Therapeutic and Prognostic Strategies Based on TAGLN2 and OGN in Idiopathic Pulmonary Fibrosis (IPF)

Idiopathic pulmonary fibrosis (IPF) is a rare, chronic, and progressive lung disease with a median survival of three years from diagnosis. Although the antifibrotic drugs Pirfenidone and Nintedanib slow respiratory functional decline, they have not yet improved mortality rates. IPF onset is due to micro-injuries to the alveolar epithelium, aberrant tissue repair mechanisms and fibroblast activity, that lead to excess extracellular matrix production, scarring and ticking of the lung parenchyma followed by impaired gas exchange. This study investigates the molecular mechanism and the functional role of transgelin 2 (TAGLN2) and osteoglycin (OGN) proteins, that we found overexpressed in IPF patients’ lung tissue, in primary lung fibroblasts from different subset of IPF patients characterized by different clinical parameters. As a first step, we will establish a structured database collecting histological samples from patients diagnosed with IPF over the two-year study period. These samples will undergo immunohistochemical staining using OGN and TAGLN2 antibodies to optimize detection and protocol standardization. Clinical data from each patient will also be integrated. The resulting dataset, will serve as a valuable resource for future digital pathology applications, including the training of AI-based tools for automated quantification of marker expression and fibrosis assessment. Indeed, we will proceed with molecular and cellular investigations using proteomics and single-cell RNA sequencing on patient-derived fibroblasts to explore their role in fibrosis progression. Based on the previously established database, we will isolate primary fibroblasts from cryobiopsy and surgical lung biopsy (SLB) of IPF patients and select based on OGN and TAGLN2 expression and clinical history (2 early-stage and 2 late-stage fibrosis). These samples will undergo single-cell RNA sequencing to identify fibroblast subsets with differential expression of OGN and TAGLN2. We will then assess correlations between these markers and pro- fibrotic gene signatures. This approach will help define functionally distinct fibroblast populations and their role in fibrosis progres-sion. To assess protein expression levels, we will perform proteomic analyses using LC-MS/MS coupled with data-dependent acquisition (DDA) on the same fibroblast samples. Given the roles of TAGLN2 and OGN in actin cytoskeleton and ECM dynamics—key processes in fibrosis—we hypothesize that their inhibition may reduce fibroblast activation and fibrosis progression. Using stable shRNA, we will downregulate TAGLN2 and OGN in primary lung fibroblasts over-expressing these proteins. Functional assays will assess proliferation, migration, 3D spheroid formation, and ECM deposition. Results will be com-pared to normal fibroblasts and cells treated with antifibrotic drugs (Nintedanib, Pirfenidone). This will help evaluate the therapeutic potential of targeting these biomarkers.