Les adénovirus humains (HAdVs) provoquent généralement une infection bénigne chez l'hôte sain. En revanche, chez les patients immunodéprimés et immunocompétents, ils peuvent causer des infections sévères à létales. Les vecteurs HAdVs sont couramment utilisés dans les domaines de la thérapie génique et de la vaccination. L'immunité pré-existante de l’hôte protège généralement contre les infections de type sauvage mais peut entraîner des effets indésirables lors du relargage des virus tel que l’induction de processus inflammatoire locale ou général. Après l'infection, l'inflammation va principalement entraîner le recrutement de cellules dendritiques (DCs), de monocytes et de neutrophiles. Les DCs ont la capacité unique de présenter l'antigène et d’activer les cellules T, qui, par la suite, aideront les cellules B à produire des anticorps. En plus de leurs activités phagocytaires, des peptides antimicrobiens dérivés des neutrophiles (AMPs) jouent un rôle central dans l'immunité innée. Les AMPs peuvent neutraliser les microbes infectieux et / ou activer différents types de cellules immunitaires. Dans ce contexte, nous avons étudié l'interaction ex vivo entre le facteur hôte (anticorps anti-HADV et AMPs) contre les HAdVs dans les DCs ainsi que le rôle des DCs activés indirectement (indir-DCs) lors de la réponse immunitaire. Nous avons caractérisé le profil des cytokines et des chimiokines sécrétées par les DCs stimulées par différents HAdVs, AMPs et les combinaisons qui en découlent. Enfin, nous avons constaté que l’opsonisation d’HAdV5 par les IgG accroît la capacité de capture antigénique des MoDCs (cellules dendritiques dérivées de monocytes) et induit la mort cellulaire par pyroptose. Je me suis donc concentrée sur les caractéristiques et la fonction des indir-DCs dans l’immunité contre les HAdVs. Afin de mieux comprendre les propriétés et les phénotypes des indir-DCs et DCs activés directement (dir-DCs), nous avons caractérisé leurs profils de maturation, les facteurs influençant cette maturation, et leur capacité fonctionnelle de recruter les leucocytes. Ainsi nous avons pu mettre en évidence que les dir-DCs empêchent le recrutement leucocytaire tandis que les indir-DCs favorisent la migration des monocytes. L’ensemble de ces données contribue à comprendre comment l'immunité pré-existante contre les HAdVs peut impacter l’efficacité des traitements contre les maladies HAdVs ainsi que la conception de vaccins. / Human adenoviruses (HAdV) generally cause mild infection in healthy host, but in immunocompromised and immunocompetent patients, they cause severe on lethal infections. HAdV vector are also commonly used for gene transfer and vaccination. We know that pre-existing immunity can protect from wild type infection and cause adverse effects during vector delivery including local and system inflammation. Following HAdV infection on vector use, inflammation leads to the recruitment of dendritic cells (DCs), monocyte and neutrophils. DCs have the unique ability to present antigens and activate T cells, that subsequently aid B cells to produce antibodies. In addition to their phagocytic activities, neutrophil-derived antimicrobial peptides (AMPs) play a central role in innate immunity. AMPs can kill invading microbes and/or activate various cell types. Here I studied the ex vivo interaction between host factor (anti-HAdV antibodies and AMPs) to HAdV in DCs and the role of indirect-activated DC (indir-DCs) in immune response. I characterized the profile of cytokines and chemokines of DC stimulated with different HAdV, AMPs and their combination. We recently found that IgG-oposonization of HAdV5 increase the update by MoDCs (monoctyes-derived dendritic cells) and induced pyroptotic cell death. I therefore focused on the characteristic and function of indir-DCs in anti-HAdV immunity. To better understand the properties and phenotypes of indir-DCs and direct-activated DCs (dir-DCs), we characterize their maturation profile, the factors influencing their maturation, and the functional ability to recruit leukocyte. We found that dir-DC prevent leucocyte recruitment while indir-DC increases monocyte migration. These data contribute to understand how the pre-existing immunity to HAdV impacts treatment for HAdV diseases and vaccine design.

