View Abstract

A7200 - Investigation of Dipeptidyl-Peptidase IV Function in Airway Epithelial Type 2 Inflammation
Author Block: A. M. Stoner1, S. Sajuthi1, J. L. Everman1, C. Rios1, M. T. Montgomery1, C. Eng2, E. G. Burchard2, M. A. Seibold1; 1Center for Genes, Environment, and Health, National Jewish Health, Denver, CO, United States, 2Department of Medicine, University of California-San Francisco, San Francisco, CA, United States.
Rationale
A subgroup of asthmatics, characterized by atopy and type-2 cytokine-driven airway inflammation, has been shown to have increased dipeptidyl-peptidase-IV (DPP4) gene expression in their airway epithelium. Although DPP4 is one of the most upregulated genes in the airways of type-2-high asthmatics, its role in airway inflammation is poorly understood.
Methods
Human primary tracheal airway epithelial cells were used to develop a CRISPR-Cas9 knockout of DPP4. Cells were differentiated at air-liquid-interface (ALI) and paired cultures were mock-treated or IL-13-treated daily for the final 10 days of differentiation. DPP4 knockout was assessed using flow cytometry, immunofluorescence staining, and DPP4 enzyme activity assays. RNA was collected from both 10 day mock- and IL-13-treated cultures, as well as cultures treated with the DPP4 enzyme inhibitor Linagliptin, for RNA-seq gene expression analysis. Single cell sequencing of DPP4 KO airway epithelial cells was performed with the WaferGen ICELL8 instrument.
Results
DPP4 gene expression is strongly induced by IL-13, with a 48-fold increase compared to control treated ALI cultures (p=1.83E-08). Minimal DPP4 protein expression was detected in unstimulated ALI culture controls, while there is a 70.2% increase in DPP4 upon IL-13 stimulation as measured by flow cytometry. DPP4 protein expression is found within non-ciliated cells, particularly co-localizing with 97% of MUC5AC+ goblet cells during IL-13 stimulation. Using a luciferase assay, DPP4 enzyme activity was detected in the apical secretions of the epithelium with a 152-fold increase in activity upon IL-13-stimulation. DPP4 enzymatic activity is also detected in the basolateral media with a 29-fold increase in activity among IL-13 stimulated cultures. CRISPR-Cas9 knockout cultures for DPP4 exhibit a >90% protein knockout measured by flow cytometry and >90% knockout as judged DPP4 enzymatic activity. Single cell sequencing demonstrates many differentially expressed genes in secretory cells with knockout of DPP4. Inhibition of DPP4 enzymatic activity results in decreases in the fold changes of the top 100 IL-13 response genes (p=1.24E-12).
Conclusions
DPP4 is induced by IL-13 in airway epithelial cells. DPP4 expression is limited to non-ciliated cells, particularly MUC5AC+ goblet cells. CRISPR-Cas9 knockout of DPP4 shows an alteration in the IL-13 response. In IL-13-stimulated samples, DPP4 enzyme is secreted primarily apically and can be inhibited by Linagliptin. Inhibition of DPP4 enzymatic activity in tracheal cells results in a dampened response to IL-13. DPP4 appears to play an instrumental role in type-2 airway inflammation, and merits further investigation.