Induction of pro-fibrotic biomarkers in precision-cut lung slices (PCLS)

Abstract

Idiopathic pulmonary fibrosis is a severe lung disease, causing irreversible dysfunction of the organ. However, the exact pathomechanism of IPF is not yet understood and identification of specific biomarkers is urgently needed. Yet, a general shift from in vivo to ex vivo models (and from animals to humans) seems to be necessary in order to support the development of translational models. With the use of PCLS, the aim was to establish a novel experimental method to identify biomarkers of pulmonary fibrosis. PCLS were prepared from lungs of bleomycin-treated rats, NaCl-treated controls or tumor-free lung tissue from cancer patients. Human PCLS were cultured in the presence of TGF-β and TNF-α to induce a pro-fibrotic profile, rat PCLS were cultivated in medium only. Cytokine responses were measured by ELISA and genetic profiles were analyzed by RTqPCR. Analysis of the mRNA profile revealed up-regulation of important pro-fibrotic genes in lungs from bleomycin-treated rats as compared to the controls. Amongst others, extracellular matrix (ECM) genes including different forms of collagen as well as ECM remodeling enzymes (e.g. metalloproteinases) were elevated in bleomycin treated rat lungs as compared to control animals. A similar pattern of up-regulated genes was found in human PCLS stimulated with TGF-β and TNF-α. Furthermore, the pro-inflammatory cytokine IL-1β, involved in the initial wound repair mechanism and early fibrotic response, was elevated on mRNA as well as protein level in TGF-β- and TNF-α-stimulated human PCLS. Overall, this novel ex vivo method shows great potential to investigate important signaling pathways and mechanisms of early onset fibrosis for the identification of pro-fibrotic biomarkers.