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.

Oral lichen planus (OLP) är en idiopatisk kronisk inflammatorisk sjukdom som drabbar munslemhinnan hos ca 2 % av den svenska befolkningen. Amalgamfyllningar kan framkalla lichenoida kontaktlesioner (cOLL), som kliniskt kan vara svåra att särskilja från OLP. Det är dessutom inte möjligt att skilja mellan OLP och cOLL histologiskt. Det är viktigt att kunna särskilja OLP och cOLL då behandlingen av dem skiljer sig.Toll-like receptorer (TLR) finns på flera av kroppens celler. De är en del av det medfödda immunförsvaret men de har också kopplats till autoimmuna sjuksomar. En ökad förekomst av TLR i skivepitel har påvisats vid OLP.Syftet med denna studie är att undersöka variationer i uttrycket av TLR4 och TLR9 i OLP och cOLL. Vår hypotes är att en histologisk skillnad i OLP och cOLL ska kunna observeras p.g.a. skillnader i patogenesen mellan OLP och cOLL.Metod: Vävnadsprov med histologiskt verifierad lichenoid reaktion valdes från Biobanken, Oral Patologi, Malmö från patienter med de kliniskt ställda diagnoserna OLP (10) och cOLL (12). TLR4 och TLR9 identifierades med hjälp av immunhistokemisk färgning varefter reaktionens lokalisation och intensitet jämfördes mellan de två grupperna.Resultat: En signifikant skillnad observerades i infärgningen av TLR4 hos fibroblaster, lymfocyter och makrofager, där TLR4 var mer positiv i cOLL. Uttrycket av TLR9 hos lymfocyter var starkare vid OLP än cOLL.Slutsats: Våra resultat visade att det finns en skillnad i uttrycket av TLR4 och TLR9 i cOLL och OLP. Resultaten bekräftar att OLP och cOLL har olika patogenes, men ytterligare studier behövs för att klargöra hur. / Oral lichen planus (OLP) is an idiopathic chronic inflammatory disease that affects the oral mucosa in approximately 2% of the Swedish population. Amalgam fillings may induce contact oral lichenoid lesions (cOLL) that can be difficult to clinically distinguish from OLP. It is not possible to histologically distinguish between OLP and cOLL. As their treatments differ, the correct diagnosis is vital.Toll-like receptors (TLR) are expressed by most of the body's cells and are part of the innate immune system, however they have also been linked to certain autoimmune diseases. OLP exhibits an increased amount of TLR in the epithelium.The purpose of this study is to investigate the variations in the expression of TLR4 and TLR9 in OLP and cOLL. Our hypothesis is that a histological difference in OLP and cOLL can be observed due to TLRs different roles in maintaining the immune response.Method: Tissue samples with histologically confirmed lichenoid reactions were chosen from Biobank, Oral Pathology, Malmö, from patients with the clinical diagnosis OLP (10 subjects) and cOLL (12 subjects). TLR4 and TLR9 were identified by immunohistochemical staining and compared between the two groups.Results: A significant difference was observed in TLR4 staining of fibroblasts, lymphocytes and macrophages where the antibody was less expressive in OLP. In TLR9 staining lymphocytes were stronger expressed in OLP compared to cOLL.Conclusion: Our results showed that there was a difference in the expression of TLR4 and TLR9 in cOLL and OLP, this could be a result of OLP being an autoimmune disorder. Further studies on this subject are recommended on this subject.

