Hepatitis C virus (HCV) establishes a chronic infection in about 70% of infected individuals that is associated with the development of liver cirrhosis and hepatocellular carcinoma. The mechanisms by which HCV avoids clearance by the host immune response are not fully understood. The first aim of this project was to determine whether immune cell subsets could become infected by HCV in vitro. None of the haematopoietic subsets analysed expressed all of the required entry factors, CD81, SR-BI, claudin-1 and occludin. Also, PBMCs were not susceptible to infection with HCVpp and HCVcc expressing glycoproteins of hepatotropic strains. Infection by a supposedly lymphotropic strain (SB) was found to be inefficient. The second aim was to identify in vitro immunomodulatory effects of HCV on innate immune cells that may impact on the immune response activated in acute infection. Crosslinking of CD81 on NK cells by antibody was found to have a minor inhibitory effect on their activation via CD16, but CD81 crosslinking by viral particles had no detectable effect. In contrast to other viruses, HCVcc elicited very little interferon-α production by pDC. HCVcc also did not affect pDC or mDC responses to TLR ligation. Systemic cytokine and chemokine responses were analysed in subjects with primary acute HCV infection and in HCV-infected patients undergoing liver transplantation (LT). Interestingly, induction of systemic type I and type III interferon was not observed in either group. Marked perturbations in systemic cytokine and chemokine levels were detected in uninfected LT patients, precluding use of HCV-infected LT patients to study the innate immune response activated in response to acute viral replication. Together, these results suggest that HCV may principally evade innate immune cell responses by avoidance rather than impairment strategies.

Les virus du Chikungunya (CHIKV) et de la dengue (DENV) sont deux virus émergents qui sévissent dans les régions tropicales et subtropicales du monde entier et qui sont transmis par les moustiques du genre Aedes. Ces dernières années, leur transmission a surtout progressé dans les zones urbaines et périurbaines touchant des millions d’individus et faisant de ces deux pathogènes des sujets majeurs de préoccupation pour la santé publique. Le Chikungunya et la Dengue sont des infections dites aiguës entrainant une mise en place rapide de la réponse immunitaire innée qui joue un rôle majeur dans le contrôle et l’évolution de la maladie. Les cellules Natural Killer (NK) représentent une population cellulaire clé de la réponse innée et jouent un rôle crucial dans les mécanismes de défense mis en place. A travers une étude ex vivo et in vitro, nous nous sommes intéressées à la caractérisation des cellules NK à travers (i) une étude phénotypique et fonctionnelle des cellules NK chez des patients infectés en phase aiguë par le CHIKV, DENV-2 ou par les deux virus et (ii) à la caractérisation des interactions entre les cellules NK et les cellules cibles infectées par le virus. L’ensemble de ces données contribue à mieux identifier l’implication des cellules NK dans le contrôle des infections par le CHIKV et DENV-2 permettant ainsi de mieux comprendre les mécanismes à l’origine des dérèglements de la réponse immunitaire. Au cours des dernières épidémies, plusieurs cas de patients coinfectés par les deux virus ont été répertoriés. De plus, l’expansion géographique des moustiques Aedes pourrait amener à une augmentation du nombre de cas de coinfections sans que les mécanismes sous jacents aux coinfections ne soient étudiés. Afin de pouvoir réponse à certaines questions concernant ce phénomène, nous avons mis en place un modèle expérimental de coinfection par CHIKV et DENV-2 chez le macaque Rhésus. / Chikungunya (CHIKV) and Dengue (DENV) virus are both re-emerging viruses transmitted by Aedes mosquitoes and responsible of widespread outbreaks in tropical and subtropical country. Recently, transmission of both viruses had emerged in urban and peri-urban area infecting millions of persons. Chikungunya and Dengue are both acute infections where innate immunity rapidly takes place and play a crucial role in the control and in the evolution of the disease. Natural Killer cells (NK) represent one of the major cellular population of innate immunity and play a crucial role in defense mechanism. By way of ex vivo and in vitro studies, we characterized NK cells by (i) a phenotypic and functional study of NK cells in CHIKV, DENV-2 infected patients or CHIKV/DENV-2 co-infected patients and (ii) characterization of NK cells interactions with infected target cells. During last outbreaks, several cases of co-infected patients were reported. Moreover, geographic spread of Aedes mosquitoes could increase number of coinfection cases without underlying mechanisms being explored. In order to respond to certain questions regarding coinfections, we realized a co-infected CHIKV and DENV-2 experimental model in Rhesus macaques.Together, these data will contribute to better identify NK cells implication in the control of CHIKV and DENV-2 infections allowing a better comprehension of mechanisms that causes immune system disorder.