Submitted by Viviane Lima da Cunha (viviane@biblioteca.ufpb.br) on 2017-06-01T14:19:33Z No. of bitstreams: 1 arquivototal.pdf: 3473301 bytes, checksum: 4d22636d8d38c872a66d2aa8bf1d2e3f (MD5) / Made available in DSpace on 2017-06-01T14:19:33Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3473301 bytes, checksum: 4d22636d8d38c872a66d2aa8bf1d2e3f (MD5) Previous issue date: 2016-12-15 / Monocytes and macrophages represent keys components of the immune response. Based on the expression of LPS co-receptor CD14 and CD16 expression, FCγ III receptor, monocytes are classified into 3 subtypes: classical monocytes, which are CD14hiCD16-; intermediate monocytes, which are CD14hiD16+; and non-classical monocytes, CD14lowCD16+. AD is a chronic inflammatory cutaneous disease, with multifactorial etiology. Our group performed comparative analysis of monocytes subtypes through the study of the frequency and medium fluorescence intensity of surface molecules (HLADR, CCR5, CD80, CD86, PD1L) and cytokine (IL-6, TNF, IL10) in adults and children by using flow cytometry. The study was performed in two stages. First we compared the subtypes of monocytes from healthy adults and children, and then between healthy children and children with atopic dermatitis (AD). The role of monocyte activation and modulation of the inflammatory process is the subject of our investigation. The results of this study showed that: (1) the relative frequency of monocytes subtypes was similar in adults, healthy children and children with AD, with a predominance of classical monocytes; (2) Classical, intermediate and nonclassical monocytes of children with AD have higher HLA-DR expression when compared to those of healthy children, and monocytes of healthy children have higher expression than healthy adults; (3) adults, healthy children and DA children have higher frequency and expression of CCR5 in intermediate and nonclassical that in classical monocytes; (4) the frequency and expression of CD80 was higher in intermediate and nonclassical monocytes both in children and healthy adults and CD86 expression was more pronounced in intermediate monocytes, in these two groups, beyond wich the expression of CD80 and CD86 molecules on classical monocytes of children with AD was higher than in healthy children; (5) PD1L frequency in monocytes subtypes was similar in adults and children, however, there is a higher expression of this molecule in children classical monocytes when compared to healthy adults classical monocytes. In addition, atopic children have higher expression of this molecule on classical monocytes than healthy children; (6) intermediate and nonclassical monocytes of healthy adults and children have greater inflammatory activity than classical monocytes when evaluating the frequency and expression of IL-6 and TNF-α, contrary to what is observed in children with AD who have greater frequency and expression of IL-6 and TNF-α in classical monocytes compared to healthy children; (7) intermediate and nonclassical monocytes have increased IL10 production than classical monocytes in healthy adults and children. Thus, our results revealed that the relative monocytes frequency is constant in the three studied population groups, but the frequency and expression of surface molecules and cytokines presented significant peculiarities. Summarizing, healthy children have greater expression of HLA-DR molecule than healthy adults, and atopic children have greater expression than healthy children. Monocytes subtypes more involved inflammatory response in healthy adults and children are intermediate and nonclassical monocytes, while classical monocytes in atopic children are more involved in inflammatory response than the same subtype in healthy children. These findings revealed changes in the innate immunity of children with atopic dermatitis extremely important for understanding the pathophysiology of disease. / Os monócitos e macrófagos representam componentes fundamentais da resposta imune. Com base na expressão do co-receptor de LPS CD14 e na expressão do CD16, receptor FCγIII, os monócitos são classificados em 3 subtipos: monócitos clássicos, que são CD14hiCD16-; monócitos intermediários, que são CD14hiD16+; e monócitos não-clássicos, ou CD14lowCD16+. A DA é uma doença inflamatória cutânea crônica, de etiologia multifatorial. Nosso grupo realizou análise comparativa entre os subtipos monocitários por meio do estudo da frequência e da média de intensidade de fluorescência (MFI) de moléculas de superfície (HLA-DR, CCR5, CD80, CD86, PD1L) e da produção de citocinas (IL6, TNFα, IL10) em