La dégénérescence des photorécepteurs dans la pathologie de la dégénérescence maculaire liée à l'âge (DMLA) est associée à une infiltration et accumulation des phagocytes mononuclées (PM). Nous avons montré précédemment que les souris déficientes pour Cx3cr1 développent une accumulation des PM sous-rétiniens avec l'âge et avec le stress, qui est associée une dégénérescence des photorécepteurs. Dans le cerveau, la déficience de Cx3cr1 dans les PM induit une augmentation de la mort des neurones via IL-1β. La raison de l'augmentation de la sécrétion d'IL-1β par les PM déficients en Cx3cr1 reste inconnue. Nous montrons que les PM déficients en Cx3cr1 surexpriment le récepteur P2RX7 qui stimule la maturation et la sécrétion d'IL-1β. L'inhibition de P2RX7 et d'Il-1β diminuent la mort des photorécepteurs dans un modèle de cocultures de monocytes/rétine et avec le modèle d'illumination in vivo. Nos résultats suggèrent que l'inhibition de P2RX7 ou d'Il-1β peut diminuer l'inflammation sous-rétinienne qui est associée à la mort des photorécepteurs dans la pathologie de la DMLA, où il n'existe aucun traitement à l'heure actuelle pour la forme atrophique. / Photoreceptor degeneration in age-related macular degeneration (AMD) is associated with an infiltration and chronic accumulation of mononuclear phagocytes (MPs). We have previously shown that Cx3cr1 -deficient mice develop age- and stress- related subretinal accumulation of MPs, which is associated with photoreceptor degeneration. Cx3cr1 -deficient MPs have been shown to increase neuronal apoptosis through IL-1β in neuroinflammation of the brain. The reason for increased IL-1 β secretion from Cx3cr1 -deficient MPs, and whether IL-1β is responsible for increased photoreceptor apoptosis in Cx3cr1 -deficient mice, has not been elucidated. Here we show that Cx3cr1 -deficient MPs express increased surface P2X7 receptor (P2RX7), which stimulates IL-1β maturation and secretion. P2RX7 and IL-1_β inhibition efficiently blunted Cx3cr1 -MP-dependent photoreceptor apoptosis in a monocyte/retina coculture system and in light induced subretinal inflammation of Cx3cr1 -deficient mice in vivo. Our results provide an explanation for increased CX3CR1-dependent IL-1β secretion and suggest that IL-1β or P2RX7 inhibition can help inhibit the inflammation-associated photoreceptor cell loss in late AMD, including geographic atrophy, for which no efficient treatment currently exists.

No description available.

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

In the gut-associated lymphoid tissues, Th17 cells mediate mucosal homeostasis and inflammation. During HIV infection, Th17 cells become depleted and functionally impaired, which is implicated in the pathogenesis of chronic inflammation in patients treated with highly active antiretroviral therapy. IL-7 is a cytokine that mediates homeostatic responses in T lymphocytes, such as proliferation and survival, which are dysregulated during HIV infection. Whether similar dysregulation occurs in Th17 cells has yet to be reported. IL-7 receptor α (CD127) expression and IL-7 responses were therefore measured in blood-derived Th17 cells from uninfected individuals and effectively treated, HIV-infected individuals by flow cytometry. Th17 cells from uninfected individuals expressed CD127 and, in response to IL-7, exhibited phosphorylation of STAT5, upregulation of Bcl-2, and proliferation. During HIV infection, expression of CD127 and pSTAT5 in Th17 cells was comparable to that observed in cells from uninfected individuals. Interestingly, expression of Bcl-2 was upregulated while proliferation was dramatically impaired. These findings may provide further insight into the mechanisms by which Th17 cells fail to become restored during HIV infection.

