Autophagy in Cystic Fibrosis Pathogenesis and Treatment

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. In epithelial cells, the CFTR channel conducts anions and plays a critical role in regulating the volume and composition of airway surface liquid. This thin layer of aqueous fluid and mucus covering the airway surface facilitates mucociliary clearance, bacterial killing, and epithelial cell homeostasis. The importance of the CFTR channel in macrophages was revealed in recent work that demonstrated that defective CFTR function is accompanied by impaired innate immune responses to specific infections. Notably, most CF-associated infections are caused by microbes that are cleared by autophagy in healthy cells. Autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens, inflammatory molecules, and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their cells exhibit limited autophagy activity. The mechanisms linking a malfunctioning ion channel function to the defective autophagy remains unclear. In this chapter, we describe and discuss the recent findings indicating the presence of several mechanisms leading to defective autophagy in CF cells. Thus, these novel 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

Caspase-11 Mediates Neutrophil Chemotaxis and Extracellular Trap Formation During Acute Gouty Arthritis Through Alteration of Cofilin Phosphorylation

Gout is characterized by attacks of arthritis with hyperuricemia and monosodium urate (MSU) crystal-induced inflammation within joints. Innate immune responses are the primary drivers for tissue destruction and inflammation in gout. MSU crystals engage the Nlrp3 inflammasome, leading to the activation of caspase-1 and production of IL-1β and IL-18 within gout-affected joints, promoting the influx of neutrophils and monocytes. Here, we show that caspase-11−/− mice and their derived macrophages produce significantly reduced levels of gout-specific cytokines including IL-1β, TNFα, IL-6, and KC, while others like IFNγ and IL-12p70 are not altered. IL-1β induces the expression of caspase-11 in an IL-1 receptor-dependent manner in macrophages contributing to the priming of macrophages during sterile inflammation. The absence of caspase-11 reduced the ability of macrophages and neutrophils to migrate in response to exogenously injected KC in vivo. Notably, in vitro, caspase-11−/− neutrophils displayed random migration in response to a KC gradient when compared to their WT counterparts. This phenotype was associated with altered cofilin phosphorylation. Unlike their wild-type counterparts, caspase-11−/− neutrophils also failed to produce neutrophil extracellular traps (NETs) when treated with MSU. Together, this is the first report demonstrating that caspase-11 promotes neutrophil directional trafficking and function in an acute model of gout. Caspase-11 also governs the production of inflammasome-dependent and -independent cytokines from macrophages. Our results offer new, previously unrecognized functions for caspase-11 in macrophages and neutrophils that may apply to other neutrophil-mediated disease conditions besides gout

An intricate link between autophagy and microRNAs in cystic fibrosis (HUM1P.262)

Abstract Each year 1,000 children and adults are diagnosed with Cystic Fibrosis (CF), a fatal genetic disorder that critically affects the lungs. Autophagy, a highly-regulated biological process, normally functions to clear dysfunctional CFTR (CF transmembrane conductance regulator) proteins that aggregate within macrophages. However, this process is defective in CF patients and CF mice, as their macrophages express limited autophagy activity thus exacerbating inflammation. Present therapies to improve autophagy are ineffective. MicroRNAs (miRNAs, miRs) are non-coding RNAs that post-transcriptionally regulate targeted mRNA expression. The objective for this study is to elucidate the role of miRNAs in CF macrophages in an effort to restore autophagy. We hypothesize CF macrophages exhibit elevated cluster expression that downregulate autophagy targets thus contributing to autophagy dysfunction. We find that, CF macrophages exhibit decreased autophagy protein expression and elevated cluster expression compared to WT. When cluster expression is absent, autophagy protein expression is restored, suggesting the canonical inverse relationship between miRNA and protein expression. Predicted autophagy targets of specific miRs comprising the cluster were validated. In vivo downregulation of specific miRs comprising the cluster increases autophagy expression. Thus, this data demonstrates, microRNA cluster expression correlates to autophagy expression which modulates the pathophysiology of CF.</jats:p

Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr ΔF508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages