Macrophages are dynamic, plastic, and heterogeneous immune cells that play an important role in host immune defense against bacterial infection. Various bacterial pathogens, such as Neisseria meningitidis and Mycobacterium tuberculosis, can modulate host immune responses by interfering with macrophage differentiation and polarization. The focus of this thesis was to understand the role of macrophages in the pathogenesis of bacteria-induced diseases, which has important implications in the search for novel therapeutic strategies to control those infectious diseases. In Paper I, we found that NhhA, a conserved meningococcal outer membrane protein, can activate macrophages through both Toll-like receptor 4 (TLR4)-dependent and -independent pathways. In Paper II, we demonstrated that NhhA activates monocytes through TLR2 and triggers autocrine IL-10 and TNF production through the ERK and JNK pathways, which skew monocyte differentiation into CD200Rhi macrophages. These immune homeostatic macrophages are associated with nasopharyngeal carriage of meningococci. In Paper III, we examined the role of human CD46, a ubiquitous transmembrane protein, in regulating macrophage apoptosis, differentiation, and functional polarization. We revealed that macrophages expressing CD46 exhibit an M1 phenotype and are prone to generate proinflammatory cytokines, such as IL-6, TNF, and IL-12, upon lipopolysaccharide challenge or meningococcal infection. The important role of these macrophages in the development of septic shock was further confirmed by in vivo studies using a CD46 transgenic mouse disease model. M. tuberculosis, a gram-positive bacterium, remains an important cause of death in developing countries. In Paper IV, we reported that murine macrophages expressing human CD46 exhibit enhanced viability and bactericidal capacity and are prone to form granulomas following chronic mycobacterial infection. Increased understanding of host factor roles in the physiopathology of tuberculosis is critical for the design of effective vaccines and new drugs. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

De nombreuses études menées chez l’homme ont montré que l’obésité est associée à un état inflammatoire chronique caractérisé par une augmentation de la sécrétion de nombreuses molécules dont la leptine et des cytokines inflammatoires comme le TNF-a et l’IL-6 (Clement et al., 2004). Des données récentes suggèrent que cette inflammation périphérique pourrait également présenter une composante au niveau cérébral se caractérisant notamment par une augmentation de l’expression de différentes cytokines inflammatoires (IL-6, TNF-a, IL-1ß…) et de l’activation de leurs voies de signalisation intracellulaire (augmentation de l’activité c-Jun-N-terminal kinase et de NFkB)(De Souza et al., 2005). De plus, l’intensité de la situation inflammatoire semble être liée au degré d’obésité. Ainsi, il est possible de distinguer différentes situations d’obésité : une obésité modérée qui ne s’accompagne pas forcément de pathologies comorbides et une obésité morbide associée à différents types de complications comme des maladies cardio-vasculaires, de l’hypertension artérielle ou un diabète de type 2. L’obésité s’accompagne également d’une forte prévalence de troubles de l’humeur (anxiété, dépression) et de la cognition. Notre laboratoire a été un des pionniers dans l’étude de l’expression et de l’action des cytokines au niveau central et de leurs conséquences, tant comportementales que neurobiologiques. Cette relation entre système de l'immunité innée et cerveau a particulièrement été étudiée dans le cadre du comportement de maladie regroupant un ensemble de symptômes non spécifiques (fièvre, activations neuroendocriniennes, anorexie, anhédonie, repli sur soi, perte d’intérêt pour l’environnement…) observés chez les individus malades et pouvant être reproduits chez l’animal en réponse à l’injection d’un inducteur de cytokines tel que le lipopolysaccharide (LPS)(Dantzer, 2001). Dans le cas d’une exposition prolongée ou non régulée de l’activation du réseau de cytokines, le comportement de maladie peut laisser place à de véritables troubles de l’humeur et de la cognition associés à une chute des taux circulants de tryptophane, un acide aminé essentiel servant de précurseur et de facteur limitant à la synthèse de sérotonine. Il a été montré que l'indoléamine 2,3-dioxygénase (IDO), une enzyme dégradant le tryptophane en réponse aux cytokines (Lestage et al., 2002; Moreau et al., 2005) est impliquée dans l’induction des symptômes de type dépressif observés notamment suite à production soutenue de cytokines et que