Fibroblasts play a sentinel role in asthmatic disease. They are the main constituents of connective tissue and are increased in number in the asthmatic lung. They are also capable of secreting a diverse repertoire of cytokines and are able to be activated by pro-inflammatory cytokines and cell-cell contact. Previously we have reported that normal human lung fibroblasts (NHLF) can be activated by monocytes (U937) through cell-cell contact to produce GM-CSF. Here we show that GM-CSF production from NHLF activated by monocyte contact is inhibited by prednisone, a synthetic glucocorticoid used in the treatment of asthma. GM-CSF is an acidic glycoprotein that potentiates development of cells in the granulocyte and macrophage lineage and is secreted at sites of peripheral inflammation. The receptor for GM-CSF was found on NHLF by flow cytometry and was able to be up-regulated by interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha and recombinant human (rh) GM-CSF. To test autocrine effects of GM-CSF on fibroblasts, rh GM-CSF was used in proliferation studies and was found to decrease fibroblast proliferation. Prednisone was used to block NF-kappaB activation and GM-CSF gene expression as well. These data indicate mechanism of action and treatment for cell-cell contact mediated inflammation of infiltrating monocytes with fibroblasts as seen in asthma and other diseases like graft versus host disease.

Human immunodeficiency virus type-1 (HIV-1) is the etiologic agent of acquired immune deficiency syndrome (AIDS). In most cases HIV-1 infection in humans, leads to AIDS, which is characterized by opportunistic infections leading to death. The role various infectable cell types play in the course of infection is unclear. However, it is becoming increasingly more evident that cells of the monocyte/macrophage lineage are very important at several stages of disease. They are involved in the transmission, establishment and dissemination of infection as well as the AIDS related complication of dementia and pulmonary dysfunction. The regulation of virus expression in monocyte/macrophages while maintaining normal cell function would be of great benefit. Retinoic acid (RA) is a bioactive metabolite of vitamin A, an essential nutrient, and acts as a transcriptional regulator of many genes. RA is also a potent modulator of myeloid cell differentiation and function; it is currently used clinically. Clinical data indicate that serum vitamin A levels are inversely correlated with various aspects of HIV-1 induced disease. Furthermore, work done by several groups has demonstrated that RA directly modulates HIV-1 replication in cells of the myeloid lineage. RA is capable of either stimulating or repressing HIV-1 replication depending on the cell type used. This dichotomy appears to depend upon the differentiation state of the cells. Changes in differentiation states are associated with the altered expression of many cellular proteins including transcriptional regulators. Experiments indicate that the TATA box of HIV-1 is required for full levels of gene expression. I hypothesized that RA was modulating replication at the level of LTR-directed gene expression, and that the differentiation state of the cell influences the RA modulation. This thesis demonstrates that the RA effect is at the level of gene expression mapping to a promoter proximal element for both HIV-1 and simian immunodeficiency virus (SIVmac.) The ability of RA to stimulate or repress expression depends upon the differentiation state of the cells. Using U937 promonocyte cells, I demonstrate that RA increases SIVmac and HIV-1 transcription. When THP-1 monocytes or primary macrophages are used, I demonstrate that RA induces repression of HIV-1 and SIVmac. This RA modulation of expression is associated with altered complexes binding to the promoter proximal regions of HIV-1 and SIVmac. There has been a great deal of interest in CD8+ T cell derived factors which modulate HIV-1 replication. Work done by Levy and colleagues over a decade ago demonstrated that factors secreted by CD8+ T cells could block HIV-1 replication. Others have shown that the β-chemokines, released by activated CD8+ T cells, can block the entry of HIV-1 into macrophages. Center and colleagues identified a lymphocyte chemoattractant factor as IL-16. IL-16 is released by activated CD8+ T cells and it's receptor is CD4. IL-16 induces the migration of CD4+ T lymphocytes, and has been shown to activate many signaling pathways in CD4+ T lymphocytes. Kurth et al. demonstrated that IL-16 blocked the replication of HIV-1 in CD8+ depleted PBMC. In these experiments, it was not determined whether IL-16 was blocking viral entry (preventing viral binding to CD4) or whether IL-16 had inhibitory effects on subsequent steps in the virus life cycle. While IL-16 and HIV-1 share CD4 as their receptor, IL-16 binding was mapped to a separate epitope on CD4 from the HIV-1 binding site. Therefore I began experiments to determine how IL-16 regulates HIV-1 expression in T cells. I hypothesized that the IL-16 signaling pathway is involved in repressing HIV-1 gene expression. Experiments presented here demonstrate that IL-16 represses LTR-directed gene expression in T cell lines in a CD4 dependent manner. The IL-16 mediated repression is dependent on a DNA binding site contained within the viral core enhancer region. The data are also consistent with IL-16 inducing a repressor which binds within or adjacent to the HIV-1 core enhancer region.