Formation of lipid-laden macrophage foam cells and inflammation are the central components in the initiation and progression of atherosclerosis. ABCA1 is well established as an anti-atherogenic factor that facilitates cellular cholesterol and phospholipid efflux, promotes reverse cholesterol transport, and suppresses pro-inflammatory cytokine secretion. Through these functions, ABCA1 is capable of reducing the lipid burden in atherosclerotic plaque. PKA signaling is an integral factor in promoting many anti-atherogenic functions of ABCA1; however, mechanistic aspects of PKA signaling associated with ABCA1 remain poorly defined. Thus, the first part of this study investigates the involvement of spatially regulated PKA signaling in ABCA1 activities through the use of st-Ht31, a PKA de-anchoring peptide. It appears that de-anchoring PKA robustly increases ABCA1-mediated microparticle release, one of the cholesterol efflux pathways of ABCA1, and reverses macrophage foam cell formation. These results highlight the significance of subcellular compartmentalization of PKA signaling in ABCA1 functions and present PKA de-anchoring as a potential therapeutic strategy for atherosclerotic lesion regression. The second part of this study provides evidence that ABCA1 activates PKA and promotes the secretion of anti-inflammatory IL-10, a cytokine crucial for inflammation resolution. Furthermore, we provide evidence that this elevated PKA activity is the underlying mechanism in which macrophage ABCA1 promotes M2-like inflammatory response. Our results also suggest that ABCA1 activates PKA by regulating cholesterol, which poises macrophages towards an anti-inflammatory or M2-activated phenotype. Collectively, we demonstrate that PKA signaling plays a crucial multifactorial role in anti-atherogenic functions of ABCA1.

