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.
- cystic fibrosis (CF)
- Rab GTPases (Rabs)
- CF-associated bacteria
- autophagy therapeutics
Cystic fibrosis (CF) is the most common life-threatening genetic disease in North America and Europe. The birth prevalence of CF is estimated to be one in 3500–4500, with 200–300 new cases each year in Europe. The typical form of CF is diagnosed during early childhood and is characterized by recurrent pulmonary infections, pancreatic insufficiency, and elevated chloride concentrations in sweat. CF is a multi-organ disorder, however infections often occur in the lungs accompanied with severe inflammation and tissue destruction . CF is directly caused by mutations in the
The previous commonly accepted hypothesis for CF pathology is the excessive secretion of thick mucus that remains in the lungs and is accompanied by impaired mucociliary clearance. This viscous mucus layer predisposes CF patients to chronic pulmonary infections. An intriguing speculation arose from the specificity of organisms that tend to infect CF patients. We hypothesized that the susceptibility of CF patients to these infectious agents is due to weak autophagic activity since most of the organisms that tend to cause chronic infection in CF are controlled by autophagy in healthy cells [4–6]. Autophagy is a highly regulated biological process that provides energy during periods of stress and starvation  and is typically induced upon glucose or amino acid starvation. Autophagy clears pathogens, inflammatory molecules, and dysfunctional protein aggregates within macrophages. Autophagy proceeds through sequential steps that begin with the formation of the phagophore or isolation membrane at a pre-autophagosomal structure (discussed in chapter of this book) . The nascent autophagic membrane elongates to form a double-membrane autophagosomes that captures regions of cytoplasm, damaged mitochondria, or aggregated proteins. Upon maturation, the autophagosome containing the isolated cargo then fuses with the lysosome to form a single membrane compartment called the autolysosome. The autophagosomal cargo is then degraded in this compartment by lysosomal acid hydrolases and other degradative enzymes. The resulting degradation products including free amino acids, fatty acids, and nucleotides are released to the cytoplasm by the action of lysosomal permeases, where they may be reutilized for anabolic pathways [9, 10]. However, this process is impaired in CF patients and CF mice, as their macrophages and epithelial cells exhibit limited autophagic activity. The mechanisms linking a malfunctioning ion channel to the defective autophagy remains unclear.
Until recently, it was believed that the production of thick mucus and the impairment of mucociliary clearance was the main underlying culprit, allowing the persistence of specific infections in the CF lung. The idea of the existence of an innate immune deficiency disorder in CF was not examined until lately. The discovery that the innate immune functions of macrophages and neutrophils are disrupted in CF was a turning point in the CF field and in understanding the pathobiology of CF. Several years were consumed to provide what is now undisputable data confirming that CF should be considered an innate immune disorder.
In this chapter, we describe and discuss the recent findings in the field, which demonstrate that CF is a newly recognized innate immune deficiency disorder. We will discuss several reports demonstrating that macrophage functions are disrupted in CF contributing to the pathobiology of the disease. This chapter, encompassing recent data in CF, suggests that targeting autophagy may be exploited as a novel strategy for treatment of CF.
2. Cystic fibrosis
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is an integral membrane glycoprotein that functions as a cAMP-activated and phosphorylation-regulated Cl channel at the apical membrane of epithelial cells. CFTR is a member of the ATP-binding cassette transporter superfamily. It is a multi-domain glycoprotein whose biosynthesis, maturation, and functions involve multi-level posttranslational modifications and complex folding processes to reach its native, tertiary conformation. The topology of CFTR includes two transmembrane-spanning domains, two nucleotide-binding domains, and a regulatory domain, which is a unique feature among ATP-binding cassette transporters (Figure 1) . The newly synthesized CFTR emerges out of the ribosome and is targeted through the signal recognition particle to the ER membrane translocon [12, 13]. The CFTR polypeptide chain emerges into the ER lumen, and its glycosylated helping stabilize the protein. CFTR follows the secretory pathway through the Golgi in order to reach the plasma membrane . The recycling of internalized CFTR channels is important for maintaining a functional pool of CFTR at the plasma membrane.
Only 20–40% of the nascent chains achieve folded conformation, whereas the remaining molecules are targeted for degradation by endoplasmic reticulum, lysosomes, or autophagy. A large number of mutations impair processing of CFTR. Growing knowledge of CFTR biosynthesis has enabled understanding of the cellular basis of CF and has brought to light various potential targets for novel and promising therapies . The most common mutation leading to CF is deletion of phenylalanine at position 508 (F508del) and is present in over 70% of the CF alleles [2, 3]. As mutant CFTR is targeted for degradation by the proteasome, the formation of protein aggregates occurs, hence provoking an unfolded protein response (UPR) . Other common mutations such as the G551D exhibit defective CFTR function, however it does not aggregate or elicit a UPR. Comparing the phenotypes and immune functions of the F508del and G551D mutants will discern if defective bacterial clearance and uncontrolled inflammation are due to defective channel function, UPR, or both.
