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

慢性阻塞性肺疾病(COPD)是21世纪日益严重的全球性健康威胁。 肺气肿是慢性阻塞性肺疾病的一种进行性和破坏性的自身免疫内型，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有关。就像那口井- 建立了香烟烟雾(CS)的有害作用，我们已经证明了纳米碳黑 烟草燃烧不完全产生的(NCB)颗粒会导致肺气肿。该机制的目的是 NCB或CS介导的肺气肿的发生包括肺髓系树突状细胞(MDCs)的激活 促进自身反应性T辅助细胞1(TH1)、TH17细胞分化和减少诱导调节性T细胞(ITreg) 肺部的细胞。我们发现let-7miRNAs是在小鼠和人类中表达的主要miRNAs 肺和免疫细胞。这一和其他初步发现表明，LET-7成员关键地确定(即， “阈值”)目标基因的不同子集取决于它们的总表达水平。我们的中央 假说认为，作为对CS的反应，让-7基因座协同调节肺气肿和协调 通过靶基因表达阈值激活MDCS和TH17细胞。我们将通过以下方式验证我们的假设 具体目标如下：1.阐明LET-7BC和LET-7AFD星团的内在要求 实验性肺气肿患者获得性免疫反应的激活。假设：LET-7BC和LET-7AFD 这些簇协同工作，在CD4+T细胞中产生目标基因表达的阈值，并协调 动态平衡TH17/iTreg平衡、肺部炎症和肺气肿。我们将全面执行 Let-7BC和let-7afd缺陷小鼠的组织病理学和细胞学研究以确定let-7机制 在NCB或CS治疗后的作用。我们将进一步确定可能抑制(IL10、Foxo1)的靶基因 或增强(STAT3，和rORγt)炎症和肺气肿的表达。2.确定LET-7afd的作用 肺气肿患者先天免疫反应的聚集性。假设：let-7用于锻炼分子 TH17驱动的肺气肿部分作用于MDCS的编程。组织病理学和分子生物学分析 条件性(let-7小鼠)和全局簇基因敲除，以及let-7转基因小鼠将允许我们 询问let-7调节Th17/iTreg动态平衡、细胞凋亡和增殖的机制(S)。 3.确定let-7afd簇在人类肺气肿中的作用。假设：协调下调Leat- 肺气肿肺MDCS中7afd簇促进TH17炎症和促炎因子上调 和共刺激靶基因。我们将明确减少let-7afd的病理生理后果 人肺MDC介导的激活(例如，诱导)中的表达和总体let-7活性 CD86、IL6)是TH17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MDCS作为抑制TH17炎症的试点策略进行过度表达。

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.