Tumor necrosis factor-alpha (TNF-£\) is a potent proinflammatory cytokine, inducing the acute-phase response that leads to physiological changes that serve to eliminate the infecting organisms. Toll-like receptors (TLRs) comprise a family of pattern-recognition receptors that detect conserved molecular products of microorganisms. To evaluate the role of TNF-£\ and TLRs in Pseudomonas aeruginosa infection-induced mortality in thermal injured mice, the wild type (WT), Tnfrsf1a-/-, and TLR4-/- mice were injected the P. aeruginosa in the back at 8 hr after 30% total body surface area burn. At 24 hr after burn, lung tissues of mice were harvested for assays of wet/dry ratio; microvascular dysfunction, myeloperoxidase (MPO) activity, NF-£eB DNA binding activity; and expression of IL-1£], iNOS, p-JNK, and TLR4. Injection of P. aeruginosa after burn induced a survival rate in Tnfrsf1a-/- 60% TBSA burn=100%, WT 60% TBSA burn=10%, WT 30% TBSA burn +P.A = 60%, Tnfrsf1a-/- 30% TBSA burn+p.A. = 30%. respectively. The high mortality in Tnfrsf1a-/- mice is related to a significant increase of pulmonary microvascular dysfunction; neutrophil infiltration, bacterial counts of blood and lung; and expression of TLR4, IL-1£], and iNOS as compared with WT mice. On the contrary, significant increase of NF-£eB DNA binding activity of lung was observed only in WT mice. When iNOS inhibitor SMT was given immediately after burn to WT and Tnfrsf1a-/- mice, the P. aeruginosa induced increases of pulmonary edema, pulmonary permeability, and lung bacterial counts were decreased significantly in Tnfrsf1a-/- mice, suggesting that TNF-£\ decreases P. aeruginosa-induced mortality in burned mice is through a negative regulation of TLR4 and iNOS.

Alcohol consumption is a significant riskactor for mortality in patients with sepsis. This study was carried to investigate the mechanisms by which acute ethanol exposure alters the course of sepsis and the effect of TLR4 signaling. Ethanol administration decreases resistance to E. coli and causes decrease in the ability to clear bacteria both from the peritoneal-cavity as well as the spleen. At early time-points, ethanol also suppresses the production of pro-inflammatory cytokines. TLR4 is dispensable for survival in E. coli sepsis but it also contributes to lethality in wild-type mice. Although TLRs have been implicated as an important element of host defense against infections, evidence indicates that these receptors may also play a crucial role in the pathophysiology of sepsis.

La biosynthèse de phénol-glycolipides (PGL) par Mycobacterium tuberculosis et M. leprae favorise l’invasion des macrophages via l'interaction de la partie saccharidique des PGL avec le domaine lectine du récepteur cellulaire au complément CR3. Les PGL inhibent également la production de cytokines inflammatoires par la cellule hôte, par un mécanisme inconnu. J’ai observé que des bactéries BCG transgéniques exprimant les PGL de M. tuberculosis ou M. leprae avaient une capacité de survie accrue dans les macrophages. Cette persistance intracellulaire était dépendante de CR3 et associée à une diminution de la production d'oxyde nitrique dans les cellules infectées. L’addition de PGL purifié suffisait à inhiber la production d’oxyde nitrique par des macrophages stimulés avec LPS/IFN-γ. J’ai montré que la liaison de PGL-1 à CR3 provoquait la dégradation post-transcriptionnelle de TIR-domain-containing adapter-inducing interféron-β (TRIF) dans les macrophages, ce qui entraînait une réduction de la signalisation TRIF-dépendante. Dans les macrophages stimulés avec LPS/IFN-γ, la dégradation de TRIF réduisait la production d’oxyde nitrique synthase, et la production TRIF dépendante de cytokines inflammatoires et des chimiokines. Mes résultats ont donc permis d’identifier un nouveau mécanisme de virulence développé par les mycobactéries pathogènes pour réprimer conjointement les réponses inflammatoires et antimicrobiennes de l’hôte / Biosynthesis of phenolic glycolipids (PGL) by Mycobacterium tuberculosis and M. leprae promotes macrophage invasion, which proceeds through