Identifying pathways & compounds regulating goblet cell metaplasia in vitro

Henry Danahay1, Roy Fox2 and Martin Gosling1
1Enterprise Therapeutics, UK; 2University of Sussex, UK

It is widely accepted that the composition of mucus in the CF airway, and the hydration status, significantly affects its clearance and thereby the potential for form plugs, restrict airflow and create a nidus for chronic microbial colonisation. A variety of largely ion channel-based strategies are being employed to promote mucosal hydration e.g. CFTR repair, ENaC blockers and TMEM16A potentiators. An alternative, but complimentary approach would be to reduce the excessive production and secretion of the mucin proteins that contribute to the solids component of the mucus gel. One approach to reduce excessive mucus production in the diseased lung is to reduce the number of mucus producing goblet cells in the airways.

To identify pathways that could regulate goblet cell formation in primary human bronchial epithelial (HBE) cells, we used ‘bronchospheres’ (Danahay et al., Cell Rep. [2015] 10(2):239) to run a screen of approximately 2,000 compounds. IL-13 treatment of the organoids increased the expression of goblet cell markers, including MUC5AC, and compounds that prevented this increase were identified as hits. This list was then refined, removing compounds that also decreased the expression of the ciliated cell marker FOXJ1, as these likely represented a non-specific, negative effect on epithelial differentiation. The remaining compounds were then validated for effects in air-liquid interface cultures of primary HBE, of which ˜40% significantly reduced an IL-13 induced increase in MUC5AC+ goblet cell numbers using quantitative immunohistochemistry.

Several of these validated hits were identified as not only preventing the IL-13 induced increase in MUC5AC+ goblet cells but also showed the potential to increase ciliated cell numbers. These compounds were prioritized and further tested in primary HBE to understand whether: 1) their pharmacology was specific to an IL-13 driven goblet cell phenotype, and 2) whether they could reverse an established goblet metaplasia.

Examples were demonstrated to attenuate the increase in MUC5AC+ goblet cells that was driven by the TLR5 agonist, flagellin as well as the type III interferon, IL-28. Furthermore, IL-28 also induced a profound increase in the number of MUC5B+ cells that could also be attenuated by some of the compounds.
A small number of the most promising compounds were then introduced to HBE that had an established goblet cell phenotype. Under conditions where HBE had been treated with IL-13 for 14 days to elevate goblet cell numbers, the introduction of test compounds between days 15-28, in the continued presence of IL-13, was able to reverse the increase in goblet cells and to also recover ciliated cell numbers.

Based on the output of this screen, we have identified compounds that can both prevent and reverse a goblet cell phenotype in primary HBE. Next steps will be to understand whether these effects can translate into in vivo models of a goblet cell metaplasia.