The epigenetic modulator 5-aza-2’-deoxycytidine reduces the fibrotic response of human lung fibroblasts to TGFß1 in a novel, long term, 3D cell culture system

DNA methylation changes in pulmonary fibrosis is a field of study which is increasingly growing in relevance. The DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine (5-aza-DC) has been shown to suppress transforming growth factor-beta-1 (TGFβ1) induced fibrotic changes. To better understand these changes, we studied the anti-fibrotic effect of 5-aza-2’-deoxycytidine on novel 3D multicellular foci derived from individual human lung fibroblast cells. Primary lung fibroblasts were derived from explant tissue of patients who did not have lung disease. These were then grown for a total of six weeks in optimal conditions to promote 3D multicellular foci formation. The multicellular foci were treated with 2.5ng/mL TGFβ1 for the final 5 weeks, and with 1µM 5-aza-DC for the final two weeks. Gross mechanical stiffness of the resulting foci was assessed by parallel plate compression testing. Alpha smooth muscle actin (αSMA) and collagen mRNA expression were assessed by RTqPCR. mRNA expression of collagen cross-linking enzymes, lysyl oxidase (LOX) and lysyl oxidase-like-2 (LOXL2), were also assessed by RTqPCR. Treatment of lung fibroblast-derived 3D foci with co-treatment of 2.5ng/mL TGFβ1 and 1µM 5-aza-DC reduced gross mechanical stiffness 3.5-fold compared to treatment with TGFβ1 alone (p<0.01). The same treatment reduced αSMA mRNA levels by 79.5% (p=0.011). Expression of LOX mRNA was reduced 2.96-fold and LOXL2 mRNA was reduced 4.76-fold (p=0.01). 5-aza-DC ameliorates both the fibrotic response and collagen cross-linking response of human lung fibroblasts to TGFβ1 cultured in this long-term cell culture model.