Investigating cigarette smoke-induced pathogenetic mechanisms in interstitial lung disease: A multi-omics approach

<bold>Rationale:</bold> Smoking can cause or deteriorate certain interstitial lung disease (ILD) including idiopathic pulmonary fibrosis (IPF), yet the precise mechanisms are unclear. The airway epithelium is a major player in ILD onset and progression, acting as a primary defense against CS. <bold>Objective:</bold> To uncover unique CS-modulated molecular pathways of relevance to ILD. <bold>Methods:</bold> We analysed bronchoalveolar lavage fluid (BALF) from chronic lung disease (CLD) patients stratified by smoking history, and CS-exposed primary human bronchial epithelial cells (phBECs) by LC-MS/MS proteomics. Pathway enrichment analysis identified potential key regulators of the CS response. Inhibition experiments in normal and IPF phBECs (+/-CS) were analysed for barrier integrity, cytotoxicity, and gene expression by TEER, LDH assay, immunofluorescent stainings, and quantitative PCR. <bold>Results:</bold> CS triggered persistent and transient protein changes in BALF with an overlap between transient changes in BALF and phBECs and macrophage-associated persistent changes. Nuclear protein 1 (NUPR1) was identified as a potential regulator of the CS response in phBECs and mainly localized to basal and ciliated cells. NUPR1 inhibition by ZZW-115 induced similar cytotoxicity in normal and CS-exposed phBECs, but altered the expression of the pro-ferroptotic gene <italic>ACSL4</italic> in a CS-dependent manner. IPF phBECs showed higher club cell proportion and greater susceptibility to NUPR1 inhibition. <bold>Conclusions:</bold> CS-induces transient and persistent protein changes in the airways. NUPR1 may protect from ferroptosis in phBECs. Finally, disease-specific differences persist in cultured IPF phBECs which are more susceptible to NUPR1 inhibition.