The stimulation of Toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) activates macrophages and dendritic cells to response to pathogens. These activated cells induce many immune-related genes, including proinflammatory cytokines which are necessary to activate immune responses against infection. TLRs and their signaling components have been linked to several human diseases, including pyogenic infection and sepsis. Sepsis often occurs in cancer patients treated with chemotherapy. The first focus of this work is to understand how TLR signal transduction pathways regulate the induction of proinflammatory cytokines. TLR stimulation triggers a signaling pathway via MyD88 and IRAK-4 that is essential for proinflammatory cytokine induction. In this study, I found that MyD88-deficient macrophages had defective c-Rel activation, which has been linked to IL-12 p40 induction. In addition, the expression of C/EBPbeta and C/EBPdelta was limited in MyD88- or IRAK-4-deficient macrophages treated with LPS. Importantly, the absence of both C/EBPbeta and C/EBPdelta resulted in the impaired induction of proinflammatory cytokines stimulated by several TLR ligands. These results identify both c-Rel and C/EBPbeta/delta as important transcription factors in a MyD88-dependent pathway that regulate the induction of proinflammatory cytokines. The second focus of this work is to understand the function of TREM2 and how TREM2 regulates TLR-mediated immune responses. TREM2 and DAP12 deficiencies were found in human patients with Nasu-Hakola disease, but the biology of TREM2 remains unclear. To study the function of TREM2 in dendritic cells, TREM2-deficient mice were generated. I found that TREM2 down-regulated the expression of proinflammatory cytokines induced by TLRs. The TREM2 ligand was expressed on activated T cells, and TREM2 enhanced the expression of IFN-gamma in antigen-specific T cells. In a mouse model of autoimmune diabetes, TREM2-deficient mice were resisted to CD8+ T cell-mediated beta-cell destruction. Therefore, TREM2 can positively or negatively regulate TLR-mediated immune responses in selective conditions. Together, the results presented in this thesis provide further understanding of how c-Rel, C/EBPbeta/delta, and TREM2 control and modulate TLR-mediated responses. Understanding these processes may ultimately provide novel therapeutic strategies to modulate immune responses in patients suffered from infectious diseases and cancer.

The stimulation of Toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) activates macrophages and dendritic cells to response to pathogens. These activated cells induce many immune-related genes, including proinflammatory cytokines which are necessary to activate immune responses against infection. TLRs and their signaling components have been linked to several human diseases, including pyogenic infection and sepsis. Sepsis often occurs in cancer patients treated with chemotherapy. The first focus of this work is to understand how TLR signal transduction pathways regulate the induction of proinflammatory cytokines. TLR stimulation triggers a signaling pathway via MyD88 and IRAK-4 that is essential for proinflammatory cytokine induction. In this study, I found that MyD88-deficient macrophages had defective c-Rel activation, which has been linked to IL-12 p40 induction. In addition, the expression of C/EBPbeta and C/EBPdelta was limited in MyD88- or IRAK-4-deficient macrophages treated with LPS. Importantly, the absence of both C/EBPbeta and C/EBPdelta resulted in the impaired induction of proinflammatory cytokines stimulated by several TLR ligands. These results identify both c-Rel and C/EBPbeta/delta as important transcription factors in a MyD88-dependent pathway that regulate the induction of proinflammatory cytokines. The second focus of this work is to understand the function of TREM2 and how TREM2 regulates TLR-mediated immune responses. TREM2 and DAP12 deficiencies were found in human patients with Nasu-Hakola disease, but the biology of TREM2 remains unclear. To study the function of TREM2 in dendritic cells, TREM2-deficient mice were generated. I found that TREM2 down-regulated the expression of proinflammatory cytokines induced by TLRs. The TREM2 ligand was expressed on activated T cells, and TREM2 enhanced the expression of IFN-gamma in antigen-specific T cells. In a mouse model of autoimmune diabetes, TREM2-deficient mice were resisted to CD8+ T cell-mediated beta-cell destruction. Therefore, TREM2 can positively or negatively regulate TLR-mediated immune responses in selective conditions. Together, the results presented in this thesis provide further understanding of how c-Rel, C/EBPbeta/delta, and TREM2 control and modulate TLR-mediated responses. Understanding these processes may ultimately provide novel therapeutic strategies to modulate immune responses in patients suffered from infectious diseases and cancer.

No description available.

