Abstract
Diacetyl (DA) is a flavoring chemical commonly found in food and beverages. When inhaled at occupationally relevant concentrations, DA can cause bronchiolitis obliterans (BO), yet the mechanisms remain poorly understood. Common to all forms of BO is airway epithelial injury, with failed epithelial cell survival contributing to BO development. The purpose of the current study was to target integrin beta 4 (ITGβ4) – one of the primary integrins that connect airway epithelial cells to the basement membrane – in DA-exposed airway epithelial cells to prevent adhesion-related cell apoptosis (‘anoikis’). Sprague-Dawley rats were exposed to 200 parts-per-million DA vapor or filtered air for 6 hours per day for 5 consecutive days, then monitored for 5 weeks post-exposure and assessed for airway remodeling using Trichrome staining and the hydroxyproline assay. ITGβ4 protein expression was assessed via western blot as well as co-immunofluorescent staining using common airway epithelial cell markers. In parallel, primary human airway epithelial cells and human bronchial epithelial cells (16HBE14o-) were grown in vitro, exposed to DA, and treated with the pan-caspase inhibitor Z-VAD-FMK or transfected with ITGB4. End-points included viability staining, extracellular caspase 3/7 activity, and ITGβ4 protein expression. Rats exposed to DA vapors developed significant airway remodeling with increased total lung collagen content and sub-epithelial airway collagen deposition. Airway epithelial ITGβ4 expression remained decreased weeks after DA exposure with expansion of pan-cytokeratin positive epithelial cells, independent of ciliated and club cell markers. In parallel, DA-exposed human airway epithelial cells exposed in vitro developed significant anoikis. Treatment with Z-VAD-FMK reduced anoikis and improved ITGβ4 cytoplasmic surface expression but failed to improve total ITGβ4 protein expression. ITGB4 overexpression failed to suppress ITGβ4 cleavage or prevent anoikis. In summary, DA exposure in both rats and human airway epithelial cells results in caspase-mediated cleavage of ITGβ4. Future studies targeting post-translational modifications of ITGβ4 may prevent airway epithelial cell anoikis and fibrotic remodeling.
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Acknowledgements
Special thanks to Dr. Michael O’Reilly and Min Yee for providing antibodies specific to airway club cells. Additional thanks to the Wilmot Cancer Center Histology Core (Director: Dr. Brian Marples; technical assistance: Eric Hernady), the Inhalation Exposure Facility (Director: Dr. Alison Elder; technical assistance: David Chalupa, MS), and the Donor families and the Biorepository for INvestigation of Diseases of the Lung (BRINDL) at the University of Rochester Medical Center. Of note, Fig. 1A was created in Biorender McGraw, M. (2025) with publication license https://BioRender.com/gy9o3ht.
Funding
Work supported by grants from NIH NIEHS P30-ES001247 (MDM) and NIEHS K08-ES033290 (MDM), NHLBI U01HL122700 and U01HL148861 (GSP).
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Conception and design (SYK, MDM); data acquisition (SYK, AP, HH, MDM); analysis and interpretation (SYK, MDM, GSP, TJM); manuscript draft, revisions and approval (SYK, AP, HH, GSP, TJM, MDM).
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All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Rochester Medical Center (URMC). All methods adhered to the National Institutes of Health Guidelines. The University of Rochester Research Study Review Board reviewed and approved a human subject exemption for the use of primary human donor cells, considering all donors were deceased (RSRB00047606).
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Kim, SY., Pitonzo, A., Huyck, H. et al. Targeting integrin beta 4 in diacetyl-induced anoikis of the airway epithelium. Cell Death Discov. (2026). https://doi.org/10.1038/s41420-026-02980-9
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DOI: https://doi.org/10.1038/s41420-026-02980-9