Oral lichen planus (OLP) är en idiopatisk kronisk inflammatorisk sjukdom som drabbar munslemhinnan hos ca 2 % av den svenska befolkningen. Amalgamfyllningar kan framkalla lichenoida kontaktlesioner (cOLL), som kliniskt kan vara svåra att särskilja från OLP. Det är dessutom inte möjligt att skilja mellan OLP och cOLL histologiskt. Det är viktigt att kunna särskilja OLP och cOLL då behandlingen av dem skiljer sig.Toll-like receptorer (TLR) finns på flera av kroppens celler. De är en del av det medfödda immunförsvaret men de har också kopplats till autoimmuna sjuksomar. En ökad förekomst av TLR i skivepitel har påvisats vid OLP.Syftet med denna studie är att undersöka variationer i uttrycket av TLR4 och TLR9 i OLP och cOLL. Vår hypotes är att en histologisk skillnad i OLP och cOLL ska kunna observeras p.g.a. skillnader i patogenesen mellan OLP och cOLL.Metod: Vävnadsprov med histologiskt verifierad lichenoid reaktion valdes från Biobanken, Oral Patologi, Malmö från patienter med de kliniskt ställda diagnoserna OLP (10) och cOLL (12). TLR4 och TLR9 identifierades med hjälp av immunhistokemisk färgning varefter reaktionens lokalisation och intensitet jämfördes mellan de två grupperna.Resultat: En signifikant skillnad observerades i infärgningen av TLR4 hos fibroblaster, lymfocyter och makrofager, där TLR4 var mer positiv i cOLL. Uttrycket av TLR9 hos lymfocyter var starkare vid OLP än cOLL.Slutsats: Våra resultat visade att det finns en skillnad i uttrycket av TLR4 och TLR9 i cOLL och OLP. Resultaten bekräftar att OLP och cOLL har olika patogenes, men ytterligare studier behövs för att klargöra hur. / Oral lichen planus (OLP) is an idiopathic chronic inflammatory disease that affects the oral mucosa in approximately 2% of the Swedish population. Amalgam fillings may induce contact oral lichenoid lesions (cOLL) that can be difficult to clinically distinguish from OLP. It is not possible to histologically distinguish between OLP and cOLL. As their treatments differ, the correct diagnosis is vital.Toll-like receptors (TLR) are expressed by most of the body's cells and are part of the innate immune system, however they have also been linked to certain autoimmune diseases. OLP exhibits an increased amount of TLR in the epithelium.The purpose of this study is to investigate the variations in the expression of TLR4 and TLR9 in OLP and cOLL. Our hypothesis is that a histological difference in OLP and cOLL can be observed due to TLRs different roles in maintaining the immune response.Method: Tissue samples with histologically confirmed lichenoid reactions were chosen from Biobank, Oral Pathology, Malmö, from patients with the clinical diagnosis OLP (10 subjects) and cOLL (12 subjects). TLR4 and TLR9 were identified by immunohistochemical staining and compared between the two groups.Results: A significant difference was observed in TLR4 staining of fibroblasts, lymphocytes and macrophages where the antibody was less expressive in OLP. In TLR9 staining lymphocytes were stronger expressed in OLP compared to cOLL.Conclusion: Our results showed that there was a difference in the expression of TLR4 and TLR9 in cOLL and OLP, this could be a result of OLP being an autoimmune disorder. Further studies on this subject are recommended on this subject. MeSH: "Dental Amalgam", "Dermatitis, Allergic Contact", "Immunohistochemistry", "Lichen Planus, Oral", "Toll-Like Receptor 4", "Toll-Like Receptor 9"

<p>Breastfeeding from HIV-infected mothers is one of the major sources of pediatric HIV-1 infection; however, an intervention that promotes exclusive breastfeeding has significantly reduced vertical HIV transmission rates and infant mortality. The mechanisms underlying this phenomenon remain unknown; however, have been closely linked to high levels of innate immune factors in breast milk. Indeed, the level of several innate factors in breast milk correlate with protection and/or have direct anti-viral properties <em>in vitro.</em> The innate immune factor, soluble TLR2 (sTLR2) is found in high concentration in breast milk and has previously been investigated for its anti-bacterial properties; however, its anti-viral properties remain poorly understood. Thus, the research presented in this thesis extended our understanding of sTLR2 by characterizing the mechanisms by which sTLR2 inhibited HIV-induced inflammation and infection. Chapter 2 examined the predominant forms of sTLR2 in breast milk from different women, its cellular source, bioavailability and kinetics postpartum. Functionally, we confirmed sTLR2’s anti-bacterial properties and extended to show, for the first time, that sTLR2 directly inhibited HIV infection <em>in vitro.</em> Chapter 3 documented a potential mechanism of sTLR2’s direct inhibition of HIV infection <em>in vitro</em> and, investigated sTLR2 and TLR2 expression in HIV uninfected compared to HIV infected breast milk and breast milk cells, respectively. Chapter 4 investigated the role of TLR2’s recognition of novel HIV pathogen associated molecular patterns (PAMPs), and whether TLR2 expression increased HIV infection and integration. Taken together, we present novel anti-viral functions of sTLR2 by demonstrating that sTLR2 bound to specific HIV PAMPs, which led to significantly decreased HIV-induced inflammation, co-receptor expression, and HIV infection. Furthermore, we demonstrated, for the first time, that TLR2 recognizes specific HIV PAMPs, which led to significantly increased pro-inflammatory cytokine production, co-receptor expression and HIV infection. Thus, sTLR2 and TLR2 represent innate immune factors that might have preventative and therapeutic applications for both infants and adults in the future.<strong><br /> </strong></p> / Doctor of Philosophy (Medical Science)

Ultra-low doses (ULD) of the opioid receptor antagonists, naloxone and naltrexone, augment the analgesic actions of morphine, block the induction of tolerance, and reverse established tolerance by an unknown mechanism. Preclinical studies demonstrate that chronic morphine administration induces spinal gliosis and that inhibition of gliosis prevents the development of analgesic tolerance to opioids. Thus, this thesis investigated the inhibition of spinal gliosis as a mechanism by which ULD antagonists attenuate analgesic tolerance and opioid-induced hyperalgesia. Immune cell activation is implicated in the etiology of morphine tolerance and intrathecal catheterization, a technique commonly used to study the spinal effects of drugs, causes profound gliosis. Thus, the first study investigated the effects of catheter-induced gliosis on acute and chronic morphine analgesic tolerance. Catheterization-induced gliosis did not alter antinociceptive responses to acute intrathecal morphine; however, tolerance to chronic morphine was exacerbated in catheterized rats compared to sham and surgery-naïve controls. The potentiation of analgesic tolerance to chronic morphine by spinal gliosis provided evidence that glia modulate opioid analgesia; therefore, inhibition of opioid-induced activation of glia was explored as a potential mechanism by which ULD antagonists prevent tolerance. The second series of experiments reported morphine-induced activation of spinal microglia and astrocytes was blocked by co-administering ULD naltrexone with morphine. These findings prompted us to elucidate the specific molecular target through which ULD antagonists attenuate opioid analgesia. Activation of glial Toll-like receptor 4 (TLR4) induces gliosis and may contribute to analgesic tolerance and/or morphine-induced hyperalgesia (MIH). Antagonism of TLR4 by the opioid receptor-inactive (+) stereoisomer of naloxone was identified as a potential mechanism by which ULD antagonists modulate opioid analgesia. Tolerance and MIH developed in mice expressing non-functional TLR4 and in wildtype controls. Analgesic tolerance was stereoselectively blocked by ULD (-)naloxone, whereas MIH was blocked by both naloxone enantiomers. Collectively, these studies demonstrate analgesic tolerance and MIH occur through distinct mechanisms. ULD naloxone attenuates analgesic tolerance likely via an opioid receptor-mediated mechanism that is TLR4-independent. ULD antagonists do not attenuate tolerance via inhibition of spinal gliosis as hypothesized. In contrast, ULD antagonists prevent MIH by inhibiting opioid-induced gliosis in an opioid receptor- and TLR4-independent manner. Immune cell activation is implicated in the etiology of morphine tolerance and intrathecal catheterization, a technique commonly used to