Les récepteurs P2Y sont exprimés à la surface de bon nombre de cellules y compris les cellules du système immunitaire. De nombreuses études réalisées in vitro suggèrent une influence des nucléotides sur la biologie de ces cellules. Toutefois pour certains d’entre eux, l’implication in vivo n’est pas clairement établie. Le présent travail, basé sur l’analyse phénotypique de souris invalidées, vise donc à étudier le rôle de trois de ces récepteurs, à savoir P2Y2, P2Y6 et P2Y12, dans la physiologie des phénomènes inflammatoires. Leur rôle a été abordé plus particulièrement dans deux types cellulaires :les macrophages et les cellules dendritiques.<p>En ce qui concerne les macrophages péritonéaux recrutés, nous avons démontré in vitro que le récepteur P2Y2 participe, avec un récepteur P1, à la production d’IL-6 en réponse au LPS. Par contre, il ne semble pas impliqué dans l’internalisation d’antigènes par macropinocytose. Le récepteur P2Y6 favorise la production d’IL-6 et de MIP-2 en réponse au LPS et stimule l’endocytose par macropinocytose de dextran-FITC.<p>Pour ce qui est des cellules dendritiques (DCs), l’étude du rôle du récepteur P2Y12 dans leur biologie in vitro avait été investigué auparavant dans notre laboratoire. Toutefois, certains paramètres devaient être confirmés ainsi que l’implication in vivo du récepteur dans l’établissement d’une réponse immune. Nous avons contribué à démontrer que l’endocytose par macropinocytose est augmentée suite à l’activation du récepteur P2Y12 par l’ADPβS au niveau de DCs spléniques murines et de DCs dérivées de monocytes humains. L’ADPβS favorise la capacité des DCs à activer les lymphocytes T spécifiques d’un antigène. Enfin, la signification de ces observations a été testé in vivo par l’immunisation de souris P2Y12+/+ et P2Y12-/-. Les résultats obtenus montrent une production d’IgG1 diminuée dans les souris invalidées pour le récepteur par rapport aux souris sauvages suggérant que le récepteur P2Y12 favoriserait le développement d’une réponse immune Th2 et ce principalement via l’augmentation de la captation d’antigènes par les DCs. <p>Suite aux données obtenues et à celles de la littérature, nous avons investigué l’implication du récepteur P2Y6 dans la réponse immune adaptative in vivo. Pour ce faire, nous avons immunisé des souris P2Y6+/+ et P2Y6-/- selon le protocole utilisé lors de notre précédente étude. Les souris P2Y6 KO montrent une diminution significative de la production d’IgG2c spécifiques de l’antigène suggérant l’implication du récepteur dans le développement d’une réponse pro-Th1. Plusieurs paramètres ont été mesurés pour expliquer ce phénomène. Nous avons tout d’abord démontré que le récepteur P2Y6 est exprimé et fonctionnel dans les cellules dendritiques dérivées de la moelle. Nous avons ensuite montré que l’activation du récepteur par l’UDP favorise la capture d’antigènes in vitro, potentialise l’expression de molécules de co-stimulation (CD80 et CD86) et la production d’IL-12 p70 et de TNF-α en réponse au CpG ODN indiquant que la modulation de la réponse immune observée in vivo s’explique, en partie du moins, par les effets du récepteur sur différentes fonctions des DCs.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

African Swine Fever Virus (ASFV) is an Asfivirus and is the only member of the family Asfarviridae. It manifests as a disease that varies from acute to sub-acute or chronic forms. A true carrier state in domestic pigs is unknown but chronically affected individuals may carry and spread the virus for extended periods. African Swine Fever (ASF) is a socio-economically important disease characterized by high morbidity and mortality affecting the livelihood of many small to big scale farmers and seriously compromising international trade. Strategic measures to control this disease are by physical containment and culling in outbreak situations. There is no vaccine available. Nevertheless, every pork producer should ideally be actively involved in having biosecurity