A artrite induzida por pristane (PIA) em camundongos AIRmax homozigotos para o alelo R e S do gene Slc11a1 foi usada para avaliar a influência do polimorfismo deste gene na resposta imune, mais especificamente na ativação de macrófagos peritoneais durante a PIA. Estudos anteriores mostraram que a presença do alelo S do gene Slc11a1 aumentou a incidência e a severidade da PIA em AIRmaxSS, sugerindo que este gene ou outro próximo esteja interagindo com o loci da inflamação para modular a PIA. O tratamento com pristane nos animais AIRmaxSS induziu infiltrado intenso composto por linfócitos, monócitos/macrófagos e neutrófilos. Macrófagos AIRmaxSS apresentaram perfis de expressão gênica e celular exacerbados durante a PIA, com expressão/produção elevada de H2O2, NO, IL-1b, IL-6, TNF-a e várias quimiocinas. Entretanto, o alelo R do gene Slc11a1 foi capaz de regular a intensidade de ativação do macrófago de forma mais eficiente que o alelo S e controlar desenvolvimento da artrite. Houve acometimento do rim, pulmão e timo durante a PIA. Nossos dados sugerem que o gene Slc11a1 modula a ativação dos macrófagos envolvidos na suscetibilidade a PIA e estas linhagens representam um modelo murino alternativo para o estudo da artrite reumatoide. / Pristane-induced arthritis (PIA) in AIRmax mice homozygous for Slc11a1 R and S allele was used in this study to characterize the role of Slc11a1 polymorphisms on immune response, more specifically in the activation of peritoneal macrophages during PIA. Previous reports showed the presence of S allele of Slc11a1 increased the incidence and severity PIA in AIRmaxSS, suggesting that this gene or another closed-linked gene interacts with inflammatory loci to modulate PIA. Pristane treatment induced intense infiltration of lymphocytes, monocytes/macrophages and neutrophils in AIRmaxSS animals. AIRmaxSS macrophages demonstrated exacerbated cellular and gene expression profiles during PIA, with higher expression/production of H2O2, NO, IL-1b, IL-6, TNF-a and chemokines. However, Slc11a1 R allele could be regulating macrophage activation intensity more efficiently than the S allele and control the development of arthritis. There was involvement of kidney, lung and thymus during PIA. Our data suggest that the Slc11a1 gene modulates macrophage activation involved in PIA susceptibility and these lines represent an alternative murine model of rheumatoid arthritis.

Avaliamos o papel dos leucotrienos (LTs), as vias de sinalização e o efeito da insulina na fagocitose via FcgR por macrófagos alveolares (MAs) de ratos sadios (RS) e diabéticos (RD). Vimos que a) MAs de RD fagocitam menos que os de RS; b) a fagocitose é dependente de LTs endógenos em RS mas não em RD; c) a adição de LTB4 ou LTD4 aos MAs em cultura aumenta a fagocitose em RS e RD; d) MAs de RS e RD produzem quantidades equivalentes de LTB4 e LTC4; e) a adição de insulina aos MAs aumenta a capacidade fagocitica em ambos os grupos; f) em RS, a fagocitose via FcgR induz fosforilação de Akt e PKC-d, que é amplificada por LTs endógenos, enquanto que em RD ocorreu fosforilação somente da PKC-d. A foforilação de Akt e PKC-d amplificada por LTs produzidos sob estímulo do FcgR em MAs de RS parece ser, de alguma forma, dependente da ação da insulina, pois MAs provenientes de RD fagocitam menos, a fagocitose não é dependente de LTs endógenos e o estímulo via FcgR não é capaz de ativar a Akt. / We evaluated the role of leukotrienes (LTs), the signaling pathways and the effect of insulin in phagocytosis via FcgR by alveolar macrophages (AMs) from healthy (HR) and diabetic (DR) rats. The results showed that: a) AMs from DR showed lower phagocytic capacity than AMs from healthy rats; b) the phagocytosis was dependent of endogenous LTs in AMs from HR but not DR; c) addition of LTB4 and LTD4 to cultures enhanced the phagocytosis by AMs from HR and DR; d) AMs from HR and DR rats produced similar levels of LTB4 and LTC4; e) addition of insulin to AMs enhanced the phagocytic capacity in HR and DR; f) in HR, the phagocytosis via FcgR induced Akt and PKC-d phosphorylation, which is amplified by endogenous LTs, whereas in DR, only PKC-d was phosphorylated. The phosphorylation of Akt and PKC-d, amplified by LTs produced under FcgR engagement in AMs from HR, seems to be, in a way, dependent of insulin action, because AMs from DR have lower phagocytic capacity, the phagocytosis is not dependent of endogenous LTs and the FcgR engagement is not capable to activate Akt.

