Role of microRNAs in B-cell dysfunction in HIV/SIV infection

 描述(由申请人提供)：B细胞功能障碍是HIV/SIV感染的一个标志，甚至在CD4T细胞耗尽之前就开始感染了。越来越多的证据表明，即使在接受抑制性抗逆转录病毒治疗的患者中，B细胞活性增强、缺乏记忆B细胞修复和疫苗诱导的抗体反应受损的情况仍然存在。尽管我们对HIV/SIV感染中B细胞动力学的了解有了很大的提高，但B细胞功能障碍的分子机制仍然知之甚少。除了细胞因子和转录因子外，最近的研究表明miRNA介导的基因调控对B细胞的分化、成熟、激活和自身免疫反应至关重要。对与B细胞缺陷相关的自身免疫性疾病的广泛研究揭示了microRNAs在疾病发病机制中的致病作用，从而使它们被确定为有希望的治疗靶点。然而，miRNAs在HIV诱导的B细胞功能障碍中的作用尚不清楚。我们对这一应用的初步研究发现，来自慢性SIV感染猕猴的纯化B细胞中，与B细胞激活和淋巴肿大相关的miRNAs显著失调。有趣的是，更重要的是，对感染SIV的猕猴进行长期THC治疗显著抑制了它们的表达，这表明它们在减弱免疫(B细胞)激活和减缓疾病进展方面具有巨大的治疗潜力。基于我们强有力的初步数据表明，在HIV/SIV感染中，miRNA在B细胞中表达异常，我们假设这导致B细胞功能障碍，从而促进免疫激活和HIV疾病的进展。此外，我们假设慢性治疗可能通过调节miRNA9的表达来减轻炎症，恢复免疫(B细胞)功能，并减缓艾滋病毒/SIV疾病的进展。这项应用的一个主要目标是确定与B细胞功能障碍和大麻素介导的抑制B细胞激活相关的miRNA机制。该项目有三个具体目标：1)验证以下假设：靶向SIRT1的miR-34a和miR-155导致乙酰化p65和乙酰化FOXO3a表达增强，分别导致B细胞过度活动和凋亡。2)在SIV感染后的三个不同时间点，确定大麻素抑制血液、淋巴结和肠道B细胞激活的miRNA机制。3)检测联合应用抗逆转录病毒治疗(CART)和慢性-9-Thc治疗对炎症、慢性免疫激活、病毒复制、miRNA表达、SIRT1水平、B细胞活化和细胞凋亡的影响。这项拟议的研究是新颖的，并应用最先进的免疫学和分子方法来解决我们在理解B细胞功能障碍的转录后机制方面的重大差距。此外，更重要的是，这一结果将加深我们对大麻素抗炎作用机制的理解，并对艾滋病毒和其他B细胞缺陷自身免疫性疾病的靶向免疫调节具有重要的治疗意义。

 DESCRIPTION (provided by applicant): B-cell dysfunction is a hallmark of HIV/SIV infection that begins very early in infection even before CD4 T cell depletion. Increasing evidence suggests that heightened B-cell activation, lack of memory B-cell restoration and impaired vaccine induced antibody responses persists even in patients receiving suppressive anti- retroviral therapy. Even though our understanding of B-cell dynamics in HIV/SIV infection has significantly improved, the molecular mechanisms underlying B-cell dysfunction remain poorly understood. In addition to cytokines and transcription factors, recent studies show miRNA-mediated gene regulation to be critical for B- cell differentiation, maturation, activation and autoimmune responses. Extensive studies in autoimmune diseases associated with B-cell defects have revealed a causative role for microRNAs in disease pathogenesis resulting in their identification as promising therapeutic targets. However, the role of miRNAs in HIV induced B-cell dysfunction is lacking. Our preliminary studies for this application identified significant dysregulation of miRNAs linked to B-cell activation and lymphomagenesis in purified B-cells from chronically SIV-infected macaques. Interestingly and more importantly, chronic THC treatment to SIV-infected macaques markedly inhibited their expression suggesting their immense therapeutic potential for attenuating immune (B-cell) activation and slowing disease progression. Based on our strong preliminary data demonstrating that miRNA expression is dysregulated in B-cells in HIV/SIV infection, we hypothesize that this leads to B-cell dysfunction which contributes to immune activation and HIV disease progression. Further, we hypothesize that chronic 9- THC treatment may reduce inflammation, restore immune (B-cell) function, and slow HIV/SIV disease progression by modulating miRNA expression. A major goal of this application is to identify miRNA mechanisms associated with B-cell dysfunction and cannabinoid mediated suppression of B-cell activation. The project has three specific aims: 1) Test the hypothesis that miR-34a and miR-155 targeting of SIRT1 leads to enhanced acetylated-p65 and acetylated-FOXO3a expression, causing B-cell hyperactivity and apoptosis, respectively. 2) Identify miRNA mechanisms of cannabinoid induced suppression of B-cell activation in blood, lymph nodes, and intestine at three different time points following SIV infection. 3) Determine the effect of combination anti-retroviral treatment (cART) in conjunction with chronic 9-THC administration on inflammation, chronic immune activation, viral replication, miRNA expression, SIRT1 levels, B-cell activation and apoptosis. The proposed research is novel and applies state of the art immunological and molecular approaches to address a significant gap in our understanding of the post-transcriptional mechanisms underlying B-cell dysfunction. Additionally and more importantly, the results will deepen our understanding of the mechanisms underlying the anti-inflammatory effects of cannabinoids and have important therapeutic implications for targeted immune modulation in HIV and other autoimmune diseases with B-cell defects.