measures in place to avoid contamination and contacting their veterinary services in case of suspicion of ASF to have appropriate samples analysed. Official veterinary services must be equipped with proper diagnostic tools in order to provide a quick response. The sensitivity of currently available diagnostic tests at the Transboundary Animal Diseases Programme, Onderstepoort Veterinary Institute was analysed in order to report the best technique available. Sensitivity to ASF virus infection and therefore diagnostic potential of cell primary cultures as bone marrow macrophages, blood macrophages and alveolar macrophages was done via comparison of titre results from inoculations of ASFV SPEC 257 as control, and ASFV MOZ 1/98. In addition, molecular detection of specific DNA fragments within the viral genome were compared using five different PCRs. Bone marrow macrophage cultures and blood macrophage cultures were the most reliable cells whereas alveolar macrophages more often showed contamination. Results show that PPA PCR and real time PCR detected the highest diluted samples, thus the lowest concentration of virus, in both trials done with ASFV MOZ 1/98 and ASFV SPEC 257. In addition, animal trials were performed by inoculating domestic pigs with four different ASFV isolates of varying pathogenicity. These viruses were all from distinct geographic origins. Non-virulent ASFV OURT 3/88 and high virulent ASFV BENIN 1/97 were previously described and used as reference viruses. ASFV MOZ 1/98, suspected of having high virulence and ASFV MKUZE, which was thought to be of low virulence were included in this study to provide further information on the pathological and clinical outcome of the disease as well as measuring viral replication in various organs and blood. The study showed that ASFV MKUZE was of intermediate virulence, whilst ASFV MOZ 1/98 was highly virulent with a high mortality rate. Results confirmed the inadequacy of ASFV MKUZE to act as vaccine opposed to ASFV OURT 3/88. Following this, a potential vaccine by use of attenuated Portuguese ASFV OURT 3/88 tested against virulent heterologous challenge with a strain now known with certainty to cause acute ASF, the isolate ASFV MOZ 1/98 collected from a diseased pig in Mozambique. Domestic commercial pigs where submitted to either one or two vaccinations before challenge. Viral load in blood and tissue samples was higher in unvaccinated animals and higher in single vaccinated than in pigs vaccinated twice. However, acute ASF afflicted all groups with severe clinical signs and post-mortem lesions. Although it did not confer total immunity it was determined that pigs vaccinated with European attenuated ASFV OURT 3/88 acquired partial protection against challenge with virulent southern Africa ASFV MOZ 1/98. / Dissertation (MSc)--University of Pretoria, 2012. / gm2014 / ab2015 / Veterinary Tropical Diseases / unrestricted

Throughout this work, we looked at P2X coupled pathways in macrophages. We worked on three different models of macrophages in order to establish the best model to understand the role of P2X4 receptors in the inflammation. Our work also consisted of further characterizing P2X7 receptor dependent pathways in these models. <p>P2X4 and P2X7 receptors are ionotropic receptors which are expressed by a variety of immune cells including macrophages. Macrophages play a very important host defense function as they are major actors in the innate immune system and they can initiate the activation of the adaptive immune system. The endogenous ligand of P2X receptors is ATP for which they share very different sensitivities. P2X4 receptors are relatively sensitive to this agonist while P2X7 receptors require concentrations > 100 μM ATP to be activated. <p>Our study supports the expression of P2X4 and P2X7 receptors in J774.2 murine macrophages and in human macrophages. Additionally, we worked on murine peritoneal macrophages for which the existence of P2X4 and P2X7 receptor