Key Role of HDAC6 Overexpression in the Migration, Proliferation and Fibrotic Remodelling by Airway Basal Cells

Background Idiopathic pulmonary fibrosis is a fatal disease with mean survival time of 3 years. Our previous work suggested a role of airway basal cells (ABC) in de novo generation of bronchial tissue/bronchiolization in progression of IPF. Recent immunohistochemistry data and single cell RNAseq analyses of IPF tissues showed airway basal cells to be shifted towards an epithelial mesenchymal transition phenotype. Histone deacetylase 6 (HDAC6) alters performance and destiny of several proteins via deacetylation and is involved in TGF-β-induced EMT (epithelial-mesenchymal transition).
Objectives We got interested in the role of EMT, HDAC6 and TGF-β signaling for airway basal cell function in IPF.
Methods We studied the expression of EMT markers and HDAC6 in lung tissues and isolated cells of 30 IPF patients. ABCs were harvested during bronchoscopy by bronchial brushing within the routine diagnostic work-up. Lung tissues were derived from explants. In addition, we tested the functional role of TGF-β, EMT and HDAC6 in our recently established 3D organoid assay. Sphere formation was counted by bright field microscopy and cell proliferation quantified with aid of the MTT Assay. 15 HDAC6 inhibitors were newly designed and generated by the University of Siena (Campiani lab) and tested in the 3D organoid assay. One candidate molecule was chosen for further experiments. We determined the half of maximal inhibitory effect of HDAC6 inhibitors on organoid formation [IC₅₀ value].
Results Immunohistochemistry revealed increased levels of HDAC6 expression in IPF tissues, 3D organoids and human airway basal cells engrafted into murine lungs. Sphere formation is dependent on EMT and increased by stimuli which increase EMT such as TGF-β. Seven of the 15 newly generated HDAC6 inhibitors completely blocked sphere formation showing a concentration dependent effect. Human airway basal cells derived from IPF patients which overexpressed HDAC6 engrafted to the alveolar compartment and induced fibrosis in Rag2^−/− mice. The half maximal inhibitory concentration (IC₅₀) of the candidate molecule is 0.182 nmol in cell proliferation and 0.220 nmol in sphere counts.
Conclusion Our data suggest that HDAC6 overexpression may confer hyperproliferative and profibrotic effects of airway basal cells in IPF. HDAC6 selective inhibitors abolishes bronchosphere formation in a concentration dependent manner in our 3D organoid model based on primary cell lines of IPF-ABCs and provides a new treatment strategy for IPF.