adultos e crianças utilizando a citometria de fluxo. A pesquisa foi executada em duas etapas. Primeiro comparamos os subtipos de monócitos entre adultos e crianças saudáveis e em seguida, entre crianças sadias e crianças portadoras de dermatite atópica (DA). O papel dos monócitos na ativação e modulação do processo inflamatório foi objeto da nossa investigação. Os resultados desse estudo mostraram que: (1) a frequência relativa dos subtipos de monócitos foi similar em adultos, crianças saudáveis e crianças portadoras de DA, com predomínio de monócitos clássicos; (2) monócitos clássicos, intermediários e não-clássicos de crianças atópicas apresentaram maior expressão de HLA-DR que os mesmos subtipos em crianças sadias e essas, que adultos sadios; (3) adultos, crianças saudáveis e crianças doentes apresentaram maior frequência e expressão de CCR5 em monócitos intermediários e monócitos não-clássicos; (4) a frequência e expressão do CD80 foi maior em monócitos intermediários e não clássicos tanto em crianças como em adultos saudáveis e a expressão de CD86 foi maior em monócitos intermediários desses dois grupos. Já a expressão das moléculas de CD80 e CD86 em monócitos clássicos de crianças portadoras de DA foi maior que em crianças saudáveis; (5) a frequência de PD1L nos subtipos de monócitos foi semelhante em adultos e crianças, entretanto, houve maior expressão dessa molécula em monócitos clássicos de crianças que em adultos saudáveis e crianças atópicas apresentaram maior expressão desta molécula em monócitos clássicos que crianças saudáveis; (6) monócitos intermediários e não-clássicos de adultos e crianças saudáveis apresentaram maior atividade inflamatória que monócitos clássicos ao se avaliar a frequência e expressão de IL-6 e TNF-α, ao contrário do que se observou em crianças com DA, que apresentaram maior frequência e expressão de IL-6 e TNF-α em monócitos clássicos que as crianças saudáveis; (7) monócitos intermediários e não-clássicos demonstraram maior produção de IL10 que monócitos clássicos em adultos e crianças saudáveis. Nossos resultados mostraram, portanto que a frequência relativa de monócitos é constante nos três grupos populacionais estudados, mas a frequência e expressão de moléculas de superfície e citocinas apresentam particularidades significativas. Em resumo, crianças atópicas apresentam maior expressão de HLA-DR que crianças saudáveis e essas, que adultos saudáveis. Os subtipos de monócitos mais envolvidos na resposta inflamatória em adultos e crianças sadias são monócitos intermediários e não-clássicos, enquanto monócitos clássicos de crianças atópicas são mais inflamatórios quando comparados ao mesmo subtipo em crianças saudáveis. Essas descobertas revelam alterações na imunidade inata de crianças portadoras de dermatite atópica de extrema importância para a compreensão da fisiopatologia da doença.

Chez les patients atteints de cancer, les cellules néoplasiques échappent au contrôle du système immunitaire en raison de leur faible immunogénicité et d'une capacité exacerbée à moduler le micro-environnement. Nous décrivons ici les effets de ce micro-environnement tumoral sur la différenciation locale et systémique des monocytes et l'impact de la présence de Macrophages-Associés aux Tumeurs (TAM) CD163+ sur la survie des patientes atteintes de cancer du sein. Par une analyse de cytométrie en flux, nous décrivons un composition hétérogène des sous-types de TAM CD163low et CD163high, où nous avons observé l'association entre une fréquence élevée de TAM CD163high et une faible infiltration des lymphocytes T CD3+. Par immunohistochimie sur une analyse rétrospective (±12 ans), nous avons démontré une forte corrélation entre la fréquence élevée de TAM CD163+ et un risque accru de progression pour les patientes (log-rank *p<0.05, n=238). In vitro, les monocytes CD14+ conditionnés par le micro-environnement tumoral présentent une différenciation biaisée en faveur des MΦ CD163highCD86lowIL-10high, que non seulement ne stimulent pas la prolifération des lymphocytes T CD4+ naïfs, mais inhibent fortement l'expansion et la production d'IFN-γ et de TNF-α par les lymphocytes T CD4+ préalablement activé. Cette différenciation de MΦ en M2-like (CD163highIL-10high) est associée à des quantités élevées de TGF-β, M-CSF et VEGF dans le micro-environnement tumoral. Par ailleurs, les monocytes circulants des patientes atteintes de cancer du sein présentent un profil cytokinique immunosuppresseur et sont biaisés vers une différenciation en MΦ et Mo-DCs qui présentant des capacités suppressives / In cancer patients, the neoplastic