CLDN18.1 Regulates Human Airway Epithelial Proliferation and Coordinates Repair Responses

Brett Wineinger; Jason Babcock; Che-Yu Hsu; Alessandra Castaldi; Zea Borok; Amy Ryan

doi:10.1152/physiol.2026.41.S1.2299765

Abstract only The mammalian airway epithelium is a dynamic barrier essential for mucociliary clearance, gas exchange, and protection from environmental injury. Tight junction (TJ) proteins maintain epithelial integrity, yet their roles in coordinating repair and stem cell behavior are poorly defined. Claudin 18.1 (CLDN18.1), a lung-specific TJ protein highly expressed in alveolar epithelial cells and subsets of airway cells, has emerged as a regulator of epithelial homeostasis. Murine Cldn18 knockout models demonstrate an expansion of alveolar type II, club, and basal stem cell populations, implicating CLDN18.1 as a potential modulator of epithelial regeneration. Understanding how CLDN18.1 influences human airway epithelial differentiation and injury responses could be important for developing new strategies to enhance lung repair in chronic lung disease. Our preliminary data suggests lower expression in cystic fibrosis donor bronchial regions compared to healthy controls. Based on this observation we hypothesized that loss of CLDN18.1 promotes goblet cell differentiation by altering club-cell fate decisions, particularly under inflammatory conditions. To test this, we generated CRISPR–Cas9–mediated CLDN18.1/18.2 knockout (KO) human primary bronchial epithelial cells (HBECs) using gRNAs targeting exon 2. Knockout efficiency was confirmed by Sanger sequencing, qPCR, and Western blot. HBECs were differentiated at air–liquid interface for 28 days, and epithelial composition was quantified by confocal microscopy and image quantification and qRT-PCR. Data shows that CLDN18.1 loss in HBEC isolated from donors with no prior evidence of lung disease does not significantly alter baseline differentiation into ciliated, club, or mucus cell lineages, contrary to expectations based on prior studies. Quantification across multiple fields revealed comparable proportions of epithelial cell types in KO and GFP controls; however, CLDN18.1-deficient cultures exhibited a significant increase in total cell number. This proliferative phenotype is consistent with CLDN18.1’s known tumor-suppressive function and supports the effectiveness of the knockout. In conclusion our data suggest that downregulation of CLDN18 supports a proliferative response important for normal epithelial repair allowing BCs to exit a barrier maintaining state and chronic CLDN18 downregulation that we have observed in end-stage disease may be due to an ongoing perception of injury in the cellular microenvironment. Ongoing experiments will clarify the temporal-spatial regulation of CLDN18.1 and how it integrates inflammatory cues to regulate proliferation and epithelial fate. Ultimately, defining CLDN18.1’s function in human airway biology may enable targeted approaches to enhance epithelial regeneration in chronic lung disease. This work was funded by an American Lung Foundation Grant, awarded to ALR This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

CLDN18.1 Regulates Human Airway Epithelial Proliferation and Coordinates Repair Responses

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