Role of microRNAs in opioid drug abuse induced persistent Inflammation and HIV di

描述(由申请人提供)：慢性药物使用与艾滋病毒感染易感性的增加有很强的相关性。在美国，慢性吸毒者约占所有艾滋病毒病例的三分之一，阿片类药物滥用者向艾滋病的进展明显加快。慢性免疫激活伴随着血清促炎细胞因子水平的升高是进展性疾病的标志。最近的研究表明，肠道细菌易位导致的血浆内毒素(LPS)升高可能是HIV感染中免疫激活的原因。已发现免疫激活和T细胞激活/耗竭之间存在相关性。有趣的是，研究表明，与不使用毒品的HIV感染者相比，静脉注射海洛因的HIV患者血清中的内毒素水平更高。同样，我们在药物滥用的小鼠模型中显示，与安慰剂治疗的动物相比，循环中的内毒素水平更高。这些效应在TATtg小鼠身上进一步增强。正如在人类患者身上观察到的那样，这些动物模型的免疫激活也延长并持续了下来。这些结果暗示了内毒素在药物滥用HIV感染者的疾病进展中所起的作用。然而，矛盾的是，有充分的证据表明，长时间的内毒素暴露会导致对内毒素的耐受，同时减少内毒素诱导的巨噬细胞刺激。然而，在HIV患者和HIV动物模型中，持续的免疫激活是疾病进展的标志。这种不一致的观察背后的机制是知识上的重大差距。在内毒素耐受中，转录和翻译抑制事件结合在一起，严格调控促炎基因。最近，microRNAs被认为是在转录后调控水平上控制各种生物过程的负调控因子。我们在初步数据中显示，在长期用脂多糖处理的动物中，两个关键的miRNAs miR-155和miR-146a的诱导作用显著。相比之下，在接受吗啡或TAT治疗的动物中，miR-155和miR-146a的表达都显著下调。脂多糖激活Toll样受体4，诱导促炎细胞因子和趋化因子的转录(32-34)。脂多糖诱导miR-155和miR-146a可能作为TLR4表达和信号的负反馈调节因子，从而防止促炎细胞因子的过度激活。我们假设，抑制内毒素诱导的miR-155和miR-146a由吗啡和TAT诱导，解除了抑制反馈环的调节，导致持续的TLR4表达和信号传递(图1)。在特定的目标1：我们将测试假设，即内毒素诱导miR-155抑制TLR4的表达，并建立吗啡和TAT介导的miR-155的减弱导致TLR4表达增加和持续。具体目标2：我们将验证一种假设，即脂多糖诱导miR-155和miR-146a作为TLR4信号的负反馈调节因子，吗啡和TAT调节miR-155和146a导致持续的TLR4信号。特异性目标3：确定吗啡和TAT调节脂多糖诱导miR-155和146a表达的机制。具体目的4：利用WT和转基因动物模型，在体内确定吗啡和TAT对脂多糖诱导的miR-155和miR-146a的调节机制和结果。这些研究的结果将有助于开发新的治疗策略，以减弱艾滋病毒感染患者和艾滋病毒感染药物滥用人群的免疫激活和逆转艾滋病毒疾病的进展。

DESCRIPTION (provided by applicant): There is a strong correlation between chronic drug use and increased susceptibility to HIV infection. Chronic drug users account for approximately one third of all cases of HIV in the USA and progression to AIDS is markedly accelerated in opiate drug abusers. Chronic immune activation with increased serum levels of proinflammatory cytokines is a hallmark of progressive disease. Recent studies show that elevated plasma endotoxin (LPS), a consequence of gut bacterial translocation, may be the likely cause of immune activation in HIV infection. A correlation has been found between immune activation and T cell activation/depletion. Interestingly, studies show that HIV patients that use intravenous heroin display higher serum levels of LPS when compared to non-drug using HIV infected patients. Similarly, we show in a murine model of drug abuse, higher circulating levels of endotoxin when compared to placebo treated animals. These effects are further potentiated in the TATtg mice. As observed in human patients, immune activation in these animal models were also prolonged and sustained. These results implicate a role for LPS in driving the disease progression in drug abusing HIV infected patients. Paradoxically, however, it is well documented that, prolonged LPS exposure leads to LPS tolerance with decreased LPS induced macrophage stimulation. Yet, sustained immune activation is a hallmark of disease progression in both HIV patients and in animal models of HIV. The mechanism underlying this discordant observation is a significant gap in knowledge. In LPS tolerance, transcriptional and translational repressive events combine to tightly regulate proinflammatory genes. More recently, microRNAs have been implicated as negative regulators controlling diverse biological processes at the level of post-transcriptional regulation. We show in preliminary data a significant induction of two key miRNAs, miR-155 and miR-146a in animals that are chronically treated with LPS. In contrast, both miR-155 and miR-146a are significantly down regulated in animals that are morphine or TAT treated. LPS activates Toll like receptor 4 to induce transcription of proinflammatory cytokines and chemokines (32-34). LPS induction of miR-155 and miR-146a may act as negative feedback regulators of TLR4 expression and signaling thereby preventing excessive activation of pro-inflammatory cytokines. We hypothesize, that suppression of LPS induced induction of miR-155 and miR-146a, by Morphine and TAT, deregulates the inhibitory feedback loop resulting in sustained TLR4 expression and signaling (Fig. 1). In Specific aim 1: We will test the hypothesis that LPS induction of miR-155 inhibits TLR4 expression and establish that morphine and TAT-mediated attenuation of miR-155 results in greater and sustained TLR4 expression. Specific aim 2: We will test the hypothesis that LPS induction of miR-155 and miR-146a acts as negative feedback regulators of TLR4 signaling and morphine and TAT modulation of miR-155 and 146a results in persistent TLR4 signaling. Specific Aim 3: Determine the mechanism by which Morphine and TAT modulate LPS induced miR-155 and 146a expression. Specific Aim 4: Determine the mechanism and consequence of morphine and TAT modulation of LPS induced miR-155 and miR-146a in vivo using WT and Transgenic animal models. The results from these studies will allow for the development of new therapeutic strategies to attenuate immune activation and reverse HIV disease progression both in HIV infected patients and in HIV infected drug abusing population.