La barrière hémo-encéphalique (BHE) est formée des cellules endothéliales microvasculaires cérébrales reliées entre elles par des jonctions serrées. Grâce à sa perméabilité restreinte et sélective, la BHE entrave le passage des molécules et cellules du sang vers le système nerveux central (SNC). Chez les patients atteints de sclérose en plaques (SEP), une maladie inflammatoire du SNC, la rupture de la BHE permet aux cellules immunes actives d'infiltrer le tissu cérébral. Il s'ensuit une réaction inflammatoire excessive au cours de laquelle d'autres leucocytes sont recrutés dans le cerveau et qui culmine par la formation des plaques de démyélinisation caractéristiques de la SEP. On dénote au niveau de ces lésions une présence importante de lymphocytes T CD4⁺ activés et de cytokines pro-inflammatoires propres à une réponse de type TH1, tels l’IFN-γ et l’IL-1. Curieusement cependant, l’inhibition de la voie TH1 n’empêche pas l’apparition de la maladie dans le modèle murin de la SEP et en aggrave même les symptômes. On attribue maintenant aux lymphocytes TH17, nommées en raison de leur capacité à produire de l’IL-17, un rôle clé dans le développement de la maladie. L’objectif de ce travail de thèse visait à caractériser les lymphocytes TH17 chez l’humain et définir leur contribution exacte dans la fragilisation de la BHE, une étape décisive dans la formation des lésions de SEP. Pour ce faire, nous avons mis au point une méthode expérimentale permettant l’expansion in vitro de populations de lymphocytes TH17 à partir de cellules mononuclées du sang de donneurs sains. Nos travaux démontrent que l’IL-23 induit la production d’IL-17, d’IL-22 et de granzyme B par les lymphocytes T CD4⁺CD45RO⁺ mémoires humains et qu’une proportion des cellules exprime de manière concomitante de l’IL-17 et de l’IFN-γ. La fréquence des lymphocytes T CD4⁺ IL17⁺, IL-22⁺ et des doubles positifs IL-17⁺IFN-γ⁺ est significativement plus élevée dans les lignées de lymphocytes TH17 provenant de patientes en poussée que dans celles de contrôles. Nos analyses démontrent que les cellules endothéliales de la BHE expriment de faibles niveaux des récepteurs de l’IL-17 et de l’IL-22 à l’état basal mais que leur présence est accrue dans le cerveau de patients atteints de SEP. L’activation du récepteur de l’IL-17 entraîne une augmentation de la perméabilité de la BHE et une perturbation de l’organisation des protéines de jonction occludine et ZO-1. Finalement, nous démontrons que la migration des lymphocytes TH17 à travers la BHE est régie en grande partie par la molécule d’adhérence ICAM-1 et que les lymphocytes qui co-expriment l’IL-17 et l’IFN-γ sont plus aptes à franchir la BHE que ceux qui produisent uniquement l’une ou l’autre de ces cytokines. Nous retrouvons d’ailleurs des cellules qui expriment simultanément les facteurs de transcription T-bet et RORC, associés respectivement aux lymphocytes TH1 et aux TH17, au sein des infiltrats péri-vasculaires des lésions actives de SEP. Les travaux présentés dans cette thèse auront permis d’affiner nos connaissances sur les mécanismes d’entrée des lymphocytes TH17 dans le SNC et les propriétés délétères des cytokines qu’ils sécrètent, notamment dans l’activation et la déstabilisation de l’endothélium cérébral. / The blood-brain barrier (BBB) plays a crucial role in protecting the central nervous system (CNS) by restricting entry of cells and molecules into the brain. In the CNS disorder multiple sclerosis (MS), breakdown of the BBB allows activated leukocytes to infiltrate the brain parenchyma, leading to the formation of the characteristic demyelinated lesions. For decades, MS was viewed as a TH1-mediated disease, a notion that was largely supported by studies in its animal model and by the abundance of prototypical TH1-associated cytokines within active MS lesions. However, over the years, accumulating evidence has highlighted the involvement of another subset of CD4⁺ T cells that express IL-17, therefore named TH17 lymphocytes, in the pathology of the disease. The goal of the work presented herein was to characterize the human TH17 lymphocyte population and define their contribution to the disruption of the BBB and leukocyte infiltration into the CNS, both important early events in the formation of MS lesions. To do so, we developed and optimized a method to successfully generate human TH17 lines in vitro from peripheral blood mononuclear cells of healthy donors. We demonstrate that in response to IL-23, human memory CD4⁺CD45RO⁺ but not naïve CD4⁺CD45RA⁺ T lymphocytes produce IL-17, IL-22, and granzyme B, with a subset of cells simultaneously expressing IL-17 and IFN-γ. Interestingly, we measure a significant increase in the percentage of T CD4⁺ IL17⁺, of IL-22⁺, and of IL-17⁺IFN-γ⁺ dual producers in TH17 cell lines expanded from the peripheral blood of acutely relapsing MS women as compared to those generated from healthy controls and remitting MS patients. We show that both IL-17 and IL-22 receptors are upregulated on BBB endothelial cells in situ during inflammation and that IL-17 enhances BBB permeability by disrupting the integrity of tight junction proteins occludin and ZO-1. Finally, we provide evidence that TH17 lymphocytes transmigrate efficiently across human brain endothelial cells via the adhesion molecule ICAM-1 and show that IL-17⁺IFN-γ⁺ double producers have an increased propensity to do so. Accordingly, we detect lymphocytes that display immunoreactivity against both the TH1- and TH17-associated transcription factors T-bet and RORC within perivascular infiltrates of active MS lesions. The work presented in this thesis has refined our understanding of the mechanisms that drive TH17 lymphocyte recruitment into the CNS and shed light on the deleterious effect of TH17-secreted cytokines, specifically in the activation and breakdown of the BBB.