Hyper-inflammation and failure to clear infection is recognized as a leading cause of lung tissue destruction in CF  that can be explained, at least in part, by incompetent autophagy machinery in cells with a dysfunctional CFTR channel . Bronchoalveolar lavages from CF patients contain high levels of the pro-inflammatory cytokine interleukin (IL)-1β [1, 19–26]. IL-1β is primarily expressed as a precursor inactive molecule that is later cleaved by caspase-1 to yield active 17-kDa IL-1β . The biological activities of IL-1β include promoting inflammatory responses and leukocyte infiltration. Autophagy directly regulates the level of pro-IL-1β in response to lipopolysaccharide (LPS) and infections [28, 29]. Interestingly, stimulation of autophagy by rapamycin dramatically reduced signs of inflammation in the murine CF lung [30, 31].
Several reports demonstrated multiple malfunctions in adaptive and innate immune responses in CF. Lack of functional CFTR in CD3+ lymphocytes leads to aberrant cytokine secretion and hyper-inflammatory adaptive immune responses  while producing high levels of IL-1β [1, 19]. Naive cystic fibrosis T cells are intrinsically predisposed to differentiate toward a Th17 phenotype . Therefore, CF is a multifaceted immune deficiency disease.
3. Rabs and the cytoskeleton: common modulators or innocent bystanders for autophagy and CFTR trafficking?
Rab (Ras-related proteins in brain) proteins are key regulators of both vesicular transport and trafficking of proteins . Several Rab GTPases have been implicated in the regulation of the intracellular transport and the plasma membrane delivery of CFTR. The trafficking of CFTR from the plasma membrane to early endosomes is controlled by RAB5 [35, 36]. RAB7 regulates the movement of CFTR away from the recycling pathway and into late endosomes and also from late endosomes to lysosomes for degradation . RAB9, however, can move CFTR away from lysosomal degradation by mediating its transport from late endosomes back to the trans-Golgi, from which CFTR may reenter the secretory pathway leading to plasma membrane insertion .
Growing knowledge of CFTR biosynthesis has enabled understanding of the cellular basis of CF and has brought to light various potential targets for novel and promising therapies . Although some
4. “Eat-me” signaling molecules: Are they trapped in CFTR aggregates?
The major molecular regulator of autophagy in response to starvation or energy depletion include the mammalian target of rapamycin complex 1 (mTORC1). Inhibition of mTORC1 by starvation or rapamycin results in the activation of autophagy and the start of autophagosome formation. The capacity of autophagy to clear intracellular pathogens such as bacteria, viruses, and parasites is collectively referred to as xenophagy , whereas the selective autophagic degradation of mitochondria is denoted as mitophagy and that of protein aggregates is termed aggrephagy. But how do autophagosomes find their targets? The modification of targets by ubiquitination represents a signal for selection of substrates to the autophagy pathway.
Mammalian cells ubiquitinate bacteria that erroneously enter the cytosol or their containing vacuole and target them for destruction by autophagy. Adaptors, including p62/SQSTM1 , optineurin (OPTN) , NBR1 (neighbor of BRCA1 gene 1) [44–46], and NDP52 (nuclear dot protein 52 kDa) , mediate “eat-me” functions by promoting autophagic sequestration of cargo. The adaptor molecule p62 is a ubiquitously expressed cellular protein and its quantity is critical for cell viability . p62 has multiple protein-protein interaction domains, including the ubiquitin-associated domain for binding of ubiquitinated cargo and a LC3 interaction region for binding Atg8/LC3 . p62 plays a role in amino acid sensing and the oxidative Stress response, in addition to its function as an autophagy receptor for ubiquitinated cargos . Most p62 protein in the healthy cell is distributed in the cytoplasm. In response to various stressors though, it is translocated to autophagy substrates such as protein aggregates, damaged mitochondria, and intracellular bacteria . Then, through its LC3-binding domain, p62 engages autophagosomes.
Autophagy is responsible for the degradation of p62. Therefore, impairment of autophagy is usually accompanied by massive accumulation of p62 followed by the formation of aggregate structures positive for p62 and ubiquitin . This accrual is a defining characteristic of impaired autophagy. Aggregation occurs due to both the predilection for self-oligomerization and the ubiquitin-binding capabilities of p62 . Notably, p62 accumulates in CF macrophages and promotes the sequestration of mutant CFTR (Figure 1). These aggregates, in turn, consume important autophagosome-needed proteins, such as BECN1 and LC3 [30, 31]. Recent reports demonstrate that the adapter protein NDP52 directly binds to ubiquitinated bacteria and facilitates the assembly of an autophagic membrane that surrounds these invaders . Interestingly, NDP52 can also bind ubiquitinated bacteria-containing vacuole when p62 is drastically reduced [30, 31]. Optineurin can mediate the removal of protein aggregates through an ubiquitin-independent mechanism. In addition, this protein can induce autophagy upon overexpression or mutation . NBR1 and p62 cooperate in the sequestration of misfolded and ubiquitinated proteins in p62 bodies and are both required for their degradation by autophagy. Recently, NBR1 was found to be necessary and sufficient for pexophagy [44–46]. Whether NBR1, optineurin, and NDP52 play important roles in CF-associated autophagy is still unknown.
5. Function of CFTR channel in epithelial cells
CFTR is an anion channel permeable to chloride and bicarbonate [2, 52]. Upon activation, CFTR transports chloride following its electrochemical gradient. In the lung, CFTR is expressed at the apical membrane of bronchial cells where it regulates chloride transport and fluid homeostasis . The absence of functional CFTR in the lung results in abnormal surface hydration and decreased airway surface fluid. Thus, mutations in the CFTR protein results in the accumulation of thick mucus at the surface of epithelial cells, leading to impairment of pathogen clearance and dysregulated inflammatory responses that in turn results in chronic infection and inflammation . In addition, epithelial cells expressing mutant CFTR exhibit weak autophagy activity.
