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

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.

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.

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.

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.