Large pH oscillations promote host defense against human airways infection (original) (raw)
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Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways
Proceedings of the National Academy of Sciences, 2013
The ability to maintain proper airway surface liquid (ASL) volume homeostasis is vital for mucus hydration and clearance, which are essential aspects of the mammalian lung's innate defense system. In cystic fibrosis (CF), one of the most common life-threatening genetic disorders, ASL dehydration leads to mucus accumulation and chronic infection. In normal airways, the secreted protein short palate lung and nasal epithelial clone 1 (SPLUNC1) effectively inhibits epithelial Na + channel (ENaC)-dependent Na + absorption and preserves ASL volume. In CF airways, it has been hypothesized that increased ENaC-dependent Na + absorption contributes to ASL depletion, and hence increased disease. However, this theory is controversial, and the mechanism for abnormal ENaC regulation in CF airways has remained elusive. Here, we show that SPLUNC1 is a pH-sensitive regulator of ENaC and is unable to inhibit ENaC in the acidic CF airway environment. Alkalinization of CF airway cultures prevented CF ASL hyperabsorption, and this effect was abolished when SPLUNC1 was stably knocked down. Accordingly, we resolved the crystal structure of SPLUNC1 to 2.8 Å. Notably, this structure revealed two pH-sensitive salt bridges that, when removed, rendered SPLUNC1 pH-insensitive and able to regulate ASL volume in acidic ASL. Thus, we conclude that ENaC hyperactivity is secondary to reduced CF ASL pH. Together, these data provide molecular insights into the mucosal dehydration associated with a range of pulmonary diseases, including CF, and suggest that future therapy be directed toward alkalinizing the pH of CF airways. bacterial permeability-increasing protein | ion channels | COPD innate defense
Background. Decreased apical bicarbonate transport into the airway surface liquid (ASL) has been associated with decreased ASL pH in some studies. Low ASL pH can have adverse respiratory effects. However, the human CF epithelium can also normalize ASL pH. We hypothesized that pH regulatory proteins other than the CF transmembrane regulator (CFTR) would be upregulated in the CF epithelium. Methods. We grew primary human nasal and bronchial epithelial cells from healthy controls and CF subjects at air-liquid interface; each culture was grown until fully mature and ciliated (~ six weeks). We used immunoblotting to measure expression of proteins that can affect pH known in the airway, renal tubule and/or gut: carbonic anhydrases (CA) 1, 2 and 12; voltage-gated proton channel (Hv1); lactate dehydrogenases (LDH) A, B, and D; dual oxidases (DUOX) 1 and 2; Na+/H+ exchange regulatory factor; potassium-transporting ATPase alpha chain 2 (ATP12A), S-nitrosoglutathione reductase (GSNOR); glutami...
Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung
Nature, 2012
Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene 1 . Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how loss of CFTR first disrupts airway host defense has remained uncertain 2-6 . We asked what abnormalities impair eradication when a bacterium lands on the pristine surface of a newborn CF airway? To investigate these defects, we interrogated the viability of individual bacteria immobilized on solid grids and placed on the airway surface. As a model we studied CF pigs, which spontaneously develop hallmark features of CF lung disease 7,8 . At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria 8 . Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly killed bacteria in vivo, when removed from the lung, and in primary epithelial cultures. Lack of CFTR reduced bacterial killing. We found that ASL pH was more acidic in CF, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defense defect to loss of CFTR, an anion channel that facilitates HCO 3 − transport 9-13 . Without CFTR, airway epithelial HCO 3 − secretion is defective, ASL pH falls and inhibits antimicrobial function, and thereby impairs killing of bacteria
International Journal of Molecular Sciences, 2021
Knowledge on the mechanisms of acid and base secretion in airways has progressed recently. The aim of this review is to summarize the known mechanisms of airway surface liquid (ASL) pH regulation and their implication in lung diseases. Normal ASL is slightly acidic relative to the interstitium, and defects in ASL pH regulation are associated with various respiratory diseases, such as cystic fibrosis. Basolateral bicarbonate (HCO3−) entry occurs via the electrogenic, coupled transport of sodium (Na+) and HCO3−, and, together with carbonic anhydrase enzymatic activity, provides HCO3− for apical secretion. The latter mainly involves CFTR, the apical chloride/bicarbonate exchanger pendrin and paracellular transport. Proton (H+) secretion into ASL is crucial to maintain its relative acidity compared to the blood. This is enabled by H+ apical secretion, mainly involving H+/K+ ATPase and vacuolar H+-ATPase that carry H+ against the electrochemical potential gradient. Paracellular HCO3− tra...
