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.

Thèse de doctorat : Biologie Moléculaire. ARN : Biogenèse et structure / fonction : Nancy 1 : 2008. / Titre provenant de l'écran-titre.

Highly antiretroviral therapies (HAART) have been implemented to slow the progression of the human immunodeficiency virus (HIV). Although these new advances in the medications for HIV-positive patients have contributed in longer life expectancy, comorbidities, such as cardiovascular disease, still cause higher number of deaths among HIV-positive patients than in the regular population. Because of the intrinsic inflammation caused by the HIV virus, atherogenesis is more likely to occur and is driven by infected macrophages. These macrophages are known to secrete cathepsins, but infection causes the macrophages not to perform their function properly as an immune agent. I hypothesize that antiretroviral drugs play an important role in arterial remodeling by affecting cells within the artery and causing an alteration of cathepsin activity, leading to an increased risk of atherosclerosis in HIV patients. To test this hypothesis, we incubated THP-1 monocytes with antiretroviral drugs efavirenz and tenofovir individually to observe any changes in cathepsin activity. These lysates were analyzed through multiplex cathepsin zymography and quantified through densitometry. We found that our hypothesis held true for efavirenz and tenofovir in THP-1 monocytes, which caused decreased cathepsin K activity compared to vehicle controls. Still, stimulation of peripheral blood mononuclear cells (PBMCs) with efavirenz and tenofovir caused differential effects. Together, our data suggest that the HAART interaction with monocytes that are physiologically relevant to our system possibly contributes to the advancement of atherogenesis in HIV+ patients.

Hematopoietic cells of the myelomonocytic lineage play a central role in orchestrating both innate and adaptive immunity. They are important in the control of infectious agents and in the pathogenesis of diseases characterized by dysregulated immune response. Like allergic asthma in human patients, recurrent airway obstruction (RAO) of horses is a disease exemplified by chronic airway inflammation in the absence of infectious agents. However, unlike allergic asthma, RAO is marked by preponderance of neutrophils rather than eosinophils in the airways. Attempts to understand the immunological basis of RAO by studying lymphocytes produced equivocal results. This thesis examined the possible role of alveolar macrophages (AM) recovered from bronchoalveolar lavage fluid (BALF) in RAO. Since macrophages are predominantly derived from circulating monocytes, the thesis investigated first the phenotypic characteristics of circulating monocytes, second those of macrophages in vitro derived from monocytes, and finally attributes of AM derived in vivo. Flow cytometric analysis following antibody staining of monocytes from 61 horses showed that the clustering pattern of human leukocytes may not always be extrapolated to horses when using this technique since clusters of granulocytes often spill over into the monocyte population. The study showed that DH24A, a monoclonal antibody directed against CD90, which recognizes T cells in other species, will specifically recognize granulocytes in horses and was therefore used to separate neutrophils from monocytes during analysis. In addition, investigation of circulating monocytes showed that expression of the hemoglobin-haptoglobin receptor CD163 on circulating monocytes is significantly increased in horses with systemic inflammation when compared with healthy horses. Evaluating cytokine and chemokine production by macrophages, it was demonstrated that CD163+ macrophages preferentially expressed IL10 while CD163- macrophages showed predominant expression of CCL17. It was, therefore, concluded that CD163+ IL10-producing macrophages of horses are homologues of the alternatively activated anti-inflammatory macrophage subset of humans. Finally, probing of alveolar macrophages for CD163 and CD206 expression showed a significant reduction in the proportion of CD163+ macrophages in horses with RAO. These findings suggest that RAO is associated with a reduction in anti-inflammatory macrophages, an observation that may in part explain the chronic airway inflammation associated with this disease.

IL-17 promotes inflammation through the recruitment of monocytes and induction of various chemokines and inflammatory cytokines. Monocytes respond to IL-17 through the heteromeric IL-17 receptor (IL-17R) composed of subunits IL-17RA and IL-17RC. Together, monocytes and IL-17 amplify inflammation. Controlling the cellular response to IL-17 is crucial to prevent hyperactivation of inflammatory responses, which could lead to chronic inflammatory diseases. The cellular response to increased IL-17 levels may be limited by controlling the receptor levels. Before we understand how monocytes respond to IL-17 during infection, we must first characterize the expression of IL-17R in these cells in response to LPS, a well-characterized pro-inflammatory signal. The aim of this study is to understand the mechanisms which regulate IL-17R levels in human monocytes. IL-17R mRNA and protein levels were measured in response to LPS by RT-PCR and Western blot analysis in primary human monocytes, peripheral blood mononuclear cells (PBMC), and the human monocytic cell line, THP-1. LPS enhanced IL-17RA and RC transcript levels in monocytes and PBMC. In contrast, IL-17RA protein levels decreased with LPS treatment in these cells. Investigation into mechanisms regulating IL-17RA protein levels lead to the observation that IL-17RA undergoes receptor degradation in response to LPS. This work identifies for the first time that 1) LPS enhances transcript levels of IL-17R and 2) after LPS treatment, IL-17RA protein levels are reduced via an endosome-dependent degradation pathway. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-06-21 11:53:28.706

