Rebuttal from Miriam F. Figueira, Megan J. Webster and Robert Tarran

Schultz and Stick (2018) based their arguments on one dataset, which measured airway surface liquid (ASL) pH in cystic fibrosis (CF) children (Schultz et al. 2017). We do not believe their ASL pH measurements are exempt from experimental errors, and their data need replicating by other groups. The authors predominantly focus their rebuttal on CF pig data (Pezzulo et al. 2012) and incorrectly state that ‘the ASL pH hypothesis is largely dependent upon an indirect link between acidic ASL and CF lung damage via innate defences to bacterial pathogens.’ More acidic pH has been measured in CF humans and base secretion is 5‐fold less across freshly isolated CF human airways (Tate et al. 2002; Cho et al. 2011; Garland et al. 2013). Importantly, their reasoning fails to consider the dynamic nature of the lung and ignores the physiological impacts of well‐recognized cystic fibrosis transmembrane regulator (CFTR)‐dependent bicarbonate transport (Poulsen et al. 1994) and pH sensitivity of the epithelial sodium channel (ENaC) (Garland et al. 2013). Indeed, SPLUNC1 is a pH‐sensitive regulator of ENaC that fails to function in CF ASL (Garland et al. 2013; Tarran & Redinbo, 2014). Thus, acidic ASL prevents SPLUNC1 from negatively regulating ENaC in the CF lung, which is predicted to cause the Na⁺ hyperabsorption and sterile inflammation described by Schultz and Stick.

Methodologically speaking, we do not understand why their pH probe is superior to existing methods (i.e. electrochemical probes and pH‐sensitive dyes). Furthermore, the statement that they are looking at ‘an unperturbed stable system’ in vitro is unjustified, since such an entity does not exist in vivo.  Indeed, due to mucociliary transport, ∼864 ml of ASL moves cephalad per day (Matsui et al. 1998). The surface area of the conducting airways is ∼2400 cm² and every cm² of epithelium is presented with 364 μl of new ASL per day. In studies at the University of North Carolina, 20–100 μl of solution was added mucosally to mimic the continual volume challenge seen in vivo (Coakley et al. 2003; Garland et al. 2013); CF ASL height rapidly fell to ∼4 μm (i.e. thin film conditions) and ASL pH was measured at 6 h and beyond (Garland et al. 2013). Thus, under the dynamic conditions seen in the lung, but also at steady state, normal vs. CF pH differences were observed. Crucially, our experiments were performed on primary airway cultures plated at high densities and never passaged. In contrast, Schultz et al. used ‘conditionally reprogrammed cells’ that are significantly passaged. In our experience, two passages leads to a >75% reduction in CFTR expression (manuscript in preparation), while conditionally reprogramming further downregulates CFTR‐mediated ion transport (Gentzsch et al. 2017).  Whilst we cannot say that primary airway epithelium cultures are ideal, they exhibit similar transepithelial voltages as measured in vivo (Tarran et al. 2006). In contrast, conditional reprogramming decreases CFTR‐dependent bicarbonate transport. Thus, the studies of Schultz et al. (2017) may suffer from hitherto unrecognized artefacts.

In conclusion, ASL is hard to sample. However, a better understanding of ASL pH has led to the development of novel therapies for the treatment of CF (Scott et al. 2017) and may continue to shed light on CF pathogenesis.

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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.