CrossTalk proposal: mucosal acidification drives early progressive lung disease in cystic fibrosis

Introduction

Cystic fibrosis (CF) is the most common life‐threatening inherited disorder affecting Caucasians. It is caused by mutations in the CFTR gene that generate impaired cystic fibrosis transmembrane regulator (CFTR) anion channels leading to a multi‐organ disease including chronic airway infection/lung deterioration and increased morbidity/mortality (Fajac & Wainwright, 2017). Despite identification of the CF gene in 1989, the initial phases of CF lung disease remain controversial (Luckie & Mauri, 2013). One of the biggest questions is how the loss of CFTR‐mediated anion secretion initiates chronic lung infections. It has been known since the early 1990s that CFTR can conduct HCO₃ ⁻ and that CF epithelia exhibit defective bicarbonate transport (Gray et al. 1990; Smith & Welsh, 1992; Poulsen et al. 1994). Based on studies in CF piglets and in primary CF airway cultures, it is thought that CF airways are not hyperinflammatory and that the observed increase in inflammation is consequent to infections (Becker et al. 2004; Stoltz et al. 2010). Multiple laboratories have concluded that mucosal acidification drives early progressive lung disease in CF (Gustafsson et al. 2012; Pezzulo et al. 2012; Garland et al. 2013). However, whether or not CF airway surface liquid (ASL) is actually acidic is controversial (Schultz et al. 2017). Nonetheless, reduced ASL HCO₃ ⁻ is receiving more recognition as a key contributor towards early CF lung pathology.

CFTR is permeable to chloride and bicarbonate

HCO₃ ⁻ permeability through CFTR is ∼20% that of Cl⁻ (${P_{\mathrm{HCO}}}_{{}_3}/\phantom{{P_{\mathrm{HCO}}}_{{}_3}{P}_{\mathrm{Cl}}}{P}_{\mathrm{Cl}}$ ∼0.2) (Gray et al. 1990; Poulsen et al. 1994). However, CFTR may have a dynamic selectivity to HCO₃ ⁻ that could differ under physiological conditions (Reddy & Quinton, 2003; Shcheynikov et al. 2004). Furthermore, CFTR can regulate and/or be regulated by the SLC26A family of Cl⁻/ HCO₃ ⁻ exchangers (Ko et al. 2002; Gray, 2004; Tuo et al. 2006) and cytokines can alter bicarbonate secretion (Gray et al. 2004; Adams et al. 2014), suggesting that HCO₃ ⁻ transport is variable. Importantly, cAMP‐regulated short‐circuit current was partially HCO₃ ⁻‐dependent in non‐CF human bronchial epithelial cultures (HBECs) but was absent in CF HBECs (Smith & Welsh, 1992).

HCO₃ ⁻ deficiency drives pancreatic pathology

The pancreas is the organ most affected by CF, and pancreatic destruction begins in utero in CF patients with severe CFTR mutations. Here, CFTR‐mediated Cl⁻ is used as the counter on during Cl⁻/HCO₃ ⁻ exchange to facilitate the HCO₃ ⁻ secretion required to neutralize digestive enzymes and prevent auto‐digestion of the pancreas (Borowitz, 2015). CF pancreatic disease correlates with the level of HCO₃ ⁻ secretion and the severity of the CFTR mutation, indicating that altered HCO₃ ⁻ transport can drive CF pathology (Choi et al. 2001).

Is there a HCO₃ ⁻ gradient?

