Une compréhension fine et détaillée ciblant les mécanismes d’action de nouvelles molécules adjuvantes sur notre système immunitaire vise de manière directe à l’élaboration de nouveaux vaccins plus ciblés et plus efficaces, mais aussi à élargir nos connaissances quant à l’induction d’une réponse immune protectrice. Au cours de cette thèse nous avons voulu comprendre les modes d’action de deux molécules lipidiques distinctes. La première est le lipide cationique diC14-amidine dont il avait été démontré une action sur les cellules dendritiques en culture par une voie qui restait à élucider. Ce lipide cationique s'organise sous forme de liposomes en milieu aqueux et peut s'associer à de nombreux antigènes. La seconde est un analogue synthétique de l'adjuvant monophosphoryl lipide A (MPL), un dérivé du LPS, nommé CRX-527. À l'instar de sa molécule parente, le CRX-527 active le récepteur TLR4 et est considéré comme un adjuvant potentiel de vaccin ou comme immunostimulant isolé. Au cours de notre travail, nous avons démontré que la diC14-amidine active les cellules cibles via le récepteur TLR4. En effet, l'absence de ce récepteur abolit les réponses induites par le lipide cationique diC14-amidine et la transfection du gène codant pour TLR4 rend répondeuses des cellules qui n'exprimaient pas ce récepteur. De plus, la diC14-amidine active et mature des cellules dendritiques, aussi bien de provenance murine qu'humaine, suggérant qu'elle puisse être utilisée en tant qu’adjuvant. Il avait d’ailleurs été précédemment décrit que l'injection d'un complexe diC14-amidine / allergène chez la souris induisait une réponse immune suffisante pour conférer une protection contre cet allergène. Dans ce contexte, nous avons caractérisé au niveau cellulaire la réponse induite suite à l'injection du complexe diC14-amidine / ovalbumine chez la souris. Cette réponse se manifeste par une production d'IFNγ lors d'une re-stimulation ex vivo par l'antigène OVA. En ce qui concerne la molécule CRX-527, nous nous sommes particulièrement focalisés sur le rôle du co-récepteur du TLR4, le CD14, dans les réponses innées induites par le CRX-527. Nous avons établi que, de manière inattendue et contrairement à la plupart des ligands TLR4, le CRX-527 induit la production de nombreuses cytokines et chimiokines en complète absence de CD14, même à faible dose. De plus, l'ajout de CD14 sous sa forme soluble ne modifie pas le niveau des réponses associées à la voie de signalisation MyD88 / NF-κB. Cependant, il semblerait que la stimulation de cellules par du CRX-527 en présence de CD14 soluble recombinant, favorise plutôt la voie TRIF / IRF3, comme le suggère l'augmentation du taux de production d'IFNβ et d'activation d'IRF3. La molécule CD14 (membranaire et/ou soluble) ne serait donc pas qu'un simple transporteur de ligands, comme il l'a été décrit par le passé, mais bien une protéine impliquée dans la modulation des réponses induites lors de l'activation du TLR4. Le CD14 jouerait donc un rôle, aussi bien au niveau de la discrimination des ligands, que celle des voies de signalisation activées.