study the spinal effects of drugs, causes profound gliosis. Thus, the first study investigated the effects of catheter-induced gliosis on acute and chronic morphine analgesic tolerance. Catheterization-induced gliosis did not alter antinociceptive responses to acute intrathecal morphine; however, tolerance to chronic morphine was exacerbated in catheterized rats compared to sham and surgery-naïve controls. The potentiation of analgesic tolerance to chronic morphine by spinal gliosis provided evidence that glia modulate opioid analgesia; therefore, inhibition of opioid-induced activation of glia was explored as a potential mechanism by which ULD antagonists prevent tolerance. The second series of experiments reported morphine-induced activation of spinal microglia and astrocytes was blocked by co-administering ULD naltrexone with morphine. These findings prompted us to elucidate the specific molecular target through which ULD antagonists attenuate opioid analgesia. Activation of glial Toll-like receptor 4 (TLR4) induces gliosis and may contribute to analgesic tolerance and/or morphine-induced hyperalgesia (MIH). Antagonism of TLR4 by the opioid receptor-inactive (+) stereoisomer of naloxone was identified as a potential mechanism by which ULD antagonists modulate opioid analgesia. Tolerance and MIH developed in mice expressing non-functional TLR4 and in wildtype controls. Analgesic tolerance was stereoselectively blocked by ULD (-)naloxone, whereas MIH was blocked by both naloxone enantiomers. Collectively, these studies demonstrate analgesic tolerance and MIH occur through distinct mechanisms. ULD naloxone attenuates analgesic tolerance likely via an opioid receptor-mediated mechanism that is TLR4-independent. ULD antagonists do not attenuate tolerance via inhibition of spinal gliosis as hypothesized. In contrast, ULD antagonists prevent MIH by inhibiting opioid-induced gliosis in an opioid receptor- and TLR4-independent manner. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-04-25 15:06:50.731

Master of Science / Division of Biology / Sherry D. Fleming / Professional phagocytes play a critical role in maintaining homeostasis within a host through phagocytic, microbicidal, and inflammatory activity. Complement receptors (CR) and toll-like receptors (TLRs) aid in phagocytosis and stimulate these cells to enhance the immune response. Environmental factors such as hypoxia, prevalent at sites of tissue damage or infection, induce a similar effect. Systemic components such as opsonins may further enhance phagocyte activity. Similar to professional phagocytes, B1 B cells exhibit a broad range of immunological activity as well as expression of CRs and TLRs. Despite extensive studies with other phagocytes, the effects of CRs and TLRs expression, hypoxic stimulation, or opsonization on B1 B cell function remain unclear. We tested the hypothesis that TLR2 stimulation, hypoxia, CR2 expression, or opsonins would enhance B1 B cell phagocytic and inflammatory activity. Negatively selected peritoneal cavity B1 B cells from the (PerC) of wild type, Tlr2[superscript]-[superscript]/[superscript]-, and Cr2[superscript]-[superscript]/[superscript]- mice, or a B1 B-like cell line, Wehi 231, were subjected to normoxia or hypoxia with or without particles for phagocytosis, TLR2 agonists, or CR2 ligands. The PerC of Tlr2[superscript]-[superscript]/[superscript]- mice contained an altered B1 B cell subset distribution while Cr2[superscript]-[superscript]/[superscript]- mice exhibited a normal repertoire. We demonstrated that hypoxia significantly downregulated inflammatory cytokine production by B1 B cells, while upregulating phagocytic activity in a TLR2 or CR2 dependent manner. TLR2 or CR2 deficiency altered constitutive production of B1 B cell associated cytokines. The CR2 ligand C3d, an opsonin, significantly enhanced the phagocytic activity of B1 B cells but failed to stimulate cytokine production. However, Cr2[superscript]-[superscript]/[superscript]- B1 B cells phagocytosed C3d-coated particles suggesting multiple CR may play a role in B1 B cell phagocytosis. Overall, the data suggest TLRs, CRs, hypoxia, and opsonization all contribute to B1 B cell effector function similar to professional phagocytes.

