Thesis (D.Sc.)--Boston University, Henry M. Goldman School of Dental Medicine, 2004 (Oral Biology). / Includes bibliography (leaves 107-151). / Lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gram negative bacteria, induces the secretion of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-a) and interleukin (IL )-1, by monocytes/macrophages. Excessive and uncontrolled secretion of these compounds leads to multiple pathological conditions, such as septic shock. LPS receptors have been shown to be CD14, TLR4 and MD-2. LPS interaction with these receptors mediates many monocyte/macrophage functions. Even though only CD14 was demonstrated to bind to LPS, and TLR4/MD-2 were capable of transducing signals, data only show that LPS and CD 14 were in close proximity to TLR4 and no direct binding was reported. Quite recently, moesin, a member of the ERM family of proteins, has been also found to function as a receptor for LPS. We have shown that anti-moesin antibody inhibited the release of TNFa by LPS stimulated monocytes. Moesin was also found to be necessary for the detection of LPS, where homozygous knockout mice exhibited 3-fold reduction in neutrophil infiltrates in LPS injected sites when compared to their wild type controls. When moesin gene expression was completely suppressed with antisense oligonucleotides, there was a significant reduction of LPS-induced TNF-a secretion. LPS stimulation of mononuclear phagocytes activates several intracellular signaling pathways including the phosphorylation of IKBa, mitogen-activated protein kinase (MAPK) pathways: extracellular signal-regulated kinases (ERK) 1 / 2 (P44/42), p38. These signaling pathways in tum activate a variety of transcription factors including NF-KB, which coordinates the induction of several genes encoding inflammatory mediators. [TRUNCATED]

Lipopolysaccharide (LPS) is the major surface molecule of Gram-negative bacteria and consists of three distinct structural domains: O-antigen, core, and lipid A. The lipid A (endotoxin) domain of LPS is a unique, glucosamine-based phospholipid that serves as the hydrophobic anchor of LPS and is the bioactive component of the molecule that is associated with Gram-negative septic shock. The structural genes encoding the enzymes required for the biosynthesis of Escherchia coli lipid A have been identified and characterized. Lipid A is often viewed as a constitutively synthesized structural molecule. However, determination of the exact chemical structures of lipid A from diverse Gram-negative bacteria shows that the molecule can be further modified in response to environmental stimuli. These modifications have been implicated in virulence of pathogenic Gram-negative bacteria and represent one of the molecular mechanisms of microbial surface remodeling used by bacteria to help evade the innate immune response. The intent of this review is to discuss the enzymatic machinery involved in the biosynthesis of lipid A, transport of the molecule, and finally, those enzymes involved in the modification of its structure in response to environmental stimuli.