Toll-like receptors (TLRs) are the main components of the innate immunity. Pathogen-activated TLRs trigger a cytoplasmic signaling cascade through adaptor proteins, with the first being the TIR domain-containing adaptor protein (TIRAP). TIRAP contains a TIR domain, which associates with TLRs and other adaptor proteins; and a N-terminal phosphoinositide-binding motif (PBM) that mediates the membrane recruitment of TIRAP. Upon ligand activation, TLRs are recruited to the phosphoinositide (PIP)-enriched region in the membrane, where TIRAP recruits other adaptors to the membrane to activate TLR signaling pathway. To investigate the mechanism of membrane targeting of TIRAP and the basis for its regulation, I functionally and structurally characterized TIRAP and its PBM using biophysical approaches. I show that TIRAP PBM adopts helical structural in dodecylphosphocholine (DPC) micelles and other membrane mimics. NMR studies reveal that TIRAP PBM binds PIPs following a fast exchange regime with a moderate affinity through two conserved basic termini. Mutation of these two basic regions abolishes PIPs binding without distorting the helical structure of the peptide. Solution NMR structure of TIRAP PBM exhibits a central relatively hydrophobic helix surrounded by the flexible N- and C-termini. Paramagnetic studies indicate that the helix is close to the micelle core, whereas two termini are located on the micellar surface. Nuclear spin relaxation experiments indicate that the two termini of TIRAP PBM become more ordered when bound to PIP, thus, we propose that the central helix in PBM is responsible for membrane insertion, whereas the two sets of basic residues interact with PIPs to stabilize TIRAP's membrane interaction. Phosphomimetic mutation of Thr28 to Asp (T28D) as well as phosphorylation in Thr28 inhibit TIRAP PBM's binding to phosphoinositides by distorting the central helical structure of the peptide. More importantly, TIRAP T28D disrupt its subcellular localization in vivo. Thus, phosphorylation can impair proper insertion of TIRAP at the plasma membrane through PBM and, consequently, it may represent the first signal that promotes TIRAP degradation. / Ph. D.

A esporotricose caracteriza-se como uma micose subcutânea causada por fungos dimórficos do gênero Sporothrix, capazes de acometer o homem e uma grande variedade de animais, dentre eles os felinos. A princípio, Sporothrix schenckii era a única espécie conhecida como responsável pela esporotricose. Após estudos genotípicos e fenotípicos de isolados ambientais, clínicos humanos e animais, verificou-se alta variabilidade entre os isolados e estabeleceu-se a existência de um Complexo Sporothrix. Dentro deste, a maior causadora de surtos epidêmicos, justificada por uma maior virulência e capacidade de evasão da resposta imune, é a espécie Sporothrix brasiliensis. Nesse sentido, dada a ausência de estudos direcionados a está espécie, objetivou-se avaliar a importância de receptores Toll like-2 (TLR-2) e Toll like-4 (TLR-4) na infecção por S. brasiliensis. Além disso, utilizando técnicas de proteômica, procurou-se elucidar proteínas diferencialmente expressas em S. brasiliensis quando comparado à espécie S. schenckii. Para avaliação da resposta imune utilizaram-se modelos in vitro e in vivo de infecção, e para a investigação das proteínas diferencialmente expressas, utilizou-se a técnica de proteômica Bottom-up. A investigação da resposta imune in vitro mostrou a dependência dos receptores TLR-2 e TLR-4 no desencadeamento da resposta imune. Os ensaios in vivo mostraram a importância desses receptores no controle da infecção e dependência dos mesmos na produção de citocinas, principalmente nos primeiros 14 dias de infecção. Na ausência do receptor TLR-2, houve a polarização de resposta Th17 na tentativa de controle da infecção. Quando avaliadas as diferenças entre as espécies S. brasiliensis e S. schenckii, em termos de proteínas expressas, verificou-se que S. brasiliensis expressa diferencialmente 60 proteínas. Dentre essas, 9 são relatadas na literatura, como importantes na virulência e escape imunológico dos principais fungos de importância médica. Os resultados encontrados no presente trabalho permitem concluir que reconhecimento de S. brasiliensis é dependente dos receptores TLR-2 e TLR-4. Estudos que investiguem a utilização de outras vias de sinalização como mecanismos compensatórios, bem como, o sinergismo desses receptores no contexto da infecção por S. brasiliensis são fundamentais na compreensão da fisiopatologia dessa doença. No que tange a caracterização proteica, estudos com mutantes para cada uma das proteínas descritas nesse trabalho devem ser avaliados. / Sporotrichosis is a subcutaneous mycosis caused by dimorphic fungi of the genus Sporothrix that affects humans and animals, predominantelly felines. Inicially, Sporothrix schenckii was the only specie associated to sporotrichosis. However, after genotypic and phenotypic studies of human and animal clinical isolates, a high variability among the isolates was found and was concluded the existence of a complex: the Sporothrix Complex. Inside the Sporothrix complex, the major cause of epidemic outbreaks, justified by a greater virulence and ability to evade the immune system, is Sporothrix brasiliensis. Concerning