Les vecteurs nanoparticulaires comme systèmes de relargage contrôlé pour des applications vaccinales font l’objet d’intenses recherches, notamment dans le domaine du VIH1. Une approche novatrice consiste à co-administrer des molécules immuno-stimulatrices avec les antigènes d’intérêt, afin d’amplifier le recrutement et l’activation des cellules dendritiques (DCs). Un tel vecteur vaccinal stimulerait l’intensité de la réponse immunitaire et une immunité au niveau des muqueuses vaginales et anales pourrait être obtenue après vaccination. Des nanoparticules de poly(acide lactique) (NP-PLA) ou de chitosane/sulfate de dextrane (NP-CSD) ont été utilisées comme véhicules et adjuvants de protéines du VIH1, gp140 et p24. Le poly(I:C), ligand de TLR3 est la molécule immuno-stimulatrice retenue pour ses propriétés adjuvantes. Les NP-PLA et NP-CSD présentent un potentiel équivalent pour l’adsorption de protéines. Par contre, si les NP-CSD permettent l'adsorption du poly(I:C) (95%), elle est moins reproductible sur les NP-PLA. Pour chaque formulation, la capacité à induire in vitro la maturation des DCs a été évaluée en suivant les marqueurs CD25, CD80, CD83, par cytométrie en flux. L’adsorption de poly(I:C) sur les NP-PLA ou les NP-CSD amplifie les capacités de maturation de ces nanoparticules, un effet synergique étant observé avec les NP-CSD. Nos travaux montrent que la co-adsorption d’un TLR ligand, avec des antigènes protéiques du VIH sur des nanoparticules biodégradables, est possible et confère à la formulation vaccinale un effet immuno-stimulant in vitro. In vivo, les formulations vaccinales contenant du poly(I:C) induisent de très forts taux d’anticorps sériques chez la souris. / Use of nanoparticulate vectors in vaccination as controlled release systems based on biodegradable polymers has been widely studied, particularly for HIV vaccine research. An innovative approach is to co-administer antigens of interest with immuno-stimulatory molecules to amplify the recruitment and activation of dendritic cells (DCs). Such a vaccine candidate could boost the intensity of the immune response, and mucosal immunity in vaginal and anal secretions could be obtained after vaccination.We used nanoparticles of poly(lactic acid) (NP-PLA) or chitosan / dextran sulfate (NP-CSD), as vehicles and adjuvants for HIV-1 proteins, gp140 and p24. Poly (I:C), TLR3 ligand molecule, is the immuno-stimulatory molecule chosen for its adjuvant properties. The NP-PLA and NP-CSD have shown their great potential as carriers of proteins. By cons, if NP-CSD allows the adsorption of poly(I:C) with a yield of 95%, the adsorption is less reproducible on NP-PLA. For each formulation, the ability to induce in vitro maturation of DCs was evaluated by following the marker CD25, CD80, CD83, by flow cytometry. Adsorption of poly(I:C) on the NP-PLA or the NP-CSD amplifies the maturation abilities of particles and has a synergistic effect with the NP-CSD.Our work shows that co-adsorption of a TLR ligand with HIV protein antigens onto biodegradable nanoparticles is possible and gives an immuno-stimulant effect to the vaccine formulation in vitro. In vivo, vaccine formulations containing poly(I:C) induce very high levels of serum antibodies in mice.

