La maladie de Fabry est une maladie de surcharge lysosomale liée au chromosome X. Elle est causée par une mutation au niveau du gène GLA qui provoque un déficit de l’enzyme α-galactosidase A. Elle entraîne une accumulation de glycosphingolipides tels le globotriaosylcéramide (Gb3), le globotriaosylsphingosine (lyso-Gb3) et galabiosylcéramide (Ga2) ainsi que leurs isoformes/analogues respectifs au niveau des tissus et des liquides biologiques des patients. Les symptômes peuvent se présenter de manière très variable soit par de l’acroparesthésie, des troubles ophtalmologiques, des angiokératomes, des troubles cardiaques, rénaux ou encore neurologiques. Les connaissances au niveau physiopathologique de la maladie de Fabry sont à ce jour, toujours déficientes. Les biomarqueurs analysés ne permettent pas de prédire la sévérité et la progression de la maladie. Afin d’apporter des connaissances plus approfondies à ce niveau, l’objectif principal de cette étude était d’analyser les biomarqueurs au niveau cellulaire pour des patients atteints de la maladie. Les objectifs secondaires étaient: 1) de développer et de valider une méthode en spectrométrie de masse en tandem pour la quantification relative et simultanée de certains biomarqueurs susmentionnés dans les leucocytes, les lymphocytes B et les monocytes de patients Fabry et contrôles sains; 2) d’évaluer les biomarqueurs dans le total des leucocytes, les lymphocytes B, les monocytes, le plasma et l’urine chez les patients Fabry et les contrôles sains appariés selon le sexe et l’âge; et 3) d’établir des corrélations entre la quantité relative de ces biomarqueurs et le génotype des patients traités et non traités. Les résultats de cette étude démontrent qu’il n’y a pas de changements significatifs dans la distribution des groupes d’isoformes/analogues du Gb3 selon le type cellulaire pour les patients et les contrôles. La découverte d’isoformes/analogues méthylés du Gb3 suggère un processus de méthylation qui se produit directement au niveau des cellules sanguines et particulièrement pour les lymphocytes B et les monocytes. Des corrélations face à la quantité de biomarqueurs et le génotype ont été établies. Ces résultats pourraient permettre une meilleure compréhension des processus biochimiques reliés à l’accumulation du Gb3 dans les cellules sanguines.

Prostaglandin (PG) E2 is an eicosanoid derived from the polyunsaturated twenty carbon fatty acid arachidonic acid (AA). PGE2 has physiological as well as pathophysiological functions and is known to be a key mediator of inflammatory responses. Formation of PGE2 is dependent upon the activities of three specific enzymes involved in the AA cascade; phospholipase A2 (PLA2), cyclooxygenase (COX) and PGE synthase (PGEs). Although the research within this field has been intense for decades, the regulatory mechanisms concerning the PGE2 synthesising enzymes are not completely established. PGE2 was investigated in human monocytes with or without lipopolysaccharide (LPS) pre-treatment followed by stimulation with calcium ionophore, opsonised zymosan or phorbol myristate acetate (PMA). Cytosolic PLA2a (cPLA2a) was shown to be pivotal for the mobilization of AA and subsequent formation of PGE2. Although COX-1 was constitutively expressed, monocytes required expression of COX-2 protein in order to convert the mobilized AA into PGH2. The conversion of PGH2 to the final product PGE2 was to a large extent due to the action of microsomal PGEs-1 (mPGEs-1). In addition, experiments with inhibitors of extracellular signal regulated kinase and p38 activation, indicated that phosphorylation of cPLA2α was markedly advantageous for the formation of PGE2. Ellagic acid, a natural polyphenolic compound found in fruits and nuts, was shown to inhibit stimuli induced release of PGE2 in human monocytes. The effect of ellagic acid was not due to a direct effect on the activities of the enzymes but rather to inhibition of the LPS-induced protein expression of COX-2, mPGEs-1 and cPLA2a.

M.Sc. / The induction of heat shock proteins (HSPs) in human monocytes during a pathogen challenge is a sophisticated selective response and plays an important role in cytoprotection from inflammation-related stress, including oxidative injury. We investigated the accumulation of the inducible isoform of the 70 kDa HSP, Hsp70, in peripheral blood monocytes from 12 healthy donors in response to Mycobacterium tuberculosis (M.tb) using flow cytometry, biometabolic labeling or Western blot analysis. Cells from each donor, prepared on two different occasions, were exposed to virulent (H37Rv) and attenuated (H37Ra) strains of M.tb at two bacterium : monocyte ratios (1:1 and 10:1) for 3 h and allowed to recover for an additional 2 h or 24 h. In spite of a prominent inter-individual variation, H37Ra (1:1, 2 h) significantly induced the mean Hsp70 accumulation (p<0.05) compared to normal cells, while H37Rv (10:1, 24 h) significantly suppressed the mean Hsp70 levels (p<0.001) in monocyte compared to normal monocytes or monocytes exposed to H37Ra. Survival of H37Rv-infected monocytes showed a significant correlation with Hsp70 levels. These results suggest a protective role of Hsp70 in host defense against mycobacterial infection. Cell death due to insufficient endogenous levels of Hsp70 implies a novel pathogenic strategy for virulence of M. tuberculosis.

Les monocytes détectent des signaux métaboliques circulants qu’ils traduisent en signaux immunologiques. ils infiltrent les tissus inflamés et sont également les précurseurs des macrophages. Dans l’obésité, la fréquence, le nombre, le phénotype et la fonctionnalité des sous populations de monocytes (CD14++CD16- [CM], CD14++CD16+ [IM] et CD14+CD16++ [NCM]) sont spécifiquement modulés. Le phénotype des monocytes est pro-inflammatoire avec une plus forte réponse à la stimulation de TLR4 et de TLR8. Nos travaux suggèrent une capacité migratoire accrue des CM et des IM. La comparaison du transcriptome des sous populations chez des sujets obèses et des témoins, révèle une signature spécifique de chaque sous population montrant un plus fort impact de l’obésité sur les CM. Ils soutiendraient l’inflammation en modulant des gènes impliqués dans la signalisation cellulaire. Ils migreraient avec les IM vers les tissus inflamés. Les NCM resteraient plus longtemps dans la circulation où leur fonction de « patrouilleurs » serait accrue. Les gènes les plus modulés par les CM et les IM, respectivement Clusterine et CDGSH iron sulfur domain 1 sont des ponts entre la réponse immunitaire, le métabolisme et la réponse au stress. L’étude des « tops » gènes dont l’expression est la plus fortement modulée et des fonctions les plus probablement modulées révèle que le métabolisme des monocytes serait modifié dans l’obésité. Ce travail ouvre des perspectives concernant : 1) une éventuelle composante auto-immune de l’inflammation de bas grade et 2) des relations entre des modifications du métabolisme des monocytes et leur fonctionnalité. / Systemic inflammation is pivotal in establishing and sustaining the obesity low-grade inflammatory status. Studying monocytes is important because they can detect metabolic cues in the circulation that are then translated into immunological factors. Monocytes infiltrating inflamed tissues are also macrophage precursors. In obesity, frequency, number, phenotype and functionality of the three monocyte-subsets (CD14++CD16- [CM], CD14++CD16+ [IM] et CD14+CD16++ [NCM]) are specifically altered. We observed a pro-inflammatory phenotype of monocytes in obesity with an increased response to TLR4 and TLR8 stimulation. Our work suggests an increased migratory capacity of CM and IM. Comparison of the transcriptome of the three subsets in obese and control subjects shows an increased imprint of obesity on CM. CM could sustain inflammation by modulating genes involved in cell signaling. They most probably migrate with the IM toward inflamed tissues. Whereas, NCM should stay longer in the circulation where their patrolling function could be increased. The most modulated genes by CM and IM, respectively Clusterin and CDGSH iron sulfur domain 1 (CISD1) are bridges between immune response, metabolism and stress response. The analysis of the top modulated genes and of the most probably modulated functions indicates that monocyte metabolism is altered by obesity. We open new paths of research: 1) a possible auto-immune trait of the low grade inflammation and 2) a possible link between altered monocyte metabolism and their functionality.