the interaction of the PGL sugar moieties with the lectin domain of cell-displayed complement receptor (CR3). PGL also limit host cell production of inflammatory cytokines by an unknown mechanism. I observed that transgenic BCG that express PGL specific to M. tuberculosis or M. leprae displayed enhanced survival within macrophages. Increased intracellular persistence of PGL-expressing BCG was CR3-dependent and correlated with the decreased production of nitric oxide in infected cells. Notably, the addition of soluble PGL to macrophages was sufficient to induce a reduction in nitric oxide production upon stimulation with LPS/IFN-γ. I showed that PGL-1 binding to CR3 causes the post-transcriptional degradation of TIR-domain-containing adapter-inducing interferon-β (TRIF) in macrophages, resulting in impaired TRIF-dependent signaling. Functionally, PGL-1-mediated degradation of TRIF resulted in the decreased induction of nitric oxide synthase, and TRIF-dependent inflammatory cytokines and chemokines in LPS/IFN-γ-stimulated macrophages. My results thus identified a virulence mechanism evolved by pathogenic mycobacteria to suppress both the inflammatory and antimicrobial responses of infected host cells

Toll-like receptors (TLRs) play a critical role in the induction of innate immune responses which have been implicated in neuronal death induced by global cerebral ischemia/reperfusion (GCI/R). The present study investigated the role and mechanisms-of-action of TLR4 signaling in ischemia-induced hippocampal neuronal death. Neuronal damage, activation of the TLR4 signaling pathway, expression of pro-inflammatory cytokines and activation of the PI3K/Akt signaling pathway in the hippocampal formation (HF) were assessed in wild type (WT) mice and TLR4 knockout (TLR4-/-) mice after GCI/R. GCI/R increased expression of TLR4 protein in the hippocampal formation (HF) and other brain structures in WT mice. Phosphorylation of the inhibitor of kappa B (p-Ik{cyrillic}B) as well as activation of nuclear factor kappa B (NFk{cyrillic}B) increased in the HF of WT mice. In contrast, there were lower levels of p-Ik{cyrillic}B and NFk{cyrillic}B binding activity in TLR4-/- mice subjected to GCI/R. Pro-inflammatory cytokine expression was also decreased, while phosphorylation of Akt and GSK3β were increased in the HF of TLR4-/- mice after GCI/R. These changes correlated with decreased neuronal death/apoptosis in TLR4-/- mice following GCI/R. These data suggest that activation of TLR4 signaling contributes to ischemia-induced hippocampal neuronal death. In addition, these data suggest that modulation of TLR4 signaling may attenuate ischemic injury in hippocampal neurons.

No description available.

No description available.

Obesity-related metabolic derangements have been linked to toll-like receptor 4 (TLR4), an innate immune system receptor, due to its role in proinflammatory pathways. Lipopolysaccharide (LPS), a gram-negative bacteria cell wall component, is the ligand for TLR4, and has been shown to be elevated in states of metabolic disease. Heightened levels of circulating endotoxin is termed metabolic endotoxemia and has been linked to systemic inflammation which is associated with obesity, type 2 diabetes mellitus (T2DM), and cardiovascular disease (CVD). Immune cells exhibit a protective ability to develop endotoxin tolerance. The objective of this study was to determine if endotoxin tolerance exists in skeletal muscle cells, and if a condition that mimics a state of over nutrition, such as elevated levels of fatty acids, affect this tolerance. To this end, L6 skeletal muscle cells were treated with low (50 pg/mL)- and high (500 ng/mL)-doses of LPS, with and without the presence of free fatty acids (FFAs). Tolerance was assessed by measuring: 1) changes in mRNA expression of interleukin-6 (IL-6) and monocyte chemoattractant-1 (MCP-1) as markers of a pro-inflammatory response; and 2) mRNA levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1-°) and mitochondrial oxidative capacity via an XF24 Flux Analyzer (Seahorse Bioscience) as measures of the metabolic response. Tolerance to LPS was observed in response to low- and high-doses with MCP-1 mRNA transcription but not IL-6. Changes in PGC1-° and mitochondrial OCR exhibited a tolerant effect in response to the high dose of LPS but not the low dose. The addition of free fatty acids to LPS treatments did not prevent the tolerant effects under any conditions. In conclusion, LPS tolerance exists in skeletal muscle cells but appears to differ depending on pro-inflammatory target and LPS concentration. Additionally, fatty acids, in the current model, have no effect on LPS tolerance. / Master of Science