Les maladies auto-immunes (MAI) correspondent à un large ensemble de pathologies cliniquement hétérogènes, affectant le plus souvent des adultes jeunes, de façon volontiers chronique. Du point de vue physiopathologique, ces maladies correspondent à la survenue d’une rupture de tolérance au soi, dont les mécanismes sont complexes et font appel à l’ensemble des acteurs du système immunitaire. Si l’implication des cellules de l’immunité adaptative est largement documentée dans ce contexte, celle des cellules appartenant à l’immunité innée, comme les cellules Natural Killer (NK) est peu étudié. A travers deux exemples de MAI systémiques, le Lupus Systémique (LS) et le Syndrome des Antisynthétases (SAS), l’objet de ce travail est de montrer l’implication des cellules NK au cours des MAI et d’étudier les mécanismes en cause.L’étude phénotypique et fonctionnelle des cellules NK chez des patients présentant une MAI révèle de nombreuses anomalies comparativement aux sujets contrôles. Ces dernières sont plus marquées chez les patients en phase active plutôt qu’en rémission. De plus, l’infiltration des tissus cibles au cours du SAS par les cellules NK d’une part, et l’activation in vitro de ces cellules par les auto-antigènes au cours du LS d’autre part, confirme l’implication des cellules NK au cours de ces deux MAI. Par ailleurs, des interactions des cellules NK avec plusieurs types cellulaires impliqués dans l’immunopathologie de ces maladies semblent conditionner les anomalies observées. Ces dernières sont différentes selon la maladie étudiée : le profil des cellules NK des patients atteints de LS étant plutôt immature et tourné vers la production de cytokines, tandis que celui des patients atteints de SAS correspond à un stade de différentiation terminal mais hypofonctionnel.L’ensemble des résultats suggère que les cellules NK participent à l’immunopathologie des MAI. Leur implication est conditionnée par l’effet de certains stimuli et certaines interactions cellulaires, qui sont de nature différente d’une MAI à l’autre. / Auto-immune diseases (AID) form a broad spectrum of heterogeneous and chronic pathologies, most commonly affecting young adults. The etiopathogenesis of AID corresponds to a breakdown of the immunological tolerance: the result of complex mechanisms, implicating every component of the immune system. While adaptive immune cells has been extensively studied in this context, the role of innate immune cells, including Natural Killer (NK) cells, is much less understood. Using Systemic Lupus Erythematosus (SLE) and Antisynthetase Syndrome (ASS) as model pathologies, the main objective of this work is to demonstrate the involvement of NK cells in AID and to study the relevant mechanisms. Patients with AID showed numerous anomalies in the phenotypical and functional analysis of their NK cells, as compared to healthy controls. These differences are more pronounced in active rather than inactive patients. Moreover, the infiltration of target tissues by NK cells in ASS as well as the activation of these cells by SLE specific auto-antigens confirm the involvement of NK cells in AID. Additionally, interactions of NK cells with different immune cells, known to be involved in AID pathogenesis, seem to be the cause of the observed anomalies. These anomalies differ among both AID: NK cells from patients with SLE are immature and devoted to cytokine production, whereas those from patients with ASS have reached a highly differentiated but hypofunctional stage. Taken as a whole, these data suggest that NK cells are involved in the immuno-pathogenesis of AID. This involvement seems conditioned by the effect of different stimuli and different cellular interactions, which are distinct from one form of AID to another.