Toll-like receptor 4 (TLR4) is an important vertebrate innate immune receptor. TLR4 recognizes both endogenous and exogenous danger signals to trigger an NF-kB dependent inflammatory response. While exogenous danger signal recognition is an essential part of pathogen response by the innate immune system, endogenous danger signal recognition by TLR4 can lead to chronic and pathological inflammation. Understanding the differences in recognition of these two types of danger signals would allow for independent modulation of pathogen and host triggered inflammatory response through TLR4. Here, we examine the evolution of activation of TLR4 by two agonists, pathogen-derived lipopolysaccharide and host-produced S100A9. We show that these two types of signals evolved earlier than previously thought. We identified TLR4 cofactors MD-2 and CD14 in amphibians and fish, and validated that zebrafish TLR4 can recognize LPS. By contrast, we find that S100 activation evolved in the ancestor of amniotes. We identified an ortholog of S100A9 in birds and reptiles capable of activating TLR4. Using comparative immunology, we found that the requirements for LPS and S100A9 activation are different. In addition to our evolutionary studies, we used molecular approaches to probe if zinc binding to S100A9 is necessary for TLR4 activation. We found that activation of TLR4 by S100A9 occurs even in the absence of zinc. Finally, we describe how our evolutionary approach led to mechanistic hypotheses regarding TLR4 activation by both LPS and S100A9. This has led to ongoing projects in the Harms lab. This dissertation includes previously published and unpublished co-authored material. / 2021-04-30

The most abundant microflora is present in the distal parts of gut and the majority of the bacteria are Gram-negative anaerobes. Toll-like receptor 4 (TLR4), one of the ¡§pathogen-recognition molecules¡¨, recognize the lipopolysaccharide (LPS), derived from Gram-negative bacteria. TLR4 recognized the intestinal microflora and triggers the inflammatory responses. Although the effect of intestinal microflora on enhancing host to response the challenge from bacteria has been established, the mechanism has not been well studied. In this study, the relationship between TLR4 expression and the inflammatory response under intestinal microflora depletion was investigated. Mice fed with antibiotics for 4 weeks to delete the intestinal commensals and supplemented with or without LPS to stimulate TLR4 at week 3 were under sham or burn treatment. Results showed that thermal injury intestinal permeability, bacterial translocation to mesenteric lymph nodes, and neutrophil deposition in lung. Also, the activation of NF-£MB, expression of HSP70 and TLR4 were induced in intestinal after thermal injury. Moreover, TLR4 expression was increased in lung and peritoneal cells, ICAM and TNF-£\ expression were increased in peritoneal cells after thermal injury. However, NF-£MB activation, expression of TLR4, ICAM, and HSP70 were decreased in intestinal mucosa of mice with microflora depletion after thermal injury. Microflora depletion also significantly decreased the MPO activity in lung, and the phagocytic activity, the TLR4, ICAM, and TNF-£\ expression of peritoneal cells after thermal injury. Interestingly, LPS supplement reversed the effect of microflora depletion, suggest that intestinal microflora can trigger the host defense through TLR4 signaling pathway in thermal injured mice.

No description available.

Morphine has been and continues to be one of the most potent and widely used drugs for the treatment of pain. Clinical and animal models investigating sex differences in pain and analgesia demonstrate that morphine is a more potent analgesic in males than in females; indeed, we report the effective dose of morphine for female rats is twice that of male rats. In addition to binding to the neuronal mu opioid receptor, morphine binds to the innate immune receptor toll-like receptor 4 (TLR4) on microglia. Morphine action at TLR4 initiates a neuroinflammatory response and directly opposes morphine analgesia. Our recent studies demonstrate that administration of chronic morphine activates microglia within the ventrolateral periaqueductal gray (vlPAG), a critical brain region for the antinociceptive effects of morphine, while blockade of vlPAG microglia increases morphine analgesia and suppresses the development of tolerance in male rats. Despite increasing evidence of the involvement of microglia in altering morphine efficacy, no studies have examined sex differences in microglia within the PAG. The present experiments seek to characterize the distribution and activity of vlPAG microglia in males and females using behavioral, immunohistochemical and molecular techniques, while demonstrating the sufficiency and necessity of vlPAG microglia to produce sex differences in morphine analgesia using site-specific pharmacological manipulation of TLR4. We also investigate a novel pharmacokinetic mechanism underlying the sexually dimorphic effects of morphine administration on microglial activity. Here, we address a fundamental gap in our current understanding of sex differences in morphine analgesia and establish a mechanistic understanding of how the activation of vlPAG microglia sex-specifically influences morphine analgesia.