Dendritic cells (DCs) comprise a distinct lineage of potent antigen-presenting mononuclear phagocytes that serve as both mediators of innate immune responses and key facilitators of the adaptive immune response. DCs play both immunogenic and tolerogenic roles through their dual ability to elicit pathogen-specific T cell immunity as well as induce regulatory T cell (Treg) responses to promote tolerance in the steady state. The aim of the work presented here is to examine the normal regulatory mechanisms of DC development and function, starting with the dissection of mechanisms behind an aberrantly activated developmental pathway, followed by the exploration of new mechanisms governed by two candidate transcription factors. The first chapter of the thesis focuses on the growth factor receptor Flt3, an essential regulator of normal DC development in both mice and humans, and concurrently one of the most commonly mutated proteins found in acute myeloid leukemia (AML). We investigated the effect of its most common activating mutation in AML, the Flt3 internal tandem duplication (Flt3-ITD), and found that this mutation caused a significant cell-intrinsic expansion of all DC populations. This effect was associated with an expansion of Tregs and the ability to dampen self-reactivity, with an inability to control autoimmunity in the absence of Tregs. Thus, we describe a potential mechanism by which leukemia can modulate T cell responses and support Treg expansion indirectly through DCs, which may compromise immunosurveillance and promote leukemogenesis. The subsequent chapters explore the basic molecular mechanisms of DC development by using Flt3 expression as a guide to uncover new candidates involved in the DC transcriptional program. We show that Myc family transcription factor, Mycl1, is largely dispensable for DC development and function, contrary to recent published findings that propose a role in proliferation and T cell priming. On the other hand, we find that conditional deletion of our second candidate gene, an Ets family transcription factor, has diverse effects on DC development, monocyte homeostasis, and cytokine production. Overall, our studies highlight an unexpected molecular link between DC development and leukemogenesis, and elucidate novel mechanisms controlling DC differentiation and function.

A atopia caracteriza-se pela tendência de um indivíduo a produzir IgE em quantidade elevada, em resposta a um alérgeno específico, levando ao desenvolvimento de asma, rinite ou eczema. Todavia, a manifestação do fenótipo da alergia depende da interação de fatores genéticos e exposição a alérgenos ambientais. Desta forma, o alérgeno é processado e apresentado aos linfócitos T, que desenvolvem uma resposta imune Th2, exacerbada característica da atopia. A principal célula que está envolvida na comunicação entre a imunidade inata e adaptativa é a célula dendrítica (DC) cuja função é capturar, processar e apresentar o antígeno aos linfócitos. As DCs imaturas capturam o antígeno e migram do tecido para o órgão linfóide periférico, onde elas se diferenciam em DCs maduras e apresentam o antígeno aos linfócitos T naive. Assim, os linfócitos T naive podem se diferenciar em subtipos de linfócitos efetores como: linfócitos Th1 e Th2. Esses linfócitos auxiliam na produção de anticorpos pelos linfócitos B na resposta imune humoral contra patógenos específicos. O sistema imune humoral compreende cinco classes de imunoglobulinas: IgG, IgM, IgD, IgE e IgA e sua produção sofre influência da imunidade celular. Desta forma, alérgenos provenientes de ácaros como, Dermatophagoides pteronyssinus, podem levar à inflamação alérgica, alterando a produção de anticorpos. Tendo em vista evidências que demonstram a interação das DCs com anticorpos, propomos investigar sua influência na apresentação dos principais alérgenos da poeira domiciliar e sua modulação sobre a resposta imune em indivíduos alérgicos e não alérgicos. / Atopy is characterized by the trend of an individual to produce high amounts of IgE in response to a specific allergen, leading to the development of asthma, rhinitis or eczema. However, the manifestation of the allergy phenotype depends on the interaction of genetic factors and exposure to environmental allergens. In this way, the allergen is processed and presented to the T lymphocytes, which develop a Th2 immune response, exacerbated characteristic of atopy. The main cell that is involved in the communication between innate and adaptive immunity is the dendritic cell (DC) whose function is to capture, process and present the antigen to lymphocytes. Immature DCs capture the antigen and migrate from the tissue to the peripheral lymphoid organ, where they differentiate into mature DCs and present the antigen to naive T lymphocytes. Thus, naive T lymphocytes can differentiate into subtypes of effector lymphocytes such as Th1 and Th2 lymphocytes. These lymphocytes assist in the production of antibodies by B lymphocytes in the humoral immune response against specific pathogens. The humoral immune system comprises five classes of immunoglobulins: IgG, IgM, IgD, IgE and IgA and their production is influenced by cellular immunity. In this way, allergens from mites such as Dermatophagoides pteronyssinus, can lead to allergic inflammation, altering the production of antibodies. In view of evidence demonstrating the interaction of DCs with antibodies, we propose to investigate their influence on the presentation of the main house dust allergens and their modulation on the immune response in allergic and non-allergic individuals.

Os cães são afetados por doenças inflamatórias e neoplásicas que compartilham diversas similaridades com as desordens em humanos, assim seu estudo representa um importante modelo animal para as condições humanas. As células dendríticas (DCs) representam a população mais potente de células apresentadoras de antígenos. As DCs representam também um novo alvo promissor de imunoterapia em cães; no entanto o uso terapêutico de DC caninas é restrito, dentre outros fatores, devido a falta de padronização nas técnicas de isolamento e limitado numero de informações específicas da espécie a esse respeito. Este projeto tem por finalidade avaliar a geração de células dendríticas caninas geradas in vitro e ativadas por diferentes estímulos biológicos na presença e ausência de extrato tumoral de melanoma canino. Os resultados demonstraram que as DCs caninas geradas na presença de extrato tumoral em grandes concentrações apresentavam atividade funcional semelhante as DCs maduras / Dogs are affected by inflammatory and neoplastic diseases that share many similarities with the disorders in humans, so their study is an important animal model for the human condition. Dendritic cells (DCs) are the most potent population of antigen presenting cells. DCs also represent a promising new target for immunotherapy in dogs; However, the therapeutic use of canine DC is restricted among others factors due to lack of standardization in isolation techniques and limited number of species-specific information in this regard. This project aims to assess the generation of canine dendritic cells generated in vitro and activated by different biological stimuli in the presence and absence of tumor extract of canine melanoma. The results showed that the canine DCs generated in the presence of high concentrations tumor extract showed similar functional activity of mature DCs

Apesar da forte ativação do sistema imune que ocorre durante a infecção pelo Plasmodium, a memória imunológica à infecção é restrita a pacientes residentes em áreas endêmicas. Dessa forma é importante a geração de métodos capazes de induzir uma resposta imune eficaz e duradoura contra o parasito. Nesse contexto o direcionamento de antígenos para células centrais do sistema imune tem se apresentado como uma alternativa promissora. Produzimos e caracterizamos um anticorpo híbrido específico para a molécula DEC205, um receptor endocítico presente nas células dendríticas, acoplado à proteína MSP-1(19) de P. chabaudi, para fins de imunização e análise da resposta imune celular e humoral. Ensaios de imunização mostraram a indução de resposta humoral em camundongos imunizados com anticorpo híbrido e seu controle isotípico, caracterizada pela produção de IgM. Nossos resultados prévios indicam que o direcionamento de antígenos aliado a outras estratégias de imunizações podem resultar na ativação da resposta imune específica ao parasita. / Despite the strong activation of the immune system that occurs during infection by Plasmodium, the immunological memory to infection is restricted to patients residing in endemic areas. Thus it is important to the generation of methods to induce an effective immune response against the parasite. In this context, the targeting of antigens to the central cells of the immune system has emerged as a promising alternative. We produce and characterize a hybrid antibody molecule specific for DEC205, an endocytic receptor present on dendritic cells, coupled to protein MSP-1(19) of P. chabaudi, for immunization and analysis of cellular and humoral immune response. Immunization tests showed the induction of humoral response in mice immunized with hybrid antibody and isotype control, characterized by production of IgM. Our previous results indicate that targeting antigens combined with other strategies for immunization may result in the activation of specific immune response to the parasite.