cette action serait dépendante du catabolisme du tryptophane via la voie de la kynurenine (O'Connor et al., 2008). L’activation de l’IDO en situation inflammatoire aboutit à la production de dérivés neurotoxiques (3-OH-kynurénine, acide quinolinique) se comportant comme des agonistes des récepteurs glutamatergiques de type NMDA (Taylor and Feng, 1991), au dépend de la production de sérotonine. Ainsi, l’activation de l’IDO par les cytokines pourrait jouer un rôle dans l’apparition de troubles cognitifs associés aux états inflammatoires via l’altération de la neurotransmission sérotoninergique et/ou glutamatergique. Ces mêmes mécanismes pourraient également sous-tendre le développement des troubles de l’humeur et de la cognition couramment observés chez les personnes obèses. L’ensemble des études réalisées dans ce travail de thèse a donc eu pour objectif général de déterminer chez la souris si l’inflammation chronique à bas bruit qui est associée à un état d’obésité entraînait le développement de troubles de l’humeur et de la cognition [...]. / Severe obesity is associated with a low grade inflammation characterized by an increased release of inflammatory markers like cytokines and leptin. It has been suggested that some of these mediators of inflammation could also be found in the brain, as manifested by the increased hypothalamic expression of inflammatory cytokines (IL-6, TNF-a, IL-1) and the activation of their intracellular pathways. Moreover, the intensity of the inflammation state seems to increase with the degree of obesity. Morbid obesity, which is accompanied by different comorbid pathologies like cardiovascular disease, hypertension, type 2 diabetes and a high prevalence of mood (anxiety, depression) and cognitive disorders, is clearly associated with peripheral inflammation. Such an association is less clear in the case of a moderate obesity which is not systematically associated with comorbid pathologies. It is clearly established that during an infection brain actions of cytokines that are released as a result of the innate immune system activation induce development of sickness behaviour. In the case of a prolonged and/or unregulated activation of the cytokine network, sickness behaviour that includes non-specific symptoms such as behavioral alterations, fever and neuroendocrine activation can lead to the development of mood and cognitive disorders. Moreover, such a development is associated with a drastic drop of circulating levels of tryptophan, the essential amino acid acting as limiting factor of the serotonin synthesis. It has been proposed that these alterations could be at least partially explained by cytokine-induced peripheral and/or central activation of the indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme that is potently induced in monocytes, macrophages and brain microglia by cytokines. IDO activation can result in the lowering of the bioavailability of tryptophan for 5-HT synthesis and the increase of neurotoxic derivates (3-OH-kynurenine, quinolinic acid). Both consequences of cytokine-induced IDO activation may play a role in the development of the cognitive and mood disorders associated with obesity. The present study aimed therefore at studying in mice the relationship between inflammation and development of mood and cognitive disorders associated with obesity. This study was performed in two different but complementary experimental conditions reproducing 1) a moderate obesity devoid of marked pathological complications (a model of diet induced obesity) and 2) a morbid obesity associated with comorbid pathologies like type 2 diabetes (db/db mice). Our results showed that: 1) The degree of obesity is correlated with the intensity of the alterations affecting innate immune system activation. 2) Obesity exacerbates the innate immune system activation as manifested by the increase of peripheral and central cytokine production, and related neurochemical, neuroendocrine and behavioral alterations. 3) The inflammation-related alterations induced by obesity are associated with impairment of cognitive abilities and emotional reactivity, as well as development of anxiety-like symptoms, although differences in their respective time-course of appearance seem to exist. Taken together, these findings showed the key role of the inflammation associated with obesity in its related mood and cognitive disorders. This work provides therefore a first important step towards the identification of new pharmacological and/or nutritional strategies aimed at ameliorating life quality of obese subjects and preventing development of related comorbidities.