Um papel importante foi atribuído ao receptor toll-like 4 (TLR4) no desenvolvimento da placa aterosclerótica. O TLR4 foi primeiramente descrito como um receptor para bactérias gram-negativas; posteriormente foi demonstrado que sua expressão está aumentada em placas ateroscleróticas e que pacientes que possuem um polimorfismo disfuncional do TLR4 são menos suscetíveis ao desenvolvimento dessa doença. Portanto, o objetivo desse estudo foi o de investigar, em um modelo experimental de aterosclerose, a influência da deleção do TLR4 na formação e morfologia da placa aterosclerótica, no perfil lipídico e em marcadores inflamatórios. Camundongos duplo knockout (DKO), deficientes no receptor de LDL e TLR4, foram gerados cruzando-se camundongos deficientes para o receptor de LDL (LDLrKO) com camundongos deficientes para o TLR4 (TLR4KO). Todos os grupos receberam dieta rica em gordura e colesterol por 12 semanas. As concentrações plasmáticas de colesterol e triglicérides foram medidas por ensaio colorimétrico. Cortes seriados da raiz aórtica foram corados com Oil red O e as áreas de lesão quantificadas por analisador de imagens. O colágeno foi medido por coloração de picrossirius. A formação de nitrotirosina e expressão de CD40L, MMP9 e iNOS nas placas foram feitas por imunohistoquímica. As comparações foram feitas por ANOVA com pós teste de Student Newman-Keuls. Os dados foram expressos como média ± EPM. Camundongos DKO desenvolveram placas menores que camundongos LDLrKO (117.6 ±1.4 vs 198.8 ± 3.3 104m2). Camundongos TLR4KO não formaram placa. As placas dos camundongos DKO apresentaram menor núcleo lipídico que as dos LDLrKO (76.2± 13.2 vs 161.7 ± 2.9 104m2). O colágeno ao redor do núcleo lipídico é maior nos camundongos DKO do que nos LDLrKO (24.9 ± 1.8 vs 16.5 ± 2.5 % da placa). A distribuição do colágeno nos camundongos DKO ocorre principalmente ao redor da placa, de forma mais organizada, enquanto que nos LDLrKO onde sua distribuição é mais difusa. As placas dos camundongos DKO apresentaram menor expressão de CD40L e iNOS do que as dos LDLrKO (13.1 ± 0.7 vs 18.5 ± 2.5 AU e 7.7 ± 0.9 vs 10.2 ± 0.4 AU, respectivamente). A expressão de MMP9 foi menor nas placas dos camundongos DKO do que as dos LDLrKO (2.99 ± 0.3 vs 1.99 ± 0.2 AU). A marcação para nitrotirosina foi maior nos camundongos LDLrKO quando comparada com as dos grupos DKO e TLR4KO (142.89 ± 208.5, 77.16 ± 227.7 e 71.73 ± 95.9 10m2, respectivamente). Todos esses resultados sugerem que o processo inflamatório é menor na ausência do TLR4. As concentrações plasmáticas de colesterol não foram diferentes entre os grupos LDLrKO e DKO mas os camundongos LDLrKO apresentaram concentrações plasmáticas de triglicérides maiores do que os camundongos DKO após a dieta (265.2 ± 27.6 vs 150.5 ± 8.8 mg/dL). O receptor toll-like 4 influencia na estrutura e formação da placa aterosclerótica independentemente dos níveis séricos de colesterol / A crucial role has been suggested for toll-like receptor 4 (TLR4) in atherosclerotic plaque formation and development. TLR4 was described primarily, as a receptor for gram-negative bacteria lipopolisacharide; later it was showed that its expression is increased in atherosclerotic plaques and patients that carries a TLR4 dysfunctional polymorphism are less susceptible to development of this disease. Therefore, the aim of this study was to investigate, in an experimental model of atherosclerosis, the influence of TLR4 deletion in atherosclerotic plaque formation and morphology, cholesterol profile and inflammatory markers. Double knockout mice (DKO), deficient in LDL receptor and TLR4, were generated by breeding LDL receptor knockout mice (LDLrKO) with TLR4 knockout mice (TLR4KO). All three experimental groups, LDLrKO, TLR4KO and DKO were fed a high fat-cholesterol diet for 12 weeks. Plasma cholesterol and triacylglicerol concentrations were measured by colorimetric assay. Cross sections of aortic sinus were stained with Oil red O and lesion areas were quantified by an image analyzer. Collagen content was measured by picrossirius staining. We also measured nitrotyrosine formation, CD40L, MMP9 and iNOS expression by immunohistochemistry. Comparisons were made by ANOVA followed by Student-Newman-Keuls post- test. Data are mean ± SEM. DKO mice developed smaller plaques than