expression had previously been shown in our lab. <p>A wide range of different macrophage phenotypes exist. Two extremes determine an array of phenotypes which are delimited by M1 pro-inflammatory macrophages and M2 anti-inflammatory macrophages while Mφ macrophages define the center of the array. Most of the work exposed in this study was carried out on pro-inflammatory macrophages which were obtained either by priming the cells with LPS alone (Mφ + LPS) or by polarizing them with LPS in association with IFNγ (M1). <p>We show in this study that LPS-primed J774.2 murine macrophages are not a good model to study the role of surface P2X4 receptor in pro-inflammatory macrophages. Additionally, we support that murine peritoneal macrophages primed with LPS are a good model to understand the hypothetical role of P2X4 receptors in the inflammation. Finally, we suggest that human M1 macrophages could be as well. Next, we also confirm that J774.2 murine macrophages, murine peritoneal macrophages and human macrophage express functional P2X7 receptors. In this study, we show that P2X7 receptors are coupled to RONS formation in J774.2 murine macrophages and to AA release through PLA2 activation in peritoneal macrophages. We show that activation of J774.2 murine macrophages with high concentrations of ATP (>600 μM) stimulates ROS formation including mitochondrial superoxide anions. In addition, our work shows the importance in using different dyes and suggests that different types of ROS play different functions in P2X7 receptors downstream pathways. <p>Next, we show that P2X7 receptor activation is coupled to an iPLA2 activity and that the release of free fatty acids mediated by 1 mM ATP is p-ERK1/2 dependent in LPS-primed murine peritoneal macrophages. <p>In addition, we have evaluated the effect of hypoxia on pathways which have been reported to be coupled to P2X7 receptor activation in pro-inflammatory human macrophages. Hypoxia does not seem to modulate P2X7 receptor functionality. However, both acute and chronic hypoxia influenced P2X7 receptors downstream pro-inflammatory coupled pathways. Finally, our work has enabled us to suggest for the first time that IFNγ plays an important function in host defense mediated by human P2X7 receptor activation in a hypoxic environment. <p>The effect of extracellular environment and thus different macrophage phenotypes have also been evaluated throughout this work in which we looked at the effect of polarization on P2X4 and P2X7 receptor functionality. Our work shows that LPS-priming does not modulate P2X4 receptor functionality in murine macrophages. Next, through our work, we suggest that polarization of human macrophages affects P2X4 receptor functionality in human macrophages. Additionally, our work shows that LPS affects ATP-mediated RONS formation in J774.2 murine macrophages but not P2X7-mediated AA release in primary murine macrophages. <p><p>Overall, first, our work has enabled us to suggest macrophage models to use in order to study the hypothetical role of P2X4 receptor in the inflammation mediated by macrophages. Second, it has allowed us to further understand how P2X7 receptors can act as important mediators of the immune system mediated by macrophages. In addition, many interesting observations were made in this study which allows us to propose several options for future directions. To finish, our work underlines the importance of the extracellular environment in some pathways mediated by ATP in macrophages.<p> / Doctorat en sciences pharmaceutiques / info:eu-repo/semantics/nonPublished

