In vitro differentiation and characterisation of the phenotypic differences between mucociliary and squamous metaplastic airway epithelium in COPD

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is one of the world’s leading causes of death, mainly caused due to smoking. One of the complications of COPD is the development of Squamous Metaplasia (SM) of the airway epithelium, with prevalence correlating with the severity of COPD. Previous work has suggested that the absence of retinoic acid (RA) can induce a SM phenotype under in vitro culture conditions. Artificial induction of SM in vitro would allow for characterisation and classification in comparison to normal differentiated epithelium from various donor sources.
Methods: Primary bronchial epithelial cells from both healthy donors and COPD donors were cultured as differentiated air-liquid interface (ALI) cultures for 21 days in either the presence or absence of RA. These cultures were harvested at day 7, 14 and 21 for mRNA extraction and qPCR analysis, with some cultures being fixed for both immunohistochemistry and immunofluorescent staining. Further cultures from both donor groups, grown in the presence and absence of RA, were stimulated basally with 10ng/μl TNFα for 24 hours to induce a proinflammatory response, measured by mRNA extraction and qPCR analysis.
Results: Phenotypically, there is little difference between healthy and COPD donors when grown in the presence of RA, with similar levels of structural protein expression and cilia visible on the surface of both donor groups. COPD donors did exhibit a significantly lower expression of the gene SCGB1A1, encoding the protein CCSP that is secreted by Club cells. The removal of RA resulted in a significant increase in the Trans Epithelial Resistance (TEER) reading as well as visible thickening of the epithelium with a loss of cilia. Decreased expression of Cytokeratin-7 and elevated Involucrin support the structural changes seen with the removal of RA. The removal of RA also alters the epithelial immune response, resulting in an attenuated response for Interleukin (IL)-6, and a decrease in expression fold induction of IL-8 and RANTES. Removal of RA also reduces expression of the antioxidant gene GCLM, but significantly increase the expression of HO1, both at baseline and post stimulation.
Conclusion: The removal of RA from ALI culture medium during the 21 days of differentiation results in an epithelium that is phenotypically similar to SM found in vivo. Altered gene expression and changes in the physical characteristics of the epithelium suggest that the epithelium is initially reacting to environmental insult to protect from further damage. The ability to generate artificial SM in vitro warrants further research to determine its exact role in the pathogenesis of COPD.

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ORCID for Donna Davies: orcid.org/0000-0002-5117-2991

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