Fibroblast-driven MMP-9/TIMP-1 imbalance in bronchoalveolar lavage reflects fibrotic progression in interstitial lung disease

Background: Interstitial lung diseases (ILDs) are characterized by progressive fibrosis, in which extracellular matrix remodelling is regulated by matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). The MMP-9/TIMP-1 balance has been implicated in fibrogenesis, but its role in human ILD remains incompletely defined. This study aimed to assess the MMP-9/TIMP-1 ratio in BAL fluids of ILD patients, its relationship with fibrotic severity, and the cellular contribution of fibroblasts/myofibroblasts. Methods: BAL samples from 48 consecutive ILD patients (non-fibrotic ILD, fibrotic ILD, idiopathic pulmonary fibrosis [IPF]) were analysed. MMP-9 and TIMP-1 concentrations were quantified by enzyme-linked immunosorbent assay (ELISA), and MMP-9 activity assessed by gelatin zymography. Fibroblasts/myofibroblasts were isolated from BAL of ILD patients and tested for TIMP-1 and MMP-9 release by ELISA, or protein expression by immunofluorescence. Published single-cell RNA sequencing datasets were reanalyzed (DESeq2 model) to define the cellular source of MMP-9 and TIMP-1. Results: The MMP-9/TIMP-1 ratio was significantly reduced in BAL of patients with more advanced fibrosis, correlating with a higher presence of fibroblastic foci (p = .002) and collagen deposition (p < .001). This reduction was driven by both decreased MMP-9 and increased TIMP-1 levels. Zymography confirmed declining MMP-9 enzymatic activity across ILD subgroups. Fibroblasts, derived from BAL of ILD patients, displayed high TIMP-1 secretion but minimal MMP-9 release, consistent with their expression in immunofluorescence. Reanalysis of two independent scRNA-seq datasets confirmed predominant TIMP-1 expression in fibroblast/myofibroblast clusters, with low but detectable MMP-9 expression. Conclusions: Fibroblast-driven MMP-9/TIMP-1 imbalance in BAL reflects fibrotic severity in ILD. The MMP-9/TIMP-1 ratio may represent a supportive biomarker for differential diagnosis and phenotyping of ILD, warranting validation in larger multicenter studies.