Organic polymers S2218600, S2429901 and S2219200 (referred to as Polymer 1, Polymer 2 and Polymer 3, respectively) of varying toxic potential, designed for use in cosmetic aerosols, were used as model substances to predict inflammatory potential. In vivo inflammogenic potential was evaluated by assessment of inflammatory cell profile (alveolar macrophage (AM), polymorphonuclear neutrophil (PMN)) of broncho-alveolar lavage fluid (BAL) 24hrs after a single instillation of either 0.5 mg or 2 mg polymer in Sprague Dawley rats. Pro-inflammatory Minusil particles and non-inflammatory titanium dioxide (TiO2) particles were used as controls. For comparison, cultured rat NR8383 AM-like cells, human THP-1 monocyte cells or human monocyte derived macrophages were treated with polymer for 24 h and supernatants analysed for indicators of cytotoxicity and inflammatory mediator release. In addition, after 6 h treatment, gene changes in the rat lung tissue and also in the rat NR8383 alveolar macrophage cell line were assessed using microarray to analyse the entire rat genome. The in vivo studies showed that Polymer 1, Polymer 3 and Minusil caused significant PMN influx into BAL. Polymer 3 and Minusil caused a significant increase in AM number in BAL. Polymer 2 and TiO2 had no effect on BAL cell profile. BAL tumour necrosis factor-α (TNFα) and macrophage inflammatory protein-2 (MIP-2) levels were significantly increased following instillation of Polymer 3 and Minusil. Thus the polymers and particles were ranked for potential to cause pulmonary inflammation: Polymer 3 > Minusil > Polymer 1 > Polymer 2 > TiO2. In vitro studies using cultured rat NR8383 AM-like cells showed that the polymers and particles could be ranked similarly for cytotoxic potential and their ability to stimulate the release of both TNFα and MIP-2. Cultured human monocyte derived macrophages detected the pro-inflammatory abilities of Polymer 3, as measured by cytotoxic potential and ability to stimulate TNFα, interleukin-8 (IL-8) and macrophage inflammatory protein-1α (MIP-1α), however, did not detect the pro-inflammatory abilities of Polymer 1. Cultured human THP-1 cells predicted the pro-inflammatory effects of Polymer 3 in rat lungs using the cytotoxicity assay and by changes in IL-1β, MIP-1α and IL-10 levels. The human THP-1 cell line did not predict the pro-inflammatory effects of Polymer 1 that were observed the rat lungs. Electron spin resonance (ESR) detected free radicals produced by the pro-inflammatory polymers and particles which had the ability to break bonds in super-coiled DNA and deplete intracellular glutathione (GSH). Microarray analysis of the canonical pathways activated by the pro-inflammatory polymers, Polymer 1 and Polymer 3, showed that 3 similar pathways were significantly activated in the instilled rats and the rat NR8383 AM-like cells following treatment. ‘Xenobiotic metabolism’, ‘IL-10 signalling’ and ‘leukocyte extravasation signalling’ pathways were significantly changed by the pro-inflammatory polymers. Use of these cell model alternatives in an industrial setting will refine and reduce in vivo testing and as these models are further developed and used alongside future new alternatives they will provide a substantial contribution towards the replacement of animal testing.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Résumé Par leur capacité de moduler la réponse immune, les rayons ultraviolets (UV) ont trouvé des applications dans le traitement de diverses maladies immunes. Leurs mécanismes d’action sont encore incomplètement définis. L’un d’entre eux comporte l’induction de cytokines immunosuppressives et antiinflammatoires. Ce processus peut être provoqué par la phagocytose de corps apoptotiques, l’apoptose constituant une des lésions cellulaires élémentaires provoquée par les UV. Le but de notre travail a été de préciser les cytokines impliquées dans la réponse aux UV, de définir certains mécanismes de leur production et de la potentialiser par des agents pharmacologiques. Notre étude a comporté deux parties: (1) l’une in vivo chez des malades souffrant de GVH chronique résistante aux traitements conventionnels et traités par photochémothérapie extracorporelle, procédure dans laquelle les leucocytes du malades, prélevés par leucaphérèse puis traités par un psoralène et par UVA lui sont finalement réinjectés; (2) l’autre in vitro où des PBMC de