Toll like receptor-4 (TLR4) is a transmembrane inflammatory receptor expressed ubiquitously on the cell surface of immune cells as well as skeletal muscle and other metabolic tissues. A compelling body of evidence shows that muscle TLR4 and the downstream cytokine signaling modulate skeletal muscle metabolism. Intriguingly, skeletal muscle has been demonstrated to gain favorable inflammatory cytokine-mediated metabolic adaptations in the context of exercise training. This paradigm suggests a role for muscle TLR4 inflammatory signaling in the regulation of exercise metabolism. As such, the question arises as to whether exercise stress response follows similar inflammatory physiological pathways to those activated by other physical and pathogenic stimuli or not. Therefore, the objective of the present study was to investigate the role of muscle TLR4 signaling in modulating skeletal muscle cytokine, also known as myokine, response and metabolic adaptations to exercise. To this end, using Cre-mediated recombination, we developed a novel muscle-specific TLR4 knockout (mTLR4-/-) mouse model on C57BL/6JJ background. The differential inflammatory and metabolic responses between mTLR4-/- mice and wild type (WT) littermates were examined following exposure to either exercise or muscle stimulus. Accordingly, different exercise and muscle contraction modalities were pursued, focusing on voluntary wheel running, forced treadmill training, and in vivo electrical muscle stimulation. Overall, this study introduces a novel muscle-specific TLR4 knockout mouse model and discloses a crucial role for mTLR4 in basal systemic cytokine homeostasis. Furthermore, our findings identify mTLR4 as a major immunomodulatory effector of exercise-induced metabolic adaptations and suggest a link between mTLR4 and physiological determinants of maximal aerobic performance. / Ph. D.

Lipopolysaccharide (LPS), a Gram-negative endotoxin, has been well-established as the trigger for the effects of sepsis and septic shock through its binding with the innate immune receptor, Toll-like receptor 4 (TLR4). High doses of LPS signal through TLR4 to produce a massive release of pro-inflammatory cytokines including IL-6, TNFα, and other. Additionally, several recent publications have demonstrated severe metabolic alterations after LPS challenge, suppressing lipid oxidation and concurrently up-regulating glucose oxidation. Unfortunately, this switch in metabolism is inefficient for the great energy demands of the host during a systemic microbial infection which can result in vital organ failure. Meanwhile, a novel concept in several chronic disease pathologies also implicates LPS, although at very low doses. The presence of subclinically elevated circulating endotoxin levels has been termed metabolic endotoxemia and is beginning to be investigated in disease pathologies including insulin resistance and type II diabetes, atherosclerosis, cancer metastasis and Parkinson's disease. These disease phenotypes all possess a component of chronic inflammation whose source has not largely been understood, but examining the effects of very low doses of LPS may provide vital information in understanding their etiologies. However, most information on LPS signaling has been obtained using high doses of LPS (10-200ng/ml) while little to no studies have been published regarding the effects of very low doses of LPS (1pg-100pg/ml) on inflammatory and metabolic alterations. Thus, we use in vivo and in vitro models to determine that both IRAK1 and JNK are critical points of crosstalk downstream of TLR4 for the metabolic and inflammatory alterations associated with metabolic endotoxemia. Additionally, we observed significant down-regulation of nuclear receptors responsible for fatty acid metabolism, including PGC1α, PPARα, and PPARγ after very low dose LPS challenge. Further, we observe phenotypic changes in fatty acid oxidation and glucose oxidation, as well as subsequent changes in cytosolic acetyl-CoA levels and acetylation of pro-inflammatory transcription factor ATF2. Overall our studies point to several mechanisms of cross-talk between metabolism and inflammation and offer significant support to the concept of metabolic endotoxemia in the development of chronic disease. / Ph. D.