Os C/EBP (CCAAT/enhancer-binding proteins) são uma família de fatores de transcrição implicados numa variedade de processos da hematopoese, regulando tanto a diferenciação terminal como a proliferação celular. Dentre estes, sabe-se que o C/EBP gamma (C/EBPG) está envolvido na maturação funcional de células Natural Killer (NK). Entretanto, os mediadores dessa regulação não são conhecidos. As células NK são linfócitos com funções efetoras de citotoxicidade e de produção de citocinas, dependentes de um equilíbrio dinâmico entre a expressão de receptores ativatórios e inibitórios bem como de receptores de citocinas. As duas funções (citotóxica e secretora), fazem das células NK importantes componentes da hematopoese, capazes de eliminar alvos susceptíveis bem como de recrutar outras células e amplificar a resposta inflamatória. Diante de incompatibilidade entre as células-alvo e células NK efetoras, os efeitos citotóxicos preponderam; enquanto na ausência de incompatibilidade, os efeitos mediados por citocinas sobre as demais células hematopoéticas se sobressaem. Por exemplo, citocinas como IFN?, TNF?, TGF?, GM-CSF e IL-10, produzidas por células NK, são potenciais reguladoras da função das células-tronco hematopoéticas (CTH). Com o objetivo de estudar a regulação das células NK pelo C/EBPG, utilizamos células NK isoladas de animais transgênicos knockout (KO) para o C/ebpg e seus controles para analisar sua expressão gênica e função diferencial, com especial foco nos genes-alvo com potencial para regulação da hematopoese. Visando identificar potenciais genes-alvo do C/EBPG, isolamos células NK deficientes para o C/ebpg e controles, por meio de sorting, e realizamos posterior isolamento do RNA seguido de análise de expressão gênica em larga escala. A validação da expressão diferencial dos genes-alvo do C/ebpg, de interesse para a função secretória das células NK, foi realizada por PCR em Tempo Real para oito genes diferencialmente expressos na análise de expressão gênica em larga escala, a saber: Il-10, Gmcsf, Ifng, Tnfa, Tgfb, Tlr4, Myd88 e Irak4. Os dados referentes à expressão basal e estimulada com IL-2, destes genes em células NK de animais KO para o C/ebpg e seus controles, revelaram uma tendência ao aumento da expressão de Myd88 nos animais KO quando comparados aos controles. Foram verificados os níveis das citocinas IL-2, IL-4, IL-6, IL-10, IL-17A, TNF? and IFN? por meio de citometria de fluxo, utilizando o sobrenadante da cultura de NK de ambos os animais, observando-se que, após a ativação com IL-2, a produção de IFN? mostrou-se diminuída em células NKdeficientes para o C/ebpg em comparação aos controles. Para caracterizar as células NK Cebpg-deficientes, analisamos a sua frequência (células linhagem-/CD3- /NK1.1+) e expressão dos receptores NKG2D, Ly49D e NKG2A, não sendo observadas diferenças numéricas ou de expressão de receptores entre células NK deficientes ou não para o C/ebpg. Os subtipos funcionais destas células foram caracterizados de acordo com a expressão de CD27 e CD11b, que permitem identificar as subpopulações de células NK imaturas secretórias, secretórias, citotóxicas e tolerantes. Os animais KO mostraram maior percentagem de células secretórias e redução percentual e numérica de células citotóxicas quando comparadas às células NK dos controles. Para demonstrar a deficiência funcional realizamos um ensaio de ativação de células. Em concordância, após a coincubação de esplenócitos totais com células YAC-1, o ensaio de detecção do CD107 revelou que as células dos animais KO para o C/ebpg são cinco vezes menos ativadas do que células NK controles. Ademais, foi realizado um ensaio de citotoxicidade por citometria de fluxo utilizando o CTO (Cell Tracker Orange) como sonda fluorescente, o qual se incorpora às células-alvo da linhagem YAC-1. Como resultado, para a razão 10:1 NK: células-alvo, as células NK C/ebpg KO foram menos