Lung Fibroblasts from Chronic Obstructive Pulmonary Disease Subjects Have a Deficient Gene Expression Response to Cigarette Smoke Extract Compared to Healthy

Martin Garcia-Ryde,1 Nicole MD van der Burg,1 Carin E Larsson,1 Anna-Karin Larsson-Callerfelt,2 Gunilla Westergren-Thorsson,2 Leif Bjermer,1 Ellen Tufvesson1 1Department of Clinical Sciences Lund, Respiratory Medicine, Allergology and Palliative Medicine, Lund University, Lund, Sweden; 2Department of Experimental Medical Science, Lung Biology, Lund University, Lund, SwedenCorrespondence: Ellen Tufvesson, Department of Clinical Sciences Lund, Respiratory Medicine, Allergology and Palliative Medicine, Lund University, BMC, D12, 221 84, Lund, Sweden, Email Ellen.Tufvesson@med.lu.seBackground and aim: Cigarette smoking is the most common cause of chronic obstructive pulmonary disease (COPD) but more mechanistic studies are needed. Cigarette smoke extract (CSE) can elicit a strong response in many COPD-related cell types, but no studies have been performed in lung fibroblasts. Therefore, we aimed to investigate the effect of CSE on gene expression in lung fibroblasts from healthy and COPD subjects.Patients and methods: Primary lung fibroblasts, derived from six healthy and six COPD subjects (all current or ex-smokers), were either unstimulated (baseline) or stimulated with 30% CSE for 4 h prior to RNA isolation. The mRNA expression levels were measured using the NanoString nCounter Human Fibrosis V2 panel (760 genes). Pathway enrichment was assessed for unique gene ontology terms of healthy and COPD.Results: At baseline, a difference in the expression of 17 genes was found in healthy and COPD subjects. Differential expression of genes after CSE stimulation resulted in significantly less changes in COPD lung fibroblasts (70 genes) than in healthy (207 genes), with 51 genes changed in both. COPD maintained low NOTCH signaling throughout and upregulated JUN > 80%, indicating an increase in apoptosis. Healthy downregulated the Mitogen-activated protein kinase (MAPK) signaling cascade, including a ≥ 50% reduction in FGF2, CRK, TGFBR1 and MEF2A. Healthy also downregulated KAT6A and genes related to cell proliferation, all together indicating possible cell senescence signaling.Conclusion: Overall, COPD lung fibroblasts responded to CSE stimulation with a very different and deficient expression profile compared to healthy. Highlighting that stimulated healthy cells are not an appropriate substitute for COPD cells which is important when investigating the mechanisms of COPD.Plain Language Summary: We investigated, for the first time, the effect of cigarette smoke extract (CSE) on gene expression in lung fibroblasts from healthy subjects and subjects with COPD. We found that fibroblasts from subjects with COPD respond very differently than from healthy subjects by changing fewer gene expressions after CSE. Pathway enrichment suggested that fibroblasts from subjects with COPD respond to CSE by affecting genes involved in apoptosis pathways, while fibroblasts from healthy subjects respond by affecting genes involved in cell senescence. This study highlights the importance of assessing stimulated COPD cells in mechanistic studies.Keywords: COPD, lung fibroblast, gene expression, cigarette smoke extract, GO term, signaling pathways