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The post human genome sequence era has begun to explore various aspects of the functional genome in relation to disease including gene expression, genetic variation and epigenetics. The genetic determinants of common and complex phenotypes are difficult to resolve even though their heritability is recognised. Recent genome-wide association studies (GWAS) for common diseases has identified many new disease susceptibility associated loci. These loci often lie in non-coding regions of the genome and disease associated genetic variants are proposed to act by modulating gene expression. This thesis investigated genetic variation as determinants of gene expression in the context of the immune system especially focused on the innate immune and inflammatory responses. Different primary human immune cell types were collected from healthy volunteers of European ancestry to achieve this. In order to identify genetic variants associating with gene expression, expression quantitative trait loci (eQTL) mapping was conducted in a cell type specific manner. The primary dataset (n=288) consists of CD19⁺ B-cells from the adaptive immune system and CD14⁺ monocytes from the innate immune system. 78% of the total cis eQTL were found to be cell type specific and include genes relating to their roles in the immune response. Trans eQTL showed greater cell type specificity and include master regulatory eQTL on the LYZ locus at chromosome 12q15 in monocytes and the KLF4 (9p31) in B-cells. The identified eQTL are implicated in association with autoimmune disease susceptibility including inflammatory bowel disease, diabetes and rheumatoid arthritis. The second analysed dataset (n=64) consists of CD14+ monocytes and macrophages differentiated ex vivo. Macrophages are involved in many inflammatory diseases as well as in the innate immune response. The differential gene expression and eQTL mapping analyses were conducted to investigate macrophages specific gene expression signatures and associations to genetic variants. Macrophage eQTL are involved in signal transduction for the inflammatory response (IL1RN and STAT4) and lipid metabolism (PPARG) with implication for metabolic disease association. The eQTL analyses using primary immune cell types provide insights into genetic variation in association to gene expression which is involved in autoimmunity and disease susceptibility.

Platelet-activating factor acetylhydrolase (PAFAH) is elevated in atherosclerosis and may play a role in pathogenesis of this disease. Molecular mechanisms regulating the expression of this lipoprotein-associated PLA2 are indistinct. Mildy oxidized low density lipoprotein (oxLDL) and monocytes (the primary source of PAFAH) are co-localized in early atheromas. Monocytes are activated by oxidized phospholipids (oxPL) in the oxLDL particle. We hypothesized that oxPL-activated monocytes are the source of increased levels of PAFAH in atherosclerosis. We found that PAFAH expression is significantly induced by OxPAPC and in particular long-chain fractions of oxPAPC in monocytes and cytokine-differentiated DC, but not cytokine-differentiated MO. Furthermore, spontaneously differentiated MO and DC from monocytes of non-periodontitis and aggressive periodontitis subjects, oxPAPC induced PAFAH in DC alone. 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphocholine (PEIPC) is a particularly bioactive component of long-chain oxPAPC fractions that binds the prostaglandin receptor subtypes DP1 and EP2. We revealed using selective agonists and antagonists of these receptors that DP1 and EP2 are required for the induction of PAFAH expression. OxPAPC stimulates IL-6 release from monocytes and this cytokine is required for oxPAPC-induced PAFAH expression. We next tested the hypothesis that oxPAPC did not induce PAFAH in MO because a key component of the signaling machinery was lacking. Flow cytometric and immunoblot analyses demonstrated that MO express very low levels of IL-6 receptor in comparison to DC and monocytes. Based on these observations, we propose that long-chain oxPL induce PAFAH expression by binding DP1 and/or EP2 and stimulating IL-6 production. These data strongly support the hypothesis that oxLDL-activated DC are the source of high PAFAH levels in atherosclerosis. Platelet activating factor (PAF) is the inflammatory phospholipids for which PAFAH is named. PAF has been shown by other investigators to induce the expression of PAFAH. In our physiologically relevant monocytes, PAF suppresses PAFAH transcription and expression. 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine (POVPC) is a short-chain oxPL that signals through the PAF receptor. Our preliminary data suggest that like PAF, POVPC suppresses PAFAH expression in monocytes. Further investigation into the effects of the short-chain oxPL are warranted. Our data support the hypothesies that oxPL-activated DC are the source of high PAFAH levels in atherosclerosis.