this, the absence of studies directed to this specific specie, the aim was to evaluate the importance of Toll like receptor-2 (TLR-2) and Toll like receptor-4 (TLR-4) during S. brasiliensis infection. In addition, was look using proteomics techniques, the proteins differentially expressed in S. brasiliensis when compared to S. schenckii. To evaluate the immune response, in vitro and in vivo tecniques were used, and for the investigation of differentially expressed proteins, the Bottom-up proteomics technique was used. The investigation of the in vitro immune response showed the dependence of TLR-2 and TLR-4 receptors on phagocytosis and the production of inflammatory mediators, such as cytokines and NO. In vivo assays showed the importance of these receptors to control the infection and their dependence on cytokine production during the first 14 days of infection. In the absence of the TLR-2 receptor, the Th17 response was polarized in an attempt to control the infection. Evaluating the differences between S. brasiliensis and S. schenckii, in terms of expressed proteins, it was verified that S. brasiliensis differentially expressed 60 proteins. Among these, 9 are reported in the literature, as important in the virulence and immune evasion among the most important medical fungi. The results found in the present study allow to conclude that S. brasiliensis recognition is dependent on TLR-2 and TLR-4 receptors. Studies investigating the use of other signaling pathways as compensatory mechanisms, as well as the synergism of these receptors in the context of S. brasiliensis infection, are fundamental to understand the pathophysiology of this disease. Regarding the protein characterization, studies with mutants for each of the proteins described in this work should be evaluated.

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. While the discrimination of host and microbial DNA is presumed to reflect TLR9-mediated recognition of CpG motifs, little information is available to verify this hypothesis. Cell stimulation experiments demonstrate preferential activation of TLR9 by CpG-containing nucleic acids, however direct binding investigations have reached contradictory conclusions with respect to the ability of TLR9 to bind nucleic acids in a sequence-specific fashion. Here we report expression of the soluble, ectodomain of human TLR9 with characterization of its ligand-binding properties. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity is mediated specifically by the absolute concentration of nucleic acids in a sequence-independent manner.<p>The bovine hsp70A promoter was used to direct the heat-regulated synthesis of the ectodomain of human TLR9 in transfected cultured bovine cells. The protein was efficiently secreted from transfected cells in a temperature-dependent manner and the recombinant receptor produced was found to be relatively pure. A stably transfected cell line with regulated expression of the protein was obtained and repeated thermal cycling of the cultures enabled high-yield production of the receptor in an active ligand-binding form. Using this recombinant receptor to study the ligand binding properties of TLR9, a model of positive cooperativity is proposed in which the sensitivity of TLR9 ligand binding is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion, while activation of TLR9 is highly dependent on DNA sequence. That is to say that TLR9 is primed for activation by interaction with non-activating sequences but activation itself occurs in a sequence-specific fashion.

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. While the discrimination of host and microbial DNA is presumed to reflect TLR9-mediated recognition of CpG motifs, little information is available to verify this hypothesis. Cell stimulation experiments demonstrate preferential activation of TLR9 by CpG-containing nucleic acids, however direct binding investigations have reached contradictory conclusions with respect to the ability of TLR9 to bind nucleic acids in a sequence-specific fashion. Here we report expression of the soluble, ectodomain of human TLR9 with characterization of its ligand-binding properties. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity is mediated specifically by the absolute concentration of nucleic acids in a sequence-independent manner.<p>The bovine hsp70A promoter was used to direct the heat-regulated synthesis of the ectodomain of human TLR9 in transfected cultured bovine cells. The protein was efficiently secreted from transfected cells in a temperature-dependent manner and the recombinant receptor produced was found to be relatively pure. A stably transfected cell line with regulated expression of the protein was obtained and repeated thermal cycling of the cultures enabled high-yield production of the receptor in an active ligand-binding form. Using this recombinant receptor to study the ligand binding properties of TLR9, a model of positive cooperativity is proposed in which the sensitivity of TLR9 ligand binding is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion, while activation of TLR9 is highly dependent on DNA sequence. That is to say that TLR9 is primed for activation by interaction with non-activating sequences but activation itself occurs in a sequence-specific fashion.