La septicémie et les troubles associés demeurent une cause majeure de mortalité dans les unités de soins intensifs. Des récents travaux ont mis en lumière un lien inattendu entre les mitochondries et les fonctions des cellules immunitaires. Des modifications des fonctions mitochondriales ont pu être observées dans les cellules sanguines périphériques lors de septicémies. Dans le cadre de ce travail de thèse, nous avons cherché à évaluer si la mitophagie pouvait avoir un impact sur les fonctions des phagocytes dans le contexte d’une infection bactérienne. La mitophagie est une autophagie dédiée aux mitochondries qui régit l'élimination des mitochondries dysfonctionnelles. Nous avons démontré ici in vivo et in vitro que les macrophages exposés aux bactéries à Gram négatif ou à leurs composants de la paroi cellulaire (Lipopolysaccharides, LPS) présentent une inhibition marquée de la mitophagie qui constitue un mécanisme de protection contre la septicémie. L'activation des macrophages avec une combinaison LPS/IFNγ entraîne une inhibition précoce de la mitophagie dépendante de PINK1 selon une voie dépendante de STAT1-Caspase 11. Cette inhibition de la mitophagie contribue à expliquer la reprogrammation métabolique observée dans les macrophages classiquement activés (macrophages M1) et conduit à une augmentation de la production de ROS mitochondriaux (mROS). En tant que molécules de signalisation, les mROS conduisent à l'activation des macrophages de manière dépendante de HIF-1α et NF-κB. En outre, ces molécules contribuent à la clairance bactérienne dans les phagocytes activés. Il est intéressant de noter que nous avons démontré in vitro et in vivo que la modulation pharmacologique de la mitophagie permet d'imiter ou de réprimer les effets du LPS sur la polarisation des macrophages, la libération des cytokines et l'activité bactéricide. Pour conclure, ce travail démontre que l'inhibition de la mitophagie est une caractéristique de l'activation LPS-dépendante des macrophages et un mécanisme de protection contre les bactéries à Gram négatif. Cette étude souligne également une relation inconnue entre la signalisation IFNγ, les caspases inflammatoires et la mitophagie. Enfin, nos travaux mettent en lumière l'impact des modulateurs pharmacologiques de la mitophagie sur la fonction des macrophages et ouvrent de nouvelles opportunités pour le développement de nouvelles stratégies thérapeutiques pour stimuler la défense de l'hôte. / Sepsis and related organ dysfunctions remain a leading cause of mortality in intensive care units. Increasing evidences have shed light on an unexpected link between mitochondria and immune cell functions. Alterations in mitochondrial functions have been reported in peripheral blood cells in sepsis. We hypothesize here that mitophagy might impact on phagocyte functions in the context of bacterial infection. Mitophagy is a mitochondria-dedicated autophagy that governs the elimination of dysfunctional mitochondria. We demonstrated here in vivo and in vitro that macrophages exposed to Gram-negative bacteria or their cell wall component LPS display a marked inhibition of mitophagy that constitutes a protective mechanism against sepsis. LPS/IFNγ-driven macrophage activation results in early inhibition of PINK1-dependent mitophagy through a STAT1-Caspase 4/11 pathway. This inhibition of mitophagy contributes to explain the metabolic reprogramming observed in classically activated macrophages and leads to a rise in mitochondrial ROS (mROS) production. As signaling molecules, mROS lead to macrophages activation in a HIF-1α- and NF-κB-dependent manner. Furthermore, these molecules contribute to bacterial clearance in activated phagocytes. Interestingly, we demonstrated in vitro and in vivo that pharmacological modulation of mitophagy allows either mimicking or repressing the effects of LPS on macrophages polarization, cytokine release and bactericidal activity. To conclude, this work demonstrates that inhibition of mitophagy is a feature of LPS-dependent macrophage activation and a protective mechanism against Gram-negative bacteria. This study also highlights an unknown relationship between IFNγ-signaling, inflammatory caspases and mitophagy. Finally, our work point out the impact of pharmacological modulators of mitophagy on macrophage function and open new opportunities for the development of novel strategies to boost host defense

