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

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.

Chronic low-grade inflammation is associated the Metabolic Syndrome (MetS) and may contribute to its development. A diet high in saturated fat (SFA) has been associated with increased inflammation and development of the MetS. SFAs have been shown to elicit pro-inflammatory signaling through proteins of innate immunity, TLR4 and Nods 1 and 2. We determined whether common polymorphisms in the genes of these proteins could modify the association between fat intake and biomarkers of the MetS. Fat intake was measured using a food frequency questionnaire and genotyping was completed using real-time PCR. The TLR4 Asp299Gly (rs4986790) polymorphism was associated with decreased insulin sensitivity while an intronic polymorphism (rs5030728) modified the association between dietary SFA and HDL-cholesterol. The Nod1 Glu266Lys polymorphism modified the association between dietary SFA and HOMA-IR. These results suggest a role for innate immunity in mediating some of the effects of dietary SFAs on factors associated with the MetS.

Chronic low-grade inflammation is associated the Metabolic Syndrome (MetS) and may contribute to its development. A diet high in saturated fat (SFA) has been associated with increased inflammation and development of the MetS. SFAs have been shown to elicit pro-inflammatory signaling through proteins of innate immunity, TLR4 and Nods 1 and 2. We determined whether common polymorphisms in the genes of these proteins could modify the association between fat intake and biomarkers of the MetS. Fat intake was measured using a food frequency questionnaire and genotyping was completed using real-time PCR. The TLR4 Asp299Gly (rs4986790) polymorphism was associated with decreased insulin sensitivity while an intronic polymorphism (rs5030728) modified the association between dietary SFA and HDL-cholesterol. The Nod1 Glu266Lys polymorphism modified the association between dietary SFA and HOMA-IR. These results suggest a role for innate immunity in mediating some of the effects of dietary SFAs on factors associated with the MetS.

Many common cancers have a predisposition for bone metastasis. Tumor occupation of bone is both destructive and a source of debilitating pain in cancer patients. As a result, cancer-induced bone pain (CIBP) is the single most common form of clinical cancer pain. Opioids remain the golden standard for the management of CIBP; however, >30% of cancer patients do not experience adequate pain relief with opioids. Furthermore, clinical reports have suggested that opioids can exacerbate bone loss and increase the likelihood of skeletal-related events. To date, there is no known direct mechanism for opioid-induced bone loss (OIBL). We hypothesized that opioid off-target activation of toll-like receptor 4 (TLR4), an innate immune receptor that is expressed in bone, mediates an increase bone loss and associated CIBP. In the 66.1-BALB/cfC3H murine model of breast cancer bone metastasis, TLR4 expression is upregulated in tumor-burdened bone. Chronic morphine treatment exacerbated spontaneous and evoked pain behaviors in a manner paralleled by bone loss: we identified an increase in spontaneous fracture and osteolysis markers including serum collagen-type I (CTX) and intramedullary receptor activator of nuclear κ-B ligand (RANKL). Administration of (+)naloxone, a non-opioid TLR4 antagonist, attenuated both exacerbation of CIBP and morphine-induced osteolytic changes in vivo. Morphine did not alter tumor burden in vivo or tumor cell growth in vitro. Importantly, morphine produced the in vitro differentiation and activation of osteoclasts in a dose-dependent manner that was reversible with (+)naloxone, suggesting that morphine may contribute directly to osteolytic activation. To improve opioid management of CIBP, we then posited and evaluated three novel adjunct therapeutic targets: cannabinoid receptor-2, adenosine 3 receptor and sphingosine-1-phosphate receptor 1. These pharmacological targets were identified as having a multiplicity of anti-cancer, osteoprotective and/or neuroprotective effects in addition to analgesic efficacy in chronic pain. Targets were tested in the 66.1-BALB/cfC3H model of CIBP and demonstrated to have stand-alone efficacy as antinociceptive agents. Taken together, this work provides a cautionary evaluation of opioid therapy in cancer-induced bone pain and seeks to mitigate opioid side effects through the identification of innovative adjunct therapies that can ultimately improve quality of life in patients suffering from cancer pain.