cells escape from the immune control because of their low immunogenicity and their exacerbated capacity to modulate the microenvironment. Here we describe the local and systemic effects of the tumor microenvironment on monocyte differentiation and the impact of the presence of Tmor Associated Macrophages (TAM) CD163+ on the survival of breast cancer patients. By flow cytometry analysis, we describe a heterogeneous composition of CD163low and CD163high TAM subtypes, where we observed the association between high frequency of CD163high TAM infiltration and low CD3+ T lymphocytes presence. By immunohistochemistry on a retrospective analysis (±12 years), we have shown a strong correlation between high frequency CD163+ TAM and an increased risk of progression for patients (log-rank *p<0.05, n= 238). In vitro, CD14+ monocytes conditioned by tumor microenvironment exhibit a biased differentiation towards a CD163highCD86lowIL-10high macrophages (MΦ) phenotype, that not only failed to stimulate the proliferation of naive CD4+ T cells, but strongly inhibited the expansion and the production of IFN-γ and TNF-α by activated-CD4+ T cells. This differentiation into M2-like MΦ (CD163highIL-10high) is associated with high levels of TGF-β, M-CSF and VEGF found in the tumor microenvironment. Furthermore, circulating monocytes of breast cancer patients produced an immunosuppressive cytokine profile and are biased towards the differentiation into MΦ and Mo-DCs that show suppressive capacities / O desenvolvimento do câncer é normalmente associado a desvios no sistema imune, principalmente devido a sua falha em perceber, reconhecer e eliminar células neoplásicas de maneira eficiente. Nesse contexto, duas Células Apresentadoras de Antígenos (APCs), Células Dendríticas (DCs) e Macrófagos (MΦ), têm um papel crucial na identificação de alterações nos tecidos e na estimulação da imunidade adaptativa antitumoral. No entanto, fatores derivados de tumores modulam essas APCs, impedindo a iniciação das respostas imunes e culminando no estabelecimento do câncer. Investigamos aqui como o microambiente tumoral poderia modular a diferenciação de monócitos em APCs in vitro e de modo sistêmico. Nossos dados revelaram que em cânceres de mama e ovário, Macrófagos-Associados a Tumores (TAMs) são a subpopulação mais frequente em leucócitos CD45+MHCII+, e são encontrados em uma frequência variável de TAMs CD163low ou TAMs CD163high. O último, (TAMs CD163high) expressaram maiores níveis de PD-L1 e elevada produção de IL-10 sob a ativação de LPS. Além disso, a análise retrospectiva por imunohistoquímica revelou uma forte correlação entre a presença de TAMs CD163+ e uma baixa taxa de sobrevida em pacientes com câncer de mama. Ainda, a alta frequência de TAMs CD163high foi correlacionada com um baixo infiltrado de células T CD3+. Monócitos saudáveis condicionados por sobrenadantes de tumores de mama tiveram sua diferenciação in vitro direcionada para um fenótipo CD163highIL-10high, células capazes de suprimir a expansão de células T naive CD4+ e a produção de IFN- γ e TNF-α via IL-10. Esse fenótipo adquirido por monócitos condicionados foi associado à presença de altos níveis de CCL22, M-CSF, TGF-β1, TGF-β3, e VEGF no microambiente tumoral. Interessantemente, avaliando os efeitos sistêmicos dos tumores, monócitos circulantes de pacientes com câncer de mama falharam em diferenciar-se em M1- MΦ na presença de GM-CSF/IFN-γ e mantiveram um fenótipo alterado CD163+/-IL-10+TNF-α+

Les syndromes septiques restent à ce jour un problème majeur de santé publique. Une importante immunodépression est observée lors des syndromes septiques, affectant notamment les lymphocytes T, acteurs majeurs de la réponse immunitaire. En effet, après avoir subi une apoptose massive ils présentent d'importantes altérations fonctionnelles et phénotypiques, associées à un mauvais pronostic. Cependant les mécanismes impliqués dans le développement de ces altérations lymphocytaires induites par le sepsis (ALIS) sont encore mal connus. Le but de ce travail était d'étudier ces mécanismes à travers la mise au point de modèles ex-vivo. Nous avons optimisé un test de mesure de la réponse proliférative des lymphocytes T, utilisable pour le diagnostic des immunodéficiences primaires, comme pour l'étude expérimentale et clinique des ALIS. Nous avons développé un premier modèle ex vivo, reposant sur l'incubation de cellules mononuclées du sang périphérique (PBMC) en présence de LPS, suivie d'une stimulation spécifique des lymphocytes T via leur TCR. Ce modèle récapitule des mécanismes indirects potentiellement impliqués dans l'induction des ALIS, impliquant les monocytes. Puis, nous avons mis au point un modèle qui repose sur l'incubation de PBMC en présence d.IL-10, suivie d'une stimulation des lymphocytes T par des anticorps anti-CD2/CD3/CD28. Ce modèle pourrait reproduire des mécanismes directs et indirects impliqués dans l'induction des ALIS. Nos résultats nous ont permis d'améliorer la compréhension des mécanismes en jeux dans les ALIS et d'en souligner la complexité. Les modèles ex-vivo présentés pourraient permettre d'évaluer de nouvelles stratégies thérapeutiques / Sepsis remains a major public healthcare issue. During sepsis, an important immunodepression develops, affecting particularly the T lymphocytes, major players of the immune response. Following a massive apoptosis, T lymphocytes display important functional and phenotypical alterations, which are associated with higher risk of secondary infections and higher mortality. Mechanisms involved in the induction of such alterations are not fully understood. The aim of this study was to analyze those mechanisms through the development of ex vivo models. We optimized a test to measure T lymphocytes proliferative response, which can be used for the diagnosis of primary immunodeficiencies, as well as to clinically and experimentally study sepsis induced T lymphocytes alterations (SILA). First, we set up a model consisting in an incubation of PBMC (peripheral blood mononuclear cells) with LPS followed by specific T lymphocytes stimulation via its TCR. This model recapitulates indirect mechanisms likely to participate in SILA induction, mediated by monocytes. Then, we set up a model consisting in PBMC incubation with IL-10 followed by T lymphocytes stimulation with anti-CD2/3/28 antibody coated beads. This model may recapitulate direct and indirect mechanisms involved in SILA induction. Our results allowed us to improve the understanding of the mechanisms involved in SILA induction and to highlight its complexity. The ex-vivo models that we developed could be used for the evaluation of new therapeutic strategies

Effectively treating infectious diseases often requires a multi-step approach to target different components involved in disease pathogenesis. Similarly, the COVID-19 pandemic has become a global health crisis that requires a comprehensive understanding of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection to develop effective therapeutics. One potential strategy to instill greater immune protection against COVID-19 is boosting the innate immune system. This boosting, termed trained immunity, employs immune system modulators to train innate immune cells to produce an enhanced, non-specific immune response upon reactivation following exposure to pathogens, a process that has been studied in the context of and clinical studies prior to the COVID-19 pandemic. Evaluation of the underlying pathways that are essential to inducing protective trained immunity will provide insight into identifying potential therapeutic targets that may alleviate the COVID-19 crisis. Here we review multiple immune training agents, including Bacillus Calmette-Guérin (BCG), β-glucan, and lipopolysaccharide (LPS), and the two most popular cell types involved in trained immunity, monocytes and natural killer (NK) cells, and compare the signaling pathways involved in innate immunity. Additionally, we discuss COVID-19 trained immunity clinical trials, emphasizing the potential of trained immunity to fight SARS-CoV-2 infection. Understanding the mechanisms by which training agents activate innate immune cells to reprogram immune responses may prove beneficial in developing preventive and therapeutic targets against COVID-19.

It is well established that continual consumption of a diet high in fat leads to the development of chronic conditions such as obesity, cardio metabolic syndrome, and atherosclerosis that are associated with high incidence of cardiovascular disease. Recent studies have identified endotoxin-derived inflammation as a major diving force for the development of these conditions. Our laboratory has recently demonstrated that consumption of a single high-fat meal results in acute postprandial endotoxemia and alters monocyte cell surface adhesion molecule expression and scavenger receptor CD36 expression. These collective projects describe our efforts to understand the physiological significance of these postprandial changes and if supplementation with spore-based probiotics are able to provide any form of protection against these responses that are associated with the onset of atherogenesis.