Células dendríticas (DCs) são componentes centrais da imunidade celular, responsáveis pelo priming de linfócitos T naïve. A polarização de linfócitos T é restrita aos sinais fornecidos durante a apresentação do antígeno. Além desses sinais, a origem e natureza de DCs que induzem diferentes perfis de linfócitos T não é totalmente compreendida. Foi investigada a capacidade de células Natural Killer (NK) de modular estágios iniciais da diferenciação de monócitos em DCs e de impactar na sua função de primar e polarizar linfócitos T naïve. DCs derivadas de monócitos pré-co-cultivados com células NK favorecem o priming de linfócitos T CD8 do tipo Tc1/Tc17, com potente capacidade de produção de IFN-γ. Este fenômeno foi dependente de interações longas via NKp30 e da maquinaria citotóxica de células NK desencadeada nas etapas inicias da sua interação com monócitos. Esta interação pode ter implicações na compreensão da imunidade mediada por linfócitos T CD8 e pode ser explorada para imunoterapia em que a produção de IFN-γ por células T CD8 é necessária ou exacerbada. / Dendritic cells (DCs) are central components of cellular immunity, responsible for the priming of naïve T cells. The polarization of T cells is restricted to signals provided during antigen presentation. Besides such signals, the origin and nature of DCs that induce different T cell profiles is not fully understood. The ability of natural killer cells (NK) to modulate early stages of monocytes differentiation into DCs and to impact on DCs function to prime and polarize naïve T cells was investigated. DCs derived from monocytes co-cultured with NK cells support the priming of type Tc1/Tc17 CD8 T cells with potent IFN-γ production capacity. NK cell-mediated cytotoxicity triggered at early stages of NKp30-dependent long-lasting monocytes-NK-cells interactions, mediated the mechanism by which this phenomenon occurred. This interaction may have implications in the understanding of CD8 T cell-mediated immunity and can be exploited for immunotherapy in which IFN-γ production by CD8 T cells is required or exacerbated.

No câncer de mama, o metabolismo tumoral, ação dos moduladores de estrógenos são fatores que podem influenciar as células dendríticas (DCs). Neste trabalho avaliou o fenótipo de DCs em amostras tumorais, a diferenciação de DCs a partir de células mononucleares do sangue periférico (PBMCs) das pacientes e comparou com voluntárias saudáveis. Os resultados mostraram que há alteração na capacidade de geração, no fenótipo, na capacidade aloestimuladora, maior produção de interleucina 10 e expressão de HSP27 nas DCs de pacientes, comparadas com as DCs de voluntárias saudáveis que produzem mais Interferon-gama. A via p38MAPK parece ser importante na diferenciação de PBMCs em DCs, entretanto, estímulos estressantes podem ativar esta via e induzir a síntese de HSP27 inibindo este processo. O tratamento com tamoxifeno parece modular a expressão de algumas moléculas de membrana. Desta forma, os resultados sugerem que as DCs diferenciadas de pacientes com câncer de mama apresentam alterações fenotípicas e funcionais causadas pelo microambiente tumoral. / In breast cancer, the tumor metabolism, the action of estrogens antagonists can influence dendritic cells (DC) generation. The aim of this work was to evaluate the frequency of DCs in tumor tissue and the differentiation of DCs derived from peripheral blood mononuclear cells (PBMCs) and compared the phenotypic and functionally of these cells from healthy individuals and breast cancer patients. The results showed that patients PBMCs were unable to generate phenotypicaly, functionally mature DCs and presented larger production of interleukin 10 and higher expression of HSP27 when compared with healthy volunteers\' DCs, presented higher production of Interferon-gamma. The p38 MAPK signaling pathway seems to be important in PBMCs differentiation into DCs, and its activation by stress can induce the synthesis of HSP27, that inhibits DC generation. The tamoxifen treatment caused modulation of membrane DC markers expression. Therefore, these results show that patients\' DCs present phenotypic and functional alterations which can be caused by tumor microenvironment.