Garland et al. (2013) reported that ASL pH was significantly different in the unperturbed ASL of primary normal and CF HBECs, similar to the pH found in nasal secretions from NL vs. CF subjects (i.e. 7.0 and 6.5, respectively). Similarly, ASL pH was lower in newborn CF vs. wild‐type piglets (Pezzulo et al. 2012). In contrast, Schultz et al. (2017) recently found no difference between normal and CF ASL pH in paediatric lower airways. The relative sensitivity of their probe vs. the filter paper and other methods of detecting pH remains to be validated by independent groups. Interestingly, they also found no difference in ASL pH in airway cultures. However, an important methodological difference here is that they did not use primary (i.e. non‐passaged cultures) and instead used conditionally reprogrammed (i.e. expanded cultures) (Schultz et al. 2017). This method leads to a significant decrease in CFTR activity (≥50% reduction in forskolin‐sensitive Cl⁻ current) and an increase in transepithelial resistance, suggesting that ion transport properties have been compromised (Gentzsch, et al. 2017). An extensive list of CF ASL pH measurements has previously been excellently compiled elsewhere (Luckie & Mauri, 2013). Airway epithelia have high water permeability (Matsui et al. 2000; Song et al. 2001) and paracellular pathways that are permeable to ions including HCO₃ ⁻ (Cotton et al. 1983; Coakley et al. 2003). One might expect ASL [H⁺] and [HCO₃ ⁻] to be isotonic with plasma, resulting in an ASL pH of 7.4 and [HCO₃ ⁻] of ∼25 mm (assuming that ASL $P_{\mathrm{C}{\mathrm{O}}_2}$ is 5%). However, based on measures of normal ASL pH (i.e. ∼7.0); (Garland et al. 2013; Schultz et al. 2017), according to the Henderson–Hasselbalch equation, with 5% CO₂, ASL [HCO₃ ⁻] should be ∼10 mm. With pH 6.5, ASL [HCO₃ ⁻] is estimated to be ∼3 mm. Thus, there is a mucosal to serosal HCO₃ ⁻ gradient in normal airways of ∼15 mm and CF epithelia may support an ∼22 mm gradient (Fig. 1). Airway epithelia can secrete protons via dual oxidases (DUOX) and the non‐gastric H⁺/K⁺‐ATPase (ATP12A) (Coakley et al. 2003; Fischer, 2009). Indeed rates of proton secretion are greater than paracellular bicarbonate diffusion in CF airway epithelia, which results in ASL acidification (Fischer, 2009). Furthermore, K⁺ gradients have been detected between ASL and plasma of ∼20 mm in vivo, suggesting that transepithelial ion gradients of 10–20 mm can be supported (Knowles et al. 1997).

Figure 1. ASL pH and HCO₃ ⁻ transport in airway epithelia.

Cartoon illustrating transcellular and paracellular HCO₃ ⁻ transport in normal airway epithelia (A) and CF airway epithelia (B). HCO₃ ⁻ traverses the basolateral membrane via the Na⁺/HCO₃ ⁻ and Cl⁻/HCO₃ ⁻ cotransporters. In normal airway epithelia, apical HCO₃ ⁻ transport is mediated via CFTR and SLC26A4. However CFTR is absent or defective in CF airway epithelia. HCO₃ ⁻, amongst other ions, is able to diffuse paracellularly. HCO₃ ⁻ becomes protonated within the ASL following the paracellular movement of and transcellular flux of H⁺ by H⁺ channels and the H⁺/K⁺‐ATPase, to produce H₂O and CO₂. Equilibrium between HCO₃ ⁻ and H⁺ is such that normal ASL pH is ∼7.0. Despite the exaggerated HCO₃ ⁻ gradient evident across CF epithelia, ASL pH is acidified to ∼6.5 due to proton flux through via dual oxidase (DUOX).

What are the consequences of acidic ASL pH?

Determining [ion] in the thin (∼7 μm) ASL in vivo is technically challenging, and even relatively abundant Cl⁻ has proved troublesome to measure (Guggino, 1999). However, despite these difficulties, pH‐sensitive changes to airway physiology are readily apparent and the absence of bicarbonate is predicted to impair epithelial function (Saint‐Criq & Gray, 2017). At the most basic level, tertiary protein structure is often pH sensitive and changes in pH can affect protein unfolding (Talley & Alexov, 2010; Moon & Fleming, 2011). Similarly, acidic pH alters protein function. For example, cathepsins are more active at acidic pH, which may lead to increased activation of the epithelial Na⁺ channel (ENaC) and greater protease‐induced damage in CF airways (Martin et al. 2010; Tan et al. 2014). Work by our group has demonstrated that the abundant innate defence protein short palate lung and nasal epithelial clone 1 (SPLUNC1) is highly pH sensitive (Garland et al. 2013). SPLUNC1's crystal structure revealed the presence of pH‐sensitive salt bridges that prevented SPLUNC1–ENaC interactions at acidic pH (Hobbs et al. 2013; Garland et al. 2013). Thus, SPLUNC1 cannot bind to and inhibit ENaC at acidic pH leading to CF ASL dehydration (Garland et al. 2013; Tarran & Redinbo, 2014). Similarly, the amiloride‐sensitive nasal potential difference (an in vivo indicator of ENaC activity) was reduced at moderately acidic pH (Garland et al. 2013).