6. Function of CFTR channel in macrophages
Macrophages are central innate immune cells that engulf invaders within a vacuole and target them to fuse with the lysosome for degradation. Therefore, lysosomes contribute to antimicrobial capacities by fusing with the pathogen-containing, intracellular vacuole . Lysosomes are acidic compartments filled with various acid hydrolases, NADPH oxidases, and oxygen radicals that degrade and break down proteins, lipids, and polysaccharides.
The lung disease seen in CF was accordingly suggested to result in part from lysosomal dysfunction , yet the exact location and function of CFTR in macrophages are still debatable. A report showed impaired bacterial killing due to impaired function of antibacterial proteins at low pH in a CF pig model . Another study demonstrated that acidification requires anion transport through CFTR . More recently, defective lysosomal acidification was also invoked as the mechanism underlying CF lysosome malfunction . Others failed to confirm the involvement of CFTR in acidification [60, 61]. Both these reports are plausible. When macrophages are infected with autophagosome-resident organisms such as
7. CFTR in neutrophils
Polymorphonuclear neutrophils (PMNs, neutrophils) are responsible for the earliest innate immune response to infection and most of their antimicrobial activity against ingested microbes is confined within phagosomes. However, exuberant neutrophil activation can culminate in extracellular release of oxidants and granule contents that leads to local damage to healthy tissue. Neutrophils function to kill microbes through compartmentalization by use of membrane-bound phagosomes, where toxic oxidants such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are generated . The azurophilic granule protein myeloperoxidase (MPO) catalyzes the oxidation of Cl− to form HOCl. Neutrophils predominate in the CF patient’s lung and is a major contributor to the inflammation and destruction of the lung . Yet, despite a successful inflammatory response, neutrophils fail to eradicate invading microbes in the CF lung . CFTR dysfunction results in impaired intraphagosomal HOCl production and neutrophil-mediated microbial killing. Notably, the
In the CF lung, there is overproduction of the neutrophil chemotactic cytokine interleukin 8 (IL-8). This leads to excessive infiltration of neutrophils . Lung infections cause significant morbidity and mortality in patients with CF, even in the presence of neutrophil infiltration into infected lungs. Thus, disturbance in innate neutrophil function in CF includes excessive recruitment , hyper-production of oxidants, and increased release of degradative enzymes . Thus, it is evident that CF is indeed an innate immune disorder involving several malfunctioning immune cells.
8. Autophagy and cystic fibrosis
Several autophagy proteins are scarcely expressed in CF cells, yet the underlying mechanism is undefined [30, 31]. This strongly suggests the presence of an epigenic regulation that targets autophagy mRNA in cells bearing mutant F508del-CFTR. Considering the strong implications of microRNAs (miRs) in autophagy [28, 29, 68–70] and given the increasing evidence showing reduced expression of essential autophagy proteins in CF cells, we performed an
In airway epithelial cells, absence of functional CFTR increases oxidative stress and transglutaminase 2 (TGM2), a calcium-dependent enzyme that creates intra- or intermolecular covalent bonds between proteins. TGM2-mediated cross-linking causes sequestration of BECN1 and its accumulation in histone deacetylase-6 (HDAC6), p62, and ubiquitin-containing cytoplasmic aggresomes. BECN1 sequestration in aggresomes results in the dislodgement of class III PtdIns3K complexes from the endoplasmic reticulum, thereby inhibiting autophagy [30, 81]. In addition, the sequestration of BECN1 within F508-CFTR protein aggregates deprives the cell for an essential factor needed for autophagosome formation.
High levels of p62 promote the aggregation of mutant F508del-CFTR sequestering several autophagy molecules such as BECN1. This p62 buildup could be due to reduced recycling in CF macrophages as a consequence of compromised autophagosome formation and maturation. Notably, p62 downregulation disassembles mutant CFTR and autophagy factors, thus improving autophagy activity and allowing the maturation and trafficking of CFTR to the cell surface in epithelial cells and bacterial clearance in macrophages [30, 31]. Similarly, genetic manipulation or autophagy stimulatory proteostasis regulators such as cystamine restore BECN1 availability and detangle SQSTM1/p62, which partially rescues F508del CFTR function in airway epithelial cells and reduces lung inflammation .
9. Cystic fibrosis and infection
There is evidence for autophagy dysregulation in a variety of disease states, including cancer, neurodegenerative diseases, chronic granulomatous diseases, infectious diseases, and autoimmune disorders [83–85]. For this reason, therapeutic modulation of autophagy is of great interest. Autophagy has emerged as a central component of the innate and adaptive immune responses where it plays roles in direct and indirect killing of intracellular and extracellular pathogens, the generation of bactericidal peptides, and antigen presentation . Functions of autophagy that are compromised in CF include bacterial clearance, degradation of protein aggregates, and the elimination of dysfunctional mitochondria. Thus, the restoration of autophagy will have positive therapeutic effects in CF.
