<p>The urinary tract is one of the most intractable mucosal surfaces for pathogens to colonize. In addition to the natural barriers at this site, potential pathogens have to contend with the vigorous local innate immune response that is initiated by engagement of surveillance molecule TLRs. TLR4 appears to be not only exclusively expressed on superficial BECs but also critical to triggering robust local innate immune responses. TLR4 recognizes Gram-negative bacterial component LPS and initiates a series of intracellular NF-kappaB associated signaling events resulting in a cytokine response. We examined intracellular signaling events in human BECs leading to the production of IL-6, a major urinary cytokine, following activation by E. coli and isolated LPS, and observed that, in addition to the classical NF-kappaB associated pathway, BEC TLR4 triggers a distinct and more rapid signaling response involving, sequentially, Ca2+, AC3 generated cAMP, and the transcriptional factor CREB. This capacity of BECs to mobilize secondary messengers and evoke a more rapid IL-6 response might be critical in their role as first responders to microbial challenge in the urinary tract. </p><p>Here, we also report two additional distinct TLR4-mediated defense mechanisms in BECs. First, BEC TLR4 inhibits bacterial invasion, a necessary step for successful infection. TLR4-mediated suppression of bacterial invasion was linked to increased intracellular cAMP levels which negatively impacted Rac-1 mediated mobilization of the cytoskeleton. Additionally, we found that BECs continue to fight UPEC even after bacterial invasion by triggering bacterial exocytosis through a distinct TLR4-mediated mechanism following activation by LPS. In addition, we reveal that Caveolin-1, Rab27b, PKA, and MyRIP are components of the exocytic compartment and that they form a complex involved in the exocytosis of bacteria. The ability of TLR4 to mediate the rapid cytokine response, the inhibition of bacterial invasion, and the expulsion of intracellular bacteria from infected cells represents three previously unrecognized functions for this innate immune receptor.</p> / Dissertation

<p>Vaccinia virus (VV) is the most thoroughly studied member of the Poxviridae family and the vaccine used to achieve the only successful eradication of a human disease. Over the years, it has proven itself as a useful tool for the study of antiviral immunity, vaccine development, and potentially cancer immunotherapy. VV is capable of eliciting a robust immune response; however the mechanisms by which VV accomplishes this task remain unknown. The overall goal of this thesis project is to determine how VV activates the innate immune system, and how this activation contributes to viral clearance in vivo. We determined that VV or VV-DNA activated the TLR8-MyD88 pathway in plasmacytoid dendritic cells (pDC), resulting in the production of type I interferons (IFN). We also demonstrated that TLR8-mediated production of type I IFN by pDC was crucial to efficient VV control and clearance in vivo. Moreover, we identified the polyA- and polyT-rich sequences in VV-DNA was the possible motif recognize by TLR8. Type I IFN, known for ability to establish the "antiviral state", are also critical mediators of NK cell activation. In the setting of VV infection, we demonstrated that direct action of type I IFN on NK cells, but not accessory cells such as DC, was necessary for NK cell activation in vivo. We further demonstrated that type I IFN-dependent activation of NK cells was required for optimal VV clearance in vivo. Given the importance of NK cells in anti-VV innate immunity, we next examined what role the TLR2-MyD88 pathway, critical for activation of cDC, played in the activation of NK cells. NK cells from TLR2-/- or MyD88-/- mice displayed a reduction in activation and cytolytic function, and this defect was independent of pro-inflammatory cytokine signaling. We were able to demonstrate that direct TLR2 signaling on NK cells was required for their optimal activation and function in response to VV infection. Moreover, we were able to demonstrate that TLR2-MyD88 signaling resulted in the activation of the PI3K-ERK pathway, which was necessary for NK cell cytotoxicity. In addition, we identified the NKG2D pathway as critical for efficient NK cell activation and function in response to VV infection, independent of the TLR2 pathway. Both the NKG2D and TLR2 pathways were crucial for optimal VV clearance and control in vivo. Collectively, this project illuminates the roles and mechanisms of the innate immune system in the control of VV in vivo.</p> / Dissertation

Thymoquinone (TQ), a volatile oil component of black seed oil (derived from Nigella sativa), has been shown to have various biological effects including disease treatment and prevention. TQ is believed to share similar properties to the benzoquinones already in use as therapeutic drugs. Based on previous reports on the anti-inflammatory properties of black seed oil and TQ, it was originally hypothesized that TQ would inhibit lipopolysaccharide (LPS)-induced cellular sialidase activity in an anti-inflammatory manner. Sialidase activity was tested on live mouse bone marrow derived primary macrophage cells, BMC-2 macrophage cells, human embryonic kidney epithelial (HEK293) cells and human fibroblast cells using an assay that measures the cleavage of the sialidase specific fluorescent substrate 2’-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid (4-MUNANA). The cleavage of 4-MUNANA causes the release of free 4-methylumbelliferone, which fluoresces at 450nm (blue) after excitation at 365nm. Unexpectedly, TQ induced sialidase activation in all three cell lines and wild type primary macrophage cells. TQ was unable to induce sialidase activity in primary macrophage cells isolated from Neu4 knockout mice suggesting that the TQ activates Neu4 sialidase enzyme. TQ-induced sialidase activity in these live cells was found to occur through intermediate GPCR-associated guanine nucleotide Gαi subunit and matrix metalloproteinase 9 (MMP9) by using specific inhibitors. In addition, TQ was found to induce sialidase activity in Toll-like receptor-deficient HEK293 cells. These latter data suggested that TQ may be activating GPCR Gαi and MMP9 signaling associated with Neu4 sialidase independent of TLRs. It is proposed that TQ-induced sialidase activity may activate Toll-like receptors in macrophage cells and the subsequent production of pro-inflammatory cytokines in the absence of LPS. Immunocytochemical staining of BMC-2 cells shows that TQ induced NFκB activation. NFκB activation was confirmed with electrophoretic mobility shift assay (EMSA) and western immunoblotting techniques. Cytokine arrays were used to test the pro-inflammatory cytokine response induced in mice by 5 hour treatment of TQ, compared to LPS. Mice treated with TQ exhibited an increase in IL-1β, IL-6 and TNF-α production, similar to LPS treatment. Taken together, the findings in these studies suggest that TQ is a novel ligand for Neu4 sialidase activation which consequently induces pro-inflammatory cytokine responses. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-04-21 17:38:10.413

Vaccination comes second in importance after introduction of clean water as a public health intervention which has largely contributed in the reduction of deaths from infectious diseases. Success in the development of a group A streptococcal (GAS) vaccine is expected to save 517 000 deaths per annum according to a recent independent review commissioned by the world health organization (WHO) and would offer an ideal means to prevent rheumatic heart disease (responsible for the greatest health burden) and other GAS-associated diseases which affect the health of 600 million. Traditional vaccine approaches (killed or live attenuated) have demonstrated great success against many bacterial and viral infectious diseases, crowned by the global eradication of smallpox announce by the WHO in 1980 and near-to-be announced eradication of polio viral disease. However, application of traditional techniques in many cases such as HIV/AIDS, malaria, GAS and Mycobacteria tuberculosis, has not shown the same success. Risk associated with the use of live–attenuated pathogens, such as recurrence of virulence (e.g. HIV), development of autoimmune diseases (e.g. GAS), and difficulties of manufacture hindered the use of such approaches. Other vaccine approaches such as subunit vaccines (recombinant proteins) and carrier conjugated vaccine are also hindered by the lack of suitable adjuvants, carriers and delivery systems. The current thesis focused on the design, synthesis and evaluation of novel adjuvants and vaccine delivery systems against GAS. The first chapter reviews recent approaches in the field of GAS vaccine design and new findings in immunology which represent the basis of our novel strategies. The second chapter describes the design, synthesis and evaluation of a novel library of lipopeptides as self-adjuvanting GAS vaccine candidates, composed of: (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2d) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment, or incorporation of P25 and J14 into a lipid-core peptide system (LCP) on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side-chain. Mucosal surface of the nasal-oral route is a primary site of GAS infections. An ideal GAS vaccine would have to elicit both mucosal as well as systemic immune responses and hence would not only prevent the development of GAS-associated diseases but also would prevent primary GAS infections. Therefore, the nasal route is considered a highly promising route of vaccine administration to provide local as well as systemic immune responses against pathogens that utilize mucosal surface as site of infection. The third chapter includes immunological assessment of the lipopeptide vaccine library described in the second chapter following intranasal immunization of B10BR (H-2k) mice. The whole library was first investigated in a small scale experiment (5 mice per group) to select promising candidates which demonstrate the best local and systemic J14-specific antibodies. Four selected lipopeptides were further investigated in a larger scale experiment (15 mice per group) followed by intranasal challenge of vaccinated mice with a virulent GAS M1 strain. The best local and systemic immune responses were demonstrated by a lipopeptide featuring a lipid moiety consisting of two 16 carbon chains incorporated at the C-terminus of the lipopeptide. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide candidate featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side-chain. A possible explanation for these results was investigated where antibodies elicited by the former candidate was found to better recognize the minimal B-cell epitope in the native p145 sequence of the M protein. Circular dichroism study of lipopeptides used in the previous experiment demonstrated that the former candidate features α-helical conformation which is required to produce protective J14-specific antibodies. Further studies are needed to explain structural features required to achieve both α-helicity and strong mucosal immune responses shown by the previously mentioned two lipopeptides. Signaling through toll-like receptors expressed by immune cells was recently shown to result in a robust immune response and was investigated as a possible mode of action for our novel lipopeptides. The fourth chapter introduces our lipopeptide vaccine approach as novel synthetic ligands targeting TLR2. A lipid moiety consisting of two alkyl chains of 16 carbons was found to achieve optimal TLR2 signaling regardless of the position of lipid attachment. Carbohydrates as polyhydroxy compounds provide an easily accessible class of compounds to design scaffolds (carriers) to attach lipids and peptide epitopes in different number and stereochemical positions which makes glycolipopeptides an attractive target for adjuvant research and structure-adjuvanticity relationships studies. The Fifth chapter reports immunological assessment of two series of glycolipopeptides as GAS vaccine candidates and novel vaccine delivery systems. The first series: lipid carbohydrate core peptide system (LCCP); represents a modification of the classical LCP system where polylysine dendrimer is replaced by different monosccharides as carriers for peptide antigens. LCCP analogues induced proper humoral immune responses against incorporated epitopes comparable to the LCP delivery system and as strong as the immune response elicited by CFA mixtures. Moreover, LCCP delivery system has been proved to be tolerant to the use of different epitopes as well as changing carbohydrate cores. Design of novel carbohydrate cores with different orthogonal protecting groups is needed to explore the potential advantage of various stereochemical arrangements provided by monosaccharides. The second series of glycolipopeptides incorporates various glycolipid moieties (self-adjuvanting activity) covalently coupled to the N-terminus of J8 (a model epitope). The new glycolipopeptide vaccine candidates (containing only one copy of J8) bear comparison with an LCP analogue (containing four copies of J8) which would improve the ease of synthesis, purification and cost of vaccine production. The slight difference in immunogenicity among these glycolipopeptides was difficult to be explained due to intervening effects of both the number and orientation of lipids on immunological activity. Further investigation is needed to determine the contribution of each factor.

Vaccination comes second in importance after introduction of clean water as a public health intervention which has largely contributed in the reduction of deaths from infectious diseases. Success in the development of a group A streptococcal (GAS) vaccine is expected to save 517 000 deaths per annum according to a recent independent review commissioned by the world health organization (WHO) and would offer an ideal means to prevent rheumatic heart disease (responsible for the greatest health burden) and other GAS-associated diseases which affect the health of 600 million. Traditional vaccine approaches (killed or live attenuated) have demonstrated great success against many bacterial and viral infectious diseases, crowned by the global eradication of smallpox announce by the WHO in 1980 and near-to-be announced eradication of polio viral disease. However, application of traditional techniques in many cases such as HIV/AIDS, malaria, GAS and Mycobacteria tuberculosis, has not shown the same success. Risk associated with the use of live–attenuated pathogens, such as recurrence of virulence (e.g. HIV), development of autoimmune diseases (e.g. GAS), and difficulties of manufacture hindered the use of such approaches. Other vaccine approaches such as subunit vaccines (recombinant proteins) and carrier conjugated vaccine are also hindered by the lack of suitable adjuvants, carriers and delivery systems. The current thesis focused on the design, synthesis and evaluation of novel adjuvants and vaccine delivery systems against GAS. The first chapter reviews recent approaches in the field of GAS vaccine design and new findings in immunology which represent the basis of our novel strategies. The second chapter describes the design, synthesis and evaluation of a novel library of lipopeptides as self-adjuvanting GAS vaccine candidates, composed of: (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2d) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment, or incorporation of P25 and J14 into a lipid-core peptide system (LCP) on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side-chain. Mucosal surface of the nasal-oral route is a primary site of GAS infections. An ideal GAS vaccine would have to elicit both mucosal as well as systemic immune responses and hence would not only prevent the development of GAS-associated diseases but also would prevent primary GAS infections. Therefore, the nasal route is considered a highly promising route of vaccine administration to provide local as well as systemic immune responses against pathogens that utilize mucosal surface as site of infection. The third chapter includes immunological assessment of the lipopeptide vaccine library described in the second chapter following intranasal immunization of B10BR (H-2k) mice. The whole library was first investigated in a small scale experiment (5 mice per group) to select promising candidates which demonstrate the best local and systemic J14-specific antibodies. Four selected lipopeptides were further investigated in a larger scale experiment (15 mice per group) followed by intranasal challenge of vaccinated mice with a virulent GAS M1 strain. The best local and systemic immune responses were demonstrated by a lipopeptide featuring a lipid moiety consisting of two 16 carbon chains incorporated at the C-terminus of the lipopeptide. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide candidate featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side-chain. A possible explanation for these results was investigated where antibodies elicited by the former candidate was found to better recognize the minimal B-cell epitope in the native p145 sequence of the M protein. Circular dichroism study of lipopeptides used in the previous experiment demonstrated that the former candidate features α-helical conformation which is required to produce protective J14-specific antibodies. Further studies are needed to explain structural features required to achieve both α-helicity and strong mucosal immune responses shown by the previously mentioned two lipopeptides. Signaling through toll-like receptors expressed by immune cells was recently shown to result in a robust immune response and was investigated as a possible mode of action for our novel lipopeptides. The fourth chapter introduces our lipopeptide vaccine approach as novel synthetic ligands targeting TLR2. A lipid moiety consisting of two alkyl chains of 16 carbons was found to achieve optimal TLR2 signaling regardless of the position of lipid attachment. Carbohydrates as polyhydroxy compounds provide an easily accessible class of compounds to design scaffolds (carriers) to attach lipids and peptide epitopes in different number and stereochemical positions which makes glycolipopeptides an attractive target for adjuvant research and structure-adjuvanticity relationships studies. The Fifth chapter reports immunological assessment of two series of glycolipopeptides as GAS vaccine candidates and novel vaccine delivery systems. The first series: lipid carbohydrate core peptide system (LCCP); represents a modification of the classical LCP system where polylysine dendrimer is replaced by different monosccharides as carriers for peptide antigens. LCCP analogues induced proper humoral immune responses against incorporated epitopes comparable to the LCP delivery system and as strong as the immune response elicited by CFA mixtures. Moreover, LCCP delivery system has been proved to be tolerant to the use of different epitopes as well as changing carbohydrate cores. Design of novel carbohydrate cores with different orthogonal protecting groups is needed to explore the potential advantage of various stereochemical arrangements provided by monosaccharides. The second series of glycolipopeptides incorporates various glycolipid moieties (self-adjuvanting activity) covalently coupled to the N-terminus of J8 (a model epitope). The new glycolipopeptide vaccine candidates (containing only one copy of J8) bear comparison with an LCP analogue (containing four copies of J8) which would improve the ease of synthesis, purification and cost of vaccine production. The slight difference in immunogenicity among these glycolipopeptides was difficult to be explained due to intervening effects of both the number and orientation of lipids on immunological activity. Further investigation is needed to determine the contribution of each factor.

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.

[Truncated abstract] The last few decades have witnessed an increase in the prevalence, morbidity and economic burden associated with asthma and allergic disease. This rising incidence cannot be completely explained by changes in genetic factors or by improvements in diagnostic procedures. Environmental factors, particularly those associated with a westernised lifestyle, are considered to be involved in this increase. In the late 1980’s Strachan was the first to link environmental factors with allergic disease, this theory became to be known as the ‘hygiene hypothesis’. This hypothesis links the “cleaner” more “healthy ” environment we now live in, with an increased risk of developing allergic disease. This effect is highlighted by studies linking farm and animal exposure (rich in microbial compounds) during early life with a decrease in allergic disease. Since then numerous studies have been undertaken to ascertain the factors present in the microbe rich environment, which elicit this protective effect. Many studies have revolved around endotoxin, however microbial components (mainly from Gram-positive bacteria) which signal through Toll-like receptor 2 (TLR2), have also shown that they can alter the allergic immune response. In mice models TLR2 has been shown to both exacerbate and inhibit allergic disease. The above research highlights the need for further studies into the effect of TLR2 ligands, and to define the mechanisms by which they exert their effects in human allergic disease. These mechanisms will be relevant to understanding the pathogenesis of allergy, but also might provide novel ways to treat allergy. The aims of the study outlined in this thesis were to determine whether in vitro exposure to TLR2 ligands could modify the established immune response to house dust mite allergen (HDM), and to examine the mechanisms by which this occurs. ... The addition of glucocorticoids to LTA enhanced the ability of this TLR2 ligand to inhibit IL-5 and IL-13 production by HDM-activated blood mononuclear cells. In conclusion, this study shows that TLR2 ligands have the ability to inhibit the Th2 response to mite allergen in previously sensitized individuals by an as yet unknown mechanism. However the findings described herein do provide an impetus for future studies designed to uncover novel mechanisms by which allergic responses can be ameliorated, and may open new treatment modalities.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2005" Includes bibliographical references.

A la naissance, le système immunitaire des nouveau-nés est encore en plein développement. La première partie du travail de thèse a consisté à étudier les spécificités du système immunitaire intestinal des nouveau-nés qui conduisent à leur plus grande susceptibilité à l’infection par Cryptosporidium parvum. Ce protozoaire constitue un excellent modèle pour étudier les réponses immunitaires mucosales. En effet, son développement est restreint à l’épithélium intestinal et est strictement relié au statut immunitaire de son hôte ce qui explique que cet agent zoonotique affecte tout particulièrement les nouveau-nés et les immunodéficients. Nous avons démontré que les cellules dendritiques (DC) CD103+ étaient indispensables au contrôle de la phase aigüe de l’infection et que leur faible représentation dans la lamina propria de l’iléon chez les nouveau-nés était responsable de leur susceptibilité à l’infection. Nous avons identifié avec précision le mécanisme CXCR3 dépendant permettant le recrutement des DC CD1O3+ dans la muqueuse infecté et leur capacité à produire de l’IL-12 et de l’IFNdz, deux cytokines majeures impliquées dans le mécanisme de protection. La deuxième partie de ce travail a consisté à utiliser une stratégie d’immunostimulation basée sur l’utilisation de ligands de TLR capables d’activer fortement les cellules dendritiques du nouveau-né. Cette approche permet un contrôle rapide et très efficace d’une infection par C. parvum. / At birth, the neonatal immune system is still developing. In the first part of the thesis we investigated the characteristics of the intestinal immune system of neonates that lead to their greater susceptibility to infection by Cryptosporidium parvum. This protozoan is an excellent model for studying mucosal immune responses. Indeed, its development is restricted to the intestinal epithelium and is strictly related to the immune status of its host which explains the particular susceptibility of neonates and immunocompromised to this zoonotic agent. We have demonstrated that CD103+ dendritic cells (DC) are essential for the control of the acute phase of infection and their low representation in the ileal lamina propria of neonates was responsible for their higher susceptibility to infection. We have accurately identified the CXCR3-dependent mechanism for the recruitment of DC CD1O3+ in the infected mucosa and their ability to produce IL -12 and IFNdz, two major cytokines involved in the mechanism of protection. The second part of this work was to use an immunostimulatory strategy based on administration of TLR ligands that can strongly activate neonatal DC of the intestine. This approach allows a fast and highly effective control of an ongoing C. parvum infection.