Helicobacter pylori are unique in their ability to colonise the human gastric mucosa. They persist lifelong in untreated individuals despite the presence of a continuous and specific immune response being mounted against it. H. pylori O-antigen is thought to be involved in immune-evasion and subversion by the bacteria and expression has been shown to facilitate colonisation and exacerbate pathology in murine models. This study investigates immuno-relevant roles of H. pylori O-antigen as a pathogen-associated molecular pattern (PAMP) and its interaction with two pattern recognition receptors (PRRs); galectin-3 and DC-SIGN. These PRRs possess distinct carbohydrate recognition domain (CRD) structures and binding affinities. Despite this, we have demonstrated that they compete for adhesion to both Lewis antigen glycoconjugates and whole cell H. pylori 26695 in solid phase binding assays. Galectin-3 significantly reduces DC-SIGN adhesion at a 2:1 stoichiometric ratio in both Lex glycoconjugate and whole cell H. pylori 26695 assays, and abrogates carbohydrate-specific binding in Lex glycoconjugate assays at a 22:1 ratio. These results suggest that galectin-3 may play a role in inhibiting or modulating the interaction between H. pylori O-antigen and DC-SIGN in vivo. Supporting this, we have shown that galectin-3 secreted by AGS cells during competitive infection with H. pylori 26695 is sequestered by H. pylori O-antigen. We have demonstrated that competitive infection of the O-antigen deficient mutant H. pylori 26695 galE in DC-SIGN expressing THP-1 cells reveals a significant reduction in intracellular survival at 8 hours compared to H. pylori 26695 Wt. Co-incubation of H. pylori 26695 Wt with 10 µg ml-1 galectin-3 reduced intracellular survival to the levels of H. pylori 26695 galE at 8 hours. Furthermore, H. pylori 26695 galE displayed rapid association of the endocytic markers Rab5 and Rab7 at 15 minutes compared to H. pylori 26695 Wt. Monoclonal antibody-mediated blocking of DC-SIGN in H. pylori 26695 Wt-THP-1 infections resulted in rapid association of the endocytic markers Rab5 and Rab7, corresponding to that of H. pylori 26695 galE, indicating that DC-SIGN-O-antigen interactions alters intracellular processing of the bacteria and reduces the rate at which these markers are recruited. Together these results elucidate novel mechanisms of H. pylori O-antigen and its interaction with galectin-3 and DC-SIGN that warrant further investigation in vivo. The identification of two PRRs competing for the same PAMP is unconventional and inspires a re-evaluation of PRRs in innate immune recognition.

Detection of evolutionarily conserved pathogen motifs by pattern recognition receptors (PRRs), particularly on dendritic cells (DCs), is crucial for adequate immune responses. Defects in DC function are known to be associated with inflammatory bowel disease (IBD). The endocannabinoid system (ECS) is the system through which exocannabinoids such as Δ⁹-tetrahydrocannabinol and cannabidiol signal. Regarding inflammation, cannabinoids generally exert anti-inflammatory effects, including on experimental colitis. However, most work has been performed in animal models and less is known about the function of this system in human immune cells, particularly DCs. Monoacylglycerol lipase (MGLL) is the key enzyme for hydrolysis of the endocannabinoid 2-arachidonoylglycerol, and a member of the serine hydrolase enzyme superfamily. This thesis defines the activity of serine hydrolase enzymes for the first time in human DCs upon stimulation by NOD2/TLR2 ligands using activity-based protein profiling (ABPP). MGLL is shown to be ubiquitously upregulated upon stimulation of DCs and in monocyte-derived macrophages. Through pharmacological inhibition studies, MGLL is demonstrated