Long-term protective immunity depends on proper initiation of professional antigen-presenting cells (APCs). Autoimmune disorders and certain infections can cause disease through modulation of APCs and thereby affecting the outcome of these diseases. This work aimed to investigate the behaviour of different APC subsets during conditions known to cause improper immune responses. In Paper I, the effects of an anti-inflammatory compound called Rabeximod, intended for treatment of rheumatoid arthritis were investigated on different subsets of APCs. The results showed that Rabeximod affected the differentiation and behaviour of inflammatory subsets of dendritic cells (DCs) and macrophages while no effects were observed on anti-inflammatory subsets. Our findings suggest that Rabeximod acts by inhibiting the functionality of inflammatory subsets of APCs. In Paper II, the effects of different malaria derived stimuli such as hemozoin (Hz) and infected red-blood cells (iRBCs) on monocyte-derived dendritic cells (MoDCs) were investigated. Both stimuli triggered activation and migration of MoDCs. MoDCs exposed to iRBCs induced allogeneic T-cell proliferation while those exposed to Hz did not. These results indicate that different malaria derived stimuli may differently affect DCs and that this could lead to improper and inefficient T-cell activation. In Paper III, innate aspects of malarial immunity were compared in children from two sympatric ethnic groups. We observed decreased activation of APCs and severely supressed TLR responses in Dogon children as compared to Fulani. This may indicate an important role for TLR and APC activation in the Fulani, known to be better protected against malaria than the Dogon. In summary, detailed knowledge of APC activation will be helpful in the understanding of specific effector immune responses. This could in turn, improve treatment of inflammatory disorders as well as the generation of efficient vaccines against infectious diseases.

Dendritic cells (DC) are antigen-presenting cells that are critical for the initiation and regulation of the immune response. Several DC subtypes within mouse spleen have previously been characterised and these include the plasmacytoid (pDC), and conventional DC (cDC) of the CD8+ and CD8- subtypes. Each subtype appears to have a specialised role in the various arms of immunity and tolerance. Less clear is the process by which these DC develop from haematopoietic precursors, of the precursor stages and branch points from bone marrow (BM) stem cells to each of the peripheral DC subtypes. The research described herein had the aim of identifying and isolating some of the intermediate precursors of DC, downstream of stem cells, and determining whether these differed in the steady-state versus inflammation. Particular was given to DC of the spleen. Experiments that sought the identity of such precursors involved both i) transfer of cell fractions that contained DC precursors into steady-state or inflamed recipient mice to assess their in vivo development at later times, and ii) analysis of an in vitro culture system to question whether it reflected development of the steady-state DC subtypes.

The host immune response to parasitic helminths is usually characterized by a Th2 phenotype. As the Jagged/Notch pathway has been implicated in driving Th2 development, it was hypothesized that host macrophages and dendritic cells (DCs) could detect helminth products and mount an appropriate response via this pathway. Schistosoma mansoni soluble egg antigen (SEA) rapidly up-regulated expression of the Notch ligand, Jagged 1, in both mouse and human macrophages, as well as in conventional mouse DCs. Other factors associated with Th cell development, including the Th1-promoting factor IL-12 p40, as well as another potential Th2-promoting factor, interleukin (IL)-33, were not transcriptionally responsive to SEA in these same cell types, thus indicating the selectivity of the response. Inducible gene expression was modified by the presence of the macrophage growth factor colony-stimulating factor (CSF)-1, which inhibited Jagged 1 induction by SEA and lipopolysaccharide (LPS), but enhanced LPS-induced IL-12p40 expression. Despite the observation that SEA upregulated Jagged 1 in both macrophages and DCs, only SEA-pulsed DCs promoted IL-4 production upon T-cell activation, suggesting that Jagged 1 induction alone is insufficient for instructing Th2 development. A recombinant form of the extracellular region of Jagged 1 did, however, enhance IFN-γ production in splenocytes, thus implying that the rapid induction of Jagged 1 in macrophages and DCs can regulate T cell responses. A potential role for SEA-induced Jagged 1 in autocrine responses in macrophages was also investigated through studies with recombinant extracellular Jagged 1, as well as ectopic expression of Jagged 1 in macrophages. A comparison of the responses initiated in macrophages by SEA and the bacterial endotoxin lipopolysaccharide (LPS) revealed common activation of extracellular signal regulated kinase-1/2 (ERK-1/2) and p38 phosphorylation. However, only LPS triggered IκB degradation, phosphorylation of c-Jun N-terminal kinase (JNK) and phosphorylation of Tyr701 of signal transducer and activator of transcription 1 (STAT1). SEA robustly activated signalling in HEK293 cells expressing either Toll-like receptor 2 (TLR2) or TLR4/MD2, as well as variably in cells expressing TLR3. Jagged 1 upregulation by SEA was not abrogated in TLR4 knockout macrophages, in contrast to the LPS response. Pharmacological inhibition of the ERK-1/2 pathway impaired both SEA- and LPS-inducible Jagged 1 expression in macrophages. In conclusion, the data within this thesis suggests that Jagged 1 is an ERK-dependent target of TLR signalling that has a macrophage-specific function in the response to SEA.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Sept. 16, 2008). Includes bibliographical references.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 49 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph.D.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