BACKGROUND A subarachnoid hemorrhage (SAH) is a bleed into the subarachnoid space surrounding the brain. This disease affects roughly 30,000 Americans each year and approximately one in six affected individuals die at the time of the ictal event. Individuals that do survive suffer from many complications including delayed cerebral vasospasm (DCV), cerebral edema, fever, and increased intracranial pressure (ICP) amongst others. These patients often suffer from brain damage due to neuronal apoptosis as a consequence of excess neuroinflammation. Microglia, the resident macrophage of the central nervous system, and Toll-like Receptor-4 (TLR4), a pro-inflammatory transmembrane receptor, have both been shown to play a role in the neuroinflammation seen in SAH. RBC components have been shown to activate microglial TLR4, and this event is suggested to trigger downstream mechanisms leading to neuronal apoptosis. The presented research takes a closer look at the role of microglial TLR4 in early neuronal apoptosis seen in an SAH model. METHODS All mice used were 10- to 12-week-old males on a C57BL/6 background: TLR4−/−, MyD88−/−, TRIF−/− and wild type (WT). To induce an SAH, a total of 60 ul of arterial blood from a donor WT mouse was injected for over 30 seconds into another mouse. For in vitro experiments, either primary microglia (PMG) or murine microglial BV2 cells were used. Microglia were separated from murine neuronal HT22 cells by 3um cell culture inserts or transwells, before being stimulated with lipopolysaccharide (LPS), red blood cells (RBCs), or RBC components including hemin (structurally similar to heme) and hemoglobin. In vivo samples were studied using either immunohistochemistry (IHC) or Fluorescence Activated Cell Sorting (FACS), and in vitro cells were studied using IHC and Light Microscopy. Neuronal cell death was measured using TUNEL and/or FloroJade C (FJC) assays. Cognitive function after SAH was measured using the Barnes Maze protocol. RESULTS In a 24-hour time course, more death occurred in the HT22 cells associated with BV2s treated with RBCs for 12-hour and 24-hour incubation time points as compared to 1-hour and 3-hour time points. Similar results were seen in the WT PMGs, as HT22 apoptosis increased in the RBC treated WT groups as the incubation time points increased. The WT PMG and MyD88−/− RBC treated PMGs showed significant death as compared to a WT untreated control (p<0.05) using a FJC assay, and both showed more death in a TUNEL assay as compared to an untreated control. WT mice treated with whole blood and hemoglobin had significantly more apoptosis as compared with a normal saline (NS)-treated control mouse (p<0.05). WT PMGs treated with whole blood and hemoglobin had more apoptosis as compared with an untreated control. MyD88-/- treated with RBC, hemoglobin, and hemin had more HT22 cell death compared with other genotypes treated with the same component. For the Barnes Maze, TLR4−/− mice performed significantly less total errors than WT mice on POD5 and 6 (p<0.01), and took significantly less time to reach the goal chamber on POD4, POD5 (p<0.05), and POD6 (p<0.01). CONCLUSION Our experimental results suggest that a microglial TLR4-dependent, MyD88-independent pathway is involved in neuronal apoptosis very early in an SAH model via RBC and hemoglobin activation, and that neuronal cell apoptosis due to TLR4 expression may be related to SAH-related cognitive and behavioral deficits. Our results suggest that TRIF may be the intracellular adaptor that is involved in this mechanism, but further experiments are needed to confirm this.

Toll-like receptor 4 (TLR4) is a transmembrane receptor, which upon activation by lipopolysaccharide (LPS) from Gram-negative bacteria, plays an important role in the induction of the innate immune response. Our lab has previously demonstrated that activation of TLR4 in skeletal muscle results in the preferential oxidation of glucose for ATP production over that of fatty acids. Currently, the exact mechanism(s) for TLR4-induced modulation of metabolism are not known. The purpose of this project was to test the hypothesis that activation of TLR4 pathway causes increased ROS production, which contributes to deceased fatty acid oxidation and altered mitochondrial respiration in skeletal muscle. To this end, skeletal muscle cells were studied following acute and chronic treatments with LPS, and a mouse model with muscle-specific over expression of TLR4 (mTLR4) was studied under chow fed conditions and following 16 weeks of high fat feeding. Acute LPS treatment of C2C12 cells resulted in mitochondrial uncoupling as evidenced by higher levels of state IV respiration, reduced maximally simulated respiration, and a robust induction of uncoupling protein 3. These observations occurred in conjunction with increased pyruvate dehydrogenase activity. The LPS-induced changes in substrate preferences and maximally-stimulated mitochondrial respiration were prevented in the presence of the antioxidants, N-acetyl-L-cyteine (NAC) and catalase. Using isolated flexor digitorum brevis (FDB) muscle fibers from C57BL/6J mice, we showed that LPS treatment results in significant increases in ROS production that are evident at 15 min and still increasing at 45 min following the addition of LPS to incubation media. Hyperpolarization of mitochondrial membrane potential was also evident at 15 min post LPS treatment in FDB fibers. Fatty acid oxidation measured in skeletal muscle whole homogenates from the mTLR4 mice was significantly reduced compared to wild-type littermates on a standard chow diet. Following a 16 week high fat diet, the mTLR4, compared to wild-type mice, gained more weight and fat mass, were glucose intolerant, and displayed elevated production of mitochondrial-derived reactive oxygen species (ROS) from complex III. In conclusion, these data show that TLR4 activation elicits a change in mitochondrial substrate preference in that acetyl-CoA derived from pyruvate oxidation is the preferred substrate for the TCA cycle over that derived from β-oxidation of fatty acids. These data also lend strong support to the idea that the TLR4-mediated change in substrate preference is dependent on the production of ROS. / Ph. D.

