Altered Differentiation of Proximal Airway Epithelium in Trisomy 21

Abstract

Rationale: Trisomy 21 (T21), resulting in Down Syndrome (DS), is the most prevalent chromosomal abnormality worldwide. Recent studies have indicated that individuals with DS present with ciliopathies, as well as impaired mucociliary clearance. Whereas these defects have been determined in children, we recently reported that structural changes initiate prenatally in T21 lungs. Thus, our goal is to determine whether proximal airway differentiation and specifically ciliary structure and function are altered during T21 lung development. Methods: Immunofluorescent (IF) staining was performed on prenatal T21 and non-T21 age and sex matched control lungs to assess proximal airway cellular composition and differentiation. Gene expression was assessed by RT-qPCR. In vitro proximal airway differentiation was performed using primary basal cells isolated from prenatal T21 and non-T21 tracheas. Cells were plated into transwells and differentiated on air liquid interface (ALI). IF staining, RT-qPCR, and trans-epithelial electrical resistance (TEER) analyses were performed on ALI cultures at Day 0, 7, 14, and 21. Results: Prenatal T21 lungs were assessed for all major proximal airway cell types (basal, club, ciliated, goblet) and compared to non-T21 controls. IF staining of club cell markers (SCGB1A1, SCGB3A2) were increased in T21 lungs, with a significantly greater number of SCGB1A1 cells (p=0.0114) (n=20). A comparable trend was observed with goblet cells, which showed a larger quantifiable area of MUC5B staining in T21 lungs as compared to non-T21. These results were confirmed by RT-qPCR, which demonstrated a trend of increased expression for both SCGB3A2 and MUC5B, and a significant increase of SCGB1A1 (p=0.0169) (n=14). IF staining of ciliated cell markers (FOXJ1, RSPH1, ARL13B) were also increased in T21 lungs as compared to non-T21. These results were validated by RT-qPCR, with a significant increase in RSPH1, a gene responsible for motile cilia and encoded on chromosome 21 (p=0.0168) (n=11). We then cultured primary T21 and non-T21 basal cells in ALI and achieved successful airway epithelium differentiation. Confluent transwell IF staining shows a steady rate of P63+ cells, and an increase of ciliated cells (ARL13B, FOXJ1) over 21 days in culture. TEER measurements were lower in T21 cultures as compared to age matched non-T21. Conclusion: Our data demonstrate that prenatal T21 lungs undergo precocious proximal airway differentiation with possibly compromised tight junctions. These aberrancies may possibly contribute to the high infection rate, ciliopathies, and faulty mucociliary clearance observed in DS. California Institute for Regenerative Medicine (CIRM) EDUC4 - 12837 (CC) and NIH NHLBI/Office of the Director R01HL155104 (SD). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.