Patients with CF are susceptible to Nontypeable
In healthy cells,
NTM strains infect between 5 and 22% of CF patients and are a growing concern among CF populations due to their increasing prevalence and multi-drug resistance. Infection is often associated with poor clinical outcomes . Although autophagy contributes to clearance of
NTHi chronically colonizes the airways of CF patients at a very young age. Recent reports suggest that autophagy may be actively subverted by NTHi by an unknown mechanism.
On the other hand, following phagocytosis, the degradation of
10. Targeting autophagy in CF improves CFTR function and bacterial clearance
Many of the opportunistic bacteria that infect the CF lung have employed mechanisms to target phagolysosomal maturation and/or autophagy, suggesting that these are major barriers that need to be overcome. Activation of autophagy through rapamycin treatment has shown efficacy in promoting clearance of certain bacteria
Rapamycin was developed as an antifungal agent but its use was abandoned due to the potent immunosuppressive and antiproliferative properties. Recently, it was found that rapamycin inhibits mTOR and therefore induces autophagy . Rapamycin promotes clearance of the CF-associated pathogens
Among the most common autophagy-stimulating compounds were the antipsychotic drugs bromperidol, metergoline, thioridazine, and chlorpromazine. However, the psychoactive nature of these compounds and their potentially life-threatening side effects limit their utility, but nevertheless provide a strong theoretical basis for future drug development [109, 110].
Metformin is a drug that activates AMPK and therefore stimulates autophagy via TORC1-dependent and TORC-1-independent methods [111, 112]. Metformin probably has many other mechanisms of action that cannot be explained by the induction of autophagy. Metformin and resveratrol activate SIRT1 that in turn activates autophagy [113, 114].
Certain anticancer drugs have also been found to stimulate autophagy. For example, Perifosine inhibits mTOR signaling through a different mechanism than classical mTOR inhibitors such as rapamycin , whereas Tamoxifen, an antagonist of the estrogen receptor, is known to induce autophagy . Tamoxifen stimulates autophagy by increasing the intracellular level of ceramide, which inhibits mTOR activation and/or stimulates expression of Atg genes.
The second generation of selective histamine H1-receptor antagonist astemizole is a potent inducer of autophagy at biologically achievable concentrations . Astemizole exhibits antifungal activity and antimalarial properties making it attractive option for CF patients even though the mechanism by which it activates autophagy is still unclear [117, 118]. Safety and drug interaction profiles of astemizole are well characterized. However, due to the availability of superior next-generation histamine receptor agonists, it is not commonly used in Europe or North America.
The potential application of the TGM2 inhibitor cystamine in CF patients has recently been reviewed. Cystamine restores normal autophagy in CFTR-deficient cells and mouse models. Cystamine also restores normal trafficking of the F508del-CFTR and stabilizes the expression of the protein at the plasma membrane of airway epithelial cells . In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and epigallocatechin gallate (EGCG) restored the levels of the autophagy molecules BECN1 and p62 and improved CFTR function from nasal epithelial cells
11. Concluding remarks
There is now strong evidence that immune cells, such as macrophages and neutrophils, are intrinsically impaired in CF. We therefore, recommend that CF be added to the list of innate immune disorders. In fact, autophagy, an intracellular degradation process that contributes to bacterial clearance, protein degradation, and cell survival, is defective in CF. Therefore, we propose that altered autophagy in CF contributes to chronic lung infection and inflammation. The CF field is in desperate need for an approach to correct autophagy in CF patients. The autophagy-correcting agents should ameliorate the marginally positive effects of correctors (therapies that correct the trafficking defect of F508del CFTR) and activators (compounds that activate the F508del CFTR that reaches the cell membrane) typically used in CF. Thus, the development of safe novel autophagy stimulating agents will improve the clinical outcome of CF and promote the clearance of infectious agents.
Berger M (2002) Inflammatory mediators in cystic fibrosis lung disease. Allergy Asthma Proc 23: 19–25.
Rich DP, Gregory RJ, Cheng SH, Smith AE, Welsh MJ (1993) Effect of deletion mutations on the function of CFTR chloride channels. Receptors Channels 1: 221–232.
Welsh MJ, Denning GM, Ostedgaard LS, Anderson MP (1993) Dysfunction of CFTR bearing the delta F508 mutation. J Cell Sci Suppl 17: 235–239.
Campoy E, Colombo MI (2009) Autophagy in intracellular bacterial infection. Biochim Biophys Acta 1793: 1465–1477.
Amer AO (2013) The many uses of autophagosomes. Autophagy 9: 633–634, in press.
Abdelaziz HAD, Khalil H, Cormet-Boyaka E, Amer A (2015) The cooperation between the autophagy machinery and the inflammasome to implement an appropriate innate immune response: Do they regulate each other? Immunological Reviews 265: 194–204. Accepted.
Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, et al. (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12: 1–222.
Mizushima N, Noda T, Yoshimori T, Tanaka Y, Ishii T, et al. (1998) A protein conjugation system essential for autophagy. Nature 395: 395–398.
Zhang XD, Qi L, Wu JC, Qin ZH (2013) DRAM1 regulates autophagy flux through lysosomes. PLoS One 8: e63245.
Kroemer G, Jaattela M (2005) Lysosomes and autophagy in cell death control. Nat Rev Cancer 5: 886–897.