to regulate cellular and secreted lipids, not limited to endocannabinoids. However, overall DC function is independent of this enzyme suggesting that the effects of lipid modulation may be on bystander cells. Challenging the current literature, MGLL inhibition with a novel inhibitor worsens murine Citrobacter rodentium colitis. Finally, ABPP demonstrates a rich serine hydrolome in colonic tissue from human IBD with many enzymes previously undefined in this disease. Gene expression of ECS components suggests the enzymes ABHD12 and DAGLα/β may be potential markers of field change in IBD.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease where failure to remove apoptotic cells, due to a defect in phagocytic cells, or deficient opsonisation, leads to secondary necrosis and the release of DNA and chromatin. The nuclear constituents from apoptotic cells are targeted by autoantibodies, which form immune complexes. Immune complex-mediated TLR9 activation of plasmacytoid dendritic cells (pDCs) and subsequent secretion of interferon (IFN-α) is thought to drive inflammation in SLE. It is currently believed that pDCs do not normally respond to apoptotic cells, as self-DNA is hidden from TLR9. However, DNA and chromatin expressed on membrane bound apoptotic bodies is essential for inducing IL-10 secreting regulatory B cells through TLR9 stimulation. The overall objective of this thesis was to understand how apoptotic cells influence immune responses in health and in patients with SLE. Splenic mouse pDCs were activated with the synthetic TLR7 agonist R848 and TLR9 agonists CpGB and CpGA and were co-cultured with apoptotic cells, or with freeze-thawed necrotic cells. PDCs co-cultured with apoptotic cells down-regulated the expression of CD40 and CD86. When pDCs were activated by R848 or CpGB, IL-10, IFN-γ and IL-6 secretion was significantly induced in the presence of apoptotic cells. PDCs so cultured induced T cells to secrete immune-regulatory IL- 10. In contrast, co-culturing apoptotic cells with pDCs activated by CpGA, augmented IFN-α secretion. These cytokine responses by pDCs were only stimulated by DNA on whole apoptotic cells; not by free nucleic acids derived from necrotic cells. This data demonstrates that the inflammatory context in which pDCs sense whole apoptotic cells is crucial to determining the threshold of tolerance to apoptotic self. It questions the perception that pDCs see all apoptotic cells and their necrotic cellular debris as dangerous and suggests that there may be something intrinsically different about SLE apoptotic cells, which causes inflammation. SNPs near ATG5, a protein of the cell survival pathway autophagy, have been linked to SLE susceptibility, but the role of autophagy in SLE pathogenesis is unclear. We hypothesised that dysfunctional autophagy is linked to abnormal apoptosis of SLE lymphocytes. Western blotting revealed that ATG5-ATG12 protein complex expression was significantly reduced in SLE lymphocytes and they failed to convert LC3-I to LC3- II, the hallmark of a functioning autophagy pathway, which caused accelerated secondary necrosis. Apoptotic SLE lymphocytes had an impaired ability to stimulate IL-10 secreting regulatory B cells and they induced pro-inflammatory cytokine secretion by monocyte-derived macrophages. Phagocytosis of apoptotic SLE lymphocytes by healthy macrophages was also impaired; however this was independent of ATG5 protein expression. The novel findings of this thesis suggest SLE apoptotic lymphocytes are intrinsically pro-inflammatory, which may be caused by diminished autophagy leading to an inability of lymphocytes to correctly execute apoptosis. Furthermore, inefficient clearance of SLE apoptotic cells results from a defect in the apoptotic cell, rather than the phagocytic cell.

