MECHANISM AND FUNCTION OF LET-7, A NOVEL MODULATOR OF TH17-DEPENDENT EMPHYSEMA

慢性阻塞性肺疾病（COPD）是21世纪全球日益严重的健康威胁。 肺气肿是一种进行性和破坏性的COPD自身免疫性内型，通常表现为 高碳酸血症和运动受限与显著的发病率和死亡率相关。就像那口井- 建立香烟烟雾（CS）的有害影响，我们已经证明，纳米炭黑， (nCB)由烟草不完全燃烧产生的颗粒可导致肺气肿。机制 nCB或CS介导的肺气肿发展包括肺髓样树突细胞（mDC）的活化， 促进自身反应性辅助性T细胞1（TH 1）、TH 17细胞的分化和减少诱导型调节性T细胞（iTreg） 肺部的细胞。我们已经发现let-7 miRNAs是小鼠和人类中表达的主要miRNAs， 肺和免疫细胞。这一和其他初步调查结果表明，let-7成员批判性地确定（即， “阈值”）的靶基因的不同子集，这取决于它们的聚集表达水平。我们的中央 一种假说认为，在对CS应答中，let-7基因座协同调节肺气肿并协调 通过阈值靶基因表达激活mDC和TH 17细胞。我们将测试我们的假设， 具体目标如下：1。阐明let-7 bc和let-7afd簇在 实验性肺气肿中获得性免疫应答的激活。假设：let-7 bc和let-7afd 簇串联工作，以产生CD 4 + T细胞中靶基因表达的阈值， 稳态TH 17/iTreg平衡、肺部炎症和肺气肿。我们将全面执行 let-7 bc和let-7afd缺陷小鼠的组织病理学和细胞研究以确定let-7机制 nCB或CS治疗后的作用。我们将进一步确定潜在抑制（IL 10，Foxo 1）的靶基因。 或增强（Stat 3和RORγt）炎症和肺气肿表达。2.确定let-7afd的作用 肺气肿先天免疫反应的研究。假设：Let-7用于调节分子 TH 17驱动的肺气肿的编程部分地在mDC中起作用。组织学和分子分析 条件性（let-7 floxed小鼠）和全局簇敲除，let-7转基因小鼠将允许我们 探究let-7调节TH 17/iTreg稳态、凋亡和增殖的机制。 3.确定Let-7afd簇在人类肺气肿中的作用。假设：Let- 肺气肿肺mDCs中的7afd簇促进TH 17炎症和促炎性细胞因子的上调 和共刺激靶基因。我们将辨别减少Let-7afd的病理生理学后果， 表达和总Let-7活性在控制人肺mDC介导的活化中的作用（例如，诱导 CD 86、IL 6），其是TH 17细胞分化所必需的。我们还将研究Let-7afd的作用 使用肺气肿的人mDC作为先导策略过度表达来抑制TH 17炎症。

Chronic obstructive pulmonary disease (COPD) is a major rising global health threat in the 21st century. Emphysema, a progressive and destructive autoimmune endotype of COPD that often presents with hypercapnia and exercise limitation, is associated with significant morbidity and mortality. Like the well- established noxious effects of cigarette smoke (CS), we have demonstrated that nano-sized carbon black (nCB) particles, generated by incomplete combustion of tobacco, can cause emphysema. The mechanism for nCB or CS-mediated emphysema development includes activation of lung myeloid dendritic cells (mDCs) that promote differentiation of autoreactive T helper 1 (TH1), TH17 cells and reduced inducible regulatory T (iTreg) cells in the lungs. We have found that let-7 miRNAs are the dominant miRNAs expressed in mouse and human lung and immune cells. This and other preliminary findings suggest that let-7 members critically determine (i.e., “threshold”) distinct subsets of target genes depending on their aggregate expression levels. Our central hypothesis states that in response to CS, let-7 loci cooperate to modulate emphysema and orchestrate activation of mDCs and TH17 cells by thresholding target gene expression. We will test our hypothesis through the following Specific Aims: 1. Elucidate the intrinsic requirement of the let-7bc and let-7afd clusters in activation of acquired immune responses in experimental emphysema. Hypothesis: The let-7bc and let-7afd clusters work in tandem to generate thresholds in target gene expression in CD4+ T cells and orchestrate homeostatic TH17/iTreg balance, lung inflammation, and emphysema. We will perform comprehensive histopathological and cellular studies of let-7bc- and let-7afd-deficient mice to determine the let-7 mechanism of action after nCB or CS treatment. We will further identify target genes that potentially suppress (IL10, Foxo1) or enhance (Stat3, and RORγt) inflammation and emphysema expression. 2. Determine the role of let-7afd cluster on innate immune responses in emphysema. Hypothesis: Let-7 serves to temper molecular programming of TH17 driven emphysema acting in part in mDCs. Histopathological and molecular analysis of conditional (let-7 floxed mice) and global cluster knockouts, and let-7 transgenic mice will allow us to interrogate the mechanism(s) by which let-7 modulates TH17/iTreg homeostasis, apoptosis, and proliferation. 3. Determine the role of Let-7afd cluster in human emphysema. Hypothesis: Coordinate downregulation of Let- 7afd cluster in emphysematous lung mDCs promotes TH17 inflammation and upregulation of pro-inflammatory and co-stimulatory target genes. We will discern the pathophysiological consequence of reduced Let-7afd expression and overall Let-7 activity in the control of human lung mDC-mediated activation (e.g., induction of CD86, IL6) that is necessary for TH17 cell differentiation. We will also examine the role of Let-7afd overexpression using emphysematous human mDCs as a pilot strategy to inhibit TH17 inflammation.