LDLrKO mice (117.6 ±1.4 vs 198.8 ± 3.3 104m2). TLR4KO mice developed no plaque. Plaques from DKO mice have also a smaller lipid core than the ones from LDLrKO mice (76.2± 13.2 vs 161.7 ± 2.9 104m2). Collagen content around the lipid core is higher in DKO mice compared to LDLrKO mice (24.9 ± 1.8 vs 16.5 ± 2.5 % of the whole plaque). Interestingly, collagen distribution in DKO mice seems to occur mainly on the plaque periphery, in a more organized manner, while in LDLrKO mice it is fuzzier, being present also inside the plaque. Plaques from DKO present lower expression of CD40L and iNOS than LDLrKO mice (13.1 ± 0.7 vs 18.5 ± 2.5 AU and 7.7 ± 0.9 vs 10.2 ± 0.4 AU, respectively). MMP9 expression is lower in DKO mice as compared to LDLrKO mice (2.99 ± 0.3 vs 1.99 ± 0.2 AU). Nitrotyrosine staining was higher in LDLrKO mice as compared to DKO and TLR4KO groups (142. 89 ± 208.5, 77.16 ± 227.7 and 71.73 ± 95.9 10m2, respectively). All together, these findings suggest that inflammatory process is milder in the absence of TLR4. Serum cholesterol were not different between LDLrKO and DKO mice but LDLrKO presented higher triacylglicerol serum levels after 12 weeks on high fat high cholesterol diet as compared to DKO mice (265.2 ± 27.6 vs 150.5 ± 8.8). Toll like receptor 4 influences atherosclerotic plaque formation and structure independently from serum cholesterol levels

背根神經節（DRG）上的初級感覺神經元通常負責感覺從環境中有害的刺激，但新出現的證據表明，它亦負責對危險的感覺。Toll-樣受體-4（TLR4）通常見於小膠質細胞，它是負責識別病原體相關分子模式（PAMPs）或損傷相關分子模式（DAMPs）並誘發炎症。奇怪的是，儘管TLR4在中樞神經系統通常見於神經膠質細胞，在DRG發現的TLR4僅見於初級感覺神經元，但從未見於周邊的衛星膠質細胞（SGC）。而重要的是，在感覺神經節中激活TLR4是會導致神經病理性疼痛的，但我們仍然未知道初級感覺神經元上的TLR4是否導致疼痛的唯一來源。本研究旨在探討在DRG細胞的TLR4信號傳導的分子和細胞機理，並探討在DRG的神經元和膠質細胞上TLR4活動的差異，在生物學上有甚麼意義。 / 為了研究在DRG神經元和膠質細胞的相互作用，我們首先在一個既定的混合DRG細胞培養模型上研究了谷氨酰胺合成酶（ GS ）的表達模式。GS是一種只會在SGC上表達的特異性酶，並於神經元和神經膠質細胞之間的谷氨酰胺 - 谷氨酸循環產生相互作用。在典型的DRG細胞培養，神經元通過擴散因子促進了GS在神經膠質細胞上的表達，然而，GS的表達亦受到TLR4激動劑，即脂多醣（LPS），的抑制。這表明DRG神經元和神經膠質細胞的關係受到TLR4介導的炎症之影響。在混合DRG細胞中，我們對TLR4-免疫反應（IR）進行了鑑定，發現TLR4最主要的是表達在神經元細胞的表面。另外，LPS（ 1微克/毫升，2小時）會刺激混合DRG細胞，通過在DRG細胞中MyD88依賴性信令，誘導環加氧酶-2（COX -2），白細胞介素-1β（ IL-1β）和腫瘤壞死因子-α（TNFα）的轉錄。此外，在DRG細胞， LPS（ 1微克/毫升， 24小時）亦會觸發依賴COX-2 的前列腺素E2（PGE₂）和的前列環素（PGI₂）的產生。但在LPS刺激後，我們發現DRG神經元和神經膠質細胞都對 COX-2-IR呈陽性反應。這證明DRG神經膠質細胞對TLR4誘發的神經炎症也擔任一定的角色。 / 為了純粹研究神經膠質細胞有沒有任何TLR4活性，我們把神經元從混合DRG細胞中除去，從而把神經膠質細胞純化。出乎意料的是，在純化後，大約80的神經膠質細胞對TLR4 -IR呈陽性反應。而且，時間和濃度依賴性的研究表明，純化後的神經膠質細胞對LPS刺激的COX-2表達反應在有效性和效率上比混合DRG細胞的顯著更高。明顯地，神經元對神經膠質細胞的TLR4活性有抑制作用。我們並且發現，神經元的抑制作用是透過由細胞與細胞之間的接觸介導，而不是由擴散因子介導。 / 重要的是， LPS也能誘導純化後的神經膠質細胞去產生依賴COX-2活性的前列腺素E2（PGE₂）。反過來， PGE₂能區別地調節依賴TLR4的炎症基因轉錄，說明在DRG 由TLR4介導的神經炎症是受多重複雜的機理控制。然而有趣的是，從受熱休克性損害的感覺神經元所收集的培養基可以激活純化膠質細胞，並通過對TLR4局部依賴性的方式，誘導COX-2的轉錄。此外，我們利用斑馬魚作為疼痛行為反應的模型，發現COXs的活性與瞬時受體電位通道亞家族V1（ TRPV1）有密切關係。斑馬魚幼蟲的疼痛行為反應是一個適合於篩選新型鎮痛化合物的體內模型。 / 總括來說，透過細胞與細胞之間的接觸和擴散因子，感覺神經元可以控制神經膠質細胞的表型。我們的研究確定感覺神經元是在DRG中表達TLR4的主要細胞類型，但當神經元施加的抑制被削弱，SGC可以成為完全勝任TLR4信息傳遞的細胞。因此我們推測TLR4的活性在DRG中被嚴格調控，以防止不必要的神經炎症發生。至於未來，我們認為在DRG中的TLR4/COX-2/PGE₂信號通路可以成為研究方的新型鎮痛化合物的方向。而轉基因斑馬魚則可用作篩選新型鎮痛化合物的工具。 / Primary sensory neurons of the dorsal root ganglia (DRG) are classically responsible for the detection of physiological stimuli from the environment, but emerging evidences suggests that they are also involved in the sensation of danger. Toll-like receptor 4 (TLR4) is commonly found on microglia for the recognition of pathogen- or damage- associated molecular patterns (PAMPs or DAMPs) and to the activation of TLR4 leads to inflammation. Curiously, while commonly found in glial cells in central nervous system, TLR4 expression was only found in primary sensory neurons but not the satellite glial cells (SGCs) in the DRG. Importantly, activation of TLR4 in sensory ganglia mediates neuropathic pain, but it remains unknown whether neurons are the only source of TLR4 activity. The present study aims to study the cellular and molecular mechanism(s) of TLR4 signalings and explore the biological significance of differential cellular TLR4 activity in the DRG. / To investigate neuron-glia interactions in the DRG, the expression of glutamine synthetase (GS), a SGC-specific enzyme in the glutamine-glutamate shuttle between neuron and glia, was studied in an established model of mixed DRG cells culture. In typical mixed DRG cell cultures, neurons promoted the GS expression in glial cells through diffusible