The mammalian target of rapamycin (mTOR) signalling pathway takes part in both extracellular and intracellular signals. It is a major regulator of cell metabolism, growth, proliferation and survival. mTOR also regulates critical processes such as cytoskeletal organization, ribosomal biogenesis, transcription and protein synthesis. The mTOR pathway has been implicated in many diseases such as cancer, neurodegeneration and diabetes, which impact homeostasis and cellular functions. Moreover, mTOR has also been shown to play a critical role in immune cell regulation of T and B cells together with neutrophils and antigen presenting cells, as it integrates signals between them extending to the entire immune microenvironment. The aim of my study was to investigate the role of a component of the mTOR complex 1, Raptor, in myeloid cells. My findings show that the absence of Raptor knock out (KO) does not affect bone marrow derived macrophage (BMDM) differentiation and maturation. However, the absence of Raptor influences BMDM polarisation towards an inflammatory phenotype, at least at the level of transcription as observed by increases in mRNA expression of inflammatory cytokines such as TNFα, IL-12β, and IL-6. This finding was consolidated by an increase in NFκΒ pathway signalling in Raptor KO BMDMs. Downstream intracellular signalling in myeloid cells was affected by deletion of Raptor as I found reduced S6K phosphorylation in Raptor KO BMDMs compared to wild type (WT) BMDMs. As a consequence of Raptor absence in BMDMs, STAT3 phosphorylation was also reduced. Raptor deletion did not impact the PI3K/Akt signalling pathway, but decreased phosphorylation of ERK. BMDMs lacking Raptor had reduced phagocytic activity as they were also observed to migrate less towards a pancreatic cancer cell line. However preliminary experiments in pancreatic cancer models did not indicate a major role for Raptor in the activity of tumour associated myeloid cells. My results demonstrate that Raptor and by implication mTORC1, is involved in macrophage polarisation and function.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Lectinas sÃo proteÃnas que atravÃs de ligaÃÃes a resÃduos de carboidratos podem interagir com sistemas biolÃgicos elicitando uma diversidade de efeitos. As lectinas vegetais tÃm sido utilizadas como ferramentas no estudo da inflamaÃÃo devido a sua capacidade de reconhecer resÃduos de carboidratos presentes nas membranas das cÃlulas inflamatÃrias atravÃs de seus domÃnios lectÃnicos. Assim, investigou- se neste trabalho o possÃvel efeito prÃ-inflamatÃrio da lectina de sementes de Dioclea rostrata; ligadora de α-metil-D-manosÃdeo sobre a migraÃÃo de neutrÃfilos (MN) [(in vivo e in vitro)]. Os modelos utilizados foram: peritonite, edema de pata e bolsa de ar subcutÃnea (in vivo), quimiotaxia de neutrÃfilos e cultura de macrÃfagos (in vitro). Foi verificado que a lectina apresentava atividade prÃ-inflamatÃria em todos os estudos realizados. O aumento do nÃmero de macrÃfagos atravÃs do prÃ-tratamento dos animais com tioglicolato potencializou a MN induzida por Dros; a depleÃÃo de mastÃcitos atravÃs do tratamento com o composto 48/80 nÃo interferiu na MN da lectina. Sendo assim sugeriu-se o envolvimento de macrÃfagos e desconsiderou-se o de mastÃcitos. Na modulaÃÃo farmacolÃgica no modelo de peritonite feita atravÃs do prÃ-tratamento dos animais com drogas anti-inflamatÃrias, observou-se que indometacina, dexametasona e talidomida inibiram a MN. A lectina induziu quimiotaxia in vitro, estimulou a sÃntese/liberaÃÃo de citocinas como TNF-α e IL-1, IL-10, desta maneira sugere-se que estudos mais detalhados sejam realizados em continuidades a este trabalho para verificar se esta lectina pode ser utilizada em modelos de imunosupressÃo. O α-metil-D-manosÃdeo reverteu a MN induzida por Dros, desta forma parece que a Dros desencadeia resposta inflamatÃria atravÃs da interaÃÃo de seus domÃnios lectÃnicos com aÃucares presentes na