Cystic fibrosis (CF) is both the most common and most lethal genetic disease in the Caucasian population. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is characterized by the accumulation of thick, adherent mucus plaques in multiple organs, of which the lungs, gastrointestinal tract and pancreatic ducts are the most commonly affected. A similar pathogenesis cascade is observed in all of these organs: loss of CFTR function leads to altered ion transport, consisting of decreased chloride and bicarbonate secretion via the CFTR channel and increased sodium absorption via epithelial sodium channel upregulation. Mucosa exposed to changes in ionic concentrations sustain severe pathophysiological consequences. Altered mucus biophysical properties and weakened innate defense mechanisms ensue, furthering the progression of the disease. Mucins, the high-molecular-weight glycoproteins responsible for the viscoelastic properties of the mucus, play a key role in the disease but the actual mechanism of mucus accumulation is still undetermined. Multiple hypotheses regarding the impact of CFTR malfunction on mucus have been proposed and are reviewed here. (a) Dehydration increases mucin monomer entanglement, (b) defective Ca2+ chelation compromises mucin expansion, (c) ionic changes alter mucin interactions, and (d) reactive oxygen species increase mucin crosslinking. Although one biochemical change may dominate, it is likely that all of these mechanisms play some role in the progression of CF disease. This article discusses recent findings on the initial cause(s) of aberrant mucus properties in CF and examines therapeutic approaches aimed at correcting mucus properties.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/ppul.24530

Mucus, mucins, and cystic fibrosis.

The mucus clearance system is the dominant mechanical host defense system of the human lung. Mucus is cleared from the lung by cilia and airflow, including both two-phase gas liquid pumping and cough-dependent mechanisms, and mucus transport rates are heavily dependent on mucus concentration. Importantly, mucus transport rates are accurately predicted by the gel-on-brush model of the mucociliary apparatus from the relative osmotic moduli of the mucus and periciliary-glycocalyceal (PCL-G) layers. The fluid available to hydrate mucus is generated by transepithelial fluid transport. Feedback interactions between mucus concentrations and cilia beating, via purinergic signaling, coordinate Na+ absorptive vs Cl- secretory rates to maintain mucus hydration in health. In disease, mucus becomes hyperconcentrated (dehydrated). Multiple mechanisms derange the ion transport pathways that normally hydrate mucus in muco-obstructive lung diseases, e.g., CF, COPD, NCFB, and PCD. A key step in muco-obstructive disease pathogenesis is the osmotic compression of the mucus layer onto the airway surface with the formation of adherent mucus plaques and plugs, particularly in distal airways. Mucus plaques create locally hypoxic conditions and produce airflow obstruction, inflammation, infection, and, ultimately, airway wall damage. Therapies to clear adherent mucus with hydrating and mucolytic agents are rational, and strategies to develop these agents are reviewed.

Physiology and Pathophysiology of Human Airway Mucus.

Key points Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca2+ elevation by TRPV4 stimulation leads to CFTR channel activation. Abstract TMEM16A is a Ca2+ -activated Cl- channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short-circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4 stimulation induced activation of the CFTR Cl- channel. Our study reveals the existence of separate Ca2+ signalling pathways linked to different Cl- secretory processes.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/JP278784

TRPV4 and purinergic receptor signalling pathways are separately linked in airway epithelia to CFTR and TMEM16A chloride channels.

Cystic fibrosis (CF) is one of the most common life-limiting recessive genetic disorders in Caucasians, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CF is a multi-organ disease that involves the lungs, pancreas, sweat glands, digestive and reproductive systems and several other tissues. This debilitating condition is associated with recurrent lower respiratory tract bacterial and viral infections, as well as inflammatory complications that may eventually lead to pulmonary failure. Immune cells play a crucial role in protecting the organs against opportunistic infections and also in the regulation of tissue homeostasis. Innate immune cells are generally affected by CFTR mutations in patients with CF, leading to dysregulation of several cellular signalling pathways that are in continuous use by these cells to elicit a proper immune response. There is substantial evidence to show that airway epithelial cells, neutrophils, monocytes and macrophages all contribute to the pathogenesis of CF, underlying the importance of the CFTR in innate immune responses. The goal of this review is to put into context the important role of the CFTR in different innate immune cells and how CFTR dysfunction contributes to the pathogenesis of CF, highlighting several signalling pathways that may be dysregulated in cells with CFTR mutations.