Horses are prone to potentially lethal endotoxemia due to their surrounding fecal containing environment and their predisposition to colic. Their gastrointestinal tract and feces naturally contain gram-negative bacteria. These bacteria express lipopolysaccharide (LPS) on their cell membranes, which is recognized by Toll-like receptor 4 (TLR4). In cases where epithelial barriers are compromised or breached LPS has the potential to enter circulation and cause the inflammatory symptoms seen with endotoxemia. The objective of this study was to determine TLR4 presence and functionality on equine B cells. TLR4 expression on B lymphocytes has been studied in mouse, human and many other mammals, but has not been well characterized in the horse. Humans are highly sensitive to LPS but their B cells express non-functional TLR4. Mice in contrast are highly tolerant of LPS yet their B cells express functional TLR4. Studies in horse have perhaps been limited by the limited array of antibody markers available for use in horse. Anti-human CD21 has previously been shown to mark equine B lymphocytes. We show rat anti-mouse CD45R(B220) mAbs also accurately labels equine B lymphocytes. To investigate TLR4 expression in horses 12 Thoroughbred geldings, ages 5-10, were used for blood collection. By using the density gradient, Lympholyte, lymphocytes were separated from peripheral blood and incubated with or without LPS. B lymphocyte proliferation, TLR4 expression and mRNA changes were examined before or after culture in the presence or absence of LPS. We demonstrate TLR4 is expressed on equine B lymphocytes through the use of a mouse anti-human TLR4 antibody, clone 76B357.1, not previously used in horse. We demonstrated equine B cells fail to proliferate under LPS challenge as opposed to highly proliferative mouse B lymphocytes. However, transcriptional changes were observed in the equine cells within the TLR4 pathway upon treatment with LPS. / Master of Science / Horses are prone to potentially lethal infections due to their surrounding fecal containing environment and their predisposition to colic. Their gastrointestinal tract and environment naturally contain gram-negative bacteria such as E.coli. The gram-negative bacterial cell membrane expresses a molecule called lipopolysaccharide (LPS) which can cause inflammation by binding to an immune cell receptor called toll-like receptor 4 (TLR4). In cases where epithelial barriers are compromised or breached, LPS has the potential to enter the circulation and cause a severe inflammatory response capable of progressing to shock and death. Humans are also highly sensitive to LPS yet their B lymphocytes express non-functional TLR4. Mice, in contrast, are highly tolerant of LPS but their B lymphocytes express functional TLR4. The objective of this study was to determine TLR4 presence and functionality on equine B lymphocytes. Studies in horse have perhaps been limited by the small number of antibody markers available for use in horse. Two B lymphocyte markers not previously assessed in horse tissue were used in this experiment to enrich for B lymphocytes. A TLR4 marker, also not previously described in the horse, was used in conjunction with the two B lymphocyte markers determined TLR4 is present on equine B lymphocytes. However, equine B lymphocytes failed to proliferate in the presence of LPS similiar to human B cells. Transcriptional changes were observed in the equine cells in the TLR4 pathway upon treatment with LPS.

Ingeniero Civil en Biotecnología / Enfermedades como la sepsis, artritis reumatoide, tendinitis, entre otras, están ligadas a respuestas pro-inflamatorias anómalas. En ellas participan citoquinas pro-inflamatorias y factores de transcripción activados a partir de la vía de señalización mediada por receptores Tipo Toll y proteínas adaptadoras afínes. Estos receptores inician una cascada de señalización a partir de su homodimerización al ser inducidos por señales microbianas. Luego prosigue el reclutamiento de una serie de adaptadores proteicos que terminan finalmente con la transcripción de factores y producción de citoquinas proinflamatorias. En particular, este proyecto se enmarca en el estudio de las vías de señalización en las cuales está involucrado el receptor TLR4. Las enfermedades mencionadas pueden ser tratadas mediante el uso de fármacos, pero su uso resulta poco efectivo en etapas medio-avanzadas de la enfermedad y la presencia de efectos secundarios serios como osteoporosis, obesidad centrípeta, hipertensión, diabetes mellitus, entre otros. En vista de esto se plantea que la descripción de las interacciones del sistema mediado por TLR4 puede permitir diseñar nuevos tratamientos para estas enfermedades caracterizadas por respuestas inflamatorias anómalas. En función de lo anterior, se define el objetivo general de este trabajo que corresponde al estudio y reconstrucción in silico del acoplamiento e interacciones proteína-proteína producidas en la vía pro-inflamatoria activada por TLR4 y proteínas adaptadoras MAL, TRAM y MyD88. Por medio del uso de algoritmos de acoplamiento y herramientas de dinámica molecular se estudia el acoplamiento de los dominios TIR del homodímero de TLR4 y los adaptadores MAL, MyD88 y TRAM. Las interacciones propuestas son caracterizadas determinándose: área de contacto, tipos de interacciones y energía libre de los complejos. De manera adicional, se comparó los resultados con otros modelos presentados en publicaciones anteriores (Nuñez et al. (2007), Basith et al. (2011)). Los resultados obtenidos muestran que los adaptadores MAL y TRAM interactúan con el receptor TLR4 mediante la unión a la nueva superficie formada por la homodimerización de los dominios TIR de TLR4. En ambos casos, se observa que tanto MAL como TRAM se acoplan al receptor TLR4 orientando frente a frente sus lazos BB, lo que confirma la relevancia de este sector en las interacciones de esta vía. Otra zona importante de interacción corresponde al lazo CD del adaptador MAL y TRAM, el que según los resultados interactuaría con la Hélice B de TLR4. Por su parte, el estudio del complejo TLR4-MAL-MyD88, muestra que el adaptador MyD88 se une preferentemente a la nueva superficie que se produce a partir de la interacción TLR4-MAL. A partir de la estimación de la energía libre de unión vía MM-PBSA, se demuestra que los contactos y el acoplamiento de los modelos propuestos muestran estructuras de mayor estabilidad termodinámica que las presentadas por Nuñez (2007). Los resultados demuestran que zonas como los lazos BB, CD, hélice A y C son transversalmente relevantes dentro de la cadena de señalización y que presentan características generalizables al tipo de interacciones que se produce en esta vía mediada por el receptor TLR4. Finalmente, se destaca que la metodología planteada en este trabajo se transforma en un procedimiento novedoso y no descrito en trabajos anteriores relacionados a la descripción de interacciones vía TLR. Más aún, el conjunto de herramientas utilizadas permiten modelar las interacciones, probar hipótesis de interacción, brindar explicación a la acción de mutantes y encontrar sectores potenciales de interacción en cualquier tipo de sistema proteína-proteína.