volontaires sains ont été irradiés avec 10J/m2 de rayons UVC qui ne nécessitent pas de photosensibilisation par psoralène. Deux cytokines, l’IL-10 et l’IL-1Ra ont été évaluées par RT-PCR dans un système de coculture autologue entre PBMC et PBL rendus apoptotiques par irradiation. L’évolution du processus apoptotique déclenché par les UV a été mesurée par cytomètrie de flux. Celui-ci concernait essentiellement les lymphocytes, les monocytes/macrophages révélant une résistance relative à l’apoptose, il était progressif, culminant entre la 24ème et la 48ème heures. Lors des cocultures entre PBMC et PBL irradiés, un accroissement très significatif du nombre de copies d’ARNm, concernant les deux cytokines étudiées, l’IL-10 et l’IL-1Ra était observé. L’induction d’IL-1Ra était dépendante de l’IL-10. Une préactivation par du LPS était nécessaire pour la révélation du phénomène. Ensuite, nous avons évalué l’implication sur la synthèse de cytokines du processus de phagocytose de lymphocytes rendus apoptotiques par irradiation UV et divers moyens pharmacologiques pour la potentialiser. La préincubation du matériel irradié pendant une nuit (16h) à 37° dans le but d’accroître la proportion de cellules en voie d’apoptose avant mise en contact avec les PBMC a permis d’obtenir un accroissement très marqué sans nécessiter de LPS, portant essentiellement sur la production d’IL-1Ra tant sur l’ARNm que la protéine secrétée; l’induction d’IL-10 était cette fois négligeable. L’implication de la phagocytose dans le processus a été démontrée par deux agents bloquants (a) l’anticorps monoclonal anti-CD36 (corécepteur avec l’intégrine V3 de la thrombospondine) activant la production d’IL-1Ra et mimant par ce fait le processus phagocytaire et (b) la cytochalasine E la bloquant. Nous avons testé diverses substances pharmacologiques dont l’action activatrice de l’IL-1Ra est connue, en l’occurrence les immunoglobulines G à usage IV (IgIV) et le GM-CSF. L’adjonction d’IgIV (1mg/ml) ou GM-CSF (10 ng/ml) une heure après le début de la coculture exerce sur la sécrétion d’IL-1Ra un effet additif avec les UV. Selon la concentration utilisée, les IgIV peuvent agir par deux mécanismes. Outre l’effet d’activation macrophagique lié au récepteur Fc, nous avons démontré à haute concentration un mécanisme nouveau, du à la présence dans les IgIV d’anticorps naturels antiFas induisant l’apoptose des lymphocytes. Une incubation de 16h des lymphocytes avec 25 mg/ml d’IgIV avant mise en culture provoque outre une apoptose importante une augmentation significative de l’IL-1Ra. Dans ce cas, le processus est indépendant du fragment Fc, la fraction F(ab’)2 gardant la capacité d’induire l’apoptose et de provoquer la production d’IL-1Ra. En conclusion, nous avons mis en évidence un mécanisme nouveau d’induction d’IL-1Ra, non décrit auparavant et défini diverses modalités qui pourraient accroître sa production: - L’incubation de 16h du matériel irradié permet d’orienter le système en accroissant la production de l’IL-1Ra sans que la production de l’IL-10 soit modifiée et sans nécessiter de LPS. Nous attribuons cet effet à l’accroissement du processus apoptotique qui en résulte. - Nous avons potentialisé la production d’IL-1Ra par deux agents pharmacologiques, le GM-CSF et les IgIV. Les mécanismes d’action des IgIV dépendent de la concentration utilisée. 1. Aux concentrations de l’ordre de 1mg/ml, les IgIV exercent, avec les UV un effet additif sur l’induction d’IL-1Ra par une action dépendant du fragment Fc. 2. Aux concentrations élevées de 25mg/ml, un effet apoptotique attribuable à l’action d’anticorps anti-Fas agonistes est observé. Une préincubation de 16h de lymphocytes avec cette concentration d’ IgIV avant mise en culture avec les PBMC autologues provoque outre l’apoptose importante des lymphocytes un accroissement significatif de la production d’IL-1Ra. Le processus est indépendant du fragment Fc, la fraction F(ab’)2 gardant la capacité d’induire l’apoptose et la production d’IL-1Ra.