Lors d’un rapport sexuel, le VIH-1, sous forme libre ou associé aux cellules, pénètre dans les tissus du tractus génital féminin. Les premières étapes de l’infection par le VIH-1 dans ces tissus sont très controversées. Afin d’analyser ces premiers évènements impliqués dans la transmission sexuelle, nous avons développé au cours de ma thèse, deux modèles d’infection de cellules isolées de tissu. Nous avons montré que les cellules dendritiques sont préférentiellement infectées et que les anticorps neutralisants inhibent ces premiers évènements de transmission du VIH-1. Dans le cadre de nouvelles stratégies vaccinales, l’inhibition de l’infection des cellules dendritiques sera à considérer afin de prévenir la transmission par voie sexuelle. / During intercourse, HIV free virus particles and cell-associated virus, penetrate into the female genital mucosa. The first events following HIV transmission in tissues are still controversial. In order to analyze these first events of transmission, two different models were developed during my thesis: a heterologous model of HIV transmission with cells generated from blood and a model with cells isolated from cervico-vaginal tissues. We found that dendritic cells are preferentially infected. Moreover, neutralizing antibodies efficiently inhibit these first events of HIV transmission. Future HIV prophylactic vaccine design should take into account the potential infection of dendritic cells and new strategies should be developed to prevent the infection of these particular cell populations.

Les nanoparticules (NP) ont été introduites en médecine pour développer des médicaments intelligents ou des agents d'imagerie. En raison de leur petite taille (<200 nm), les NP permettent la mise en place de thérapies ciblées, augmentent la diffusion et l’efficacité des drogues, tout en facilitant les modes d’administration et en diminuant les couts de santé publique. Malgré les promesses que présentent les NP pour la médecine, les risques potentiels pour la santé humaine associés à une exposition aux NP restent mal documentés ; en particulier en ce qui concerne leurs effets sur le système immunitaire. Les cellules présentatrices d’antigène (CPA) (comprenant les macrophages et les cellules dendritiques) sont recrutées au site d’inflammation induite par des pathogènes et constituent une ligne de défense majeure pour notre organisme. Les CPA sont dotées d’une activité de phagocytose et endocytose conduisant à une forte internalisation des NP. Ainsi, ces cellules seront parmi les plus affectées par une exposition aux NP et sont un modèle expérimental pertinent pour l’étude du devenir cellulaire des NP et de leurs effets sur l’hôte.Dans cette étude, nous avons étudié si les fonctions de ces cellules pourraient être modifiées par une exposition aux NP. Comme modèles de NP, nous avons choisi l'or (AuNP) et le gadolinium-polysiloxane (GdSi) utilisés comme agent de contraste ou de théranostique, le poly lactic-co-glycolic acid (PLGA) et une nano émulsion lipidique (LNP) développés comme plateforme de délivrance d’antigènes ou de médicaments. Tout d'abord, en utilisant des microsphères fluorescentes comme sonde, nous avons montré que toutes les NP testées n'altèrent pas la capacité de phagocytose de la lignée cellulaire de macrophages J774. Ensuite, l’activation des cellules a été analysée par cytométrie de flux, basée sur l'expression des marqueurs de surface CD-86 et MHC-II. Nous avons établi que l'exposition aux NP n'active pas les cellules dendritiques dérivées de la moelle osseuse (BMDC). Dans le même sens, aucune de ces NP n’induit par elle-même de sécrétions de cytokines par les BMDC. En outre, l’activation de ces cellules par des activateurs connus, tels que le lipopolysaccharide bactérien (LPS) n'est pas modifiée par les NP. Cependant, l'exposition aux AuNP diminue l'expression des cytokines IL-6, IL-12 et IL-23 par les BMDC activées par LPS. Or, ces cytokines sont impliquées dans la polarisation des lymphocytes T CD4 + vers le phénotype T helper approprié (Th). Nous avons analysé si ces modifications de cytokines pourraient modifier la réponse Th. Dans un modèle in vitro de présentation d'antigène, les BMDC ont été incubés avec un antigène modèle (ovalbumine (OVA)) et co-cultivés avec des cellules T spécifiques de l'OVA. L'exposition aux AuNP a conduit à une augmentation des cytokines spécifiques des lymphocytes T: IL-13 (indiquant un déplacement de la balance Th1 / Th2 classique vers Th2) et IL-17 (permettant de diriger les cellules T vers Th17). L'exposition des BMDC aux autres NP de l'étude ne modifie que très faiblement leurs sécrétions de cytokines inflammatoires, et n'a donc pas d'impact sur le destin des lymphocytes T après la présentation de l'antigène.L’ensemble de ces résultats démontrent que GdSi, PLGA et LNP ne modifient pas la phagocytose, l'activation des DC et la présentation antigénique. Cependant, l'exposition aux AuNP modifie les réponses inflammatoires des DC et le devenir des cellules T vers les phénotypes Th2 et Th17. Ces modifications pourraient entraver la physiologie du système immunitaire et contribuer aux maladies chroniques ou à l'auto-immunité. / Nanoparticles (NP) have been introduced in medicine to develop intelligent drugs or imaging agents. Due to their small size (<200 nm), NPs allow the development of targeted therapies, increase drug diffusion and effectiveness while facilitating modes of administration and decreasing public health costs. Nevertheless, the potential risks for human health associated to NP exposure remain poorly documented; especially about their effects on the immune system. Antigen-presenting cells (APC) (including macrophages and dendritic cells) are recruited at the site of pathogen-induced inflammation and constitute to the maintenance of body integrity, engulfing pathogens and delivering signals to other components of the immune system. Due to their internalization abilities, APC accumulate NP in their cytoplasm. Thus, these cells will be among the most affected by exposure to NP and constitute a relevant experimental model for the study of the cellular fate of NP and their effects on the host.In this study, we investigated whether the functions of these cells could be modified by an exposure to NP. As models of NP, we selected gold (AuNP) and gadolinium-polysiloxane (GdSi) used as contrast agent for therapeutic and diagnostic applications, and poly lactic-co-glycolic acid (PLGA) and lipid nano emulsion (LNP) developed as a platform for the delivery of antigens or drugs.First, using fluorescent microspheres as probe, we have shown that all of the tested NP did not alter the phagocytosis capacity of the J774 macrophage cell line. Then, cell activation was analyzed by flow cytometry, based on the expression of the surface markers CD-86 and MHC-II. We have established that NP exposure did not activate bone marrow derived dendritic cells (BMDC). In this way, none of these NP induced cytokine secretions by the BMDC. Furthermore, activation of these cells by known activators, such as bacterial lipopolysaccharide (LPS) was not modified by NP.However, in this case, the cytokine response was altered by AuNP exposure, showing reduced inflammatory cytokine production such as IL-6, IL-12 and IL-23. Interestingly, these cytokines are involved in the polarization of CD4 + T lymphocytes to the appropriate T helper phenotype (Th). In a model of antigen presentation in vitro, this cytokine profile resulted into an altered development of specific immune responses. AuNP exposure increased T cell specific cytokines: IL-13 and IL-4 (indicating a shift of classical Th1/Th2 balance towards Th2) and IL-17 (standing for an alteration of T-cell fate towards Th17). The exposure of BMDC to the other NP of the study only very slightly altered their inflammatory cytokine secretions and therefore did not affect the fate of T lymphocytes after antigen presentation.All together, these results demonstrate that GdSi, PLGA and LNP do not modify phagocytosis, DC activation and antigen presentation. However, exposure to AuNP alters the DC induced inflammatory responses and polarizes the T cell fate towards Th2 and Th17 phenotypes. These changes could impair the physiology of the immune system and contribute to chronic diseases or autoimmunity.