TNF-a is an important cytokine mediator of inflammation which suggests that inhibition of TNF-a activity may provide potential for clinical application. Recent data indicated that treatment of both human and mouse cells with Kavain significantly modulates P. gingivalis- and LPS-induced TNF-α expression. In order to obtain a selective analog with optimized biological activity and structural physico-chemical properties of Kavain, Kavain analogs were designed and synthesized and found one Kavain analogue (named Kav001) that is similar to Kavain but soluble and does not induce a significant toxicity. Both studies in vitro and in vivo treatment by Kav001 showed stronger biological function as compared to Kavain. Furthermore, most mouse bone marrow macrophages up-regulated Bcl-6 while down-regulating LITAF expression after treatment with Kav001 for 36 h. Consequently, this led to an extension of macrophage pseudopods due to its immune response to P.g. infection/ LPS stimulation. we further found that Kav001 not only inhibits TNF-α, but also IL-1β, IL-6, caspase 1 and neutrophil infiltration in response to LPS. However, this phenomenon cannot be observed when macrophages were treated with LPS plus Kavain. We believe that Kav001 may mediate a novel link between Kav001 and LPS-induced inflammation and may be used as a key inhibitor to LPS-induced inflammation/inflammatory disease.

by Chan Shuk Chong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 135-142). / Statement --- p.i / Acknowledgment --- p.ii / Abbreviations --- p.iii / Abstract --- p.iv / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Haematopoiesis --- p.1 / Chapter 1.1.1 --- Sites of haematopoiesis / Chapter 1.1.1.1 --- Bone marrow stroma / Chapter 1.1.1.2 --- Thymus / Chapter 1.1.1.3 --- Spleen and lymph node / Chapter 1.1.1.3.1 --- Spleen / Chapter 1.1.1.3.2 --- Lymph Nodes / Chapter 1.1.2 --- Blood Cell / Chapter 1.1.2.1 --- Development of T and B cells / Chapter 1.1.2.1.1 --- T cells / Chapter 1.1.2.1.2 --- B cells / Chapter 1.1.2.2 --- Development of Granulocytes and monocytes / Chapter 1.2 --- White Cell Disorder -Leukemia --- p.13 / Chapter 1.2.1 --- Leukemia - general concept / Chapter 1.2.1.1 --- Classification of leukemia / Chapter 1.2.1.2 --- Pathophysiology and Clinical features / Chapter 1.2.1.3 --- Etiology of myeloid leukemia / Chapter 1.2.2 --- Genetic basis of leukemia / Chapter 1.3 --- Acute myeloid leukemia (AML) cell model --- p.19 / Chapter 1.3.1 --- Cell Model for human acute myeloid leukemia / Chapter 1.3.2 --- Murine leukemia cell lines / Chapter 1.4 --- Induction of leukemia cell differentiation --- p.21 / Chapter 1.4.1 --- Overview of different inducers / Chapter 1.4.2 --- Cytokines as Inducers / Chapter 1.5 --- Objectives and Research Strategy --- p.26 / Chapter 1.5.1 --- Objectives / Chapter 1.5.2 --- Research strategy / Chapter Chapter 2 : --- Materials and Methods / Chapter 2.1 --- Materials --- p.29 / Chapter 2.1.1 --- Cell line / Chapter 2.1.2 --- Tissue culture medium / Chapter 2.1.3 --- Tumor necrosis Factor - alpha (TNF-α) / Chapter 2.1.4 --- Interleukin 1- alpha (IL-lα)and Interleukin 1- beta (IL-1β) / Chapter 2.1.5 --- "Monoclonal hamster anti-mouse IL-lα monoclonal hamster anti-mouse IL-1β, and Polyclonal rabbit anti-mouse TNF-α antibodies" / Chapter 2.1.6 --- Lipopolysaccharides (LPS) / Chapter 2.1.7 --- Buffers and solutions / Chapter 2.2 --- Methods : --- p.33 / Chapter 2.2.1 --- Cell culture / Chapter 2.2.2 --- Cytotoxicity assay / Chapter 2.2.3 --- Proliferation assay / Chapter 2.2.4 --- Cell morphology / Chapter 