A74-36 Dissecting Early Fibro-inflammatory Pathways in Human Lung Tissue Ex Vivo

Abstract Rationale Idiopathic pulmonary fibrosis (IPF) arises from a complex interplay of inflammatory and tissue-remodeling signals that drive progressive extracellular matrix (ECM) deposition and structural disorganization of the lungs. Yet, the initiating factors and pathways leading to pro-fibrotic activation remain poorly understood. To identify molecular triggers and cytokine combinations that induce early fibrotic signatures, we exposed non-fibrotic human lung tissue to an IPF-relevant cytokine cocktail (IPF-RC) composed of mediators elevated in bronchoalveolar lavage or sputum of IPF patients. The specific contribution of TGF-β1, an important canonical fibrosis driver, was dissected by comparing TGF-β1 stimulation alone, IPF-RC, and IPF-RC lacking TGF-β1. Methods Precision-cut lung slices (PCLS) were generated from non-fibrotic (non-PF) human lungs and cultured for up to 72h. To induce fibrotic responses, non-PF tissue was stimulated with IPF-RC, TGF-β1 alone, or IPF-RC lacking TGF-β1. In addition, the effect of antifibrotic treatment (nintedanib and pirfenidone) was tested. Subsequently, tissue RNA was analyzed by RT-qPCR, and cytokine levels were quantified in the culture supernatants. Results Stimulation with the complete IPF-RC induced a broad pro-fibrotic transcriptional and protein signature encompassing fibrosis-associated genes (COL1A1, COL3A1, COL6A1, FN1), myofibroblast activation (ACTA2), and transcriptional regulation of fibroblast phenotype and epithelial-mesenchymal transition (SNAI2, ZEB2). Antifibrotic treatment with nintedanib and/or pirfenidone selectively decreased the collagen/ECM and ACTA2 genes, whereas SNAI2 and ZEB2 remained unaffected. At the protein level, IPF-RC induced pronounced fibro-inflammatory remodeling with marked increases in pro-fibrotic and ECM-modulating markers (pro-Collagen1a1, TIMP1, PAI-1, MMP7), inflammatory mediators (CCL18, IL-6), and VEGF, indicative of angiogenesis and tissue remodeling. Comparative analyses revealed that TGF-β1 predominantly drove canonical collagen synthesis and matrix remodeling markers (pro-Collagen1a1 and fibronectin) whereas the cocktail without TGF-β1 selectively triggered inflammatory and TGF-β-independent matrix modulating factors (CCL18, IL-6, TIMP1, MMP7, and PAI-1). Importantly, PAI-1 secretion appeared to be synergistically induced by IPF-RC compared to IPF-RC lacking TGF-β1 or TGF-β1 alone. Additionally, secretion of CCL18, IL-6, MMP7, and TIMP1 was markedly enhanced by non-PF PCLS following IPF-RC stimulation compared to IPF-RC lacking TGF-β1, while TGF-β1 treatment alone did not elicit a detectable response. Conclusion Our findings highlight that pro-fibrotic activation can arise from multiple, partially TGF-β-independent cytokine networks, integrating inflammatory and matrix-regulatory factors. The identified additive and synergistic cytokine patterns provide new mechanistic insights into how early fibro-inflammatory signaling cascades converge to initiate lung remodeling in IPF. These results establish a framework to dissect triggers of fibrosis and for evaluating targeted antifibrotic interventions that intercept disease initiation. This abstract is funded by: None