Goblet cells use Ca²⁺ to cross‐link and densely pack mucins. Following the exchange of Ca²⁺ for Na⁺, mucins expand ∼800 times due to the lack of cross linking (Verdugo, 1991). However, HCO₃ ⁻ can influence Ca²⁺ activity and may hinder the Ca²⁺/Na⁺‐exchange process and limit mucin expansion, causing mucins to be secreted without fully unfolding, leading to impaired rheology and reduced mucus clearance (Dorschner et al. 2006; Quinton, 2017).

A neutral ASL pH is also optimal for antimicrobial actions (Bahar & Ren, 2013; Berkebile & McCray, 2014). In addition to reports of antimicrobial peptides being pH sensitive (Zasloff, 1992; Burkhard, 1996; Bals et al. 1999), ASL antimicrobial activity was also pH sensitive and impaired in CF piglet ASL (Pezzulo et al. 2012), suggesting that a moderately acidic pH will also impair anti‐bacterial actions in the lung.

Thus, three arms of the lung's innate defence system, hydration, mucus rheology and antimicrobial activity, can be impaired by small reductions in ASL pH. Importantly, it is likely that the mucus clearance system, which requires coordinated CFTR and ENaC activity as well as correct mucin expansion, and the antimicrobial system work in concert to prevent bacterial colonization. That is, mucus clearance physically clears bacteria while antimicrobial proteins/peptides prevent bacterial growth, and pH‐sensitive inhibition of all of these processes would be more serious for CF pathogenesis than if just one arm of the innate defence system were abrogated.

Gastro‐oesophageal reflux and CF lung disease

Gastro‐oesophageal reflux occurs in up to 60% of adults over any 12 months (Zhao & Encinosa, 2006). In CF, there is a similar incidence (Robinson & DiMango, 2017). During gastro‐oesophageal reflux, the highly acidic stomach contents reflux into the airways, especially at night when subjects are prone. Irrespective of the absolute difference in ASL pH, Coakley et al. (2003) noted that CF HBECs recovered from a mucosal acidic challenge more slowly than non‐CF HBECs in the absence of CFTR‐mediated bicarbonate secretion, indicating that CF subjects are more at risk of gastro‐oesophageal reflux‐induced airway acidification since they cannot rapidly neutralize their airways after an acid challenge. Thus, transient ASL acidification due to gastro‐oesophageal reflux may temporarily exacerbate pH‐sensitive innate lung defence, leading to lasting lung damage.

Closing remarks

In summary, a little change in pH goes a long way. Compelling information from the airways shows how small changes in the pH can alter multiple processes, leading to chronic infections in CF lungs. Given the ‘noise’ that is inherent in most in vivo measurements, a ∼15 mm difference in bicarbonate may be difficult to detect, but the physiological impact of this change is readily apparent.

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Additional information

Competing interests

None.

Author contributions

All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

This work was funded by Emily's Entourage and the Cystic Fibrosis Foundation.

Biographies

Miriam Figueira is a postdoctoral research associate in the Tarran Lab in the Marsico Lung Institute at the University of North Carolina. Her work focuses on studying how peptides can regulate the epithelial Na⁺ channel in severe cystic fibrosis lung disease.

Megan Webster is a postdoctoral research associate in the Tarran Lab in the Marsico Lung Institute at the University of North Carolina. Her work focuses on the effects of the cystic fibrosis microenvironment on ion transport.

Robert Tarran is an associate professor of cell biology and physiology at the University of North Carolina and a member of the Marsico Lung Institute. He has a long standing interest in studying cystic fibrosis and tobacco‐related lung disease. His work currently focuses on the role of ion channels and secreted proteins in innate defence of the lung.