2.2.5 --- Phagocytosis assay / Chapter 2.2.6 --- Preparation of undifferentiated and differentiated murine leukemia WEHI3B (JCS) cells for cell lysate / Chapter 2.2.7 --- Isolation of total cellular RNA / Chapter 2.2.8 --- Extraction of the total RNA / Chapter 2.2.9 --- Spectrophotometry / Chapter 2.2.10 --- Electrophoresis of RNA in agarose gel containing formaldehyde / Chapter 2 2.11 --- First strand cDNA synthesis / Chapter 2.2.12 --- Cytokines phenotyping of the uninduced and induced WEHI 3B (JCS) by The Reverse Trancription Polymerase Chain Reaction method / Chapter 2.2.13 --- Gel electrophoresis of PCR- product / Chapter 2.2.14 --- Southern blot / Chapter 2.2.15 --- Dot blot / Chapter 2.2.16 --- Hybridization with oligonucleotides / Chapter 2.2.17 --- Chemiluminescent detection / Chapter Chapter 3 : --- Growth Inhibitory and Differentiation Effects of Lipopolysaccharides ( LPS ) on WEHI 3B (JCS) cells / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.1.1 --- Chemical structure of LPS / Chapter 3.1.2 --- Biological activity of LPS / Chapter 3.2 --- Results --- p.55 / Chapter 3.2.1 --- Anti-proliferative effects of LPS / Chapter 3.2.2 --- Differentiation inducing effect of LPS on WEHI 3B (JCS) cells / Chapter 3.2.3 --- Phagocytic activity LPS treated WEHI 3B (JCS) cells / Chapter 3.2.4 --- Anti-proliferative effect of TNF-α / Chapter 3.2.5 --- Differentiation inducing effect of TNF-α / Chapter 3.2.6 --- Phagocytic activity of TNF-α treated WEHI3B (JCS) cells / Chapter 3.3 --- Discussion --- p.67 / Chapter 3.4 --- Summary --- p.69 / Chapter Chapter 4 : --- The Cytokine Genes Expression of the TNF-α and LPS Treated WEHI 3B (JCS) cells / Chapter 4.1 --- Introduction --- p.70 / Chapter 4.1.1 --- Differentiation of leukemia cell line / Chapter 4.1.2 --- Study of the cytokine genes expression of WEHI 3B (JCS) cells / Chapter 4.2 --- Results --- p.72 / Chapter 4.2.1 --- Isolation of total RNA from uniduced and induced WEHI 3B (JCS) cells / Chapter 4.2.2 --- The cytokine genes expression during differentiation / Chapter 4.2.2.1 --- "Up-regulation of IL-lα, IL-1β,TNF-α and IFN-γ in both TNF-α induced and LPS induced WEHI 3B (JCS) cells" / Chapter 4.2.2.1.1 --- Southern blot / Chapter 4.2.2.1.2 --- Semi-quantitation of PCR-products by gel electrophoresis and dot-blot hybridization / Chapter 4.2.2.2 --- up-regulation of GM-CSF and G-CSF in LPS induced WEHI 3B (JCS) cells / Chapter 4.3 --- Discussion --- p.92 / Chapter 4.4 --- Summary --- p.95 / Chapter Chapter 5 : --- Growth inhibitory and Differentiation Inducing Effect of IL-l( IL-1α and IL-1β) on WEHI 3B (JCS) cells / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.1.1 --- The interleukin 1 (IL-1) family / Chapter 5.1.1.1 --- Structure of IL-1 / Chapter 5.1.1.2 --- The biological function of IL-1 / Chapter 5.1.2 --- Tumor necrosis factor - alpha ( TNF-α) / Chapter 5.1.2.1 --- Structure of TNF-α / Chapter 5.1.2.2 --- Biological functions of TNF-α / Chapter 5.1.3 --- The similarity between TNF and IL-1 / Chapter 5.2 --- Results --- p.102 / Chapter 5.2.1 --- Anti-proliferative effect of IL-1 / Chapter 5.2.2 --- Differentiation inducing effect of IL-1 / Chapter 5.2.3 --- Phagocytic activity of IL-1 treated JCS cells / Chapter 5.2.4 --- "Role of endogenously produced IL-lα, IL-1β and TNF-α in LPS cytokines differentiation of WEHI 3B (JCS) cells" / Chapter 5.2.4.1. --- "Effect of neutralizing anti- ILl-α,anti - IL-l-β, and anti-TNF-α antibodies on the growth inihbition of the treated WEHI 3B (JCS) cells" / Chapter 5.2.4.2 --- "Effects of neutralizing anti-IL-lα, anti- IL-1β, and anti-TNF-α antibodies on differentiation of the treated WEHI 3B (JCS) cells" / Chapter 5.3 --- Discussion --- p.124 / Chapter 5.4 --- Summary --- p.127 / Chapter Chapter 6 --- : Concluding Discussion --- p.128 / References --- p.135