Dissertation (Ph.D.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 72-96.

Made available in DSpace on 2014-06-11T19:23:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-20Bitstream added on 2014-06-13T20:29:44Z : No. of bitstreams: 1 pagliarone_ac_me_botib.pdf: 396538 bytes, checksum: 70c1ba1ab2097248edf4d300ec8ca0ae (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O estresse pode exercer atividades imunossupressoras, acarretando o desenvolvimento de doenças como câncer, inflamações crônicas, neurodegeneração, autoimunidade, além de maior susceptibilidade a infecções por microrganismos. Os recém-descobertos receptores Toll-like (TLRs) têm sido extensamente investigados recentemente por reconhecer microrganismos patogênicos, ativando, consequentemente, a resposta imune. Devido ao pouco conhecimento quanto ao papel da própolis no sistema imune desafiado pela ação do estresse, o objetivo deste trabalho foi verificar o possível efeito deste apiterápico sobre a produção de citocinas pró-inflamatórias (IL-1β e IL-6) e de padrão Th1 (IFN-γ e IL-2) e Th2 (IL-4 e IL-10), e sobre a expressão do receptor TLR-4 por células esplênicas de camundongos submetidos a estresse. Camundongos BALB/c foram divididos em 3 grupos: G1 (controle), G2 (estresse) e G3 (própolis + estresse). G2 foi submetido a estresse por imobilização por 3 dias consecutivos, e G3 foi tratado com própolis e submetido a estresse. Após o sacrifício, o sangue foi coletado para a dosagem de corticosterona, como indicador de estresse. O baço de cada animal foi extraído e culturas celulares foram estimuladas com LPS por 24h ou com Con A por 48 h para determinação da produção de citocinas. Parte do baço foi utilizada para a realização de PCR quantitativo em tempo real a fim de verificar a expressão gênica de TLR-4 pelas células esplênicas. Os grupos submetidos a estresse, tratados ou não com própolis, apresentaram aumento na concentração de corticosterona. Houve inibição na produção de IL-1β e IL-6 nestes mesmos grupos, em comparação ao controle. Não houve alterações na produção de IL-2 em nenhum dos grupos, enquanto que a de IFN-γ esteve inibida nos grupos submetidos a estresse, tratados ou não com própolis. Já a produção de IL-4... / Stress can exert immunossupressive activities, which lead to the development of diseases such as cancer, chronic inflammation, neurodegenerative dysfunctions, autoimmunity and a higher susceptibility to infectious microorganisms. Recently, Tolllike receptors (TLRs) have been widely investigated for recognizing pathogenic microorganisms and, as a consequence, activating the immune response. Since little is known about propolis effects on the immune system challenged by stress, the aim of this work was to verify the possible effect of this bee product on pro-inflammatory (IL- 1β and IL-6), Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-10) cytokines and on TLR-4 expression by spleen cells from stressed mice. BALB/c mice were divided into 3 groups: G1 (control), G2 (stress) and G3 (propolis + stress). G2 was submitted to restraint stress for 3 consecutive days and G3 was treated with propolis and immediately submitted to stress. After sacrifice, blood was collected for corticosterone determination as a stress indicator. Spleens of all groups were removed and cell cultures were stimulated with LPS for 24h or with Con A for 48 h to further cytokines determination. A portion of the spleens was used for quantitative PCR real time assay in order to verify TLR-4 gene expression. An increased corticosterone concentration was seen in stressed groups, treated or not with propolis. IL-1β and IL-6 production was inhibited in these same groups, comparing to control. No alterations were found in IL-2 production in the experimental groups, while IFN-γ was inhibited on stressed group even when treated with propolis. IL-4 production was inhibited on stressed mice, but propolis treatment was able to antagonize this inhibition. There were no alterations in IL-10 production between the experimental groups. TLR-4 expression was inhibited in stressed mice, but propolis treatment exerted... (Complete abstract click electronic access below)

Orientador: José Maurício Sforcin / Banca: Sandra Cordellini / Banca: Glenda Nicioli da Silva / Resumo: O estresse pode exercer atividades imunossupressoras, acarretando o desenvolvimento de doenças como câncer, inflamações crônicas, neurodegeneração, autoimunidade, além de maior susceptibilidade a infecções por microrganismos. Os recém-descobertos receptores Toll-like (TLRs) têm sido extensamente investigados recentemente por reconhecer microrganismos patogênicos, ativando, consequentemente, a resposta imune. Devido ao pouco conhecimento quanto ao papel da própolis no sistema imune desafiado pela ação do estresse, o objetivo deste trabalho foi verificar o possível efeito deste apiterápico sobre a produção de citocinas pró-inflamatórias (IL-1β e IL-6) e de padrão Th1 (IFN-γ e IL-2) e Th2 (IL-4 e IL-10), e sobre a expressão do receptor TLR-4 por células esplênicas de camundongos submetidos a estresse. Camundongos BALB/c foram divididos em 3 grupos: G1 (controle), G2 (estresse) e G3 (própolis + estresse). G2 foi submetido a estresse por imobilização por 3 dias consecutivos, e G3 foi tratado com própolis e submetido a estresse. Após o sacrifício, o sangue foi coletado para a dosagem de corticosterona, como indicador de estresse. O baço de cada animal foi extraído e culturas celulares foram estimuladas com LPS por 24h ou com Con A por 48 h para determinação da produção de citocinas. Parte do baço foi utilizada para a realização de PCR quantitativo em tempo real a fim de verificar a expressão gênica de TLR-4 pelas células esplênicas. Os grupos submetidos a estresse, tratados ou não com própolis, apresentaram aumento na concentração de corticosterona. Houve inibição na produção de IL-1β e IL-6 nestes mesmos grupos, em comparação ao controle. Não houve alterações na produção de IL-2 em nenhum dos grupos, enquanto que a de IFN-γ esteve inibida nos grupos submetidos a estresse, tratados ou não com própolis. Já a produção de IL-4... (resumo completo, clicar acesso eletrônico abaixo) / Abstract: Stress can exert immunossupressive activities, which lead to the development of diseases such as cancer, chronic inflammation, neurodegenerative dysfunctions, autoimmunity and a higher susceptibility to infectious microorganisms. Recently, Tolllike receptors (TLRs) have been widely investigated for recognizing pathogenic microorganisms and, as a consequence, activating the immune response. Since little is known about propolis effects on the immune system challenged by stress, the aim of this work was to verify the possible effect of this bee product on pro-inflammatory (IL- 1β and IL-6), Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-10) cytokines and on TLR-4 expression by spleen cells from stressed mice. BALB/c mice were divided into 3 groups: G1 (control), G2 (stress) and G3 (propolis + stress). G2 was submitted to restraint stress for 3 consecutive days and G3 was treated with propolis and immediately submitted to stress. After sacrifice, blood was collected for corticosterone determination as a stress indicator. Spleens of all groups were removed and cell cultures were stimulated with LPS for 24h or with Con A for 48 h to further cytokines determination. A portion of the spleens was used for quantitative PCR real time assay in order to verify TLR-4 gene expression. An increased corticosterone concentration was seen in stressed groups, treated or not with propolis. IL-1β and IL-6 production was inhibited in these same groups, comparing to control. No alterations were found in IL-2 production in the experimental groups, while IFN-γ was inhibited on stressed group even when treated with propolis. IL-4 production was inhibited on stressed mice, but propolis treatment was able to antagonize this inhibition. There were no alterations in IL-10 production between the experimental groups. TLR-4 expression was inhibited in stressed mice, but propolis treatment exerted... (Complete abstract click electronic access below) / Mestre

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