Naren AP, Cormet-Boyaka E, Fu J, Villain M, Blalock JE, et al. (1999) CFTR chloride channel regulation by an interdomain interaction. Science 286: 544–548.
Pitonzo D, Yang Z, Matsumura Y, Johnson AE, Skach WR (2009) Sequence-specific retention and regulated integration of a nascent membrane protein by the endoplasmic reticulum Sec61 translocon. Mol Biol Cell 20: 685–698.
Sadlish H, Skach WR (2004) Biogenesis of CFTR and other polytopic membrane proteins: new roles for the ribosome-translocon complex. J Membr Biol 202: 115–126.
Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, et al. (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63: 827–834.
Pranke IM, Sermet-Gaudelus I (2014) Biosynthesis of cystic fibrosis transmembrane conductance regulator. Int J Biochem Cell Biol 52: 26–38.
Lukacs GL, Chang XB, Bear C, Kartner N, Mohamed A, et al. (1993) The delta F508 mutation decreases the stability of cystic fibrosis transmembrane conductance regulator in the plasma membrane. Determination of functional half-lives on transfected cells. J Biol Chem 268: 21592–21598.
Bruscia EM, Bonfield TL (2016) Innate and adaptive immunity in cystic fibrosis. Clin Chest Med 37: 17–29.
Hartl D, Gaggar A, Bruscia E, Hector A, Marcos V, et al. (2012) Innate immunity in cystic fibrosis lung disease. J Cyst Fibros 11: 363–382.
Armstrong DS, Hook SM, Jamsen KM, Nixon GM, Carzino R, et al. (2005) Lower airway inflammation in infants with cystic fibrosis detected by newborn screening. Pediatr Pulmonol 40: 500–510.
Rosenfeld M, Gibson RL, McNamara S, Emerson J, Burns JL, et al. (2001) Early pulmonary infection, inflammation, and clinical outcomes in infants with cystic fibrosis. Pediatr Pulmonol 32: 356–366.
Reiniger N, Lee MM, Coleman FT, Ray C, Golan DE, et al. (2007) Resistance to Pseudomonas aeruginosachronic lung infection requires cystic fibrosis transmembrane conductance regulator-modulated interleukin-1 (IL-1) release and signaling through the IL-1 receptor. Infect Immun 75: 1598–1608.
Levy H, Murphy A, Zou F, Gerard C, Klanderman B, et al. (2009) IL1B polymorphisms modulate cystic fibrosis lung disease. Pediatr Pulmonol 44: 580–593.
Corvol H, Fitting C, Chadelat K, Jacquot J, Tabary O, et al. (2003) Distinct cytokine production by lung and blood neutrophils from children with cystic fibrosis. Am J Physiol Lung Cell Mol Physiol 284: L997–L1003.
Bonfield TL, Panuska JR, Konstan MW, Hilliard KA, Hilliard JB, et al. (1995) Inflammatory cytokines in cystic fibrosis lungs. Am J Respir Crit Care Med 152: 2111–2118.
Greally P, Hussain MJ, Vergani D, Price JF (1993) Serum interleukin-1 alpha and soluble interleukin-2 receptor concentrations in cystic fibrosis. Arch Dis Child 68: 785–787.
Kronborg G, Hansen MB, Svenson M, Fomsgaard A, Hoiby N, et al. (1993) Cytokines in sputum and serum from patients with cystic fibrosis and chronic Pseudomonas aeruginosainfection as markers of destructive inflammation in the lungs. Pediatr Pulmonol 15: 292–297.
Abdelaziz DH, Gavrilin MA, Akhter A, Caution K, Kotrange S, et al. (2011) Apoptosis-associated speck-like protein (ASC) controls Legionella pneumophilainfection in human monocytes. J Biol Chem 286: 3203–3208; PMCID:3030324.
Fesus L, Demeny MA, Petrovski G (2011) Autophagy Shapes Inflammation. Antioxid Redox Signal. 11: 2233–2234.
Levine B, Mizushima N, Virgin HW (2011) Autophagy in immunity and inflammation. Nature 469: 323–335.
Abdulrahman BA, Khweek AA, Akhter A, Caution K, Kotrange S, et al. (2011) Autophagy stimulation by rapamycin suppresses lung inflammation and infection by Burkholderia cenocepaciain a model of cystic fibrosis. Autophagy 7: 1359–1370.
Abdulrahman BA, Khweek AA, Akhter A, Caution K, Tazi M, et al. (2013) Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr DeltaF508 mutation improves the delivery of Burkholderia cenocepaciato the autophagic machinery. J Biol Chem 288: 2049–2058.
Mueller C, Braag SA, Keeler A, Hodges C, Drumm M, et al. (2011) Lack of cystic fibrosis transmembrane conductance regulator in CD3+ lymphocytes leads to aberrant cytokine secretion and hyperinflammatory adaptive immune responses. Am J Respir Cell Mol Biol 44: 922–929.
Kushwah R, Gagnon S, Sweezey NB (2013) Intrinsic predisposition of naive cystic fibrosis T cells to differentiate towards a Th17 phenotype. Respir Res 14: 138.
Feng Y, Press B, Chen W, Zimmerman J, Wandinger-Ness A (2001) Expression and properties of Rab7 in endosome function. Methods Enzymol 329: 175–187.
Jouret F, Devuyst O (2009) CFTR and defective endocytosis: new insights in the renal phenotype of cystic fibrosis. Pflugers Arch 457: 1227–1236.