Orientador: Lício Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T06:28:45Z (GMT). No. of bitstreams: 1 Santos_AndressaCoopedos_D.pdf: 9572929 bytes, checksum: ce4c4fb2095c6ffe99868ec77f19d5b3 (MD5) Previous issue date: 2011 / Resumo: A ativação da sinalização através do toll-like receptor-4 (TLR4) e a indução de estresse de retículo endoplasmático (ERE) são importantes mediadores da resistência à insulina na obesidade e em outras situações nas quais há um excesso de ácidos graxos saturados. Em um estudo recente, demonstrou-se que sinalização através de TLR4 pode, per se, induzir a ativação de ERE, sugerindo que a ativação do TLR4 é o evento inicial para a indução do estresse celular que contribui para o aumento da expressão de genes de resposta inflamatória. No entanto, os mecanismos que conectam essas duas vias distintas são desconhecidos. As chaperonas GRP78 e GRP94 exercem uma função importante no processamento das moléculas recém-traduzidas do TLR4. Além disso, a chaperona GRP94 é responsável por sua translocação e conteúdo na superfície celular. Durante uma ativação prolongada da via de sinalização do TLR4, a demanda por novas moléculas sintetizadas aumenta, e consequentemente, a demanda por novas chaperonas. Por esta razão, nós aventamos a hipótese de que sob uma ativação extrema da via do TLR4, a síntese de proteínas sobrepujaria a expressão de chaperonas, dessa forma induzindo ERE. Para testar essa hipótese, monócitos da linhagem THP-1 foram incubados com LPS e foi avaliada a expressão e ativação de proteínas responsivas ao ERE por real-time PCR, citometria de fluxo, imunoprecipitado e western blot. Em alguns experimentos, as células foram privadas de glicose ou tratadas com siRNA para aumentar ou diminuir, respectivamente, a expressão das chaperonas. Experimentos de time-course revelaram que o LPS induz um aumento de 2,5 vezes na expressão do TLR4, iniciando após 8 h, com um pico após as 24 h e permanecendo significantemente aumentado após 48 h. A expressão de GRP78 foi aumentada em três vezes com um aumento acentuado após 24 h sem aumento às 8 h, enquanto o GRP94 aumenta apenas 1,5 vezes com um pico após 2 h que retorna aos valores basais após 8 h do estímulo. Não houve aumento da expressão protéica das chaperonas após 48 h. A indução de ERE por LPS foi detectada antes de 4 h do estímulo observado pela avaliação da via da PERK/eIF2a, IRE1 e ATF6 e se mantém ativado após 48 h. Adicionalmente, a privação de glicose em células THP-1 aumenta a expressão de GRP94 e GRP78 em 2,5 e 11 vezes, respectivamente. Na ausência de glicose, o tratamento com LPS não induz ERE. A inibição da expressão das chaperonas por siRNA anula o efeito da privação de glicose em proteger as células do desenvolvimento de ERE induzido por LPS. Portanto, a hiperexpressão das chaperonas GRP78 e GRP94 protegem as células do ERE induzido por LPS. Assim, defeito na expressão das chaperonas induzido por TLR4 é um mecanismo envolvido na integração da sinalização do TLR4 e ERE / Abstract: TLR4 activation and the induction of endoplasmic reticulum stress (ERS) are two of the most important mechanisms connecting excessive fat with insulin resistance. Recently, it was shown that activation of TLR4 can, per se, induce ERS, suggesting that TLR4 is a primary event in the induction of the cellular stress that contributes to increased inflammatory gene expression. However, the mechanisms linking these molecular events are unknown. The chaperones GRP78 and GRP94 play an important role during the assembly of newly translated TLR4 molecules. In addition, the chaperone GRP94 escorts the protein to the cell membrane. Under prolonged activation, the demand for newly synthesized TLR4 molecules increases, and thus, the demand for new chaperones. Therefore, we hypothesized that under increased activation of TLR4, the synthesis of the protein would not be matched by the expression of chaperones, thus, triggering ERS. To test this hypothesis, the monocyte cell line THP-1 was incubated with LPS and the expression/activation of proteins involved in ERS was determined by real-time PCR, flow-cytometry, immunoprecipitation and immunoblot. In some experiments, cells were deprived of glucose or treated with siRNA to increase or decrease, respectively, the expression of the chaperones. Time-course experiments revealed that LPS led to a 2.5-fold increase of TLR4 expression starting as early as 8h, peaking after 24h and remaining significantly increased after 48h. The expression of GRP78 underwent a 3-fold increase with a sharp rise at 24h (no increase at 8h), while GRP94 increased by only 1.5-fold with a peak at 2h and an early return to base-line levels. None of the chaperones were increased after 48h. LPS-induced ERS was detected as early as 4h after stimulus as detected by the evaluation of PERK/eIF2?, IRE1 and ATF6 pathways. Strong signals of ERS were still present after 48h. The pre-incubation of THP-1 in glucose-deprived medium produced 2.5 and 11-fold increases of GRP94 and GRP78, respectively. Upon glucosedeprivation, LPS could no longer induce ERS. Inhibition of chaperone expression by siRNA completely abrogated the effect of glucose deprivation to protect cells from LPS-induced ERS. Thus, the hyperexpression of GRP78 and GRP94 protect cells from LPS-induced ERS. Defective TLR4-induced chaperone expression is a mechanism involved in the integration of TLR4 signaling and ERS / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica