Made available in DSpace on 2018-08-01T21:34:59Z (GMT). No. of bitstreams: 1 tese_10421_Dissertação_Emmanoel Loss Dias.pdf: 1989385 bytes, checksum: 17589b2593a44ffae93864b2f8f67fe7 (MD5) Previous issue date: 2016-09-05 / PAM3CSK4 (PAM) é um agonista sintético de TLR1/2 composto por um lipopeptídeo triaciletado que imita a lipoproteína de bactérias e que apresenta uma potente capacidade em induzir atividade pró-inflamatória mediada por ativação de NF-kB. No presente estudo, foi investigada a capacidade da imunização intranasal com antígenos totais de promastigotas de L. amazonensis (LaAg) associados ao adjuvante PAM em potencializar a resposta imune imunogênica antígeno em camundongos. Assim, camundongos BALB/c foram imunizados por via intranasal (instilação nasal) com 20 μg de LaAg associados com 20 μg do adjuvante PAM em doses intervaladas por 7 dias. PBS, LaAg ou PAM sozinhos foram utilizados como controle. A dosagem das transaminases e da creatinina no soro animais demonstraram a biocompatibilidade e segurança da combinação LaAg/PAM. Os camundongos vacinados com LaAg/PAM demostraram maior reação de hipersensibilidade cutânea tardia (DTH) 24 e 48 horas, respectivamente, após a injeção de 20 μg de LaAg na pata traseira, quando comparado ao grupo controle. Além disso, em comparação aos outros grupos, os esplenócitos de animais vacinados com LaAg/PAM produziram mais IFN-γ, TNF-α e IL-4 depois do reestimulo in vitro com o antígeno LaAg (50 μg/mL). Células analisadas ex vivo, demonstraram que a combinação LaAg/PAM não alterou a frequência das populações de células CD4+ e CD8+ de memória e totais no baço. A vacinação com LaAg/PAM foi capaz de induzir a redução no parasitismo do fígado, em comparação com o grupo não tratado. Os animais vacinados com LaAg/PAM demostraram um aumento significativo na produção de IFN-γ e a diminuição na produção de IL-4 nos sobrenadantes do baço. Além disso, observou-se um aumento significativo na produção de NO nos sobrenadantes do fígados dos animais vacinados com LaAg/PAM. Em conjunto, nossos dados demonstram, pela primeira vez a possibilidade de imunização intranasal com os antígenos totais de L. amazonensis (LaAg) associados com PAM como um mecanismo eficaz de indução de imunogenicidade.

Interleukin-10 (IL-10) is a pleiotropic cytokine which limits inflammatory responses by balancing the hosts immune response against infection. Mammalian Toll-like receptors (TLRs) are pattern recognition receptors that recognize specific molecular pattens on microbial pathogens and activate intracellular signaling via the transcription factors NF-B and IRF-3. In this study we evaluate the contribution of the TLR ligands Poly I:C, Pam3CSK4, LPS and LTA to IL-10 receptor dysfunction in murine peritoneal macrophages (PM). We examine how these ligands are able to alter IL-10 mediated STAT3 phosphorylation and CCR5 gene expression in PM. The ability of Poly I:C and Pam3CSK4 to alter the immunosuppressive activity of IL-10 in C2-ceramide stimulated PM is also examined. The results of our study indicate a delayed inhibition of IL-10 mediated activation of STAT3 by LPS, LTA, Poly I:C and Pam3CSK4. The CCR5 gene expression experiments demonstrate that LPS was able to down-regulate IL-10 induced CCR5 mRNA expression in PM.

Sepsis is the body’s response to an overwhelming infection and is a serious consequence of critical illness. It can cause tissue damage, organ failure, and death. Sepsis continues to have an unacceptably high mortality rate, due to the lack of effective treatments. Specific therapeutic targets for sepsis remain elusive since the complex functional changes that result in a septic state remain poorly understood. Macrophage Scavenger Receptor A (SRA, CD204) is a surface receptor that binds negatively charged, endogenous and exogenous ligands. We have discovered that SRA plays a significant role in the pathophysiology of sepsis. We have shown that mice with SRA have increased inflammation, decreased survival, and increased bacterial burden compared to SRA deficient mice. We have also found an increase in the expression of SRA on monocytes and macrophages in septic wild type mice. To determine the mechanism responsible for increased SRA expression in sepsis we treated a mouse macrophage cell line, (J774a.1), with mediators that stimulate toll like receptors (TLRs), innate immune receptors which are activated in sepsis. The cells were cultured with ultra pure LPS (a TLR 4 ligand), PAM3CSK4 (a TLR 2 ligand), glucan (a Dectin-1 ligand), ultra pure LPS and PAM3CSK4, or ultra pure LPS and glucan for 24 hours. The cells were stained with an SRA antibody, and flow cytometry was used to measure the SRA expression for each treatment group. LPS treatment alone resulted in a significant increase in SRA expression when compared to control cells. Specifically, LPS increased SRA expression by 53.4% compared to media alone (p