Diabetes mellitus (DM) is the 4th leading cause of mortality in Taiwan. Chronic persistent inflammation as demonstrated by higher proinflammatory mediators in blood has been correlated to cardiovascular complications of type 2 DM. The cellular and molecular mechanism of chronic inflammation in type 2 DM remains to be determined. This study was conducted to explore altered innate immunity in toll-like receptor (TLR) expression and signaling of monocytes from type 2 DM patients. Blood leukocytes from type 2 DM patients were counted and studied for TLR2 and TLR4 expression and signaling. Each experiment was run with 1 to 2 type 2 DM patients, simultaneously with 1 to 2 age-matched normal adults as controls. 31 type 2 DM patients and 37 normal age-matched controls completed the study. Results showed that blood monocytes from type 2 DM patients had a significantly higher TLR4 but not TLR2 expression. Using a TLR4 ligand, lipopolysaccharide (LPS), to trigger TNF£\ production, a significantly higher TNF£\ production by blood leukocytes from type 2 DM patients than age-matched controls was found. The higher TNF£\ production by blood leukocytes from type 2 DM patients was associated with down-regulation of suppressor of cytokine signaling 1and 3 (SOCS-1 and SOCS-3) expression. We have further postulated that increase of oxidative stress or decrease of IFN-£\ production in type 2 DM patients was related to the alteration of TLR-4 response. Correction of SOCS-1 expression by addition of antioxidant, superoxide dismutase (SOD), but not IFN-£\, significantly decreased TNF£\ production in blood leukocytes from type 2 DM patients. This study is the first in the literature to identify an alteration of TLR4 expression associated with depressed SOCS-1 expression in leukocytes of type 2 DM patients. Results from this study highlight a potential pathway to improve chronic inflammation of type 2 DM patients via modulation of TLR4 expression and SOCS-1 mRNA expression of leukocytes.

A esporotricose caracteriza-se como uma micose subcutânea causada por fungos dimórficos do gênero Sporothrix, capazes de acometer o homem e uma grande variedade de animais, dentre eles os felinos. A princípio, Sporothrix schenckii era a única espécie conhecida como responsável pela esporotricose. Após estudos genotípicos e fenotípicos de isolados ambientais, clínicos humanos e animais, verificou-se alta variabilidade entre os isolados e estabeleceu-se a existência de um Complexo Sporothrix. Dentro deste, a maior causadora de surtos epidêmicos, justificada por uma maior virulência e capacidade de evasão da resposta imune, é a espécie Sporothrix brasiliensis. Nesse sentido, dada a ausência de estudos direcionados a está espécie, objetivou-se avaliar a importância de receptores Toll like-2 (TLR-2) e Toll like-4 (TLR-4) na infecção por S. brasiliensis. Além disso, utilizando técnicas de proteômica, procurou-se elucidar proteínas diferencialmente expressas em S. brasiliensis quando comparado à espécie S. schenckii. Para avaliação da resposta imune utilizaram-se modelos in vitro e in vivo de infecção, e para a investigação das proteínas diferencialmente expressas, utilizou-se a técnica de proteômica Bottom-up. A investigação da resposta imune in vitro mostrou a dependência dos receptores TLR-2 e TLR-4 no desencadeamento da resposta imune. Os ensaios in vivo mostraram a importância desses receptores no controle da infecção e dependência dos mesmos na produção de citocinas, principalmente nos primeiros 14 dias de infecção. Na ausência do receptor TLR-2, houve a polarização de resposta Th17 na tentativa de controle da infecção. Quando avaliadas as diferenças entre as espécies S. brasiliensis e S. schenckii, em termos de proteínas expressas, verificou-se que S. brasiliensis expressa diferencialmente 60 proteínas. Dentre essas, 9 são relatadas na literatura, como importantes na virulência e escape imunológico dos principais fungos de importância médica. Os resultados encontrados no presente trabalho permitem concluir que reconhecimento de S. brasiliensis é dependente dos receptores TLR-2 e TLR-4. Estudos que investiguem a utilização de outras vias de sinalização como mecanismos compensatórios, bem como, o sinergismo desses receptores no contexto da infecção por S. brasiliensis são fundamentais na compreensão da fisiopatologia dessa doença. No que tange a caracterização proteica, estudos com mutantes para cada uma das proteínas descritas nesse trabalho devem ser avaliados. / Sporotrichosis is a subcutaneous mycosis caused by dimorphic fungi of the genus Sporothrix that affects humans and animals, predominantelly felines. Inicially, Sporothrix schenckii was the only specie associated to sporotrichosis. However, after genotypic and phenotypic studies of human