Airway surface liquid pH is not acidic in children with cystic fibrosis
Nature communications, 2017
Modulation of airway surface liquid (ASL) pH has been proposed as a therapy for cystic fibrosis (CF). However, evidence that ASL pH is reduced in CF is limited and conflicting. The technical challenges associated with measuring ASL pH in vivo have precluded accurate measurements in humans. In order to address this deficiency, ASL pH was measured in vivo in children using a novel luminescent technology integrated with fibre-optic probes. Here we show that ASL pH in children with CF is similar to that of children without CF. Findings were supported by highly controlled direct pH measurements in primary human airway epithelial cell culture models, which also suggest that the potential ASL pH gradient produced by defective apical ion transport is balanced out by paracellular shunting of acid/base. Thus, reduced baseline ASL pH is unlikely to be an important pathobiological factor in early CF lung disease.
Molecular Biology of the Cell, 2009
Organellar acidification by the electrogenic vacuolar proton-ATPase is coupled to anion uptake and cation efflux to preserve electroneutrality. The defective organellar pH regulation, caused by impaired counterion conductance of the mutant cystic fibrosis transmembrane conductance regulator (CFTR), remains highly controversial in epithelia and macrophages. Restricting the pH-sensitive probe to CFTR-containing vesicles, the counterion and proton permeability, and the luminal pH of endosomes were measured in various cells, including genetically matched CF and non-CF human respiratory epithelia, as well as cftr ؉/؉ and cftr ؊/؊ mouse alveolar macrophages. Passive proton and relative counterion permeabilities, determinants of endosomal, lysosomal, and phagosomal pH-regulation, were probed with FITC-conjugated transferrin, dextran, and Pseudomonas aeruginosa, respectively. Although CFTR function could be documented in recycling endosomes and immature phagosomes, neither channel activation nor inhibition influenced the pH in any of these organelles. CFTR heterologous overexpression also failed to alter endocytic organellar pH. We propose that the relatively large CFTR-independent counterion and small passive proton permeability ensure efficient shunting of the proton-ATPase-generated membrane potential. These results have implications in the regulation of organelle acidification in general and demonstrate that perturbations of the endolysosomal organelles pH homeostasis cannot be linked to the etiology of the CF lung disease.
A Novel Host Defense System of Airways Is Defective in Cystic Fibrosis
American Journal of Respiratory and Critical Care Medicine, 2007
The respiratory tract is constantly exposed to airborne microorganisms. Nevertheless, normal airways remain sterile without recruiting phagocytes. This innate immune activity has been attributed to mucociliary clearance and antimicrobial polypeptides of airway surface liquid. Defective airway immunity characterizes cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator, a chloride channel. The pathophysiology of defective immunity in CF remains to be elucidated. Objective: We investigated the ability of non-CF and CF airway epithelia to kill bacteria through the generation of reactive oxygen species (ROS). Methods: ROS production and ROS-mediated bactericidal activity were determined on the apical surfaces of human and rat airway epithelia and on cow tracheal explants.
Salt-Independent Abnormality of Antimicrobial Activity in Cystic Fibrosis Airway Surface Fluid
American Journal of Respiratory Cell and Molecular Biology, 2001
The link between the genetic defect in cystic fibrosis (CF) and the recently described breach in pulmonary host defense has focused on the role of salt and water metabolism in the airways. Using a human bronchial xenograft model we demonstrate a salt-independent abnormality in bacterial killing in CF airway surface fluid (ASF). Biochemical characterization implicates the absence or dysfunction of a molecule critical to the constitution of normal bacterial killing. Our study suggests that CF transmembrane conductance regulator (CFTR) deficiency causes a primary abnormality in the composition of ASF that leads to a salt-independent defect in host defense. Importantly, this defect is corrected by adenovirus-mediated gene transfer of CFTR.
Journal of Biological Chemistry, 2009
Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. In order to perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) Clchannel as a determinant in lysosomal acidification (1). Lysosomes and phagosomes in murine cftr-/-AMs failed to acidify and the cells were deficient in bacterial killing compared to wild-type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between CFTR genotype and disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, ΔF508, is found in 70% of CF patients. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR ΔF508 and G551D on a set of core intracellular functions including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild-type, cftr-/-, as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing and agonist-induced secretory responses all of which would be expected to contribute to a significant impact on microbial clearance in the lung. Macrophages and neutrophils are key cells of