Le syndrome métabolique est caractérisé par un ensemble de perturbations métabolique. Il inclut la dyslipidémie, l’obésité abdominale, la résistance à l’insuline et l’hypertension. L’association de ces facteurs de risques est liée à une augmentation du risque de développer un diabète de type-2. Les acides gras polyinsaturés de la famille des oméga-3 ont plusieurs effets biologiques et modulent les facteurs de risques du syndrome métabolique par l’intermédiaire de multiples mécanismes. Cependant, leurs impacts sur la résistance à l’insuline et le diabète de type 2 sont encore inconnus.Au cours de ce travail, nous avons étudié l’effet d’Odontella aurita (OA), une microalgue riche en EPA (AGPI oméga-3) et antioxydants, sur la prévention de l’obésité et la résistance à l’insuline induites par un régime riche en acides gras saturés High-Fat (HF). En effet, nous avons montré que le régime HF soumis aux rats pendant 8 semaines conduit à une résistance à l’insuline qui se caractérise par une augmentation de l'insulinémie ainsi qu'à une diminution de l'expression protéique du récepteur de l'insuline. De plus, le régime HF provoque une diminution de la sensibilité du récepteur à l'insuline en inhibant son activité tyrosine kinase. Le régime HF conduit également à une augmentation de l'expression du récepteur TLR4, qui joue un rôle dans l'induction de la résistance à l'insuline par l'intermédiaire de l'activation des voies proinflammatoires par la résistine et le LPS. En effet, l'augmentation de l'expression de TLR4 est associée avec l'activation des MAPK proinflammatoires JNK et P38. Cependant, l'enrichissement du régime HF avec la microalgue normalise l'insulinémie et les niveaux d'expression du récepteur à l'insuline. Son activité tyrosine kinase est aussi restaurée. Et d'une manière intéressante, la supplémentation du régime HF avec la microalgue conduit à une réduction de l'expression du récepteur TLR4 ainsi qu'une inhibition des voies proinflammatoires prévenant ainsi la résistance hépatique à l’insuline.Le récepteur TLR4 et l’activation des voies pro-inflammatoires jouent un rôle important dans l’induction de la résistance à l’insuline. Afin d'explorer les mécanismes moléculaires impliqués dans la régulation de l’expression de TLR4 et déterminer les voies proinflammatoires impliquées dans l'induction de la résistance à l'insuline par les acides gras saturés, ainsi que la mise en évidence des mécanismes insulino-sensibilisateurs des AGPI oméga-3, nous avons utilisé les cellules SH-SY5Y (cellules de neuroblastome humain). En effet, les cellules SHSY5Y ont été exposées pendant 4h à l’acide palmitique (PA, acide gras saturé) ou au DHA (oméga-3) puis traitées avec la résistine. Tout d'abord, nous avons analysé l'effet de la résistine, le PA et le DHA sur les marqueurs de l'inflammation. Seule la résistine est capable d'activer NFkB et augmenter la phosphorylation d’Akt et de p38 MAPK. Toutefois, le prétraitement avec le PA augmente l'expression des cytokines inflammatoires (IL-6 et TNF-a), similaire à la résistine. D’une manière intéressante, le prétraitement au DHA supprime l’effet d PA et la résistine et prévient l’augmentation de l’expression d'IL-6 et TNF-α. Nous avons ensuite étudié la possibilité d'un effet synergique entre la résistine et le PA sur l’expression de TLR4. En effet, le prétraitement avec le PA augmente l'expression de TLR4 alors que le prétraitement au DHA n'a aucun effet. Nous avons montré aussi que le prétraitement au PA potentialise les effets de la résistine. En effet la résistine est le ligand de TLR4, et le PA, en augmentant l’expression de TLR4 favorise et amplifie les effets de la résistine.En