and animal clinical isolates, a high variability among the isolates was found and was concluded the existence of a complex: the Sporothrix Complex. Inside the Sporothrix complex, the major cause of epidemic outbreaks, justified by a greater virulence and ability to evade the immune system, is Sporothrix brasiliensis. Concerning this, the absence of studies directed to this specific specie, the aim was to evaluate the importance of Toll like receptor-2 (TLR-2) and Toll like receptor-4 (TLR-4) during S. brasiliensis infection. In addition, was look using proteomics techniques, the proteins differentially expressed in S. brasiliensis when compared to S. schenckii. To evaluate the immune response, in vitro and in vivo tecniques were used, and for the investigation of differentially expressed proteins, the Bottom-up proteomics technique was used. The investigation of the in vitro immune response showed the dependence of TLR-2 and TLR-4 receptors on phagocytosis and the production of inflammatory mediators, such as cytokines and NO. In vivo assays showed the importance of these receptors to control the infection and their dependence on cytokine production during the first 14 days of infection. In the absence of the TLR-2 receptor, the Th17 response was polarized in an attempt to control the infection. Evaluating the differences between S. brasiliensis and S. schenckii, in terms of expressed proteins, it was verified that S. brasiliensis differentially expressed 60 proteins. Among these, 9 are reported in the literature, as important in the virulence and immune evasion among the most important medical fungi. The results found in the present study allow to conclude that S. brasiliensis recognition is dependent on TLR-2 and TLR-4 receptors. Studies investigating the use of other signaling pathways as compensatory mechanisms, as well as the synergism of these receptors in the context of S. brasiliensis infection, are fundamental to understand the pathophysiology of this disease. Regarding the protein characterization, studies with mutants for each of the proteins described in this work should be evaluated.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2010-2011 / Le TLR2 et le TLR9 sont membres de la famille des ±Toll-like receptors¿ (TLRs), récepteurs de l'immunité innée, pouvant reconnaître divers motifs de microorganismes. Il est connu que ces deux récepteurs peuvent lier différentes composantes de certains virus de la famille des Herpesviridae. Le TLR2, exprimé à la surface de la cellule, peut reconnaître l'herpès simplex, le cytomegalovirus et varicella zoster. Quant au TLR9, il est exprimé dans les vésicules intracellulaires et peut lier l'ADN double brin étranger présent notamment chez les virus herpétiques. Ces données nous ont poussés à vérifier si une telle interaction était observée entre ces récepteurs et le virus Epstein-Barr (EBV). Nous avons tout d'abord établi, dans un modèle in vitro de cellules HEK293 transfectées avec le TLR2 et un plasmide rapporteur NF[kabba]B-LUC, qu'EBV pouvait activer la voie NF-[kabba]B et ce, dépendamment du TLR2. En effet, l'initiation de la voie NF-[kabba]B par le virus peut être abolie par l'utilisation d'anticorps bloquant/neutralisant contre le TLR2 ou la glycoprotéine 350 (gp350) virale. De plus, nous avons aussi démontré que'EBV pouvait activer la sécrétion de MCP-1 par les monocytes primaires humains, un processus pouvant être inhibé par l'utilisation d'ARNs bloquants contre le TLR2 (SiRNA TLR2). Dans un deuxième temps, nous avons examiné si le génome d'EBV pouvait être reconnu par le TLR9. Les résultats indiquent clairement que les particules intactes d'EBV, ainsi son génome purifié, peuvent induire la sécrétion d'IL-8 par les monocytes humains. Cette induction par l'ADN viral semble de plus être dépendante de l'acidification des endosomes. Le traitement des cellules par un SiRNA TLR2, un ODN inhibiteur du TLR9 ou une combinaison des deux diminue la production d'IL-8, ceci indiquant une coopération probable de ces récepteurs lors de la détection d'EBV. Concernant les cellules dendritiques plasmacytoïdes (pDCs), qui expriment majoritairement le TLR7 et le TLR9, la stimulation avec EBV ou l'ADN viral purifié induit la production d'IFN-α. Une combinaison d'antagonistes du TLR7 et du TLR9 élimine complètement la sécrétion d'IFN-α, supportant ainsi notre hypothèse voulant que le TLR7 puisse aussi contribuer à cette réponse. En conclusion, les résultats présentés dans ce manuscrit démontrent que la famille des TLRs joue un rôle important dans le déclenchement d'une réponse pro-inflammatoire et antivirale suite à la reconnaissance d'EBV, et que l'activation de multiples TLRs par diverses composantes virales permet probablement de potentialiser l'efficacité de celle-ci.