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. In the present study, we examined the effect of Toll-like receptor 2 (TLR2) ligands, peptidoglycan (PGN), and Pam3CSK4 (Pam3) on cardiac function in cecal ligation and puncture (CLP)-induced sepsis in mice. We also investigated whether the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is involved in the effect of TLR2 ligands on cardiac function in CLP mice. PGN was administered to C57B6/L mice 1 h before the induction of CLP. Sham surgically operated mice served as a control. Cardiac function indexes (rate of change in left ventricular pressure, stroke work, cardiac output, and ejection fraction) were examined by a microconductance pressure catheter. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with shamoperated control. In contrast, PGN administration attenuated CLPinduced cardiac dysfunction. Importantly, the therapeutic treatment with Pam3 1 h after CLP also significantly attenuated cardiac dysfunction in CLP mice. However, the beneficial effect of TLR2 ligands on cardiac dysfunction in CLP-mice was abolished in TLR2-deficient mice. PGN administration significantly increased the levels of phospho-Akt and phospho-GSK-3β in the myocardium compared with the levels in untreated CLP mice. PI3K inhibition abolished the PGNinduced attenuation of cardiac dysfunction in CLP mice. In conclusion, these data demonstrate that the administration of TLR2 ligands, PGN, or Pam3 attenuates cardiac dysfunction in septic mice via a TLR2/PI3K-dependent mechanism. More significantly, Pam3 therapeutic treatment will have a potential clinical relevance.

The brain's resistance to ischemic injury can be transiently augmented by prior exposure to a sub-lethal stress stimulus, i.e. preconditioning. It has been reported that Toll-like receptors (TLRs) are involved in the preconditioning-induced protective effect against ischemic brain injury. In this study, we investigated the effect of preconditioning with a TLR2 specific ligand, Pam3CSK4, on focal cerebral ischemia/reperfusion (I/R) injury in mice. Pam3CSK4 was administered systemically 24 h before the mice were subjected to focal cerebral ischemia (1 h) followed by reperfusion. Cerebral infarct size was determined, blood brain barrier (BBB) permeability was evaluated, and expression of tight-junction proteins were examined after focal cerebral I/R. Results showed that pre-treatment with Pam3CSK significantly reduced brain infarct size (1.9 ± 0.5% vs 9.4 ± 2.2%) compared with the untreated I/R group. Pam3CSK4 pre-treatment also significantly reduced acute mortality (4.3% vs 24.2%), preserved neurological function (8.22 ± 0.64 vs 3.91 ± 0.57), and attenuated brain edema (84.61 ± 0.08% vs 85.29 ± 0.09%) after cerebral I/R. In addition, Pam3CSK4 pre-treatment preserved BBB function as evidenced by decreased leakage of serum albumin (0.528 ± 0.026 vs 0.771 ± 0.059) and Evans Blue (9.23 ± 0.72 μg/mg vs 12.56 ± 0.65 μg/mg) into brain tissue. Pam3CSK4 pre-treatment also attenuated the loss of the tight junction protein occludin in response to brain I/R injury. These results suggest that TLR2 is a new target of ischemic preconditioning in the brain and preconditioning with a TLR2 specific ligand will protect the brain from I/R injury.

Innate immune and inflammatory responses contribute to myocardial and cerebral ischemia/reperfusion (I/R) injury. Toll-like receptors (TLRs) play a critical role in the induction of innate immune and inflammatory responses via activation of nuclear factor kappa B (NF-κB). We have shown that activation of NF-κB contributes to myocardial and cerebral I/R injury. Indeed, inhibition of TLR4-mediated NF-κB activation significantly decreased myocardial and cerebral I/R injury via activation of PI3K/Akt signaling. PI3K/Akt signaling is an important pathway in regulating cellular survival and inflammatory responses. Therefore, an important question is how to differentially modulate PI3K/Akt signaling and TLR/NF-κB-mediated signaling pathway during I/R injury? We demonstrated that pretreatment of mice with Pam3CSK4, a specific TLR2 ligand, significantly decreased cerebral I/R injury and improved neuronal functional recovery. Importantly, therapeutic administration of Pam3CSK4 also markedly decreased cerebral I/R injury. The mechanisms involved suppression of NF-κB binding activity and activation of PI3K/Akt signaling. We also demonstrated that CpG-ODN, a specific TLR9 ligand, induced protection against cerebral I/R injury via activation of PI3K/Akt signaling. Our findings were consistent with our previous reports showing that administration of Pam3CSK4 or CpG-ODN protected against myocardial I/R injury via a PI3K/Akt-dependent mechanism. In addition, we demonstrated for the first time that TLR3 located in endosomes played a deleterious role in myocardial I/R injury via activation of NF-κB. To investigate how to activate PI3K/Akt signaling during I/R injury, we examined the role of microRNA (miRs) in regulating PI3K/Akt signaling during myocardial ischemic injury. We discovered that Pam3CSK4 or CpG-ODN treatment significantly increased the expression of miR-130a in the myocardium, while myocardial infarction markedly decreased the levels of miR-130a in the myocardium. The data indicated that miR-130a served a protective role in myocardial ischemic injury. Indeed, we demonstrated for the first time that increased expression of miR-130a significantly attenuated cardiac dysfunction and promoted angiogenesis after myocardial infarction. The mechanisms involved activation of PI3K/Akt signaling via targeting PTEN expression by microRNA-130a. This dissertation discovers novel mechanisms of cerebral and myocardial ischemic injury and provides solid basis for developing new approaches for the treatment and management of stroke and heart attack patients.

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