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. TAFIa also possesses anti-inflammatory properties. Although liver is the main source of plasma TAFI, platelet-derived TAFI has also been reported. An alternatively spliced TAFI variant resulted from the skipping of exon 6 and a 52-base deletion in exon 10 of CPB2 mRNA (∆6+10) was described to be brain specific. This TAFI variant is reputed to possess a secretase-like activity that cleaves β-amyloid precursor protein to form β-amyloid, a process involved in the onset of Alzheimer's disease. In this thesis, we report the identification of CPB2 mRNA and TAFI protein in various vascular and inflammatory cells. Specifically, we describe the expression of CPB2 mRNA in the megakaryocytic cell lines MEG-01 and Dami, the monocytic cell line THP-1, and peripheral blood mononuclear cells. TAFI protein was detected in differentiated Dami and THP-1 cells. We next describe the effect of external stimuli such as phorbol myristate acetate (PMA) on CPB2 expression in Dami and THP-1 cells. We found that PMA treatment increases both CPB2 mRNA abundance and promoter activity in Dami cells, and decreases both CPB2 mRNA abundance and promoter activity in THP-1 cells. Deletion analysis of the CPB2 promoter indicated cell-type specific regulation of CPB2 gene expression. Finally, we evaluated the expression of alternatively spliced CPB2 mRNA variants in hepatic and non hepatic cells. We found that exon 6 skipping variants are expressed in all cell types of interest. The variant previously reported to be brain specific was also found to be expressed in platelets. We found that the alternatively spliced TAFI variants accumulated inside the cells in a non-secretable, hypoglycosylated form and showed no carboxypeptidase activity. Taken together, this thesis provides further evidence supporting the hypothesis that platelet-derived TAFI is originated from CPB2 gene expression in megakaryocytes. Moreover, our data imply a potential for site-specific anti-inflammatory control provided by macrophage-derived TAFI. Alternative splicing of the CPB2 mRNA may give rise to variants with an intracellular role, perhaps as a peptidase chaperone, and may modulate the synthesis of secretable TAFI. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-09-26 21:22:33.348

Every year there are approximately 300 million new cases of malaria with 2 million deaths. The majority of deaths occur in African children between the ages of 1 and 4 years and are caused by the parasite Plasmodium falciparum. Approximately R90-million is spent by the South African government each year to control malaria. Peripheral blood monocytes are the first line of defence during infection and they perform many functions, such as phagocytosis, intracellular and extracellular killing by the generation of reactive oxygen intermediates and the production of cytokines. During malaria infection some of these functions are suppressed or elevated by phagocytosis of hemozoin, fever conditions (heat shock) and the presence of anti-malarial drugs in the bloodstream of the patient. Under normal conditions phospholipase A₂ (PLA₂) is down regulated by heat shock protein 70 (HSP70) but in severe malaria PLA₂ is elevated. Two antigenic peptides were selected from the highly conserved human HSP70 and HSC70 proteins. Anti-peptide antibodies raised in chickens were affinity purified and were able to recognize the free peptide in an ELISA and the native proteins in human and canine heat shocked lymphocyte lysates on western blots. Antibodies against HSP70 detected two major proteins at 70 kDa and 33 kDa, which are most likely native HSP70 and a possible breakdown product of HSP70 respectively. The anti-HSC70 antibodies detected two proteins, an as yet unidentified 100 kDa protein and the 70 kDa HSC70. Due to the monocytes being activated during the isolation procedure, HSP70 was expressed at both 37°C and 44°C in this study. Electron-probe X-ray microanalysis enables determination of the elemental composition of any sample under the electron microscope. When the electron beam interacts with a specimen, X-rays are generated and can be used to identify and quantify the elements in the cell. Canine monocytes were analysed using this technique after incubation with therapeutically relevant concentrations of anti-malarial drugs, β-hematin and under fever conditions. The concentrations of the elements in normal canine monocytes were: Na (518.2 mmoles/kg), Mg (199.1 mmoles/kg), P (439.7 mmoles/kg), S (316.3 mmoles/kg), Cl (279.7 mmoles/kg), K (204 mmoles/kg) and Ca (81.3 mmoles/kg). All the drugs (quinine, chloroquine, primaquine, pyrimethamine, artemisinin, tetracycline, doxycycline, dapsone and suramin), phagocytosis of latex beads and β-hematin as well as heat shock, altered the elemental concentrations of canine monocytes in a unique way. Quinine, artemisinin and suramin were the most influential drugs in altering the concentrations of elements in the cells.Suramin substantially increased the concentration of Ca (356%) after 18 h and decreased K concentration (64%) after 18 h. Quinine decreased the concentrations ofNa (47%), Cl (70%), and K (67%). The concentrations of P (52%) and Ca (72%) were increased by quinine after 10 min. Artemisinin induced small increases in Mg (21 %) and K (38%) concentrations within 10 min and large increases in the concentrations of Na (291%) and Cl (389%) after 18 h. Chloroquine induced a large increase in S (212%). Quinine induced major changes after 10 min whereas artemisinin, suramin chloroquine induced huge changes after 18 h. Although artemisinin did increase the concentrations certain elements after 10 min, it was by much smaller amounts than after 18 h. Quinine, suramin and pyrimethamine altered the P/K ratios by the greatest margins whereas artemisinin had no significant effect. The P/K ratio was increased by quinine (348%) after 10 min and suramin (261%) after 18 h. Pyrimethamine decreased the P/K ratio after 18 h by 49%. The findings suggest that further investigations into the alterations in the elemental concentrations of monocytes by anti-malarial drugs, fever and hemozoin may lead to a greater understanding of the influence of these conditions in a patient during a malaria infection and its treatment. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

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