CrossTalk opposing view: mucosal acidification does not drive early progressive lung disease in cystic fibrosis (2018)

Whether airway mucosal acidification drives early progressive lung disease is controversial. Our argument that other factors are more important in the early pathogenesis of cystic fibrosis lung disease is supported by unique data obtained from very young children with cystic fibrosis and age-matched controls. So, what of the counter view? Data from a newborn porcine model suggest that airway mucosal pH is low in cystic fibrosis piglets (Pezzulo et al. 2012). The authors argue that the pathogenic link between low airway surface liquid pH and the development of cystic fibrosis lung disease is through defective innate immune responses of the airway epithelium. They showed that airway surface liquid from newborn wild-type pigs rapidly kills bacteria in vivo. In transgenic cystic fibrosis piglets, Pizzulo and colleagues found that the airway surface liquid pH was more acidic than in wild-type pigs and that antimicrobial activity was inhibited. Finally, increasing the pH rescued bacterial killing in the cystic fibrosis piglets. Thus, the airway surface pH hypothesis is largely dependent upon an indirect link between more acidic airway surface liquid pH and progressive lung damage in cystic fibrosis via innate defences to bacterial pathogens. There are a number of important observations in young children that render this assertion, derived from the porcine in vivo model and limited in vitro experiments, inadequate to explain the pathobiology of early progressive lung disease in humans. Prospectively collected cross-sectional and longitudinal data obtained through the Australian AREST CF early surveillance programme demonstrate that by the age of 3 months 85% of children diagnosed with cystic fibrosis after newborn screening have evidence of lung disease (Sly et al. 2009, 2013). Only a small proportion of these have evidence of lower airway infection using standard clinical laboratory methods, and furthermore, pathogenic phyla are largely absent from lower airway microbiota early in life (Pittman et al. 2017, Muhlebach et al. 2018). The most consistent observation in humans with cystic fibrosis is the strong association between early progressive lung disease and neutrophilic inflammation (Sly et al. 2009, 2013; Stick et al. 2009; Mott et al. 2010; Garratt et al. 2015; Esther et al. 2016). In fact, the presence of neutrophil elastase (Sly et al. 2013) and metabolomic markers of neutrophilic inflammation (Esther et al. 2016) in bronchoalveolar lavage fluid from young children with cystic fibrosis predict subsequent structural lung disease, identified by chest computed tomography, independently of the presence of lower airway pathogens. Thus it seems that sterile inflammation is a significant early contributor to progressive lung disease. The question then arises of how inflammation occurs in the absence of infection? Our understanding of the role of cystic fibrosis transmembrane regulator (CFTR) in airway surface liquid homeostasis and the consequences of CFTR dysfunction provide important clues. Mutant CFTR fails to act as a chloride channel and regulator of the epithelial sodium channel (Boucher, 2007). Dysfunctional CFTR protein results in dehydration of the airway surface liquid and subsequent hyperconcentration of the airway surface mucus layer that in turn results in osmotic compression of the periciliary liquid layer. Compression of the periciliary layer inhibits mucociliary clearance which then predisposes to infection and/or results in the build-up of inflammatory particles on the airway surface (Button et al. 2012; Henderson et al. 2014). Importantly, the combination of airway surface liquid dehydration and adherent airway mucus plaques has been shown to cause steep local epithelial hypoxic gradients that can stimulate inflammation and promote biofilm formation by pathogens (Worlitzsch et al. 2002). Additional in vivo evidence that airway dehydration with subsequent mucus plugging, hypoxia and epithelial necrosis can cause significant lower airway inflammatory responses comes from experiments from transgenic mice that overexpress the β-subunit of the epithelial sodium channel (β-ENaC) (Mall et al. 2008). In mice, ENaC is a rate-limiting pathway for absorption of sodium and fluid across the airway epithelium. Airway-specific, overexpression of β-ENaC results in a characteristic phenotype that includes impaired mucus clearance, airway mucus obstruction, goblet cell metaplasia, chronic neutrophilic inflammation with increased levels of the IL-8 homologue KC, and reduced clearance of bacterial pathogens. In a series of experiments in β-ENaC mice with and without conditional interleukin-1 receptor (IL-1R) knock-down, Fritzsching et al. (2015) demonstrated that hypoxic cell death triggers neutrophilic inflammation via IL-1R signalling. These observations, therefore, provide an important direct link between known pathogenic mechanisms in cystic fibrosis and sterile inflammation (Montgomery et al. 2017). Ultimately, the assertion that mucosal acidification drives early progressive lung disease in cystic fibrosis rests on whether the observation of low pH in a newborn piglet model (Pezzulo et al. 2012) is relevant to progressive lung disease in young humans with cystic fibrosis. The in vivo measurement of pH in the airway surface liquid layer of the lung has been a challenge for multiple reasons including the following: (1) the airway surface liquid layer of the lower airways is extremely thin, (2) the lower airway is hard to reach with measuring instruments, (3) movement of the airway wall during breathing can complicate measurements, and (4) pH can be extremely responsive to changes in temperature and other ambient conditions such as carbon dioxide concentrations. These limitations have hampered previous measurements in humans and bring into question the validity of observations which are inconsistent and difficult to interpret. We took account of all of these issues when designing a series of experiments and developing a novel optical pH probe that could fit down the instrument channel of a small-diameter bronchoscope. The output from the probe was a pH-dependent digital phase shift