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2017-06-22T17:47:52Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016CamileWunsch.pdf: 1221188 bytes, checksum: d4f164f1168ed40c958561be1e994dc5 (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2017-06-26T18:42:58Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016CamileWunsch.pdf: 1221188 bytes, checksum: d4f164f1168ed40c958561be1e994dc5 (MD5) / Made available in DSpace on 2017-06-26T18:42:58Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016CamileWunsch.pdf: 1221188 bytes, checksum: d4f164f1168ed40c958561be1e994dc5 (MD5) Previous issue date: 2017-06 / CAPES / Introdução: A doença arterial coronariana (DAC) é a principal causa de morbidade e mortalidade no mundo, sendo caracterizada como uma doença inflamatória crônica, multifatorial, cuja fisiopatologia é a aterosclerose. Considerando a alta herdabilidade da doença, vários polimorfismos genéticos vêm sendo estudados e associados com o surgimento da DAC e, entre eles, destacam-se variantes nos genes CD14, TLR4, NFKB1 e TNFα, os quais regulam a via de sinalização celular do sistema imune inato e desencadeiam o processo inflamatório na DAC. Objetivo: O objetivo principal deste estudo é verificar a possível associação de polimorfismos nos genes CD14 (rs2569190), TLR4 (rs4986790 e rs4986791), NFKB1 (rs28362491) e TNFα (rs1800629 e rs361525) com a DAC. Metodologia: A amostra foi composta por 707 indivíduos adultos submetidos ao exame de cateterismo cardíaco no Hospital Bruno Born, de Lajeado, RS. Todos os indivíduos assinaram um termo de consentimento livre e esclarecido e responderam a um questionário semiestruturado. Os indivíduos foram classificados entre casos e controles, por um médico cardiologista, com base no seguinte critério: presença de estenose, com comprometimento maior do que 50%, em pelo menos uma das artérias coronárias. Foram também coletadas amostras de sangue periférico para análises bioquímicas e moleculares. A extração de DNA foi realizada pelo método de salting out. Os polimorfismos dos genes CD14, TLR4 e TNFα foram genotipados pelo sistema de discriminação alélica TaqMan, em equipamento de reação em cadeia da polimerase (PCR) em Tempo Real (StepOnePlus®). O polimorfismo rs28362491, no gene NFKB1, foi amplificado através da técnica convencional de PCR. Resultados: Identificamos uma associação dos polimorfismos rs2569190, localizado no gene CD14, e rs28362491, no gene NFKB1, com a DAC. Além disso, foram detectados efeitos dos polimorfismos dos genes TLR4 e TNFα nos níveis glicêmicos e dos polimorfismos nos genes TLR4, NFKB1 e TNFα no perfil lipídico. Conclusão: Nossos achados sugerem a participação de polimorfismos nos genes CD14 e NFKB1 no desenvolvimento da DAC na nossa amostra, corroborando evidências prévias do envolvimento de genes do processo inflamatório nessa patologia. / Introduction: Coronary artery disease (CAD) is the main cause of morbidity and mortality in the world, being characterized as a chronic, multifactorial inflammatory disease, whose pathophysiology is atherosclerosis. Considering the high heritability of the disease, several genetic polymorphisms have been investigated and associated with CAD and, among them, there are variants in the CD14, TLR4, NFKB1 and TNFα genes, which regulate cell signaling pathways of the innate immune system and inflammatory process in CAD. Objective: The main objective of this study is to verify the association beteween polymorphisms in the CD14 (rs2569190), TLR4 (rs4986790 and rs4986791), NFKB1 (rs28362491) and TNFα (rs1800629 and rs361525) genes and CAD. Methods: The sample group was composed of 707 adult individuals, recruited at the time when they were bought in for coronary angiography procedures at the Hemodynamic Center of the Hospital Bruno Born, City of Lajeado, Rio Grande do Sul. The individuals were classified between cases and controls by a cardiologist, based on the following criteria: presence of stenosis, greater than 50% of the luminal diameter, in at least one of the coronary arteries. Peripheral blood samples were also collected for biochemical and molecular analyzes. DNA extraction was performed using the salting out method. The polymorphisms in the CD14, TLR4 e TNFα genes were genotyped by Taqman® allelic discrimination assays. The rs28362491 polymorphism in the NFKB1 gene was amplified by polymerase chain reaction (PCR). Results: We identified an association between the polymorphisms rs2569190, located in the CD14 gene, and rs28362491, located in the NFKB1 gene, and CAD. In addition, there were detected significant effects of TLR4 and TNFα gene polymorphisms on glycemic levels and TLR4, NFKB1 and TNFα gene polymorphisms on the lipid profile. Conclusion: Our findings suggest a role for the polymorphisms in CD14 and NFKB1 genes in CAD susceptibility in our sample, corroborating previous evidence of the envolviment of inflamotory process genes in this patology.