IL-12, an immunoregulatory cytokine, plays a key role in the development of cell-mediated immune responses. However, very little is known about the regulation and induction of the other members of this family, particularly IL-23 and IL-27. The regulation of these cytokines was studied in the human primary monocytes and monocyte-derived macrophages (MDMs) as they play a key role in innate and adaptive immune responses. THP-1 promonocytic cells were employed as a model system to confirm the results obtained with monocytes and MDMs. Two stimuli IFN-γ and LPS were used as both are strong inducers of IL-12 family cytokines. My results show that IFN-γ induced the production of IL-12/23p40 and IL-23p19 mRNA as well as IL-12p40 and IL-23 proteins in primary human monocytes isolated by positive selection. IFN-γ-induced IL-23 and IL-12/23p40 expression was positively regulated by the p38 mitogen-activated protein kinases (MAPK), independent of the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) signaling. In contrast, IL-12 and IL-23 were negatively regulated by the Jak/STAT, phosphoinositide-3 kinase (PI3K) and the c-Jun-N-terminal kinase (JNK) MAPKs in IFN-γ-stimulated monocytes. LPS significantly stimulated IL-23p19 and IL-12/23p40 mRNA expression as well as IL-12/23p40 and IL-23 protein production in THP-1 cells, while IFN-γ stimulation alone did not affect IL-23 mRNA or protein levels. THP-1 cells were pre-treated with ERK, JNK or p38 MAPK inhibitors and then stimulated with LPS. LPS-induced IL-12p40 and IL-23 proteins were positively regulated by the p38 and JNK MAPKs and PI3K, whereas LPS-induced IL-23p19 mRNA expression was negatively regulated by these kinases. These results were confirmed using siRNA in LPS-stimulated THP-1 cells. My results also show that IFN-γ/LPS-induced IL-23 expression is not regulated through MAPK or PI3K signaling pathways in human MDMs. My results also show for the first time that IFN-γ alone without any second stimulus induced IL-27p28 gene expression and IL-27 protein production in human monocytic cells. I investigated the signalling pathways governing the regulation of IL-27 protein and its subunit IL-27p28 following stimulation with IFN-γ in primary human monocytic cells. IFN-γ-mediated IL-27 protein, but not IL-27p28 gene expression was positively regulated by JNK MAPK and PI3K, independent of JAK/STAT signaling in primary human monocytes. I also investigated the signalling pathways governing the regulation of IL-27 and its α subunit, IL-27p28 following stimulation with IFN-γ alone or IFN-γ-primed LPS-stimulated macrophages (IFN-γ/LPS) and THP-1 cells. A differential regulation of IL-27p28 and IL-27 in response to stimulation by either IFN-γ or IFN-γ/LPS was observed. IFN-γ- and IFN-γ/LPS induced IL-27 expression was positively regulated by the JNK, p38 MAPK and PI3K, independent of Jak/STAT signaling in human MDMs and THP-1 cells. Taken together, my results show that IL-23 induction is differentially regulated by different pathways in response to different stimuli, whereas IL-27 expression is regulated by JNK, p38 MAPK and PI3K regardless in the stimulus in human myeloid cells. These results may provide additional strategies aimed at targeting disease, autoimmune disorders and cancer.

Listeria monocytogenes is a facultative intracellular bacterium that causes foodborne disease in humans. L. monocytogenes invade the gut mucosa and then disseminate, causing systemic infections associated with high mortality rates in immunocompromised individuals. It is unknown how L. monocytogenes traffic to the mesenteric lymph nodes, which represent an important bottleneck for systemic spread. In addition, little is known about the gastrointestinal stage of infection due to the general resistance of mice to oral infection with L. monocytogenes. Our laboratory developed a novel foodborne mouse model of listeriosis utilizing a murinized strain of L. monocytogenes to investigate the gastrointestinal stage of infection. First, we found that the majority of L. monocytogenes isolated from the intestinal tissue and MLN were extracellular; however, the minimal fraction of intracellular L. monocytogenes was vital for persistence in the gut and spread to the MLN. The vast majority of cell-associated L. monocytogenes in the MLN were adhered to inflammatory monocytes, but these cells did not support the intracellular growth of L. monocytogenes. A minor proportion of L. monocytogenes were associated with migratory dendritic cells in the intestinal lamina propria and MLN, but like monocytes, these cells did not appear to serve as an intracellular growth niche for L. monocytogenes. Lastly, extracellular L. monocytogenes were observed migrating in mesenteric lymphatic vessels that drain from the intestine to the MLN, suggesting that L. monocytogenes can spread beyond the intestinal mucosa independent of migratory immune cells. Overall, these studies are the first to characterize the interaction of L. monocytogenes with immune cells in the intestine and MLN following foodborne infection and suggest that extracellular, and not cytosolic L. monocytogenes, primarily drive innate immune responses in the gut.

Inflammation plays a crucial role in protecting the host from infection and tissue injury. However, uncontrolled inflammation contributes to the pathogenesis of major auto-inflammatory diseases. Interleukin-1 (IL-1), a pleiotropic pro-inflammatory cytokine, is a pivotal mediator of many of these diseases. The best characterised IL-1 family members, IL-1α and IL-1β, are produced as precursor forms of 31 kDa in size. Both precursors are cleaved and secreted, activating transmembrane IL-1 receptors on IL-1-responsive cells. Many studies that focused on IL-1α have shown that the precursor and processed mature Ct peptide, as well as its N terminus (Nt) form, can elicit a signal. However, with IL-1β, only the processed mature Ct form is known to elicit an inflammatory response and no immunological activity has been attributed to Nt fragments of pro-IL-1β. Therefore, the first objective of this study was to produce recombinant human Nt-IL-1β fragments in bacterial and mammalian expression system to investigate their possible immunomodulatory functions. Recombinant His-tagged N-terminus fragments (10 and 14 kDa) of pro-IL-1β were cloned into the bacterial expression vector pET-22(+) and expressed in E. coli BL21(DE3) followed by purification using three consecutive columns (IMAC, SEC and AEC). Purification analysis of eluted proteins from columns indicated that the recombinant proteins were always co-purified with some other bacterial proteins. The Nt fragments of pro-IL-1β were cloned into the mammalian expression plasmid, pcDNA3.1(+). Expression of these proteins was monitored by transfection of two mammalian cell lines: Human Embryonic Kidney (HEK) 293 cells and monkey kidney cells (COS-7). No protein expression was observed with either construct. These limitations urged us to investigate the expression and degradation of endogenous IL-1 in vitro. Previous studies have shown that the transcription of cytokine genes in response to lipopolysaccharide (LPS) is usually rapid and begins to decline within a few hours after stimulation. The proteasome is the major cellular proteolytic apparatus and controls the turn-over of cellular proteins. We investigated the intracellular stability of IL-1α and IL-1β in LPS-stimulated mouse J774 macrophages and primary mouse bone marrow derived macrophages (BMDMs). Exposure of LPS-stimulated J774 and BMDMs to three different classes of proteasome inhibitors (peptide alhedyde (ALLN), peptide boronate (MG262) and non-peptide inhibitor (β-lactone)) prevented the degradation of intracellular IL-1α and IL-1β in a concentration and time dependent manner. Furthermore, the release of IL-1 into the culture media was not affected by any of these inhibitors in LPS-stimulated J774 cells. However, in LPS-stimulated BMDMs, β-lactone increased the release of both IL-1α and IL-1β and ALLN only increased IL-1α release into culture supernatant compared to control. MG262 had no effect on the release of either. These data suggest that the proteasome plays an important role in controlling the amount of IL-1α and IL-1β by restricting the intracellular levels of these cytokines in activated monocytes and macrophages. Therefore, this study provides evidence in support of the hypothesis that the proteasome is involved in the degradation of IL-1α and IL-1β and may offer a potential therapeutic target in inflammatory diseases.

Os alimentos desempenham papel fundamental na nutrição e também na imunidade, pois podem conter substâncias que interagem direta ou indiretamente com o sistema imune, principalmente o de mucosas. Alguns polissacarídeos não-amido (PNAs) originados de plantas, algas e fungos comestíveis podem modular a função imune, contribuindo para a manutenção da saúde. A banana apresenta composição monossacarídica da parede celular similar à de plantas com efeitos imunomoduladores, permitindo formular a hipótese de que os PNAs dessa fruta também tenham essas propriedades. Assim, o objetivo foi extrair, purificar e caracterizar PNAs hidrossolúveis da polpa de banana das cultivares Nanicão e Thap Maeo e testá-los em cultivos de macrófagos RAW 267.4, células THP-1, macrófagos diferenciados por PMA THP-1 e células HL-60 in vitro avaliando a ação imunomoduladora através da dosagem de citocinas, NO e ensaios de fagocitose. As frações hidrossolúveis foram caracterizadas quanto ao seu conteúdo monossacarídico e ligações por hidrólise enzimática, identificando homogalacturonanos, mananos, arabinogalactanos, xilogalacturonanos e galactoglucomananos. As frações foram testadas para ausência de endotoxinas e viabilidade celular por MTT. Foram estabelecidas as doses não-tóxicas e ensaiadas as concentrações de 10, 50 e 200 &#181;g.ml-1 das frações polissacarídicas para os testes in vitro. Não houve toxicidade para macrófagos e monócitos, enquanto que para a linhagem de células pro-mielóciticas de leucemia humana HL-60, as frações se mostraram citotóxicas. Houve aumento de atividade fagocítica nas frações WSP, UFP e HTP quando comparadas ao controle negativo de células, assim como a produção de citocinas inflamatórias como IL-1&#946;, TNF-&#945;, IL-6, além da quimiocina IL-8 nas células oriundas de THP-1 e diminuição nos marcadores TLR-2 com aumento de CD14 nas células THP-1, além do aumento do tamanho celular promovido pelas frações polissacarídicas mostrando diferenciação para série granulocítica. Portanto, PNAs de ambas as cultivares de banana possuem potencial imunomodulador seja inflamatório, anti-inflamatório ou de diferenciação de células THP-1 imaturas in vitro. / Food plays a major role in nutrition as in immunity, as they may contain substances that interact directly or indirectly with the immune system, especially those located on mucous membranes. Some non-starch polysaccharides (NSPs) originate from plants, algae, and edible fungi can modulate immune function, contributing to the maintenance of health. The banana presents cell wall monosaccharide composition like the other plant polysaccharides with immunomodulatory effects allowing to formulate the hypothesis that this fruit NSPs also have these properties. The objective was to extract, purify and characterize water-soluble banana pulp NSPs of Nanicão and Thap Maeo cultivars and test them in vitro on cell lineages of RAW 267.4 macrophages, PMA differentiated THP-1 macrophages, THP-1 monocytes, and HL-60, evaluating their immunomodulatory action by the dosage of cytokines, NO and phagocytosis assays. Water-soluble fractions were characterized as to their content and monosaccharide linkages by enzymatic hydrolysis, identifying homogalacturonans, mannans, arabinogalactans, xylogalacturonan, and galactoglucomannans in its composition. Fractions were tested for the absence of endotoxins and cell viability by MTT. Non-toxic concentrations doses of 10, 50 and 200 &#181;g.ml-1 of the polysaccharide fractions were established for in vitro testing. The NSPs fractions WSP, UFP, and HTP tested promoted an increase in phagocytic activity in THP-1 PMA derived macrophages compared to negative control cells, as well as the production of inflammatory cytokines such as IL-1&#946;, TNF-&#945;, IL-6. An increase in the THP-1 CD14 cell receptor and decrease in CD33, CD123, CD11c and TLR-2 receptors were promoted by the banana polysaccharides. Besides, there was an increase in cell size showing cell differentiation towards granulocyte line and demonstrating that NSPs from both cultivars were immunomodulatory with a capacity of immature cell differentiation and proving that in vitro screening test is an efficient method to test other foodborne substances with immunomodulatory potential.

The application of nanoparticles in the field of theranostics requires knowledge of the specific interaction of nanoparticles with the immune system. One of the first cells with which nanoparticles interact when given to the body are cells of the mononuclear phagocytic system. The aim of this diploma thesis is to prepare an in vitro study that describes the effect of two types of gold and three types of silicon ultra-small nanoparticles on immune cells. Immune cells are presented in the form of primary PBMCs isolated from whole blood , and cells of monocytic cell line THP-1 in the form of monocytes and differentiated macrophages. During the experiments with primary cells, emphasis is placed on maintaining the concept of personalized protein corona. After characterization of the immune cells used, cells are subsequently stimulated with ultra-small nanoparticles and the influence of these nanoparticles on cell metabolism, viability, degree of differentiation and secretion of pro- and antiinflammatory cytokines is monitored. The outcome takes into account further use of the tested nanoparticles in the field of biomedicine. Key words: primary monocytes, cell lines, differentiation, macrophages, cytokines, cytotoxicity

The Huntington disease (HD) is a hereditary neuro-degenerative disorder caused by a mutation of the huntigtin gene that codes a protein of the same name. The mutated form of the huntigtin gene plays its part in many pathological interactions and influences a number of cellular mechanisms, including the immune system that could serve as a modifier of the neuropathology of the disease. The cells of the monocyte-macrophage system express cytokines whose production changes in relation to the activation of the cell. The presence of the mutated huntingtin protein in these cells renders them hyper-responsive to immunity incentives leading to changes in the production of cytokines. These differences are discernible a few years prior to the appearance of the symptoms. Therefore, the changes in the levels of certain cytokines could serve as appropriate biomarkers for monitoring of the onset of the disease and its progression. The HD pathogenesis includes an inflammation of the central neutral system. Inflammatory changes in peripheral tissues could reflect inflammatory processes in the central neural system. A miniature TgHD pig could represent an appropriate model organism for studying of the impact of the mHtt on the immune system. This model enables to observe a slow progression of the disease. Changes in...