signal between two light frequencies. The instrument was meticulously validated in vitro and when used in vivo the operator was blinded to the pH reading with only the raw digital output displayed from the instrument to indicate when the airway surface liquid had entered and when a stable plateau reading was obtained. The airway surface liquid pH was similar in children with cystic fibrosis and age-matched non-cystic fibrosis controls (Schultz et al. 2017). Furthermore, in children with cystic fibrosis there was no association between airway surface liquid pH and measures of airway inflammation or infection in bronchoalveolar lavage fluid obtained from the corresponding lung lobe where pH was measured. There was no relation between airway surface liquid pH and the age of participants, with the youngest participants 12 months of age. The pH probes used were highly responsive to changes in pH brought about by changes in the environment, e.g. temperature and CO2 levels. These observations suggested that airway surface liquid pH in young children with and without cystic fibrosis controls is closely regulated in a relatively narrow physiological range, i.e. the mean ± SD pH for CF and non-CF was 6.98 ± 0.15 and 7.00 ± 0.12, respectively. These in vivo observations were supported by highly controlled direct pH measurements of airway surface liquid pH in primary human airway epithelial cell culture derived from patients with cystic fibrosis and healthy controls. These studies suggested that that any potential airway surface liquid pH gradient produced by defective apical ion transport by mutant CFTR is balanced by effective paracellular shunting of acid/base (Schultz et al. 2017). Whilst our in vivo results were supported by our in vitro findings, our results contrasted with previous in vitro studies that found no difference between CF and non-CF airway surface liquid pH. Indeed, we based much of our experiments on significant work described by others (Coakley et al. 2003) but with the luxury of hindsight aimed to address some limitations that we recognized in previous studies. The apparent discordance between our in vitro work and that of previously published work can therefore be ascribed, in part, to methodological differences. Firstly, some previous studies examined dynamic responses to changes in the airway surface liquid that are non-physiological (Coakley et al. 2003; Tang et al. 2005; Garland et al. 2013), e.g. adding 20 μl of phosphate-buffered saline to apical cell culture surfaces before measuring pH. We went to great lengths to measure pH in unperturbed cell cultures under stable conditions (Schultz et al. 2017). Secondly, some previous studies did not use bicarbonate in the basolateral medium (Pezzulo et al. 2012; Tang et al. 2016). We demonstrated with ATP12A-overexpressing cell cultures that the paracellular path effectively shunts out bicarbonate gradients (Schultz et al. 2017). Therefore, the presence of bicarbonate in the basolateral medium of cell cultures is essential to simulate normal physiology. Thirdly, some studies measured pH using fluorescent dyes dissolved in the airway surface liquid (Pezzulo et al. 2012; Tang et al. 2016). Calibration of such pH-sensitive dyes would have been challenging and interaction of the dye with proteins in the ASL would have been likely. Such protein–dye interactions would have been limited in our study as the dyes were embedded in a hydrogel matrix (Schultz et al. 2017). A particular strength of our study is the use of similar pH technology for both in vitro and in vivo measurements with confirmation of our in vitro results in a separate laboratory on the other side of the world using a similar stable, highly controlled environment but a conventional micro pH probe. Furthermore, we are confident that all sources of subjective bias were excluded because the operator was always blinded to the pH outcome of the measurements. In conclusion, evidence from the porcine cystic fibrosis model suggests an indirect pathobiological link between airway surface liquid acidification, defective innate immunity to pathogens and progressive lung disease in cystic fibrosis. In contrast, our observations provide strong human, in vivo evidence, that airway inflammation occurs before lower airway infection with pathogenic bacteria occurs and there are additional strong data in support of a direct causal pathway between airway dehydration, mucus obstruction of airways and neutrophilic inflammation, the hallmark of early, progressive cystic fibrosis lung disease. Finally, when measured in vivo, airway surface liquid pH is not reduced in young children with cystic fibrosis. Readers are invited to give their views on this and the accompanying CrossTalk articles in this issue by submitting a brief (250 word) comment. Comments may be submitted up to 6 weeks after publication of the article, at which point the discussion will close and the CrossTalk authors will be invited to submit a `LastWord'. Please email your comment, including a title and a declaration of interest, to jphysiol@physoc.org. Comments will be moderated and accepted comments will be published online only as `supporting information' to the original debate articles once discussion has closed. Stephen Stick graduated in medicine from Cambridge University, UK. Following training in paediatrics he moved to Perth, Western Australia, where he completed specialist respiratory training and a PhD in infant respiratory physiology. He is a career clinical researcher and lead investigator for the Australian Respiratory Early Surveillance team for Cystic Fibrosis (AREST CF), Director, Telethon Kids Institute Respiratory Research Centre and respiratory specialist, Perth Children's Hospital. André Schultz is the Director of the Cystic Fibrosis Centre at Princess Margaret Hospital for Children where he leads a multidisciplinary team who care for over 200 children and young people with cystic fibrosis. He is a Respiratory Physician and Honorary Research Fellow at the Telethon Kids Institute, University of Western Australia. He is a clinician-scientist with an interest in rare paediatric lung disease. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. None declared. Both 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.