Pathological features of pulmonary fibrosis include accumulation of myofibroblasts and increased extracellular matrix (ECM) deposition in lung tissue. Contractile α–smooth muscle actin (α–SMA)–expressing myofibroblasts that produce and secrete ECM are key effector cells of the disease and therefore represent a viable target for potential novel anti–fibrotic treatments. We used primary normal human lung fibroblasts (NHLF) in two novel high–throughput screening assays to discover molecules that inhibit or revert fibroblast–to–myofibroblast differentiation. A phenotypic high–content assay (HCA) quantified the degree of myofibroblast differentiation, whereas an impedance–based assay, multiplexed with MS / MS quantification of α–SMA and collagen 1 alpha 1 (COL1) protein, provided a measure of contractility and ECM formation. The synthetic prostaglandin E1 (PGE1) alprostadil, which very effectively and potently attenuated and even reversed TGF–β1–induced myofibroblast differentiation, was identified by screening a library of approved drugs. In TGF–β1–induced myofibroblasts the effect of alprostadil was attributed to activation of prostanoid receptor 2 and 4 (EP2 and EP4, respectively). However, selective activation of the EP2 or the EP4 receptor was already sufficient to prevent or reverse TGF–β1–induced NHLF myofibroblast transition. Our high–throughput assays identified chemical structures with potent anti–fibrotic properties acting through potentially novel mechanisms.

In the progression phase of idiopathic pulmonary fibrosis (IPF) the normal alveolar structure of the lung is lost and replaced by remodeled fibrotic tissue and by bronchiolized cystic airspaces. Although these are characteristic features of IPF, knowledge of specific interactions between these pathological processes is limited. Here, the interaction of lung epithelial and lung mesenchymal cells was investigated in a co-culture model of human primary airway epithelial cells (EC) and lung fibroblasts (FB). Single-cell RNA sequencing (sc-RNA-seq) revealed that the starting EC population was heterogenous and enriched for cells with a basal cell signature. Furthermore, fractions of the initial EC and FB cell populations adopted distinct pro-fibrotic cell differentiation states upon co-cultivation, resembling specific cell populations that were previously identified in lungs of IPF patients. Transcriptomic analysis revealed active nuclear factor NF-kappa-B (NF-κB) signaling early in the co-cultured EC and FB cells and the identified NF-κB expression signatures were also found in "HAS1 High FB" and "PLIN2+ FB" populations from IPF patient lungs. Pharmacological blockade of NF-κB signaling attenuated specific phenotypic changes of EC and prevented FB-mediated interleukin-6 (IL6), interleukin-8 (IL-8) and C-X-C motif chemokine ligand 6 (CXCL6) cytokine secretion, as well as collagen alpha-1(I) chain (COL1A1) and alpha-smooth muscle actin (α-SMA) accumulation. Thus, we identified NF-κB as a potential mediator, linking epithelial pathobiology with fibrogenesis.