Plasma neopterin is an excellent marker of inflammation and is found in elevated levels in plasma of patients with cardiovascular disease. Neopterin originates as the oxidation product of 7,8-dihydroneopterin (7,8-NP), which is secreted by human macrophages when stimulated with interferon-y during inflammation. 7,8-NP has been shown to be a very efficient free radical scavenger and a potent antioxidant which can protect macrophages from a range of oxidative stresses. The uptake of oxidised low density lipoprotein (oxLDL) by macrophages which lead to the formation of foam cells is a hallmark of early atherosclerotic lesions. OxLDL-induced cell death is also considered to be an important process in the formation of necrotic lipid rich plaques and in atherosclerotic plaque destabilisation. This thesis examined the extent of oxLDL-induced damaged to HMDMs and whether 7,8-NP can inhibit oxLDL-mediated cell death in HMDMs. Foam cells had previously been defined as cholesteryl ester (CE) macrophages that stained positive with oil red-O. This thesis shows that the foamy appearance and presence of lipid droplets stained with oil red-O was not dependent on accumulation of CE which raises the suitability of using oil-red-O staining to identify the foam cells. In addition, HPLC but not GC analysis showed an increased in CE levels of the macrophages when the macrophages were incubated with oxLDL. The HPLC approach spared the samples of lengthy manipulations that might cause ex vivo oxidation. It also avoided subjecting the samples to high temperature treatment that could alter the lipid composition and therefore quantification of the lipid contents. Previous studies showed that 7,8-NP is a potent antioxidant and cytoprotective agent. Exposure of HMDMs to 1 mg/ml oxLDL caused 50% loss of cell viability as measured by the MTT reduction and trypan blue exclusion assays. The development of apoptotic features including caspase-3 activity, cytochrome c release from mitochondria and phophatidyserine (PS) exposure was examined. OxLDL did not cause caspase-3 activation as shown by Western Blot analysis and did not cause DEVD-AMC cleavage in HMDMs. However, cytochrome c release and phosphatidylserine exposure were observed when HMDMs were incubated with oxLDL as shown by Western Blot analysis and Annexin V-FITC staining respectively. Dihydroethidium (DHE) staining showed that oxLDL treatment caused mitochondrial superoxide generation in HMDMs. OxLDL-induced oxidative stress appeared to cause a rapid loss of HMDMs' intracellular glutathione (GSH) as analysed by HPLC technique. Incubation of HMDMs' with buthionine sulfoximine (BSO) and diethyl maleate (DEM) caused similar loss in GSH as incubation with oxLDL but did not result in HMDMs' death. This showed that oxLDL-induced decrease in GSH alone was not sufficient to cause cell death. The loss of cell viability by oxLDL was inhibited by 7,8-NP in the concentration range of 50 to 200 lM. HMDMs' GSH loss caused by oxLDL was similarly inhibited by 7,8-NP supporting the idea that preventing the cellular GSH loss will protect the HMDMs from death. Incubation of HMDMs with 7,8-NP showed reduction in DHE fluorescence intensity staining suggesting that 7,8-NP inhibited or scavenged oxLDL-dependent generation of superoxide. 7,8-NP also effectively inhibited oxLDL-induced PS externalisation to the outer membrane but failed to inhibit the oxLDL-induced release of cytochrome c from mitochondria to the cytosol. The labelling of oxLDL with DiI showed that 7,8-NP significantly inhibited the uptake of oxLDL. However, the inhibitory effect was only measured at non-toxic concentration of oxLDL. The ability of 7,8-NP to inhibit oxLDL uptake raised the possibility that 7,8-NP protective effect against oxLDL involved modulation of the scavenger receptors'expression in particular SRA and CD36. The Western Blot analysis showed that incubation of HMDMs with 7,8-NP did not affect HMDMs' SRA protein expression. In 50% of the experiments, it was demonstrated that certain isoforms of CD36 protein were significantly down regulated by 7,8-NP suggesting that various factors might interact with 7,8-NP or CD36. The ability of 7,8-NP to protect HMDMs from oxLDL-induced death provides further evidence that this antioxidant is secreted by HMDMs to protect them against the oxidative damage in the highly oxidative environment of atherosclerotic plaque.