O lipopolissacarídeo (LPS) é uma endotoxina de bactérias gram-negativas utilizada experimentalmente para estimular processos inflamatórios e ativar o sistema imune através da liberação de citocinas pró-inflamatórias no sistema nervoso central (SNC). A administração experimental de LPS a ratas prenhes resulta em alterações que podem ser verificadas tanto nos animais submetidos ao tratamento quanto na prole destas fêmeas. O objetivo deste estudo foi o de verificar a existência alterações relacionadas à aprendizagem e à memória na prole masculina de ratas submetidas ao LPS durante a gestação. Para tanto, foi administrado LPS (100mg/kg) no 9,5º dia da prenhes de ratas Wistar. O presente trabalho avaliou os efeitos desta administração no reconhecimento dos animais utilizando o olfato tanto na infância quanto na idade adulta, assim como os níveis de neurotransmissores no bulbo olfatório. A atividade geral dos animais foi analisada no campo aberto e também o comportamento no labirinto em cruz elevado. A aprendizagem dos animais foi observada na caixa de Skinner e também foi analisado reconhecimento de objetos dos animais. Os níveis de neurotransmissores do hipocampo na idade adulta também foram dosados. Os resultados mostraram que a administração de LPS no 9,5º de gestação causou: 1) diminuição do reconhecimento do odor materno nos filhotes na infância e que essa alteração não permanece na idade adulta; 2) diminuição nos níveis de dopamina no bulbo olfatório dos animais tratados pré-natalmente; 3) ausência de interferência na atividade geral e de locomoção dos animais quando comparados com o grupo controle; 4) ausência de alterações na aquisição do aprendizado resultando porém em diminuição no tempo de reversão do aprendizado; 5) nenhuma diferença no reconhecimento de novos objetos assim como ausência de alterações nos níveis de neurotransmissores no hipocampo comparado ao grupo controle. Esses dados sugerem que infecções maternas podem interferir no ambiente intra-uterino e desta forma interferir no desenvolvimento e na atividade do SNC da ninhada em longo prazo / The lipopolysaccharide (LPS) is an endotoxin of gram-negative bacteria used experimentally to stimulate inflammatory processes and activate the immune system through the release of pro-inflammatory cytokines in the central nervous system (CNS). The experimental administration of the LPS in pregnant rats results in changes that can be viewed both in animals subjected to the treatment and the offspring of these females. The aim of this study was to verify the changes related to learning and memory in male offspring of rats submitted to LPS during gestation. It was administered LPS (100mg/kg) in 9,5th day of pregnant of Wistar rats. This work has investigates the effects of this administration in recognition of animals using the olfaction in childhood and adulthood as well as levels of neurotransmitters in the olfactory bulb. The general activity of the animals was analyzed in the open field task and also the behavior in the elevated pluz maze. The learning process of the animals was observed in the Skinner box and also studied animal objects recognition. The levels of neurotransmitters of hippocampus were also analyzed. The results showed that the administration of LPS on 9,5 gestation day caused: (1) decrease of the smell recognition in childhood and do not remains in adulthood; (2) decrease in levels of dopamine in the olfactory bulb of the animals treated; (3) not interfered in general activity of animals when compared with the control group; (4) there was no difference in the acquisition of learning but presented decrease in time reversal of learning; (5) did not make differences in recognition of the new objects as well as not submitted changes in levels of neurotransmitters in hippocampus compared to the group control. These data suggest that maternal infection can interfere in the intrauterine environment and thus interfere with development and activity of the SNC

Galectina-3, uma proteína que se liga a -galactosídeos, é expressa por neutrófilos e inúmeras evidências indicam que esta molécula atua como uma possível reguladora da resposta imune. Sabe-se que galectina-3 ao ligar com LPS pode levar a formação de oligômeros, que podem alterar o limiar de ativação de células da resposta imune inata. Apesar de existirem diversos estudos que mostram a influência de galectina-3 na resposta de neutrófilos frente a componentes bacterianos, os resultados são em sua maioria contraditórios e inconclusivos. Para elucidar a influência da galectina-3 na reposta imune inata a patógenos periodontais, o presente trabalho avaliou a atividade antimicrobiana in vitro de neutrófilos, isolados de camundongos selvagens (WT) ou geneticamente deficientes de galectina-3 (Gal-3KO), previamente estimulados com LPS de Aa e Pg. Os resultados não evidenciaram diferenças significativas no número de unidades formadoras de colônia (UFC) recuperadas das culturas de neutrófilos provenientes de animais deficientes de galectina-3 e do grupo controle (WT). Contudo, a estimulação de neutrófilos com LPS por 18 horas levou a redução no número de UFC recuperadas das culturas, quando comparado com as culturas estimuladas com LPS por apenas 3 horas. / Galectin-3, a protein that binds -galactosides, is expressed by neutrophils and numerous evidences indicate that this molecule acts as a possible regulator of the immune response. It is known that galectin-3 binding to LPS can lead to the formation of oligomers and thus changing the activation threshold of cells of the innate immune response. Although there are several studies that show the influence of galectin-3 in neutrophil response against bacterial components, the results are conflicting and inconclusive in their majority. To elucidate the influence of galectin-3 in the innate immune response to periodontal pathogens, the present study evaluated the in vitro antimicrobial activity of neutrophils, isolated from wild-type or galectin-3 deficient mice, previously stimulated with LPS of Aa and Pg. The results showed no significant differences in the number of colony forming units (CFU) recovered from cultured galectin-3 deficient neutrophils or control group. However, in a 18 hours time course of LPS stimulation, we observed reduction in the number of CFU, when compared to 3 hours of LPS stimulation.

Thesis (Ph.D.)--McMaster University (Canada), 1992. / Source: Dissertation Abstracts International, Volume: 54-08, Section: B, page: 4119. Adviser: D. B. MacLean.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Örebro : Örebro universitet, 2006. / Härtill 4 uppsatser.