Background and Objectives: The pathophysiology of post-operative pneumonia following lung resection is poorly understood despite it being the most common complication which may lead to death. The role of the acute inflammatory response following lung resection, in particular innate immune cells, was investigated and used to identify biomarkers for post-operative pneumonia. Comparison of inflammatory responses to resection undertaken by video-assisted thorascopic surgery (VATS) and thoracotomy was also evaluated. Methods: Patients undergoing lung resection for suspected bronchogenic carcinoma were recruited. Objective pre-defined criteria were used to diagnose pneumonia. Bronchoalveolar lavage (BAL) was conducted in the contra-lateral lung pre- and postoperatively to measure cellular composition and cytokines. Blood was sampled preoperatively and 6-, 24- and 48-hours post-operatively primarily to assess neutrophil phagocytic capacity, monocyte subsets, monocyte cytokine responses to lipopolysaccharide (LPS) stimulation and serum cytokine responses. Exhaled nitric oxide (eNO) was also measured at these time points. Patient groups were compared using paired or student t-tests together with ANOVA/ANCOVA modelling. The predictive strength of the biomarkers identified was tested. Results: 40 patients were recruited. 26 patients (65%) underwent major lung resection using VATS and 14 (35%) thoracotomy. There was a post-operative blood monocytosis (p<0.0005) with an absolute expansion of classical and intermediate monocytes (p=0.001) and a relative fall in non-classical monocytes (p<0.005). Post-operatively blood monocytes became more pro-inflammatory with an overall significant increase in IL-8 (p=0.034) and TNF-α (p=0.028) together with an increase in IL-6 (p=0.028) and IL-10 by 48 hours (p=0.010). VATS was associated with a smaller release of IL-10 only (p=0.011). There was a general trend towards post-operative reduction in neutrophil phagocytosis of zymosan (in suspension) on ANOVA modelling (p=0.047). Lung resection led to an increase in serum cytokines IL-6, IL-8 and IL-10 which peaked at 24hrs before falling (p<0.0005). ANOVA modelling confirmed significantly lower levels of serum cytokines in VATS patients compared with thoracotomy (p=0.026 for IL-6, p=0.018 for IL-8 p=0.047 for IL-10). No significant post-operative change was found for IL-1β, TNF-α and IL-12p70 (p>0.05). Bronchoalveolar lavage fluid (BALF) and blood samples demonstrated a relative post-operative leucocytosis due principally to neutrophilia. A relative blood lymphopenia and thrombocytopenia developed postoperatively (p<0.0005). VATS was associated with a lower fall in serum albumin (p=0.001). BALF from the non-operated lung became more pro-inflammatory immediately post-operatively with an increase in IL-6 (p<0.0005), IL-8 (p=0.017), IL- 10 (p=0.018) and IL-1β (p=0.002). eNO tended to fall post-operatively which reached significance at 48 hrs (p=0.029). 14 patients developed pneumonia. Pre-operatively, a blood neutrophil count above 5.04x109/L had a relative risk (RR) for pneumonia of 3.3 (95% confidence interval (CI95) 1.1-10.1), and a BAL cell count of greater than 1.04x105/ml had a RR of 3.4 (CI95 1.3-9.0), whilst LPS-stimulated monocyte secretion of IL-12 of less than 0.15 pg/ml/μg protein had a RR of 3.0 (CI95 1.2-7.3). At 24 hours post-operatively, LPS-stimulated release from monocytes of IL-10 greater than 1.99 pg/ml/μg protein (RR 4.1, CI95 1.3- 12.3) and IL-6 greater than 414 pg/ml/μg protein (RR 3.1, CI95 1.2-8.1) were predictive of pneumonia. Conclusion: Lung resection is associated with significant early pro- and antiinflammatory responses. VATS resection invoked significantly lower levels of serum cytokines and albumin changes compared with thoracotomy suggesting VATS lobectomy should be the surgical treatment strategy of choice for early stage lung cancer. No difference in neutrophil function or monocyte function was however observed between the surgical groups. Clinical benefits of this reduced inflammation need to be evaluated in a larger cohort of patients. Relative pre-operative leucocytosis in blood and BAL together with monocyte hyper-responsiveness in the early postoperative period is associated with the development of pneumonia. These findings warrant further investigation for their predictive power in accurately identifying postoperative pneumonia. Ultimately, they may be incorporated into a risk stratification model enabling targeted prophylactic or earlier therapeutic intervention.