/pdf/dysregulated-signalling-pathways-in-innate-immune-cells-with-1memvjv7fi.pdf

Dysregulated signalling pathways in innate immune cells with cystic fibrosis mutations

TMEM16A Ca2+-activated Cl- channel inhibition ameliorates acute pancreatitis via the IP3R/Ca2+/NFκB/IL-6 signaling pathway.

The inflammatory airway disease cystic fibrosis (CF) is characterized by airway obstruction due to mucus hypersecretion, airway plugging, and bronchoconstriction. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is dysfunctional in CF, leading to defects in epithelial transport. Although CF pathogenesis is still disputed, activation of alternative Cl- channels is assumed to improve lung function in CF. Two suitable non-CFTR Cl- channels are present in the airway epithelium, the Ca2+ activated channel TMEM16A and SLC26A9. Activation of these channels is thought to be feasible to improve hydration of the airway mucus and to increase mucociliary clearance. Interestingly, both channels are upregulated during inflammatory lung disease. They are assumed to support fluid secretion, necessary to hydrate excess mucus and to maintain mucus clearance. During inflammation, however, TMEM16A is upregulated particularly in mucus producing cells, with only little expression in ciliated cells. Recently it was shown that knockout of TMEM16A in ciliated cells strongly compromises Cl- conductance and attenuated mucus secretion, but does not lead to a CF-like lung disease and airway plugging. Along this line, activation of TMEM16A by denufosol, a stable purinergic ligand, failed to demonstrate any benefit to CF patients in earlier studies. It rather induced adverse effects such as cough. A number of studies suggest that TMEM16A is essential for mucus secretion and possibly also for mucus production. Evidence is now provided for a crucial role of TMEM16A in fusion of mucus-filled granules with the apical plasma membrane and cellular exocytosis. This is probably due to local Ca2+ signals facilitated by TMEM16A. Taken together, TMEM16A supports fluid secretion by ciliated airway epithelial cells, but also maintains excessive mucus secretion during inflammatory airway disease. Because TMEM16A also supports airway smooth muscle contraction, inhibition rather than activation of TMEM16A might be the appropriate treatment for CF lung disease, asthma and COPD. As a number of FDA-approved and well-tolerated drugs have been shown to inhibit TMEM16A, evaluation in clinical trials appears timely.

/pdf/tmem16a-in-cystic-fibrosis-activating-or-inhibiting-3t9zagimx3.pdf

TMEM16A in Cystic Fibrosis: Activating or Inhibiting?

Previous analysis of CFTR-knockout (CFTR−/−) in piglets has provided important insights into the pathology of cystic fibrosis. However, controversies exist as to the true contribution of CFTR to the pH balance in airways and intestine. We therefore compared ion transport properties in newborn wild-type (CFTR+/+) and CFTR-knockout (CFTR−/− piglets). Tracheas of CFTR−/− piglets demonstrated typical cartilage malformations and muscle cell bundles. CFTR−/− airway epithelial cells showed enhanced lipid peroxidation, suggesting inflammation early in life. CFTR was mainly expressed in airway submucosal glands and was absent in lungs of CFTR−/− piglets, while expression of TMEM16A was uncompromised. mRNA levels for TMEM16A, TMEM16F, and αβγENaC were unchanged in CFTR−/− airways, while mRNA for SLC26A9 appeared reduced. CFTR was undetectable in epithelial cells of CFTR−/− airways and intestine. Small intestinal epithelium of CFTR−/− piglets showed mucus accumulation. Secretion of both electrolytes and mucus was activated by stimulation with prostaglandin E2 and ATP in the intestine of CFTR+/+, but not of CFTR−/− animals. pH was measured inside small bronchi using a pH microelectrode and revealed no difference between CFTR+/+ and CFTR−/− piglets. Intracellular pH in porcine airway epithelial cells revealed only a small contribution of CFTR to bicarbonate secretion, which was absent in cells from CFTR−/− piglets. In contrast to earlier reports, our data suggest a minor impact of CFTR on ASL pH. In contrast, enhanced amiloride-sensitive Na+ absorption may contribute to lung pathology in CFTR−/− piglets, along with a compromised CFTR- and TMEM16A-dependent Cl− transport.

/pdf/transport-properties-in-cftr-knockout-piglets-suggest-normal-1btlseip73.pdf

Melanie Janda

Papers

TMEM16A in Cystic Fibrosis: Activating or Inhibiting?

Transport properties in CFTR−/− knockout piglets suggest normal airway surface liquid pH and enhanced amiloride-sensitive Na+ absorption