Les cellules dendritiques plasmacytoïdes (pDC) sont infectées par le VIH-1 et la diminution de leur nombre dans la circulation sanguine est corrélée avec la virémie des patients. Au cours de mes travaux de thèse, nous avons montré que les anticorps neutralisants (AcN) spécifiques du VIH-1 inhibent l’infection des pDC par des isolats primaires de VIH-1. Contrairement aux mDC, le mécanisme d’inhibition de l’infection des pDC est indépendant du RFcγII présent à leur surface. En parallèle, nos résultats indiquent que les pDC produisent de l’interféron-α et d’autres cytokines et chimiokines en réponse au VIH-1, même lorsque l’infection des cellules est inhibée par les AcN. Enfin, nous avons observé l’inhibition du transfert en cis et en trans du VIH-1 des pDC aux lymphocytes T CD4 par les AcN.Dans un contexte d’induction d’AcN par vaccination, l’inhibition de la réplication du VIH-1 dans les pDC associé au maintien de la sécrétion de cytokines pro-inflammatoire par ces cellules pourrait favoriser l’élimination du virus et ralentir sa dissémination dans l’organisme. / Plasmacytoid dendritic cells (pDC) are able to replicate HIV-1, and the decrease of pDC number in blood is correlated with HIV-1 viremia in patients. During my thesis, we showed that HIV-1-specific neutralizing antibodies (NAb) inhibited the infection of pDC by HIV-1primary isolates. Unlike mDC, the mechanism of inhibition of pDC infection was independent of FcγRII expressed on these cells. In parallel, our results indicated that pDC produce interferon-α and other cytokines and chemokines in response to HIV-1, even when HIV-1 infection of these cells was inhibited by NAb. Finally, we showed that NAb were able to inhibit HIV-1 transfer in cis and trans from pDC to CD4 T cells.In the context of antibodies induction by vaccination, the inhibition of HIV-1 replication in pDC associated with the maintenance of pro-inflammatory cytokines released by these cells may help to eliminate the virus and impede its dissemination in the body.