Thesis (MScMedSc) –Stellenbosch University, 2014. / ENGLISH ABSTRACT: HIV-infection is characterized by depletion of CD4+ T-cells from the gut-associated lymphoid tissue (GALT) which causes irreparable gastrointestinal tract damage and subsequent microbial translocation of bacterial products such as lipopolysaccharide (LPS), a component of Gram-negative bacteria, into systemic circulation. HIV infection also affects the functions and relative population sizes of various immune cells, such as monocytes. Monocytes are important innate immune cells as they are often the first cells recruited to sites of infection and inflammation. They then either promote inflammatory processes; elicit adaptive immune responses, through their antigen presenting ability; aid in pathogen and debris clearance or aid in damage repair. This cross-sectional study investigated functional changes to monocytes and monocyte subsets (CD14+CD16- and CD14+CD16+) in HIV+, treatment naïve individuals and healthy uninfected controls, using whole blood assays and isolated monocytes. A number of chemokine receptors associated with function and homing, and specific gut-homing receptors, were investigated. Monocyte activation, apoptotic potential and intracellular monocyte cytokine production were also investigated. All markers were evaluated using multi-parameter flow cytometry. Monocyte responsiveness to in vitro LPS stimulation and expression of the afore-mentioned chemokine receptors to viral load, CD4+ count and CD38/8 T-cell expression was also assessed. During HIV-infection monocytes appeared primed to exit systemic circulation and migrate towards the gut, as seen through elevated CD62-L (p < 0.005) and CCR7 (p < 0.005), whereas the CD14+CD16+ subset was increased (p = 0.0461) and exhibited a higher activation status through increased CD69 expression (p < 0.005) compared to the CD14+CD16- subset. An interesting observation was the significantly increased IL-10 production by the CD14+CD16+ subset (p < 0.005). An elevated CCR5 expression in total monocytes (p < 0.005) was also seen. After LPS stimulation, the HIV+ group displayed unique and significant percentage increases in the total monocyte population. The findings of the current study suggest that monocyte functionality may be retained during HIV-infection and that CD14+CD16+ monocytes play a vital role during HIV-infection evidenced by their preferential expansion and priming for GALT migration. The production of IL-10 by this subset further highlights their importance and emphasizes the need for future studies on the role of these cells in chronic stable HIV-1 infection and whilst disease progresses. / AFRIKAANSE OPSOMMING: MIV-infeksie word gekenmerk deur die uitputting van CD4+ T-selle, veral uit die derm-verwante limfweefsel (GALT). Dit veroorsaak onherstelbare skade aan die spysverteringskanaal en die daaropvolgende mikrobiese translokasie van bakteriële produkte soos LPS, „n komponent van Gram-negatiewe bakterieë, wat gaan binne sistemiese sirkulasie. MIV-infeksie beinvloed die funksies en relatiewe bevolkingsgrootte van verskeie immuun selle, insluitend monosiete. Monosiete is belangrike ingebore immuun selle en is dikwels die eerste selle wat gewerf word na areas van infeksie en inflammasie. Monosiete kan inflammatoriese prosesse bevorder of aanpabare immuunstelsel reaksies ontlok deur middel van hul antigeen aanbiedings vermoë of help met patogeen en puin klaring en skade herstel. In hierdie deursnee-studie het ons veranderinge aan monosiete (CD14+CD16+ en CD14+CD16-) ondersoek in MIV+ behandelde naïef individue en gesonde onbesmette kontroles, deur die gebruik van hele bloed toetse en geïsoleerde monosiete. 