Orientador: Deilson Elgui de Oliveira / Banca: Ricardo Sobhie Diaz / Banca: Celso Teixeira Mendes Júnior / Resumo: A história natural de infecção pelo HIV e a progressão para a aids podem variar entre diferentes indivíduos, possivelmente devido a fatores genéticos, entre eles os polimorfismos de nucleotídeo simples (SNPs). SNPs localizados em regiões promotoras de genes que codificam citocinas podem afetar a síntese e a regulação dessas moléculas, resultando em alterações nas respostas imunitárias. O presente estudo buscou avaliar as frequências e os possíveis efeitos de SNPs nas posições -589 e -1098 da região promotora do gene da IL-4 e SNPs nas posições -238 e -862 da região promotora do gene do TNF-α em pacientes portadores do HIV. Amostras de DNA de 157 pacientes foram obtidas através de células mononucleares de sangue periférico e a genotipagem dos SNPs foi realizada pela técnica de High Resolution Melting (HRM). Foi observado que pacientes portadores de TT em SNP/pIL-4 -589 apresentaram contagem de linfócitos T CD8 + menor em relação aos portadores de CC (p=0.0104). Além disso, portadores de TT em SNP/pIL-4 -1098 apresentaram contagem de linfócitos T CD8 + maior em comparação aos portadores de GT (p=0.0053). Em relação a SNP/pTNF-α -238, as proporções de pacientes portadores de GG e GA diferiu entre os pacientes sem HAART e pacientes com HAART e sem falha terapêutica (p=0.0205). Assim, os resultados obtidos no presente estudo fortalecem a hipótese de que SNPs em genes de citocinas podem alterar a história natural da infecção pelo HIV e o curso clínico da doença, principalmente devido a alterações no balanço da produção de citocinas pro- e antiinflamatórias. / Abstract: The natural history of HIV infection and its progression towards aids may vary considerably among different individuals, possibly due to genetic factors, such as single nucleotide polymorphisms (SNPs). In cytokines genes promoters, SNPs may affect protein synthesis and regulation, resulting in more or less efficient immune responses against HIV. The present study evaluated the frequencies and possible effects of SNPs in the IL-4 gene promoter at positions -589 and -1098 and in the TNF-α gene promoter at positions -238 and -862 in HIV-infected patients from Brazil. DNA samples from 157 patients were obtained from peripheral blood mononuclear cells, and SNPs genotyping was performed by High Resolution Melting analysis (HRM). Patients carrying TT at SNP/pIL-4 -589 had lower circulating T CD8 + cells compared to CC carriers (p=0.0104). Moreover, carriers of TT at SNP/pIL-4 -1098 had more circulating T CD8 + cells compared to GT carriers (p=0.0053). Regarding SNP/pTNF-α -238, GG and GA proportions were significantly different between patients without HAART and patients on HAART without therapeutic failure (p=0.0205). In conclusion, these results provide compelling evidence that the presence of SNPs in cytokine-coding genes do modify the natural history of HIV infection, mainly due to changes in the balance between pro- and anti-inflammatory cytokines. / Mestre

Human immunodeficiency virus (HIV) and substance abuse (drug and/or alcohol) independently impair the immune system; importantly, the combination of HIV infection and substance abuse might produce more than an additive effect on this system. Tumor necrosis factor-alpha (TNF-?) and Interferon gamma (IFN?) are pro-inflammatory cytokines involved in differentiation of Th0 cells into Th1 cells. Interleukin 4 (IL-4) and Interleukin 10 (IL-10) are anti-inflammatory cytokine involved in differentiation of Th0 cells to Th2 cells. Unbalanced Th1 and Th2 cells can lead to immune suppression. Thus, changes in these cytokines could have important implications for people infected with HIV (HIV+). Resistance training can counteract muscle wasting, improve strength, and improve muscle mass. The purpose of this study was to examine the effect of resistance training on resting concentrations of circulating TNF-?, IFN-?, IL-4, and IL-10. Sixteen men (42 ± 11 years, 180.4 ± 9.1 cm, 89.2 ± 20.7 kg) infected with HIV and enrolled in an intensive 60-day in-patient substance addiction/abuse treatment program were recruited shortly after admission to the treatment facility. Participants were assigned to one of two groups using randomization: supervised resistance exercise 3 times per week using a progressive and non-linear periodized program (Exercise) or no exercise training (Non-Exercise) for six weeks. Before (Pre) and after (Post) the 6-week period, resting and fasted blood samples were obtained and analyzed for serum TNF-?, IFN-?, IL-4, and IL-10 concentrations using a high-sensitivity ELISA. TNF-? did not change following the 6-week period for Exercise (Pre: 4.8 ± 2.7 pg·ml-1; Post: 4.6 ± 2.4 pg·ml-1) or Non-Exercise (Pre: 3.0 ± 1.3 pg·ml-1; Post: 2.7 ± 0.8 pg·ml-1). IFN-?, IL-4, and IL-10 concentrations were below detectable limits. No adverse effects of the intervention were reported. A six-week resistance training program does not elicit changes in circulating TNF-? concentrations in men infected with HIV and undergoing an intensive in-patient substance addiction/abuse treatment program. Concentrations of IFN-?, IL-4, IL-10 were below detectable levels. Six weeks of resistance training was not sufficient to affect circulating TNF-?, nor enough to increase concentrations of IFN-?, IL-4, IL-10 to detectable levels. The lack of adverse effects shows that adding resistance training to the current in-patient treatment regimen for substance abuse among men infected with HIV is safe.

Growing tissues are communities of cells that cooperate to form a robust, functional organ. Cooperative behavior is enforced by cell competition, wherein comparisons of fitness lead to selective elimination of cells sensed as relatively less healthy. Elimination of these ‘loser’ cells from Drosophila wing imaginal discs results in cell death induced by deployment of a genetic module consisting of the secreted Toll ligand Spätzle (Spz), several Toll related receptors, and NFkB factors. How signaling by this module is activated and restricted only to competing cells is unknown. Here, we investigate the signaling role of Spz in Myc-induced cell competition. We demonstrate that elimination of wild-type loser cells requires local synthesis and activation of Spz in the wing disc. We identify Spätzle Processing Enzyme (SPE) and Modular Serine Protease (modSP) as upstream mediators of Spz-mediated loser cell elimination, and show that an increase in SPE in ‘winner’ cells is required for Spz to kill loser cells. Finally, we show that Spz requires both Toll and Toll-8 to induce apoptosis of wing disc cells. Our results indicate that during cell competition, Spz-mediated signaling is strictly confined to the imaginal disc, allowing errors in tissue fitness to be corrected without compromising organismal physiology.

