&#945;, 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-&#945; 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-&#945; 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.

A melatonina é uma indolamina sintetizada principalmente pela glândula pineal, cuja função está associada à marcação do escuro. Além deste papel cronobiótico, a melatonina também tem papel na defesa e é sintetizada por outros sítios podendo exercer ação parácrina e autócrina, como em células imunocompetentes. Concentrações substancialmente elevadas de melatonina são encontradas no liquido cefalorraquidiano (LCR) que tem sido vinculada à síntese de melatonina por células do sistema nervoso central (SNC), como as microglias. Sabendo-se que estas células são os macrófagos residentes no SNC e que a síntese de melatonina por estes fagócitos já é comprovada, nosso trabalho teve por objetivo avaliar se microglias cerebelares sintetizam essa indolamina e se esta atua potencializando a fagocitose destas células. Nossos resultados mostram que o bloqueio dos receptores de melatonina com o antagonista luzindol, diminuiu tanto a fagocitose induzida por melatonina exógena, quanto à fagocitose basal, indicando que há síntese de melatonina por microglias cerebelares que, por sua vez, age na fagocitose. Esses resultados são relevantes e indicam que a melatonina sintetizada pela microglia, pode estar relacionada com a homeostase do ambiente neural. Sendo assim, nossos dados podem contribuir com estudos que estabeleçam novas estratégias terapêuticas para doenças neuroinflamatórias. / Melatonin is a indolamine synthesized primarily by the pineal gland, whose function is associated with the marking of the dark phase. Beyond this chronobiotic function, melatonin also plays a role in defense and is synthesized by other sites. It may exert paracrine and autocrine action, like in immunocompetent cells. High substantially concentrations of melatonin are found in the cerebrospinal fluid (CSF) that has been linked to the synthesis of melatonin by the central nervous system cells (CNS), such as microglia. Knowing that these cells are the resident macrophages in the CNS and that melatonin synthesis by these phagocytes is proven, our study aims to assess whether cerebellar microglia synthesize this indolamine and whther this acts enhancing phagocytosis of these cells. Our results show that blocking the melatonin receptors with the antagonist, luzindole, both the exogenous melatonin-induced phagocytosis and the basal phagocytosis decreased, indicating that there is melatonin synthesis by cerebellar microglia which acts on phagocytosis. These results are significant and indicate that melatonin synthesized by microglia may be related to the neural environment homeostasis. In this way, our data can contribute, for example, in studies to establish new therapeutic strategies for neuroinflammatory diseases.

Orientador : Alexandra Ivo de Medeiros / Banca: Vania Luiza Deperon Bonato / Banca: Carlos Rossa Junior / Resumo: A fagocitose de células apoptóticas é um processo dinâmico importante para a homeostase dos tecidos após injúria. Os macrófagos além de atuarem na remoção de células apoptóticas, também colaboram na defesa contra microrganismos. Existem ao menos duas populações de macrófagos classificadas como macrófagos M1 (pró-inflamatórios) e macrófagos M2 (anti-inflamatórios). Estes leucócitos diferem quanto ao fenótipo e funções efetoras dependendo, primordialmente, do microambiente e dos microrganismos com que interagem no tecido. Em uma situação de injúria pulmonar estéril (inalação de agentes tóxicos), há acúmulo de células apoptóticas sem a presença de agente microbiano. Por outro lado, durante uma infecção pulmonar bacteriana, ocorre intensa migração de células para o local da infecção na tentativa de conter a proliferação bacteriana, resultando em intenso acúmulo de células apoptóticas infectadas neste tecido. Portanto, nesse trabalho foi avaliado, in vitro e in vivo, o efeito da fagocitose de células apoptóticas infectadas (AC-Sp) ou estéreis (AC) na polarização de macrófagos M1/M2, bem como suas funções efetoras, e o efeito da PGE2 nesse contexto de eferocitose. Macrófagos M0 co-cultivados com AC ou AC-Sp apresentam um fenótipo intermediário entre M1 e M2, com secreção de altos níveis de IL-6, TNF-α, PGE2, TGF-β e NO e a expressão de genes relacionados ao perfil M1, iNOS, e ao perfil M2, Arginase 1. Além disso, macrófagos M0 cultivados com AC ou AC-Sp, apresentam supressão da função microbicida quando desafiados com S. pneumoniae. A presença de PGE2 dirige a polarização de macrófagos M0 a um perfil M2 e a presença de inibidores de COX promove a reversão do fenótipo de polarização destes macrófagos. Os resultados in vivo demonstram que a instilação de AC ou AC-Sp promove distintos microambientes no pulmão destes animais, que influenciam na resolução da infecção..... / Abstract: The phagocytosis of apoptotic cells is dynamic and crucial for homeostasis after injury. Macrophages act on the clearance of apoptotic cells and collaborate with defense against microorganisms. There are at least two distinct macrophage populations classified as M1 macrophages (pro-inflammatory) and M2 macrophages (anti-inflammatory). Both cells differ on the state of polarization and effectors function depending primarily on the microenvironment and microorganisms that interact into the tissue. In a situation of sterile lung injury (inhalation of toxic agents), there are accumulation of apoptotic cells without the presence of pathogen. On the other hand, during a bacterial pulmonary infection there is intense cell migration to the infection site in an attempt to impair bacterial growth, resulting in a large accumulation of infected-apoptotic cells in the tissue. Therefore, this study has evaluated in vitro and in vivo, the effect of the phagocytosis of infected-apoptotic cells (AC-Sp) or sterile cells (AC) on the polarization of M1/M2 macrophages, as well as in their effector functions and the effect of PGE2 in the context of efferocytosis. M0 macrophages co-cultured with AC or AC-Sp show an intermediate phenotype between M1 and M2 with high secretion levels of IL-6, TNF-α, PGE2, NO and TGF-β, and gene expression profile related to M1, iNOS, and M2, arginase 1. Furthermore, M0 macrophages cultured with AC or AC-Sp show strong suppression of the macrophage microbicidal function when challenged with S. pneumonia. The presence of PGE2 drives the polarization of M0 macrophages to a M2 profile, and in the presence of COX inhibitors, it reverses the polarization of this macrophage phenotype. The in vivo results demonstrate that the instillation of AC or AC-Sp promotes distinct microenvironments in the lungs of these animals, which influence the resolution of the infection. All these results suggest that the presence of AC or AC-Sp promotes, ... / Mestre