Dora Angyal

The work presented in this thesis focuses on bicarbonate transport mediated by the CFTR channel in primary cell cultures, with a specific interest in its role in the etiology of pancreatitis and its improvement by CFTR modulators. The first part centers on CFTR-mediated anion transport in subjects carrying CFTR variants, whereas the second part centers on the development of models for studying pancreatic CFTR function.

In chapter 2, we reviewed the current knowledge of CFTR-dependent bicarbonate transport in pancreatic ductal cells, and its role in the development of CF, and CFTR-related and acquired forms of pancreatitis.

The production of alkaline pancreatic juice is strongly dependent on CFTR, which is activated upon postprandial stimulation of ductal cells by secretin. This hormone triggers cyclic AMP (cAMP) production, and protein kinase-mediated phosphorylation and activation of CFTR. Through the coordinate action of CFTR and other ion transport mechanisms, the bicarbonate concentration in human pancreatic juice can reach levels of up to 140 mmol/L. These base equivalents serve to neutralize gastric acid entering the duodenum, and aid nutrient digestion.

In view of the important role of CFTR in the production of alkaline pancreatic juice, it is perhaps not surprising that when loss of CFTR function occurs, as in cystic fibrosis (CF), exocrine pancreatic function is severely compromised. As in other hereditary forms of pancreatitis, environmental factors seem to have little influence on disease progression in this scenario. Conversely, for those carrying CFTR variants that permit residual channel function and do not cause CF, environmental factors are increasingly important in determining disease liability. In fact, pancreatitis in this cohort is often triggered by an external stressor, e.g. the ingestion of a fatty meal or alcohol. The high incidence of CFTR mutations observed in the pancreatitis population, indicates that the exocrine pancreas is highly vulnerable to even small perturbations in ductal CFTR activity. Initially silent mutations, although not causing overt CF-like pathology, may sensitize the pancreas to a second hit. Such CFTR alleles may only have a small effect on the individual risk, but on the population level they may significantly increase the risk of developing pancreatitis.

Part I Pancreatitis in carriers of CFTR variants
None of the bioassays currently used for diagnostic purposes can accurately assess CFTR-mediated bicarbonate transport. Therefore, because of the particular relevance of CFTR-dependent bicarbonate transport in the pancreas, and because CFTR mutations may reduce the bicarbonate permeability of CFTR, for the work contained in this thesis, we employed assays that were designed to specifically assess CFTR-mediated bicarbonate transport in patient tissues. In chapters 3-5, we used these assays to screen a range of (mostly) rare CFTR mutations, and also tested the effect of modulators on CFTR-mediated bicarbonate vs. chloride transport.

Among various other CFTR variants evaluated, this analysis included four carriers of the D1152H allele, one of the purported CFTR-BD variants (chapter 4). These four subjects carried this variant in trans of a minimal function allele, i.e. a class I mutation. While the levels of residual CFTR-mediated chloride and bicarbonate transport between these different subjects with similar CFTR genotype varied, in all the D1152H lines assayed, we observed that chloride transport was lowered compared to wild type controls. Moreover, in all, modulator treatment, apart from enhancing bicarbonate transport, also markedly improved chloride transport function. Collectively, this argues against the contention that the D1152H mutation confers a specific defect in bicarbonate permeability of CFTR. In fact, we found that none of the variants investigated in chapters 3-5 demonstrated a specific defect in bicarbonate transport, and thus were unable to confirm their existence.

Our studies included an analysis of CFTR activity in a cohort of pancreatitis patients carrying mutations in CFTR (chapter 5). It showed that CFTR dysfunction in pancreatitis patients carrying CFTR variants is relatively prevalent, but no specific defect in CFTR-mediated bicarbonate transport was detected. Importantly, some subjects in this cohort with a lowered CFTR activity carried a wild type CFTR allele, suggesting that disease-causing mutations may also be located outside the CFTR locus. The study showed that patient-derived organoids may be used to assess the impact of CFTR genotype on epithelial bicarbonate and chloride transport, and the response to CFTR modulator therapy.

Part II Pancreatic ductal models to study CFTR function
The function of CFTR in human pancreatic ducts of people with CF has not been assessed, mainly because of the current lack of suitable in vitro or in vivo bioassays (1). Therefore, in the second part of this thesis, we explored the use of pancreatic tissue to culture ductal organoids and perform CFTR assays.

To model the pancreatic ductal tree during pancreatitis, we prepared organoids from porcine pancreas, and used these to assess the effect of inflammation on ductal CFTR-dependent anion transport (chapter 6). Our results indicate that select cytokines that are key mediators of the pancreatic inflammatory response markedly increased expression of CFTR in ductal epithelium, and stimulated CFTR-mediated ductal anion secretion. As was observed for other epithelia (e.g. airways), CFTR induction required a combination of pro-inflammatory cytokines (2). Stimulation of CFTR activity, by enhancing the secretory capacity of the ductal epithelium, may mitigate the effects of external (metabolic) stresses located within the ducts, attenuating or preventing further tissue injury and containing the inflammatory response in the initial stages of a pancreatitis event. These results point to a more central role of the ductal compartment, and CFTR in particular, in the etiology of pancreatitis.

Pancreatitis may not only be linked to CFTR mutations, but may also result from environmental factors that reduce (wild type) CFTR function. We exploited the organoid model to test the hypothesis that ethanol and/or unsaturated fatty acids affect ductal CFTR function (chapter 7). We found that ethanol and unsaturated fatty acids had only marginal effects on ductal cell viability, and even prolonged exposure did not attenuate ductal, CFTR-mediated anion secretion, which argues against a direct involvement of acquired CFTR dysfunction in the etiology of alcohol-related pancreatitis.

To assess pancreatic CFTR function in patients with pancreatitis, we set out to establish ductal organoids from patient material (chapter 8). Tissue was collected by per-oral pancreatoscopy, from the main pancreatic duct using a biopsy forceps, when subjects diagnosed with calcifying chronic pancreatitis underwent electrohydraulic lithotripsy. We found that such tissue samples allowed the culture of ductal epithelial cells. Importantly, these cultures displayed CFTR-dependent anion and fluid transport, and we found that CFTR modulators improved ductal anion and fluid secretion in one of these cases. Therefore, we propose that this model may be exploited for drug testing, ultimately to improve treatment of CP.