Arthritis is a pathological condition whereby a persistent inflammatory response leads to breakdown of articular cartilage in synovial joints. Cartilage is a specialised avascular tissue containing chondrocytes embedded in an extracellular matrix. The cartilage matrix is composed of collagen to provide strength with aggregated proteoglycan to facilitate hydration. Cartilage has been reported to loose proteoglycans with concordant loss of integrity observed in arthritic disease pathology. Proteoglycans loss from cartilage has also been reported in in vitro models. Application of interleukin-1 (lL-1β) to cartilage in vitro has been demonstrated to increase loss of proteoglycans and modulate production of inflammatory mediators such as Nitric Oxide (NO) and Prostaglandin-E2 (PGE2). NO and PGE2 have also been associated with cartilage breakdown. Other cytokines such as colony stimulating factors (CSFs) may regulate cartilage function. The aim of this study was to select a cartilage explant system and compare the effects of interleukin-1 (lL-1) with those of colony stimulating factors (CSFs) by measuring the production of NO and PGE2 and release of proteoglycans. It was found that IL-1β increased PGE2 and NO production, but not loss of proteoglycans from rat cartilage explants. Granulocyte-CSF (G-CSF) and lL-3 increased production of NO and PGE2, respectively. When combined, IL-1β / Granulocyte-Macrophage (GM-CSF) increased production of PGE2 and G-CSF / IL-1β produced increased proteoglycan loss from explants. The model was then modified by integrating Swiss 3T3 Fibroblasts monolayers with explants. Fibroblasts were initially screened to determine their separate response to these cytokines. Fibroblasts did not release proteoglycans into the culture media, but produced elevated concentrations of NO and PGE2 in response to IL-lβ.·Fibroblast-cartilage co-cultures treated with IL-lβ produced increased NO, PGE2 and proteoglycan release. G-CSF, GM-CSF and IL-3 caused increased levels of PGE2 in co-cultures, however, IL-1β was required to generate significant proteoglycan loss from cartilage explants. Finally, extra-cellular signal related protein kinases I and 2 (ERK 1&2) and p38 intracellular signalling pathways were shown to be involved in IL-1β mediated production of NO fibroblasts and explants. These studies show that IL-1β has increased potential to mediate cartilage breakdown when interacting with other cytokines, such as G-CSF, and other cell types, such as Swiss 3T3 fibroblasts. IL-1β has defined intracellular signalling pathways that may produce a range of responses in cartilage explants and fibroblasts. These studies may relate to production of inflammatory processes and loss of cartilage integrity and function in pathological conditions.

Orientador: Vera Fernanda Martins Hossepian de Lima / Coorientador: João Paulo Elsen Saut / Banca: Ricarda Maria dos Santos / Banca: Lindsay Unno Gimenes / Banca: Maria Emília Franco Oliveira / Banca: Érica Azevedo Costa / Resumo: De modo geral, o objetivo deste estudo foi determinar se as células endometriais bovinas responderam a análogos virais de padrões moleculares associados a patógenos (PAMPs) mediante a produção de citocinas pró-inflamatórias após ativadas pelos receptores "Toll-Like" (TLRs) no endossoma celular e no citoplasma celular pelo genes indutores de ácido retinóico tipo I (RIG-I). No primeiro experimento, amostras uterinas de vacas de corte mestiças pós-púberes foram dissectadas para obtenção de células endometriais epiteliais e estromais. Um controle negativo e quatro PAMPs: LPS, ssRNA, Poly I:C (LMW), Poly (I:C) HMW foram utilizados. Dois grupos de tratamentos (transfectados e não transfectados) foram analisados durante 24 horas. Em outro experimento, células endometriais foram tratadas apenas com o PAMP Poly (I:C) LMW e um grupo Controle Negativo. Neste, os grupos foram incubados às 0, 2, 6, 12, 24, 36, 48 e 72 horas. Sobrenadantes foram colhidos para desenvolver o teste de ELISA para IL-6 e IL-8. Células epiteliais produziram IL-6 em resposta ao Poly I:C (HMW) quando comparadas com o Controle (Grupo DOTAP positivo; P< 0.05), enquanto que o LPS induziu produção de IL-6 e IL-8 em células estromais (P< 0.05). O uso de um reagente de transfecção entre as células e tratamentos demonstrou efeito (P> 0.05). Ainda, células estromais tratadas por Poly I:C (LMW) demonstraram uma maior produção de IL-6 às 48 e 72 horas (P< 0.05), e para