Cystic Fibrosis (CF) is a Mendelian recessive ?monogenic? disorder caused by mutations in CFTR, with a broad range of lung disease severity. Heritability studies show that genetic variation (?modifier genes?), rather than allelic variation in CFTR, is responsible for most of the variability in CF lung disease severity (heritability estimate = 0.54). In 134 CF patients with genomic SNP data, we performed respiratory (nasal) epithelial transcriptomic mRNA studies to determine modifier genes. This analysis identified heritable differential expression of genes in pathways pertinent to the pathophysiology of CF lung disease, highlighting that dysregulated inflammation is detrimental in CF. However, the mechanism of pathogenesis for these candidate modifier genes remains incompletely understood. miRNA regulation is one undefined mechanism of effect for modifier genes on CF lung disease, and we have now isolated miRNAs from secreted extracellular vesicles (EVs) in readily accessible CF nasal lavage. Building on candidate gene modifiers identified in our transcriptomic studies, we will test the hypothesis that extracellular vesicle (EV) miRNAs are modifiers of inflammatory signaling that contribute to the severity and progression of CF lung disease. We will take a global approach to characterize differential expression of EV miRNAs in CF respiratory secretions, as well as query the mechanistic role of specific candidate EV miRNAs on CF airway disease in vitro.
In Aim 1 we will perform miRNASeq on already obtained nasal lavage biospecimens from 134 CF patients, and identify miRNAs associated with CF lung disease severity. We will determine the effects of these identified miRNAs on gene regulation, using our existing intra-subject nasal mucosal RNAseq data, plus validate candidate miRNAs in an independent CF cohort. In our transcriptomic studies, we have already shown that increased expression of genes in the methionine salvage pathway (also implicated in CF GWAS) is associated with worse CF lung disease. However, the mechanism of influence for this pathway in the pathogenesis of CF remains unknown, presenting the importance of mechanistic validation studies. We show that the key substrate of this pathway, methylthioadenosine (MTA), reduces levels of a top-ranked candidate miRNA-21 (identified by our in silico analysis), in THP-1 macrophage secreted extracellular vesicles (EV). Importantly, miRNA-21 is known to stimulate inflammatory signaling.
In Aim 2, we will test the hypothesis that MTA treatment decreases secreted EV miRNA-21 levels and results in reduced inflammatory cytokine production directly in macrophages, which are critical to CF inflammation. We will further test the effect of these secreted EVs, and the direct effects of reduced miRNA-21, on attenuating the inflammatory response to LPS in human CF airway epithelial cells (AECs) cultured at air-liquid interface. Our proposed studies will elucidate candidate miRNAs as modifiers of CF lung disease severity, and also shed light on novel potential therapeutic targets in CF.
