Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages

Abstract

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection

“…Nevertheless, WT macrophages successfully restrict B. cenocepacia growth, as functional autophagy machinery is restored in WT but not in CF cells [5,41]. Reduced autophagy activity has been implicated in dysfunctional cellular immunity to intracellular pathogens including the bacteria L. pneumophila, M. tuberculosis, L. monocytogenes , and parasites including Toxoplasma gondii [7,42,43].…”

“…B. cenocepacia ΔT6SS was kindly provided by Dr. Valvano at Queen’s University, Belfast, UK. All bacterial strains were grown overnight in LB medium at 37°C and 200 rpm as previously described [5,14,41,65]. …”

“…For the generation of primary bone marrow-derived macrophages from mice, tibias and femurs were flushed with IMDM medium (Thermo Fisher Scientific, 12,440,053) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, 16,000,044), 50% L cell-conditioned medium [66,67], 0.6x MEM non-essential amino acids (Thermo Fisher Scientific, 11,140,050) and 0.1% penicillin and streptomycin (Thermo Fisher Scientific, 15,140,122) as previously described [5,16,25,30,41,65]. Cells were cultivated at least 6 days at 37°C in a humidified atmosphere containing 5% CO 2 .…”

“…To stain intracellular B. cenocepacia strain MH1K, live macrophages were incubated with 1 μg/mL Hoechst in PBS for 20 min and subsequently fixed with 4% paraformaldehyde. Images were captured using a laser scanning confocal fluorescence microscope with a 60X objective (Olympus Fluoview FV10i) as previously described [25,41,65]. Intensities of target proteins were measured using ImageJ Software.…”

“…Corresponding secondary antibodies conjugated with horseradish peroxidase (Cell Signaling Technology, 7074; Santa Cruz Biotechnology, sc-2006) and in combination with enhanced chemiluminescence reagent (Amersham, RPN2209,) were used to visualize protein bands. Densitometry analyses were performed by normalizing target protein bands to their respective loading control (GAPDH) using ImageJ software as previously described [16,25,41]. For CASP4, graphs display average values of the upper and lower band.…”