'n Aantal chemokine reseptore, wat verband hou met homing en funksie was ondersoek in toevoeging tot spesifieke derm-homing reseptore. Monosiet aktivering, apoptese potensiaal en intrasellulêre monosiet sitokien produksie was ook ondersoek. Alle merkers is ondersoek deur multi-parameter vloeisitometrie. Die beoordeel reaksies van monosiete na in vitro LPS stimulasie en die uitdrukking van die merkers met merkers van algemene immuun aktivering en MIV-siekte patogenese was ook ondersoek. CD14+CD16+ monosiete was gedurende MIV-infeksie verhoog (p-waarde = 0.0461). Daar was 'n hoër algehele monosiet uitdrukking van verskeie chemokine merkers soos CD69 (p-waarde < 0.005) uitdrukking; CD62-L (p-waarde < 0.005), en CCR7 (p-waarde < 0.005) uitdrukking in die CD14+CD16+ subgroep. Daar was ook „n toename in IL-10 produksie, veral in die CD14+CD16+ subgroep (p-waarde < 0.005). Hoewel baie funksionele merker reaksies dieselfde was, het die MIV+ groep „n unieke en beduidende persentasie verhooging in die totale monosiet bevolking getoon. Ons algehele bevindinge dui op 'n voorkeur uitbreiding van CD14+CD16+ monosiete tydens MIV-infeksie. Die CD14+CD16+ monosiet subgroep blyk ook bevoordeel word met betrekking tot voorbereiding vir migrasie na limfknope en die GALT. Die toename in geaktiveer de CD14+CD16+ monosiete op siekte webwerwe is waarskynlik 'n groot bydraende faktor tot aanhoudende immuun aktivering wat op sy beurt virale replikasie bevorder. Hierdie resultate beklemtoon die behoefte om die rol van hierdie selle en in veral die CD14+CD16+ subgroep, in kroniese stabiele MIV-1 infeksie verder te studeer en terwyl siekte bevorder.

L'immunité innée et l'immunité adaptative constituent les deux volets du système immunitaire. Contrairement à l'immunité adaptative qui utilise un vaste répertoire de récepteurs spécifiques obtenus par réarrangements géniques, l'immunité innée utilise un nombre réduit de récepteurs appelés " Pattern Recognition Receptors " ou PRR. Les PRR reconnaissent des motifs hautement conservés et exclusivement exprimés par les micro-organismes, et appelés " Pathogen-Associated Molecular Patterns " ou PAMP. Les PRR sont soit associés aux cellules (impliqués dans l'endocytose et/ou la signalisation), soit solubles (impliqués dans l'élimination des micro-organismes et du soi modifié). Les pentraxines courtes (SAP et CRP) et longues (PTX3) sont des PRR solubles impliqués dans la capture du non-soi et/ou du soi modifié et facilitent leur élimination par les phagocytes. La protéine Tat du VIH-1 est indispensable à la synthèse du génome viral. Sa forme soluble, sécrétée par les cellules infectées, régule de nombreuses fonctions cellulaires. Cependant, la nature des structures de fixation de Tat soluble aux cellules reste mal connue. Nous avons montré que Tat interagit sélectivement avec les pentraxines SAP, CRP et PTX3. Cependant, et de manière inattendue, nous avons observé que seule la protéine CRP augmente la transcription in vitro du VIH-1 induite par Tat en favorisant son accumulation nucléaire. Nous avons également observé une influence significative de Tat et des complexes Tat/pentraxines sur la différenciation de monocytes en cellules dendritiques (DC). L'ensemble de ces données met en évidence un nouveau mécanisme mis en jeu par le VIH pour exploiter le bras humoral de l'immunité innée et échapper au système immunitaire. Des données préliminaires suggèrent également un rôle potentiel de Tat dans l'acquisition d'un phénotype immunosuppresseur par les cellules dendritiques. En parallèle, dans le cadre d'un projet fédératif portant sur la régulation d'expression du FcγRIII (CD16), nous avons montré que les cytokines IL-1β, IL-6, IL-10 et IL-21 induisent non seulement l'augmentation d'expression de CD16 sur les monocytes/macrophages et son induction sur les DC, mais également son acquisition par la sous population monocytaire CD14+ CD16-. Ces données suggèrent donc un rôle de ces cytokines dans la modulation d'expression de CD16 par les cellules myéloïdes.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.