Villella VR, Esposito S, Bruscia EM, Vicinanza M, Cenci S, et al. (2013) Disease-relevant proteostasis regulation of cystic fibrosis transmembrane conductance regulator. Cell Death Differ 20: 1101–1115.
Gentzsch M, Chang XB, Cui L, Wu Y, Ozols VV, et al. (2004) Endocytic trafficking routes of wild type and DeltaF508 cystic fibrosis transmembrane conductance regulator. Mol Biol Cell 15: 2684–2696.
Saxena SK, Kaur S (2006) Regulation of epithelial ion channels by Rab GTPases. Biochem Biophys Res Commun 351: 582–587.
Girard E, Chmiest D, Fournier N, Johannes L, Paul JL, et al. (2014) Rab7 is functionally required for selective cargo sorting at the early endosome. Traffic 15: 309–326.
Huynh KK, Plumb JD, Downey GP, Valvano MA, Grinstein S (2010) Inactivation of macrophage Rab7 by Burkholderia cenocepacia. J Innate Immun 2: 522–533.
Lopez de Armentia MM, Amaya C, Colombo MI (2016) Rab GTPases and the autophagy pathway: Bacterial targets for a suitable biogenesis and trafficking of their own vacuoles. Cells 5.
Ichimura Y, Komatsu M (2010) Selective degradation of p62 by autophagy. Semin Immunopathol 32: 431–436.
Ying H, Yue BY (2016) Optineurin: The autophagy connection. Exp Eye Res 144: 73–80.
Deosaran E, Larsen KB, Hua R, Sargent G, Wang Y, et al. (2013) NBR1 acts as an autophagy receptor for peroxisomes. J Cell Sci 126: 939–952.
Lamark T, Kirkin V, Dikic I, Johansen T (2009) NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets. Cell Cycle 8: 1986–1990.
Kirkin V, Lamark T, Johansen T, Dikic I (2009) NBR1 cooperates with p62 in selective autophagy of ubiquitinated targets. Autophagy 5: 732–733.
Ivanov S, Roy CR (2009) NDP52: the missing link between ubiquitinated bacteria and autophagy. Nat Immunol 10: 1137–1139.
Komatsu M, Ichimura Y (2010) Physiological significance of selective degradation of p62 by autophagy. FEBS Lett 584: 1374–1378.
Zheng YT, Shahnazari S, Brech A, Lamark T, Johansen T, et al. (2009) The adaptor protein p62/SQSTM1 targets invading bacteria to the autophagy pathway. J Immunol 183: 5909–5916.
Bjorkoy G, Lamark T, Johansen T (2006) p62/SQSTM1: a missing link between protein aggregates and the autophagy machinery. Autophagy 2: 138–139.
Ichimura Y, Kumanomidou T, Sou YS, Mizushima T, Ezaki J, et al. (2008) Structural basis for sorting mechanism of p62 in selective autophagy. J Biol Chem 283: 22847–22857.
Hanrahan JW, Tabcharani JA, Becq F, Mathews CJ, Augustinas O, et al. (1995) Function and dysfunction of the CFTR chloride channel. Soc Gen Physiol Ser 50: 125–137.
Sheppard DN, Welsh MJ (1999) Structure and function of the CFTR chloride channel. Physiol Rev 79: S23–S45.
Sarkadi B, Bauzon D, Huckle WR, Earp HS, Berry A, et al. (1992) Biochemical characterization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis epithelial cells. J Biol Chem 267: 2087–2095.
Gong L, Devenish RJ, Prescott M (2012) Autophagy as a macrophage response to bacterial infection. IUBMB Life 64: 740–747.
Lamothe J, Valvano MA (2008) Burkholderia cenocepacia-induced delay of acidification and phagolysosomal fusion in cystic fibrosis transmembrane conductance regulator (CFTR)-defective macrophages. Microbiology 154: 3825–3834.
Pezzulo AA, Tang XX, Hoegger MJ, Alaiwa MH, Ramachandran S, et al. (2012) Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature 487: 109–113.
Di A, Brown ME, Deriy LV, Li C, Szeto FL, et al. (2006) CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol 8: 933–944.
Teichgraber V, Ulrich M, Endlich N, Riethmuller J, Wilker B, et al. (2008) Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis. Nat Med 14: 382–391.
Haggie PM, Verkman AS (2007) Cystic fibrosis transmembrane conductance regulator-independent phagosomal acidification in macrophages. J Biol Chem 282: 31422–31428.
Barriere H, Bagdany M, Bossard F, Okiyoneda T, Wojewodka G, et al. (2009) Revisiting the role of cystic fibrosis transmembrane conductance regulator and counterion permeability in the pH regulation of endocytic organelles. Mol Biol Cell 20: 3125–3141.
Qadri YJ, Cormet-Boyaka E, Benos DJ, Berdiev BK (2011) CFTR regulation of epithelial sodium channel. Methods Mol Biol 742: 35–50.
Painter RG, Valentine VG, Lanson NA, Jr., Leidal K, Zhang Q, et al. (2006) CFTR Expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis. Biochemistry 45: 10260–10269.
Witko-Sarsat V (2013) Neutrophils in the innate immunity conundrum of cystic fibrosis: A CFTR-related matter? J Innate Immun 5: 195–196.