membrana de macrÃfagos / Lectins are proteins possessing a carbohidate moiety that are able to interact with biological systems eliciting a variety of effects. Vegetal lectins have been used as tools in the study of inflammation due to its ability to recognize carbohydrate residues in inflammatory cell membranes by means of its lectin domain. The present work studied the pro-inflammatory effects of the lectin from Dioclea rostrata seeds (Dros), a binder of α-methyl-D-manoside, on neutrophil migration ( NM ) [(in vivo and in vitro)]. The models of peritonitis, paw edema, and subcutaneous air pouch (in vivo), neutrophil chemotaxy and macrophage culture (in vitro) were utilized. It was found that Dros showed a pro-inflammatory activity in all of the above models. The increase in the number of macrophages by the pre-treatment of the animals with thioglycolate potentialized the Dros-induced NM. Also, the depletion of mast cells by the use of the substance 48/80 did not interfere in the lectin-induced NM. The above data suggest the involvement of macrophages but not mast cells in the mechanisms studied. The pre-treatment of the peritoneum with anti-inflammatory drugs like indomethacine, dexamethasone and thalidomide inhibited the NM. Dros induced chemotaxy in vitro and stimulated the synthesis / release of cytokines as TNF-α and IL-1 in addition to IL-10 and this way he/she suggests himself that more detailed studies are accomplished in continuities to this work to verify this lectin can be used in imunosupression models. In the paw edema model the lectin promoted an intense edema and a significant increase in the mieloperoxidase activity (when compared to the control group). The α-methyl-D-manoside reversted the Dros-induced NM and so it seems that Dros triggers the inflammatory response by means of the interaction of its lectin domain with sugars in the macrophage membrane. The present data suggest that Dros could be used as tools in biochemical and pharmacological studies.

Camundongos AIRmax e AIRmin exibem diferenças significativas no número médio de leucócitos migrantes e no conteúdo protéico do exsudato inflamatório produzido por partículas de poliacrilamida. Um dos fatores preponderantes para a maior capacidade inflamatória da linhagem AIRmax é a maior produção de neutrófilos maduros pela medula óssea. Assim, considerando a diferente capacidade de produção leucocitária, entre as linhagens AIRmax e AIRmin, nos propomos a estudar comparativamente nestas linhagens, o processo de mielopoese in vitro em resposta ao GM-CSF e ATRA. Verificamos que as células da medula óssea dos animais AIRmax apresentaram maior potencial proliferativo e maiores níveis de expressão de genes envolvidos nos estágios iniciais da mielopoese, do que os animais AIRmin. Além disso, o conteúdo protéico das células em cultura revelou diferenças quantitativas e qualitativas de proteínas provavelmente envolvidas no processo de mielopoese. / Mice AIRmax and AIRmin exhibit significant differences in the average number of migrating leukocytes and in the protein content of inflammatory exudate produced by polyacrylamide particles. This higher inflammatory capacity of the AIRmax mice is due to three convergent factors: higher local production of chemotactic factors, increased resistance of locally infiltrated neutrophils to spontaneous apoptosis and larger production of mature neutrophils by the bone marrow. Thus, considering the differential capacity of leukocyte production between AIRmax and AIRmin mice, we are studying comparatively the myelocytic differentiation process in vitro. We verified that the BM cells of AIRmax mice showed higher proliferation levels. In addition by qPCR technique it was verified a larger expression of genes involved in the initial stages of myelopoiesis in the AIRmax BM cultures. The analysis of BM cellular protein content by 2D gel electrophoresis revealed quantitative and qualitative differences between two mouse lines.

Tumor necrosis factor α (TNFα) is a pleiotropic cytokine that is predominantly produced by activated macrophages. The effects of TNFα are as diverse as the cells with which it interacts, e.g., stimulating fibroblast growth, exerting cytotoxic/cytostatic; activity against various human and murine cell lines, promoting inflammation through upregulation of endothelial adhesion molecules and IL-8 production. Yet TNFα is best known, and in fact was originally described, for its role in the bacterial-induced hemorrhagic necrosis of tumors and exacerbation of septic shock in which aberrant TNFα production leads to vascular collapse, cachexia, multiple organ failure, and ultimately death in as many as 100,000 people each year in the United States alone. LPS, a component of the outer cell wall of gram-negative bacteria, is the principal inducer of macrophage TNFα production. TNFα production can be enhanced by IFNγ which also induces upregulation of TNFα receptors allowing for the establishment of a TNFα autocrine loop. It has been hypothesized that autocrine TNFα stimulation plays a critical role in the induction of macrophage effector function, e.g., nitric oxide production. This dissertation represents efforts to evaluate the respective roles of the TNFα receptors in the induction of macrophage effector function, in addition to examining the mechanism by which autocrine TNFα exerts its effects on macrophages. Exploiting the species specificity of the murine TNFα receptor type 2 (TNF-R2), splenic macrophages were stimulated with human TNFα (which binds to TNF-R1 but not TNF-R2), in the presence of IFNγ. Human TNFα was effective in the induction of nitric oxide production, albeit at concentrations 12.5-fold greater than those required by murine TNFα to achieve the same result. Addition of anti-TNF-R1 completely inhibited the murine TNFα mediated induction of macrophage effector function. However, treatment with anti-TNF-R2 resulted in partial inhibition of macrophage activation. Taken together this data suggests that the primary TNFα mediated signals involved in macrophage activation are transduced through TNF-R1, although TNF-R2 appears to contribute to the intensity of the macrophage response. To evaluate the role of autocrine TNFα signaling in the induction of macrophage effector function, immortalized macrophages from normal C57Bl/6J mice (B6/J2) and C57Bl/6J mice containing gene targeted disruptions of the TNF-R1 and TNF-R2 genes (TRN) were stimulated under CD14-dependent and CD14-independent conditions. Although the B6/J2 and TRN clones mounted similar NO responses to LPS in the presence of serum, the TRN macrophages generated a weak nitric oxide response as compared to B6/J2 when stimulated with LPS under serum-free conditions. The involvement of TNFα autocrine stimulation in the CD14-independent activation was corroborated by the ability of soluble TNF-R1 to inhibit the response of B6/J2 macrophages to LPS in serum-free medium. CD14-independent LPS stimulation of TRN and B6/J2 resulted in equivalent levels of IL-1β, TNFα, and NOS gene expression, as determined by RT-PCR, and in release of equivalent amounts of biologically active TNFα. However, western blot analysis revealed that NOS protein production by TRN was as much as 50% less than that produced by B6/J2. These results indicate that autocrine TNFα stimulation contributes to the signaling pathways initiated by ligation of CD14-independent LPS receptors and may be involved in NOS post-transcriptional regulation.