GB virus C (GBV-C) is a lymphotropic human virus which was recently assigned to a new genus Pegivirus within the Flaviviridae family. GBV-C infection is found worldwide, and viremia prevalence is about 1% to 4% in healthy blood donors and up to 42% in HIV-infected individuals. In clinical studies, GBV-C coinfection is associated with prolonged survival of HIV-infected individuals. GBV-C infection modestly alters T cell homeostasis in vivo through various mechanisms, including modulation of chemokine and cytokine release and receptor expression, and by diminution of T cell activation, proliferation and apoptosis, all of which may contribute to improved HIV clinical outcomes. This thesis explores the interrelationship between GBV-C infection and immunoactivation and identifies potential mechanisms by which GBV-C reduces immunoactivation. Chronic HIV infection is associated with persistent immunoactivation which contributes to the immune dysfunction. In particular, T cell activation supports HIV replication and correlates with HIV viral load (VL). Persistent immunoactivation also contributes to the depletion of uninfected bystander cells by mechanisms of activation induced cell death (AICD). Although treatment with combination antiretroviral therapy (cART) reduces HIV VL, T cell activation does not return to levels found in HIVuninfected individuals. Sustained immunoactivation is also associated with lower virological response to cART suggesting therapies to reduce immunoactivation in combination with cART may benefit HIV-infected individuals. Since GBV-C infection is associated with reduced immunoactivation, understanding mechanisms by which GBV-C modulates these signaling pathways may provide insights into novel approaches to treat HIV infection and chronic immunoactivation. The effect of GBV-C infection on T cell activation and IL-2 signaling pathways were studied in a cohort of HIV-positive individuals. GBV-C viremic HIV positive individuals on cART have reduced T cell activation which was significantly associated with higher percentage of immunomodulatory CD3 +CD4-CD8-T cells. Ex vivo GBV-C infection was associated with reduced lymphocyte proliferation in response to IL-2, lower frequency of reactivation of latent HIV and protection against AICD. In vitro expression of GBV-C envelope glycoprotein E2 in CD4+ T cell lines inhibited T cell receptor (TCR) induced IL-2 secretion and inhibited IL-2 signaling pathways. This effect was mediated at least in part by reducing activation of lymphocyte specific tyrosine kinase (Lck). Through deletion mutagenesis, the inhibitory motif within the viral protein was mapped to a region that contains a predicted Lck substrate, a highly conserved tyrosine at position 87 (Y87). Lck phosphorylated GBV-C E2 protein in vitro and mutation of Y87 residue abolished the inhibitory effects of E2 protein. Synthetic peptides containing this inhibitory motif competed for Lck phosphorylation and inhibited TCR signaling in primary human T cells. The number of GBV-C infected T cells was found to be low in vivo, yet GBV-C infection reduced global TCR signaling. GBV-C RNA and E2 protein were detected in extracellular microvesicles purified from GBV-C infected human serum or the culture supernatant of E2 expressing cells, and these microvesicles inhibited TCR signaling in uninfected bystander T cells. Together, these data identify a novel mechanism by which GBV-C infection leads to global reduction in T cell activation and IL-2 signaling in the infected host, and provide a working model in which the viral envelope glycoprotein serves as a substrate for Lck and competes for Lck phosphorylation in the infected T cells and in uninfected bystander T cells.

Antigen-specific CD8 T cells provide an important protective role in response to infection by viruses, intracellular bacteria, and parasites. Pathogen-specific CD8 T cells render this protection by undergoing robust expansion in numbers while gaining the ability to produce cytokines and cytolytic machinery. Following expansion and effector differentiation, pathogen-specific CD8 T cells will contract in number while further differentiating into a highly functional population of memory CD8 T cells. These antigen-experienced cells persist in secondary lymphoid organs and the periphery in order to rapidly respond to repeated infection. Creating optimal CD8 T cell responses to infection can be critical for raising sufficient armament to provide protection against invading intracellular pathogens. Although CD8 T cells have protective value, many vaccine strategies tend to focus on creating productive B cell antibody responses to promote immunological protection. Even though antibody responses can be highly protective, coupling optimal CD8 T cell responses with B cell responses could provide higher orders of protection than either one on their own. Therefore, a deeper understanding of the pathways that ultimately guide the magnitude of CD8 T cell responses is required to achieve this potential therapeutic benefit. My studies evaluate the role of receptor signaling events in guiding the expansion of activated CD8 T cells during primary and secondary responses. Specifically, the first portion of my studies dissect the mechanism by which direct IL-12 and Type I IFN stimulation can substantially bolster primary CD8 T cell responses in vivo. Within this context, I demonstrate that direct IL-12 and Type I IFN signaling increases CD8 T cell accumulation during primary expansion by prolonging division without altering survival. IL-12/Type I IFN signaling promoted prolonged division of activated CD8 T cells by maintaining high-affinity IL-2 receptor subunit (CD25) expression and IL-2 signaling. The other portion of my work was dedicated to understanding the expression and role of the inhibitory FcgR (FcgRIIB) during primary and secondary CD8 T cell responses. FcgRIIB expression could be detected as early as the peak of the CD8 T cell response and marked activated CD8 T cells that were highly sensitive to antigen stimulation. Although FcgRIIB did not appear to play a substantial role in regulating the magnitude of primary CD8 T cell responses, it played an important role in inhibiting the expansion and cytotoxicity of memory CD8 T cells during homologous challenge. Collectively, these data highlight potential avenues that could be exploited by future therapies that aim to achieve appropriately sized CD8 T cell responses.

Les lymphocytes B régulent la réponse immunitaire par la sécrétion de facteurs proinflammatoires ou immunosuppresseurs. Dans la Leucémie Lymphoïde Chronique (LLC),une sous-population de lymphocytes B CD5⁺ présente des propriétés immunosuppressives qui l’apparente aux lymphocytes B régulateurs notamment par la production d’IL-10. La survie des cellules leucémiques est, elle, associée à la réponse antigénique et à la production de cytokines dont l’IL-6. L’objectif de ce travail a été de caractériser dans la pathologie les populations produisant les cytokines pro-survie ou immunorégulatrices et d’analyser la relevance fonctionnelle de leur sécrétion. Nous avons identifié des sous-populations de cellules B leucémiques exprimant trois facteurs immunorégulateurs l’IL-10, le TGFβ1 et pour la première fois le facteur de transcription FOXP3, La proportion augmentée de cellules exprimant l’IL10 est associée à une diminution des cellules exprimant l’IL6. De manière importante, ce travail a identifié une boucle autocrine de stimulation de l’activité métabolique des cellules par l’IL10. La cytokine en se fixant à son récepteur permet l’activation des facteurs STAT3 et induit l’expression à la fois de protéines anti- apoptotiques de la famille Bcl2 mais surtout sa propre expression. Un blocage de cette boucle au niveau du récepteur à l’IL10 suspend l’avantage de survie des cellules tumorales. L’IL-6 ne déclenche pas ces mécanismes de maintien des cellules de LLC. Ce travail montre qu’en plus de son rôle sur les cellules du microenvironnement tumoral, l’IL-10 participe au maintien autocrine de la sous-population immunorégulatrice dans la LLC. / B cells produce pro-inflammatory or immunosuppressive factors to modulate the immuneresponse. In Chronic lymphocytic leukemia (CLL), a subset of the tumor lymphocytes produces IL10 and share immunoregulatory functions with regulatory B cells. CLL cell ssurvival is driven by antigenic response and pro-survival cytokines such as IL6. This project aimed at deciphering the cytokines profile of CLL subsets and analyzing their functional relevance. We identified immunoregulatory subsets producing IL-10, TGFβ1 and for the firsttime FOXP3. In patients, the increased proportion of cells expressing IL10 was correlated with decrease in IL6⁺ cells. Importantly we described an autocrine survival loop driven by IL10 in these cells. IL10 triggering led to STAT3 activation, induction of active pro-survival factors altogether with IL10 self-induction. Interrupting this loop with a blocking ab against IL10R prevented survival of the cells. IL6 did not manage such mechanisms. In conclusion,this work demonstrates that IL10 is an important mediator in CLL; the cytokine alters immune recognition of the tumor cells and sustains leukemic cells survival via the autocrine loop.

Orientador: Daniela Ponce / Resumo: A prevalência da Lesão Renal Aguda (LRA) associada ao uso da vancomicina é muito variável e diferentes fatores relacionados ao paciente e ao tratamento (tempo e dose) podem potencializar a ocorrência da nefrotoxicidade. Estudos relacionaram o desempenho de biomarcadores urinários como preditores diagnósticos e prognósticos de LRA na insuficiência cardíaca (ICC), no pós-operatório de cirurgia cardíaca e na sepse, porém, pouco se sabe sobre o desempenho dos novos biomarcadores na LRA associada ao uso da vancomicina. / Abstract: The prevalence of acute kidney injury (AKI) related to vancomycin is variable and different risk factors related to the patient and the treatment (time and dose) may potentiate the occurrence of nephrotoxicity. Studies related the performance of urinary biomarkers as predictors of diagnostic and prognostic AKI in congestive heart failure (CHF), in the postoperative period of cardiac surgery and in sepsis, however, little is known about the performance of the new urinary biomarkers in vancomycin related AKI. / Mestre