conclusion, ces résultats montrent que les acides gras polyinsaturés oméga-3 préviennent l'inflammation et la résistance à l'insuline induite par les acides gras saturés via la régulation de la voie de signalisation de TLR4 empêchant ainsi l’installation du diabète de type 2 et du syndrome métabolique. / The metabolic syndrome is characterized by dyslipidemia, insulin resistance, abdominal obesity and hypertension, which are related to an elevated risk for type 2 diabetes mellitus. Omega-3 polyunsaturated fatty acids have extensive biological effects and modulate the risk factors for metabolic syndrome via multiple mechanisms. However their impact on insulin resistance and type 2 diabetes are still unknown.In the current study, we report that Odontella aurita, a microalga rich in the omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), prevents High saturated fat diet induced insulin resistance and inflammation in the liver of Wistar rats. High fat diet (HFD), given for 8 weeks, increased plasma insulin levels associated with the down-regulation of insulin receptor (IR) and the impairment of insulin-dependent IR phosphorylation. Furthermore, HFD increased toll-like receptor 4 (TLR4) expressions. Indeed, we have recently reported that TLR4 is implicated in resistin-induced inflammation and insulin resistance in the hypothalamus (Benomar et al, 2013). We also show that TLR4 up-regulation is concomitant with the activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38). Importantly, Odontella aurita enriched HFD (HFOA, 12%) normalized body weight and plasma insulin levels, and restores IR expression at both protein and mRNA levels. In addition, HFAO improves insulin responsiveness as estimated by in vitro phosphorylation of hepatic plasma membrane IR. Furthermore, HFOA decreased TLR4 expression and JNK /p38 phosphorylation. In conclusion, we demonstrate, for the first time to our knowledge, that omega-3 fatty acids brought by Ondontella aurita overcomes HFD-induced insulin resistance through the inhibition of TLR4/JNK/p38 MAP kinase signaling pathways.To further explore the molecular process underlying the activation of TLR4 by fatty acids, we aim to decipher the mechanisms implicated in the regulation of TLR4 expression. For this purpose, human neuroblastoma cells (SHSY-5Y) were exposed during 4h to either palmitic acid (a saturated fatty acid) or the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Cells were then treated with resistin. Firstly we analyzed the effect of resistin, palmitic acid and DHA on inflammation markers. We show that only resistin was able to activate NF-κB and to increase the phosphorylation of Akt and p38 MAPK. However, palmitic acid pretreatment increases the expression of inflammatory cytokines (IL-6 and TNF-α), similar to resistin. Interestingly, DHA pretreatment suppresses palmitic acid and resistin induced up-regulation of IL-6 and TNF-α. Secondly, we studied the possible synergistic interaction between resistin and palmitic acid on TLR-4 expression. We show that palmitic acid pretreatment increases TLR4 expression, at both protein and mRNA levels, while DHA pretreatment had no effect. Importantly, palmitic acid pretreatment potentiates resistin effects. In conclusion, we show for the first time, to our knowledge, that palmitic acid induces TLR4 expression and this leads to the amplification of resistin effects promoting then insulin resistance at the neuronal level.Taken together, these results demonstrate that omega-3 fatty acids prevent saturated fat-induced inflammation and insulin resistance through resistin/TLR4 signaling thereby preventing insulin resistance.