factors. However, GS expression was negatively regulated by theTLR4 agonist, lipopolysaccharide (LPS), indicative of a change in neuron-glia relationships by TLR4 mediated inflammation. In mixed DRG cells, cell surface TLR4-immunoreactivity (-ir) was predominantly identified on the neurons. LPS (1 μg/mL, 2 h) stimulation induced cyclooxygenases-2 (COX-2), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) transcription through MyD88-dependent signalings in DRG cells. Furthermore, LPS (1 μg/mL, 24 h) triggered COX-2-dependent production of prostaglandin E₂ (PGE₂) and prostacyclin (PGI₂) in mixed DRG cells. / To study the TLR4 activity of glial cells, glial cell cultures were purified by removing neurons from mixed DRG cell culture. Unexpectedly, approximately 80% of purified glial cells become TLR4-ir positive. Moreover, a time- and concentration-dependent study showed that the efficacy and efficiency of purified glial cells to express COX-2 in response to LPS was significantly higher than that of mixed DRG cells. We found that neuron inhibited glial cells through cell-cell contact, but not by diffusible factors. Importantly, LPS also induced COX-2 dependent PGE₂ production in purified glial cells. In turn, PGE₂ can differentially modulate TLR4-dependent gene transcription, suggestive of a complex regulation of TLR4-mediated inflammation in the DRG. Intriguingly, conditioned media from heat-shocked damaged sensory neurons activated purified glial cells to induce COX-2-transcription through a partially TLR4-dependent mechanism. Using zebrafish as a model of nocifensive behavior, we found that the activity of COXs was closely associated with the transient receptor potential channel subfamily V1 (TRPV1), and the nocifensive behavior of zebrafish larvae is suitable for in vivo screening of novel analgesic compounds. / To conclude, sensory neurons regulate the phenotypes of DRG glial cells through cell-cell contact and diffusible factors. Here, sensory neurons are confirmed to be the predominant cell type expressing TLR4 in the DRG, but SGCs become fully competent for TLR4 signalings when the neuronal inhibitions are diminished. We therefore hypothesize that TLR4 activity is tightly regulated in the DRG to prevent unwanted neuroinflammation. Future studies with genetically modified zebrafish can be used for the screening of novel analgesic compound targeting the TLR4/COX-2/PGE₂ signaling pathway. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tse, Kai Hei. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 190-222). / Abstracts also in Chinese.

L'hémophilie A (HA) est une maladie hémorragique congénitale qui se traduit par un défaut en facteur VIII (FVIII) de la coagulation. Le traitement privilégié des saignements est l'administration de FVIII exogène cependant, 30% des patients développent une réponse anticorps alloimmune contre le FVIII thérapeutique qui inhibe son activité pro-coagulante. L'endocytose du FVIII par les cellules dendritiques (DC) et sa présentation aux lymphocytes T ont été documentées. Cependant, la nature des signaux de danger (SD) responsables de la maturation des DC indispensable à l'initiation de la réponse immunitaire anti-FVIII, est inconnue. Au cours de ma thèse, j'ai cherché à identifier l'origine de ces SD et envisagé 3 possibilités: Une origine liée à la structure intrinsèque du FVIII, au contexte inflammatoire avant l'administration de FVIII, ou au contexte inflammatoire généré par l'injection de FVIII. Mes résultats ont montré que le FVIII n'était pas capable d'induire la maturation de macrophages ou d'activer directement le TLR2. J'ai également écarté l'hypothèse d'un état d'activation compensatoire des plaquettes (PLT) dans un organisme privé de FVIII. En revanche, mes travaux ont mis en évidence un rôle des PLT dans l'initiation de la réponse immunitaire anti-FVIII dans le modèle murin d'HA. Mes résultats suggèrent que l'implication des PLT passe par leur activation par la thrombine générée lors de l'administration de FVIII. L'identification des médiateurs de l'inflammation issus de l'activation plaquettaire devrait ouvrir des perspectives thérapeutiques intéressantes dans le contrôle de l'inflammation au moment de l'administration de FVIII, afin de réduire son immunogénicité.

<i>Rhodococcus equi</i> (<i>R. equi</i>) is an intracellular, gram-positive coccobacillus that causes pneumonia in foals aged 2 to 4 months. Neonatal foals are susceptible to <i>R. equi</i> infection probably due to inefficient Toll-like receptor (TLR)-2 signaling and inability to produce interferon gamma. One of the reasons for inefficient receptor signaling and recognition of <i>R. equi</i> by the foals immune system may be the inefficient sequestration of TLRs in lipid rafts, which act as signaling platforms. However, there are no protocols to isolate lipid rafts from equine cells and, therefore, no data on the association of TLRs with the lipid rafts in the lung cells of normal and infected foals. Because of the clinical importance of the disease, there is considerable interest in developing effective prophylactic methods, which in turn requires a better understanding of fundamental immunology of the foals. In this study, I have examined the effect of <i>R. equi</i> vaccination on the lung inflammation induced following challenge with <i>R. equi</i>. I also developed a protocol to isolate lipid rafts from broncho-alveolar lavage (BAL) cells and investigated the association of lipid rafts with TLRs.<p> In the first study, 15 mixed breed draft-type foals up to 7 weeks of age were studied. The foals were divided into control (n=7) and a vaccinated (n=8). The control foals were given 10 mL phosphate buffered saline intramuscularly while the vaccinated group was vaccinated on day 0 of the study followed by a booster on day 14. All the foals were challenged with <i>R. equi</i> (5x106 cells/mL into the dorso-caudal region of the right lung lobe). BAL was performed on day 14, 28 and 35 and all the foals were euthanized on day 49 of the study.<p> The study design did not leave any non-infected foal at the end of the experiment. Therefore, lung samples were obtained from two untreated control (non-vaccinated non-infected) foals from the Department of Veterinary Pathology, University of Saskatchewan were used. The data showed similar levels of lung inflammation in both the control and vaccinated foal groups based on BAL cytology, gross pathology and histopathology. Gross and histopathological studies indicated that both control and vaccinated foals developed granulomatous lesions. Immunohistology showed increased expression of TLR4, TLR2 and TNF alpha in alveolar septa and in some cases in the vascular endothelium and airway epithelium in the lungs of both groups compared to the untreated control foals. Western blots showed increased expression of TLR2 but not TLR4 in the lung extracts from both the vaccinated and the control foals. Vaccinated foals showed higher concentrations of TNF alpha(p=0.0219) in their BAL on day 28 but lower concentrations of IL-10 (p=0.0172) in their lung extracts collected on day 49 compared to the controls. There were no differences in IFN gamma and protein concentrations between the two groups.<p> To understand the role of lipid rafts in TLR4 and TLR2 signaling, I developed an efficient and simpler protocol to isolate lipid rafts from BAL cells of foals and confirmed their identity by localizing Flotillin-1 and GM-1 (fractions 6-9), which are lipid raft markers, and transferrin receptor (fractions 1-4) which is present in non-lipid raft fractions. Lung macrophages from naïve foals lacked sequestration of Flotillin-1 and GM-1 in the higher fractions compared to the vaccinated foals. Further, the data showed that while TLR4 and TLR2 were localized in most of the fractions (1-9) in control foal BAL collected on day 14 and 28, the TLR4 and TLR2 association was restricted to fractions 6-9 in the lipid rafts isolated from BAL cells of vaccinated foals. These data suggest that BAL cells of neonatal foals may not have effective signaling machinery because of lack of association of TLR2 and TLR4 with lipid rafts.<p> Taken together, the data show similar levels of lung inflammation in the control and vaccinated foals upon infection with <i>R. equi</i>. The vaccination, however, appeared to have some effect on the immunohistologic expression of TLR2, TLR4 and TNFalpha in the lung tissues, and increased association of TLR2 and TLR4 with the lipid raft fractions. Based on the higher expression of TNF alpha and lower expression of IL-10, the vaccinated foals may be more competent to mount an immune response against <i>R. equi</i>.