Infections with parasitic helminths are counted as neglected tropical diseases; they infect millions of people worldwide, causing high morbidity and economic loss. Many parasites establish long lasting infections in the host by blocking immune recognition, activation and effector pathways. To allow in depth research on their modes of immune evasion, several mouse models for parasitic helminth infections have been established. Heligmosomoides polygyrus for example is a gastrointestinal nematode of rodents exhibiting a wide spectrum of immunomodulatory effects, mediated in part by soluble molecules released by adult worms in vitro, the excretory/secretory products (HES). HES is a potent inhibitor of dendritic cell (DC) activation by Toll-like receptor (TLR) ligands, completely abolishing LPS induced IL-12 production and reducing the upregulation of cell surface activation markers. As of now, neither the modulatory molecule nor its mechanism of action are known. Here, the effect of HES on TLR ligand induced DC maturation was characterized in considerably more detail compared to previous publications. It could be shown to inhibit DC maturation induced by various TLR ligands, on both protein and mRNA levels. These effects were comparable in both C57BL/6 and BALB/c derived cells; in contrast to this HES differentially affected alternative activation of BMDC from these two mouse strains. Although for most of the experiments GM-CSF differentiated BMDC were used, HES also inhibited LPS induced activation of splenic CD11c+ cells as well as the activation of all three populations described in Flt3-L differentiated BMDC - pDCs, CD11b+ cDCs and CD24+ cDCs. Furthermore, it could be shown here that HES also inhibits LPS induced maturation in human monocyte derived DCs. In the search for the component in HES responsible for its inhibition of TLR ligand induced DC maturation, exosome depleted HES rather than exosomes was inhibitory, and the effect was heat labile. This lead to the conclusion that the modulatory molecule has a protein component which is indispensable for its effect; following this reasoning HES was subjected to fractionation, with subsequent analysis of the fraction protein contents by mass spectrometry. The top nine candidate proteins were expressed recombinantly; however, the recombinants were not able to inhibit LPS induced DC activation. In parallel, experiments to elucidate the mechanism by which HES inhibits TLR ligand induced DC maturation were performed. This led to the conclusion that HES induces changes in the cells that, while not affecting the induction of signalling downstream of TLRs, do impair its maintenance. As a complement to these experiments, the transcriptomes of LPS and LPS+HES treated cells eight hours after LPS stimulation were compared. This revealed that transcripts encoding a number of transcription factors inducing the expression of activation markers after TLR ligation were reduced upon treatment of cells with HES, as were the transcript levels of IRAK2, a kinase necessary for persistent signalling. In addition, HES increased the transcript levels for several factors known to negatively regulate DC maturation, including ATF3. Furthermore, this analysis revealed changes in transcript levels of factors like HIF-1a, indicating an even greater reliance on aerobic glycolysis if cells were treated with HES, in addition to hints at increased ER and oxidative stress. In conclusion, this work narrows down the list of potential DC modulators in HES, gives a first insight into changes in DC metabolism induced by HES and sheds light on the role of a number of signalling pathways with important roles in DC activation as targets of DC inhibition by HES.

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-04-10Bitstream added on 2014-06-13T21:01:53Z : No. of bitstreams: 1 botari_cme_me_botfm.pdf: 1339344 bytes, checksum: f4a978b0ca0056da6a691563d873a9ac (MD5) / Fundação Amaral Carvalho / The graft versus host disease (GVHD) or graft-versus-host-disease(GVHD) is a major cause of morbidity and mortality in patients who underwent hematopoietic stem cell transplantation (HSCT). It is a syndrome with various clinical, pathological and immunological. Aiming to contribute to clarifying the role of Myeloid dendritic cells, plasmacytoid cells and NK (natural killer) in chronic graft versus host disease (GVHD) and to clarify the mechanisms involved were examined microscopic sections of the oral mucosa of 26 patients with leukemia Acute Myeloid (AML) who underwent allogeneic hematopoietic stem cell in the Hospital Amaral Carvalho, Jau - SP, where 13 patients develop GVHDc oral mucosa and 13 did not develop GVHD. Microscopic sections were submitted to immunohistochemistry staining using monoclonal antibodies anti-CD1a, anti-CD56 and anti- CD123. The number of immunostained cells in the chorion (CD1a, CD123 and CD56) per square millimeter was calculated by dividing the mean of dendritic cells present in the 10 fields examined area covered by each specimen. The data were evaluated by Mann-Whitney. Results showed a statistically significant increase of myeloid dendritic cells CD1a (p = 0.02) and NK cells CD56 (p = 0.04) in patients with GVHDc compared with those without GVHDc. Analysis of CD123 immunostaining of no statistical difference between groups. It is concluded from this study that the development of chronic GVHD is participation of myeloid dendritic cells and NK cells in the chorion of oral mucosa