Ng HP, Zhou Y, Song K, Hodges CA, Drumm ML, et al. (2014) Neutrophil-mediated phagocytic host defense defect in myeloid Cftr-inactivated mice. PLoS One 9: e106813.
Zhou J, Wang J, Cheng Y, Chi YJ, Fan B, et al. (2013) NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. PLoS Genet 9: e1003196.
Downey DG, Martin SL, Dempster M, Moore JE, Keogan MT, et al. (2007) The relationship of clinical and inflammatory markers to outcome in stable patients with cystic fibrosis. Pediatr Pulmonol 42: 216–220.
Yoshizaki T, Kusunoki C, Kondo M, Yasuda M, Kume S, et al. (2012) Autophagy regulates inflammation in adipocytes. Biochem Biophys Res Commun 417: 352–357.
Jo EK, Shin DM, Choi AM (2012) Autophagy: Cellular defense to excessive inflammation. Microbes Infect 14: 119–125.
Deretic V (2009) Links between autophagy, innate immunity, inflammation and Crohn’s disease. Dig Dis 27: 246–251.
Tazi MF, Dakhlallah DA, Caution K, Ahmed A, Khalil H, et al. (2016) Elevated miR-17~92 cluster expression negatively regulates autophagy and cystic fibrosis transmembrane conductance regulator (CFTR) function in CF macrophages. Autophagy, in press.
Treiber T, Treiber N, Meister G (2012) Regulation of microRNA biogenesis and function. Thromb Haemost 107: 605–610.
Mendell JT (2008) miRiad roles for the miR-17-92 cluster in development and disease. Cell 133: 217–222.
Dakhlallah D, Batte K, Wang Y, Cantemir-Stone CZ, Yan P, et al. (2013) Epigenetic regulation of miR-17~92 contributes to the pathogenesis of pulmonary fibrosis. Am J Respir Crit Care Med 187: 397–405.
Aguda BD, Kim Y, Piper-Hunter MG, Friedman A, Marsh CB (2008) MicroRNA regulation of a cancer network: Consequences of the feedback loops involving miR-17-92, E2F, and Myc. Proc Natl Acad Sci U S A 105: 19678–19683.
Nana-Sinkam SP, Karsies T, Riscili B, Ezzie M, Piper M (2009) Lung microRNA: From development to disease. Expert Rev Respir Med 3: 373–385.
Bonauer A, Dimmeler S (2009) The microRNA-17-92 cluster: still a miRacle? Cell Cycle 8: 3866–3873.
Chen L, Li C, Zhang R, Gao X, Qu X, et al. (2011) miR-17-92 cluster microRNAs confers tumorigenicity in multiple myeloma. Cancer Lett 309: 62–70.
Grillari J, Hackl M, Grillari-Voglauer R (2010) miR-17-92 cluster: ups and downs in cancer and aging. Biogerontology 11: 501–506.
Hayashita Y, Osada H, Tatematsu Y, Yamada H, Yanagisawa K, et al. (2005) A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res 65: 9628–9632.
Luciani A, Villella VR, Esposito S, Brunetti-Pierri N, Medina D, et al. (2010) Defective CFTR induces aggresome formation and lung inflammation in cystic fibrosis through ROS-mediated autophagy inhibition. Nat Cell Biol 12: 863–875.
De Stefano D, Villella VR, Esposito S, Tosco A, Sepe A, et al. (2014) Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation. Autophagy 10: 2053–2074.
Kroemer G, White E (2010) Autophagy for the avoidance of degenerative, inflammatory, infectious, and neoplastic disease. Curr Opin Cell Biol 22: 121–123.
Shintani T, Klionsky DJ (2004) Autophagy in health and disease: a double-edged sword. Science 306: 990–995.
Huang J, Klionsky DJ (2007) Autophagy and human disease. Cell Cycle 6: 1837–1849.
Webb AK (1993) The difficulties of treating infection in adults with cystic fibrosis. Monaldi Arch Chest Dis 48: 657–661.
Hoiby N (1988) Hemophilus influenzae, Staphylococcus aureus, Pseudomonas cepacia, and Pseudomonas aeruginosain patients with cystic fibrosis. Chest 94: 97S-103S.
Vergne I, Singh S, Roberts E, Kyei G, Master S, et al. (2006) Autophagy in immune defense against Mycobacterium tuberculosis. Autophagy 2: 175–178.
Tatterson LE, Poschet JF, Firoved A, Skidmore J, Deretic V (2001) CFTR and pseudomonas infections in cystic fibrosis. Front Biosci 6: D890–D897.
Yuan K, Huang C, Fox J, Laturnus D, Carlson E, et al. (2012) Autophagy plays an essential role in the clearance of Pseudomonas aeruginosaby alveolar macrophages. J Cell Sci. 125: 507–515.
Junkins RD, Shen A, Rosen K, McCormick C, Lin TJ (2013) Autophagy enhances bacterial clearance during P. aeruginosalung infection. PLoS One 8: e72263.
Eidelman O, BarNoy S, Razin M, Zhang J, McPhie P, et al. (2002) Role for phospholipid interactions in the trafficking defect of Delta F508-CFTR. Biochemistry 41: 11161–11170.