The pathophysiology of chronic morphine on the immune system, especially on the cells of the innate immune system that leads to an immune compromise state has not been fully elucidated. The cells of the innate immune system are the first line of defense in mounting an immune response needed in infections, inflammation, cancer development, etc. One of the ways by which these innate immune cells act is by the production of cytokines with direct effects and to also recruit other immune cells, as required. A balance of pro- and anti-inflammatory cytokines is necessary for immune competence. I hypothesized that chronic morphine would act via a classical opioid receptor to stimulate the PI3K/Akt/Gsk3β pathway to produce predominantly anti-inflammatory cytokines. Cytokine gene expression levels were assessed via RT-PCR; Akt and Gsk3β protein levels measured using indirect ELISA. The data suggests that chronic morphine causes a significant reduction in IL-6 production, but does not act via the Akt/Gsk3β pathway or the classical opioid receptor to cause this effect in microglia cells.

Les macrophages sont des cellules immunitaires innées présentes dans chaque organe. Ils sont des cibles thérapeutiques dans de nombreuses maladies, dont le cancer. En dépit de travaux récents sur l'origine des macrophages, les mécanismes régulant leur différenciation sont mal définis. L'expression de Jun, membre de la famille AP-1, augmente pendant la différenciation des macrophages, mais son rôle dans ce processus n'est pas connu.Au cours de mon doctorat, nous avons caractérisé le rôle de Jun dans le développement et l'homéostasie des macrophages, dans un modèle de souris avec délétion conditionnelle de Jun dans la lignée myéloïde (JunΔCsf1r). Nous montrons que Jun contrôle la différenciation, induite par CSF1, des monocytes en macrophages. In vivo, Jun régule l'accumulation de macrophages dérivés de monocytes dans les poumons et intestins. Les macrophages associés aux tumeurs (TAMs) jouent un rôle crucial dans la progression des cancers. L’absence de Jun freine la croissance d’un mélanome et la différenciation, induite par CSF1, des TAMs dérivés de monocytes qui participent à l’angiogénèse tumorale. Cependant, lors d'une inflammation aiguë, Jun n’affecte pas le recrutement de macrophages inflammatoires.En conclusion, nos résultats identifient Jun comme un régulateur central de la différenciation des macrophages. Dans un modèle de mélanome, les macrophages Jun-dépendants exercent des fonctions pro-tumorales. Le fait que Jun soit un régulateur sélectif du développement des macrophages dépendants de CSF-1 permettra de définir de nouvelles approches ciblant sélectivement la différenciation des macrophages, sans altérer les réponses immunitaires dépendantes des monocytes. / Macrophages are immune cells present in every organ. Given their variety of functions, macrophages are therapeutic targets in many diseases including cancer. Despite the research efforts to characterise their origins, the molecular mechanisms regulating macrophage differentiation are still poorly defined. Expression of the AP-1 factor, Jun, increases during differentiation, but its role in macrophage development is not known.During my PhD, we characterised how Jun affects macrophage development and homeostasis. We developed a conditional mouse model in which Jun is deficient in the myeloid lineage (JunΔCsf1r). We showed that Jun controls CSF1-mediated monocyte to macrophage differentiation, proliferation and survival. In vivo, Jun loss limits macrophage accumulation in lungs and intestine. Tumour-associated macrophages (TAMs) play critical roles in cancer progression. We observed that Jun deficiency dampens melanoma growth and the differentiation of CSF1-dependent monocyte-derived TAMs. We further showed that Jun-dependent TAMs mediate vessel normalisation in melanoma. During inflammation, Jun was dispensable for the recruitment of monocyte-derived inflammatory macrophages.Altogether, our results identify Jun as a master regulator of macrophage differentiation, without altering monocyte effector functions. In a melanoma model, we showed that Jun-dependent TAMs play tumour-promoting roles. Therefore, Jun is a selective regulator of CSF-1-dependent macrophage development, which is redundant during inflammation; this observation should help to define novel approaches to selectively target macrophage differentiation, without altering monocyte-dependent immune responses.

Improvements in medical care over recent decades have increased the number of premature and low birth weight infants that survive hypoxic-ischemic (H-I) insults. Because there is a rising incidence in diseases associated with these events, it is critical to develop effective therapies to treat the various resulting neuropathies. Extracellular matrix constitutes the majority of brain parenchyma. Lecticans and matrix-degrading proteases including ADAMTSs (a disintegrin and metalloproteinase with thrombospondin repeats) and matrix metalloproteinases (MMPs) exert effects on cell viability and may be associated with either protective or destructive processes after H-I. Both ADAMTSs (Cross et al. 2006; Tian et al. 2007) and MMPs (del Zoppo et al. 2007; Gu et al. 2005; Rosenberg et al. 2001) have been associated with pathological states in brain, yet the relative contributions of lecticans, ADAMTSs and MMPs to inflammation and cell death remain unknown. In the present study, the first series of experiments were conducted to characterize cellular damage and neuroinflammation in the postnatal day 7 rat after exposure to H-I, and to determine if cell death and inflammation were associated with alterations in lectican expression. Data showed that reduced brevican expression occurred 4 days after H-I in lesioned hippocampus. Additionally, reduced versican expression in white matter was concomitant with pre-OL cell death at this endpoint. In contrast, both lecticans were elevated at later endpoints (14, 21 days) that were associated with increased neuroinflammation and cavitary infarction. These data suggest that lectican loss is associated with cell death at the early endpoint, whereas increased lectican deposition over time likely leads to glial scar formation and a reduced capacity for neuroplasticity. Two subsequent series of experiments were conducted to determine the relative contributions of matrix-degrading proteases to injury, and whether proteolytic activity was associated with neuroinflammatory events. The first objective was to determine whether treatment with AG3340, a selective inhibitor of gelatin-degrading MMPs, or the anti-inflammatory compound minocycline, could provide neuroprotection when administered at a delayed time point after insult, and to compare the efficacy of AG3340 with that of the well-known anti-inflammatory compound minocycline. Data showed that both compounds effectively dampened the recruitment of microglia/macrophages to the lesion site when administered 24 hrs after H-I. These effects were associated with reduced neurodegeneration, indicating that these compounds neuroprotect at a clinically relevant time point. The final series of experiments tested whether these compounds could neuroprotect in an ex vivo model of oxygen glucose deprivation (OGD) that lacks peripheral immune cell involvement, thus providing insight into the relative contributions of resident microglia and gelatinase activity to the inflammatory sequelae. Results showed that both compounds blocked the OGD-induced increase in gelatinase activity and were neuroprotective in the absence of peripheral immune cells. Taken together, these data indicate that resident microglia contribute to H-I injury through gelatinase activation. Thus, the present study demonstrates that gelatin-degrading MMPs are important targets to consider when developing therapies to combat neonatal H-I injury.

Biomaterials are becoming the most commonly used therapeutic method for treatment of lost or damaged tissue in the body. Metallic materials are chosen for high strength orthopaedic and dental applications. Titanium (Ti) implants are highly successful in young, healthy patients with the ability to fully integrate to surrounding tissue. However the main population requiring these corrective treatments will not be healthy or young, therefore further research into material modifications have been started to improve outcomes in compromised patients. The body’s immune system will generate a response to any implanted material, and control the final outcome. Among the first and most influential, cells to interact with the implant will be macrophages. Throughout this study we have 1) established the ability of macrophages to recognize and differentially activate in response to material surface properties, 2) investigated the role of integrin binding in macrophage activation to material properties, and 3) confirmed the importance of macrophage activation in vivo following Ti implant placement. The generation of a hydrophilic implant surface promoted the greatest anti-inflammatory and pro-regenerative macrophage activation. Surface wettability will control protein adsorption which can activated different integrin binding on macrophages and may be responsible for changes in activation. When integrin β3 subunit binding was prevented hydrophilic surfaces no longer promoted an anti-inflammatory macrophage activation. Additionally, when macrophage levels were reduced using two separate ablation models, MaFIA mice and clodronate liposomes, hydrophilic surfaces no longer promoted anti-inflammatory T-cell populations and cytokine profiles. There were also fewer stem cells adhered to implant surfaces at 1, 3, and 7 days when macrophage populations were compromised.