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

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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.