citotóxicas do que as células NK dos controles. Concluímos que as células NK de animais transgênicos KO para o C/ebpg têm função deficiente, menor potencial citotóxico e expressão de genes e citocinas alteradas em relação aos seus controles. Os mediadores apontados, em especial, o IFN?, são alvos importantes para a regulação da função secretória das células NK. / C/EBP (CCAAT/enhance-binding proteins) are a family of transcription factors involved in a variety of hematopoietic processes, regulating both terminal differentiation and cellular proliferation. Among these, it is known that C/EBP gamma (C/EBPG) is involved in the functional maturation of Natural Killer (NK) cells. However, the mediators of this regulation are unknown. NK cells are lymphocytes with effector functions of cytotoxicity and production of cytokines, both dependent on a dynamic equilibrium between the expression of activating and inhibitory receptors as well as cytokine receptors. The two functions (cytotoxic and secretory) make NK cells important components of hematopoiesis, able to eliminate susceptible targets as well as recruit other cells to amplify inflammatory responses. In face of incompatibility between target cells and NK effector cells, cytotoxic effects predominate; while in the absence of incompatibility, cytokine-mediated effects that influence other hematopoietic cells prevail. For example, cytokines such as IFN?, TNF?, TGF?, GMCSF and IL-10, produced by NK cells, are potential regulators of hematopoietic stem cells (HSC). With the aim of studying the regulation of NK cells by C/EBPG, we isolated NK cells from transgenic C/ebpg knockout (KO) animals and controls to analyze their differential gene expression and function, with a special focus on hematopoiesis regulation. In order to identify potential C/EBPG target genes, we isolated C/ebpg-deficient and control NK cells by the use of sorting by flow cytometry and isolated RNA for gene expression analysis. Differential expression of C/ebpg target genes was performed by Real-time PCR for eight genes differentially expressed in the microarray analysis: Il-10, Gmcsf, Ifng, Tnfa, Tgfb, Tlr4, Myd88 e Irak4. When compared to controls, non-activated and IL-2-stimulated C/ebpg KO NK cells presented a tendency to have higher expression of Myd88. Cytokine levels of IL-2, IL-4, IL-6, IL-10, IL-17?, TNF? and IFN?, obtained from NK culture supernatants, were verified by flow cytometry, after IL-2 activation. Among these cytokines, the production of IFN? by C/ebpg-deficient NK cells was found to be reduced. To further characterize NK cells, we analyzed their frequency (Lineage- /CD3-/NK1.1+ cells) and the expression of the receptors NKG2D, Ly49D and NKG2A, and both analyses presented similar expression between control or C/epbg KO NK cells. The functional subtypes of these cells were characterized according to the expression of CD27 and CD11b, which allow the identification of NK subpopulationsas immature secretory, mature secretory, cytotoxic or tolerant. The KO animals showed higher percentage of secretory cells and percentual and numerical reduction of cytotoxic cells when compared to the NK control cells. In agreement, CD107a expression was 5-times lower in C/ebpg KO splenocytes than in control splenocytes after co-incubation with YAC-1 cells. In addition, a cytotoxicity assay by flow cytometry was performed. The fluorescent probe CTO (Cell Tracker Orange) was incorporated to YAC-1 cells, used as target cells. As a result, in the 10:1 NK:target cells ratio, C/ebp KO cells were less cytotoxic than NK control cells. We concluded that C/ebpg-deficient cells are dysfunctional, have a greater secretory potential and an altered expression of genes and cytokines as compared to their controls. The potential mediators revealed by our study, in particular, IFN?, may be important targets for the regulation of NK secretory function.

Linfócitos T CD8 e células NK atuam no combate à infecções por bactérias intracelulares, vírus e células tumorais, provocando a morte dessas células por meio da secreção de grânulos citotóxicos. Proteínas RAB GTPase têm se destacado em estudos de tráfego intracelular, porém, são escassos dados sobre o papel destas proteínas em células citóxicas. Um estudo prospectivo de proteômica realizado por nosso grupo identificou a RAB2A, RAB5A, RAB17 e RAB18 em grânulos citotóxicos. Análises mais aprofundadas revelaram que a RAB2A está associada a proteínas como LAMP-1 e LAMP-2, enquanto que RAB5A, RAB17 e RAB18 estavam presentes na mesma linhagem em um contexto não contemplado neste estudo. Desenvolvemos ainda uma abordagem de silenciamento gênico da RAB2A, e por fim, adaptamos uma série de protocolos de simples execução e baixo custo para avaliar funções efetoras de células NK. O conhecimento da maquinaria secretória é fundamental, uma vez que defeitos nas vias de tráfego intracelular constituem a base de um grande número de doenças que desencadeiam quadros fatais. / CD8 T lymphocytes and NK cells fight against infections by intracellular bacteria, viruses and tumor cells by killing those cells through the secretion of cytotoxic granules. RAB GTPase has been highlighted in studies of intracellular trafficking, however there are scarce reports regarding the role of these proteins in cytotoxic cells. A proteomic study performed by our group identified RAB2A, RAB5A, RAB17 and RAB18 in cytotoxic granules. Further analysis revealed that RAB2A is associated with LAMP-1 and LAMP-2, while RAB5A, RAB17 and RAB18 were present in the same cell line, but in a context not included in this study. We also have developed a gene silencing approach for RAB2A and adapted a number of protocols, simple and low-cost, that can be used to evaluate effector functions of natural killer cells The knowledge of secretory machinery involved in the movement cytotoxic granules of cytotoxic cells is critical, since defects in intracellular trafficking pathways constitute the basis for a large number of diseases which trigger death.

There are multiple immune mechanisms identified for persistence of hepatitis B virus (HBV) infection. This thesis considers the vital role that inhibitory receptors play in contributing to impairment of the adaptive immune system in chronic hepatitis B (CHB), and the potential role they play in the innate immune system, focusing on the inhibitory receptor leucocyte-associated immunoglobulin-like receptor (LAIR)-1. The unique aspect of this work is that for the first time LAIR-1 expression has been investigated on natural killer (NK) cells in CHB. Our striking findings of increased LAIR-1 expression on peripheral NK cells in CHB and an inverse correlation between expression and effector function suggest this inhibitory receptor could have a potential role in exhaustion of NK cells in CHB. We therefore additionally explored the expression of LAIR-1 on circulating NK cells from patients with hepatocellular carcinoma (HCC) and non-alcoholic fatty liver disease (NAFLD). The particular relevance of LAIR-1 to liver disease is that one of its major ligands is collagen. We demonstrated a downregulation of LAIR-1 expression on intrahepatic NK cells, which we postulate might occur following repetitive engagement with abundant collagen within the liver. In line with this, intrahepatic NK cells with a liver-resident (CXCR6+) phenotype had even lower LAIR-1 expression than liver infiltrating (non-resident, CXCR6-) NK cells. Furthermore, preliminary experiments display attenuation of the cytotoxic degranulation capacity (CD107a) by circulating NK cells from CHB patients upon exposure to plate-bound collagen. We demonstrate differential expression of LAIR-1 on NK cells in viral hepatitis, HCC and NAFLD and between peripheral and intrahepatic NK cells. Preliminary experiments demonstrate a role in inhibiting NK cell function suggesting this as a novel therapeutic target to harness the capacity of NK cells to control chronic infection and cancer.

Natural killer (NK) cells are non-specific killer cells of the innate immune system that eliminate target cells based on discrimination between self and non-self. Activation is carefully regulated through integration of signals received through both activating and inhibitory receptors. During the course of a herpes simplex virus type-1 (HSV-1) infection, NK cells can influence host susceptibility to infection with severe infections occurring in individuals with genetic defects in the NK cell response. In response to HSV infection, NK cells are recruited to the inflammatory tissue where ensuing reciprocal interactions with accessory cells and proinflammatory cytokines induce NK cell activation, cytolytic activity, and cytokine production, contributing to innate immune response and ultimately influencing the adaptive immune response. The objective of this study was to elucidate the multiple roles of NK cells during the numerous steps in anti-HSV immune induction. Accordingly, we have demonstrated that NK cells are novel helpers that assist and influence an anti-HSV immune response via the secretion of cytokines that enhance HSV-specific CD8+ T cell effector function and cytokine production. Taken together, data from this study presented the critical importance of NK cells in mounting an essential and efficient anti-HSV immunity. The key findings of our study were: 1. In the absence of NK cells, dendritic cells have decreased capacity to prime HSV-specific T cells. 2. HSV infected NK cells can be directly activated via toll-like receptor (TLR) in a MyD88-dependent mechanism; however, interaction with HSV infected dendritic cells yields optimal NK cell activation and function (CD69 and IFNγ). 3. TRAIL-expressing NK cells eliminate antigen-bearing immature dermal DCs (CD11c+CD8α-DR5+), that migrate to draining lymphoid organs, to facilitate antigen transfer to lymphoid resident CD8α+ DC for T cell cross priming. 4. 'Helpless' CD8+ T cell function, generated in the absence of CD4+ T cells, can be partially restored to wild-type levels by NK cell supplementation. 5. Treatment of NK cells with anti-CD69 antibody results in a heightened NK activated state and augments the adaptive immune response, without increasing NK cell numbers. These findings may contribute to the potential revelation of avenues to manipulate NK cells for anti-viral therapies.

Healthy term pregnancy requires precisely timed coordination of multiple systems, including reproductive, neuroendocrine, immune and cardiovascular. Dynamic maternal alterations occur systemically as well as locally within the reproductive tract. Systemic cardiovascular changes during gestation are relatively conserved in mammals, permitting comparison. These physiological changes are relatively acute and reversible, in contrast to the pathological changes seen during cardiovascular disease development. Gestational hypertensive disorders, such as preeclampsia, are the leading causes of maternal and fetal morbidity and mortality. The pathogenesis of preeclampsia is not fully elucidated, but perturbation of the immune system is a fundamental component. The angiogenic and vascular properties of uterine NK lymphocytes have been well studied in mice and women, but their relationships to gestational blood pressure regulation and cardiovascular adaptations have not been addressed. In non-pregnant women and mice, T cells, but not B cells, have been found to alter cardiovascular functioning. NK cells in humans also possess these capabilities, but no functional studies have been completed. The aim of this thesis was to define the role of NK and T lymphocytes in cardiovascular adaptations during mouse gestation. Using chronic radiotelemetry, histology, post-mortem and other techniques, female inbred mice of differing genotypes that lack specific lymphocyte subsets were compared before and across gestation. In normal, immune competent mice, a five-phase gestational blood pressure profile was found. This dynamic profile corresponded to stages of placental development. In mice with a compound deficit in arterial modification and lymphocytes, no gestational hypertension was observed. To elevate the maternal challenge of pregnancy, studies of pregnant, autoimmune Type 1 Diabetic mice were conducted. Impaired spiral artery remodeling, dysfunctional lymphocytes and growth-restricted fetuses were identified. From mid-gestation, diabetic pregnant mice were hypotensive and bradycardic and showed signs of pre-renal failure (proteinuria and electrolyte imbalances). In pregnant mice lacking T cells, tachycardia was observed despite otherwise normal gestational outcomes. In pregnant mice lacking T cells with impaired NK cells, blood pressure was blunted and tachycardia was observed. These findings support the conclusion that impaired spiral artery remodeling is insufficient to cause gestational hypertension in mice. The data further identify a role for T and NK cells in cardiac function during gestation. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2010-09-01 20:56:15.648

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 147 pages. Includes vita. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.