o IL-8 às 6, 12, 24, 36, 48 e72 horas quando comparadas com o grupo Controle (P< 0.05). No segundo experimento, outras amostras uterinas de vacas de corte pós-púberes foram utilizadas. A obtenção de células endometriais estromais e epiteliais foram isoladas pelo mesmo protocolo do primeiro experimento. O PAMP Poly (I:C) LMW e um controle negativo foram utilizados. Proteínas para o RIG-I e p65 foram colhidas após 12, 24, 48 e 72 horas de tratamento / Abstract: In general, the objective of this study was to determine if bovine endometrial cells replied to virus analogs of pathogen associated molecular pattern (PAMPs) by production of proinflammatory cytokines after Toll-Like Receptor (TLR) activation in the cell endosome and after retinoic acid inducible gene - I (RIG-I) stimulation in the cell cytoplasm. In the first experiment, uterine samples from post pubertal cross-breed beef cows were dissected using a protocol to obtain epithelial and stromal cells. A negative control and four different PAMPs: LPS, ssRNA, Poly I:C (LMW), Poly (I:C) HMW were used. Two treatments (transfected and non-transfected) groups were investigated during 24 hours. In the other experiment, endometrial cells were treated with only Poly (I:C) LMW and a negative Control group. All incubated at 0, 2, 6, 12, 24, 36, 48 and 72 hours. Supernatants were collected to develop Elisa for IL-6 and IL-8. Epithelial cells produced IL-6 in response do Poly I:C (HMW) compared to Control (P< 0.05), otherwise, LPS induced IL-6 and IL-8 in stromal (P< 0.05). The transfection Reagent differ between cells and treatments (P> 0.05). Still, in stromal cells treated by Poly I:C (LMW) the production of IL-6 was higher at 48 and 72 hours (P< 0.05), and for IL-8 at 6, 12, 24, 36, 48 and 72 hours when compared to the Control (P< 0.05). In the second experiment, uterine samples from others post pubertal mixed-breed beef cows were used. To obtain stromal and epithelial cells, uterine samples were dissected with the same protocol as the first experiment. The PAMP Poly (I:C) LMW and a negative control were used. Proteins for RIG-I and p65 were collected after 12, 24, 48 and 72 hours. In response to Poly (I:C) LMW induction, stromal cells activated RIG-I at 48 hours (P< 0.05) were compared to the Control group. On the other hand, epithelial cells were not sufficient stimulated Poly (I:C) LMW to activate ... / Doutor

Mast cells have been implicated in the pathogenesis of both atopic and non-atopic asthma. Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a mast cell-restricted guanine nucleotide exchange factor and diacylglycerol (DAG)/ phorbol ester receptor whose function has not been deduced. RT-PCR analysis of 40 asthmatic patients and 40 non-asthmatic controls demonstrated a higher hRasGRP4 mRNA expression in a subgroup of the asthmatics. A RasGRP4-defective variant of the human mast cell line HMC-1 was used to create stable clones expressing green fluorescent protein-labeled human RasGRP4 for monitoring the movement of this signaling protein inside mast cells before and after exposure to phorbol-12-myristate 13-acetate (PMA) and for evaluating the protein???s ability to control the development, phenotype, and function of mast cells. Transcript-profiling approaches revealed hRasGRP4 constitutively regulates the expression of numerous genes in the HMC-1 cell line. For example, expression of hRasGRP4 in HMC-1 cells substantially decreased GATA-1 levels without altering GATA-2 levels, suggesting that hRasGRP4 regulates mast cell commitment of multipotential progenitors in part by controlling the intracellular levels of at least one lineage-dependent transcription factor for hematopoietic cells. hRasGRP4 resided primarily in the cytosol before HMC-1 cells were stimulated with PMA. After exposure to PMA, hRasGRP4 translocated to the inner leaflet of the cell???s plasma membrane and then to perinuclear and Golgi compartments. Extracellular signal-regulated kinases 1 and 2 were activated during this translocation process, and the PMA-treated cells transiently increased their expression of the transcripts encoding the interleukin 13 receptor IL-13R??2 and numerous other proteins. The accumulated data in our mast cell model suggest hRasGRP4 translocates to various intracellular compartments via its DAG/PMA-binding domain to regulate those signaling pathways that allow mast cells to respond quickly to changes in their tissue microenvironments.