Submission: 89 – IDENTIFYING PATHWAYS REGULATING GOBLET CELL METAPLASIA: PHENOTYPIC SCREENING WITH BRONCHOSPHERES

Henry Danahay¹, Clive McCarthy¹, Roy Fox² and Martin Gosling¹

¹Enterprise Therapeutics, UK; ²University of Sussex, UK

 

The composition of mucus in the CF airway, and in particular 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. An alternative approach would be to reduce the excessive production/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. To identify drug targets that could be regulated to achieve this, we have utilised a 3D culture model of the human airway epithelium, ‘bronchospheres’. Bronchospheres are derived from primary human airway basal cells, and can be cultured to form a well-differentiated mucociliary epithelium without the need for an air-liquid interface. Bronchospheres are cultured in a 384 well assay format that makes them amenable to medium throughput screening. Treating bronchospheres with mediators such as IL-13 induces a mucus hypersecretory phenotype with increased numbers of goblet cells and reduced numbers of ciliated cells. Our hypothesis was that the co-administration of test compounds together with IL-13 would identify compounds capable of preventing goblet cell formation with the opportunity to seed future drug discovery programs.

Bronchospheres were cultured as previously described (Danahay et al., Cell Rep. (2015) 10(2):239). On day 2 after seeding primary human airway basal cells, treatment with IL-13 ± test compounds was initiated. A library of approximately 2,000 pharmacologically active LMW compounds was used, each with a well-annotated mechanism of action. On day 8, media and treatments were topped-up and on day 14 bronchospheres were lysed and RNA isolated. QPCR was then used to assess the expression of cell-specific markers: MUC5AC (goblet cells) and FOXJ1 (ciliated cells). Compounds that induced a ≥2-fold reduction in expression of MUC5AC were classified as hits. This hit list was then refined by checking the expression of FOXJ1. Compounds that had likewise attenuated FOXJ1 expression by ≥2-fold were deprioritised as these likely represented a non-specific effect on epithelial differentiation. Compounds that either maintained or enhanced FOXJ1 expression in addition to repressing MUC5AC gene expression were prioritised for validation using traditional air-liquid interface cultures.

In total, 92 hit compounds from the bronchosphere screen were tested for effects on goblet and ciliated cell numbers in ALI HBE cultures using quantitative immunohistochemistry. Of these, 38 (41%) significantly attenuated the MUC5AC+ stained area in IL-13 treated HBE and either maintained or increased the FOXJ1+ stained area. Validated hits were then aligned based on their previously reported pharmacological activity to enable common pathways to be identified and to refine our hypotheses through further exemplification of pathway regulators.

Following this screen, we are progressing a lead optimisation program for eventual therapy in respiratory diseases associated with mucus obstruction.

It is widely accepted that the composition of mucus in the CF airway, and in particular 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 drug targets that could be regulated to achieve this, we have utilised a 3D culture model of the human airway epithelium, ‘bronchospheres’. Bronchospheres are derived from primary human airway basal cells, and can be cultured to form a well-differentiated mucociliary epithelium without the need for an air-liquid interface. As such, bronchospheres are cultured in a 384 well assay format that makes them amenable to medium throughput compound screening. Treating bronchospheres with inflammatory mediators such as IL-13 induces a mucus hypersecretory phenotype with increased numbers of goblet cells and reduced numbers of ciliated cells. Our hypothesis was that the co-administration of test compounds together with IL-13 would identify compounds capable of preventing goblet cell formation with the opportunity to seed future drug discovery programs.

Bronchospheres were cultured as previously described (Danahay et al., Cell Rep. (2015) 10(2):239). On day 2 after seeding primary human airway basal cells, treatment with IL-13 ± test compounds was initiated. A library of approximately 2,000 pharmacologically active LMW compounds was used, each with a well-annotated mechanism of action. On day 8, media and treatments were topped-up and on day 14 bronchospheres were lysed and RNA isolated. QPCR was then used to assess the expression of cell-specific markers: MUC5AC (goblet cells) and FOXJ1 (ciliated cells). Compounds that induced a ≥2-fold reduction in expression of MUC5AC were classified as hits. This hit list was then refined by checking the expression of FOXJ1. Compounds that had likewise attenuated FOXJ1 expression by ≥2-fold were deprioritised as these likely represented a non-specific effect on epithelial differentiation. Compounds that either maintained or enhanced FOXJ1 expression in addition to repressing MUC5AC gene expression were prioritised for validation using traditional air-liquid interface cultures.

In total, 92 hit compounds from the bronchosphere screen were tested for effects on goblet and ciliated cell numbers in ALI HBE cultures using quantitative immunohistochemistry. Of these, 38 (41%) significantly attenuated the MUC5AC+ stained area in IL-13 treated HBE and either maintained or increased the FOXJ1+ stained area. Validated hits were then aligned based on their previously reported pharmacological activity to enable common pathways to be identified and to refine our hypotheses through further exemplification of pathway regulators.

Based on this screen, we have identified pathways that regulates the induction and maintenance of a goblet cell metaplasia in vitro and are progressing a lead optimisation program for eventual therapy in respiratory diseases associated with mucus obstruction.

The TLR5-agonist flagellin (FLG) is the key protein component of bacterial flagella expressed by a number of organisms including Pseudomonas aeruginosa. The proinflammatory activity of bacterial FLG has been well documented in leukocytes although the effects on airway epithelial function are less clear.

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