Goldberg JB, Pier GB (2000) The role of the CFTR in susceptibility to Pseudomonas aeruginosainfections in cystic fibrosis. Trends Microbiol 8: 514–520.
Marks MI (1990) Clinical significance of Staphylococcus aureusin cystic fibrosis. Infection 18: 53–56.
Goss CH, Muhlebach MS (2011) Review: Staphylococcus aureusand MRSA in cystic fibrosis. J Cyst Fibros 10: 298–306.
Mestre MB, Colombo MI (2012) Staphylococcus aureuspromotes autophagy by decreasing intracellular cAMP levels. Autophagy 8: 1865–1867.
Schnaith A, Kashkar H, Leggio SA, Addicks K, Kronke M, et al. (2007) Staphylococcus aureussubvert autophagy for induction of caspase-independent host cell death. J Biol Chem 282: 2695–2706.
Jarry TM, Cheung AL (2006) Staphylococcus aureusescapes more efficiently from the phagosome of a cystic fibrosis bronchial epithelial cell line than from its normal counterpart. Infect Immun 74: 2568–2577.
Catherinot E, Roux AL, Vibet MA, Bellis G, Ravilly S, et al. (2013) Mycobacterium aviumand Mycobacterium abscessuscomplex target distinct cystic fibrosis patient subpopulations. J Cyst Fibros 12: 74–80.
Junkins RD, McCormick C, Lin TJ (2014) The emerging potential of autophagy-based therapies in the treatment of cystic fibrosis lung infections. Autophagy 10: 538–547.
Assani K, Tazi MF, Amer AO, Kopp BT (2014) IFN-gamma stimulates autophagy-mediated clearance of Burkholderia cenocepaciain human cystic fibrosis macrophages. PLoS One 9: e96681.
Zullo AJ, Jurcic Smith KL, Lee S (2014) Mammalian target of Rapamycin inhibition and mycobacterial survival are uncoupled in murine macrophages. BMC Biochem 15: 4.
Zullo AJ, Lee S (2012) Mycobacterial induction of autophagy varies by species and occurs independently of mammalian target of rapamycin inhibition. J Biol Chem 287: 12668–12678.
Foldenauer ME, McClellan SA, Berger EA, Hazlett LD (2013) Mammalian target of rapamycin regulates IL-10 and resistance to Pseudomonas aeruginosacorneal infection. J Immunol 190: 5649–5658.
Abraham RT, Wiederrecht GJ (1996) Immunopharmacology of rapamycin. Annu Rev Immunol 14: 483–510.
Berney T, Secchi A (2009) Rapamycin in islet transplantation: friend or foe? Transpl Int 22: 153–161.
Ferrer IR, Araki K, Ford ML (2011) Paradoxical aspects of rapamycin immunobiology in transplantation. Am J Transplant 11: 654–659.
Saunders RN, Metcalfe MS, Nicholson ML (2001) Rapamycin in transplantation: a review of the evidence. Kidney Int 59: 3–16.
Cheng HW, Liang YH, Kuo YL, Chuu CP, Lin CY, et al. (2015) Identification of thioridazine, an antipsychotic drug, as an antiglioblastoma and anticancer stem cell agent using public gene expression data. Cell Death Dis 6: e1753.
Shin SY, Lee KS, Choi YK, Lim HJ, Lee HG, et al. (2013) The antipsychotic agent chlorpromazine induces autophagic cell death by inhibiting the Akt/mTOR pathway in human U-87MG glioma cells. Carcinogenesis 34: 2080–2089.
Feng Y, Ke C, Tang Q, Dong H, Zheng X, et al. (2014) Metformin promotes autophagy and apoptosis in esophageal squamous cell carcinoma by downregulating Stat3 signaling. Cell Death Dis 5: e1088.
Tomic T, Botton T, Cerezo M, Robert G, Luciano F, et al. (2011) Metformin inhibits melanoma development through autophagy and apoptosis mechanisms. Cell Death Dis 2: e199.
Delmas D, Solary E, Latruffe N (2011) Resveratrol, a phytochemical inducer of multiple cell death pathways: apoptosis, autophagy and mitotic catastrophe. Curr Med Chem 18: 1100–1121.
Song YM, Lee YH, Kim JW, Ham DS, Kang ES, et al. (2015) Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway. Autophagy 11: 46–59.
Fu L, Kim YA, Wang X, Wu X, Yue P, et al. (2009) Perifosine inhibits mammalian target of rapamycin signaling through facilitating degradation of major components in the mTOR axis and induces autophagy. Cancer Res 69: 8967–8976.
de Medina P, Silvente-Poirot S, Poirot M (2009) Tamoxifen and AEBS ligands induced apoptosis and autophagy in breast cancer cells through the stimulation of sterol accumulation. Autophagy 5: 1066–1067.
Jakhar R, Paul S, Bhardwaj M, Kang SC (2016) Astemizole-Histamine induces Beclin-1-independent autophagy by targeting p53-dependent crosstalk between autophagy and apoptosis. Cancer Lett 372: 89–100.
Chong CR, Chen X, Shi L, Liu JO, Sullivan DJ, Jr. (2006) A clinical drug library screen identifies astemizole as an antimalarial agent. Nat Chem Biol 2: 415–416.
Tosco A, De Gregorio F, Esposito S, De Stefano D, Sana I, et al. (2016) A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR. Cell Death Differ. 23: 1380–1393.