Abstract Rationale Increased extracellular matrix (ECM) stiffness is a hallmark of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Monocyte-derived macrophages (MDMs) contribute to fibrosis progression and exhibit enhanced oxidative phosphorylation (OXPHOS), which is required to sustain their profibrotic phenotype. However, whether matrix stiffness regulates OXPHOS and drives profibrotic macrophage polarization remains unknown. Methods Human macrophages were cultured on functionalized silicone substrates of defined stiffness. Transcriptional changes were assessed by RNA sequencing. Metabolic alterations were quantified by untargeted metabolomics (GC-MS/MS) and OXPHOS was evaluated using the Seahorse Bioanalyzer. Precision-cut lung slices (PCLS) served as an ex vivo model of pulmonary fibrosis. Bronchoalveolar lavage macrophages from IPF patients and healthy controls were analyzed to verify key findings. Results Metabolic flux analysis demonstrated that matrix stiffness enhances OXPHOS in macrophages. Among the three major pathways fueling OXPHOS, glutaminolysis emerged as the dominant source supporting mitochondrial metabolism, compared with fatty acid oxidation and glycolysis. IPF BAL macrophages showed altered glutamine metabolism consistent with enhanced glutaminolysis observed in stiffness-activated macrophages. Matrix stiffness increased the expression and activity of glutaminase (GLS), the rate-limiting enzyme of glutaminolysis. Glutaminolysis supplies the tricarboxylic acid (TCA) cycle with α-ketoglutarate (α-KG), a co-factor for the histone demethylase KDM6B, which regulates STAT6-dependent and other profibrotic transcriptional programs. KDM6B activity was elevated in macrophages from patients with pulmonary fibrosis. Stiffness-induced metabolic reprogramming promoted profibrotic MDM activation and increased secretion of secreted phosphorprotein 1 (SPP1), whereas pharmacologic inhibition of GLS in stiffness-activated macrophages suppressed SPP1 production. When PCLS were cultured with conditioned media from stiffness-activated macrophages, fibroblast activation marker CTHRC1 was increased; neutralizing SPP1 in conditioned media abrogated CTHRC1 induction. Conclusions ECM stiffness enhances glutaminolysis to drive TCA flux and OXPHOS, thereby promoting profibrotic macrophage polarization through α-KG-dependent activation of KDM6B. This mechanotransduction-metabolism-epigenetic axis regulates SPP1 production and fibroblast activation, representing a conserved pathway linking the fibrotic microenvironment to macrophage immunometabolism. Targeting this stiffness-sensing metabolic circuit may provide a novel therapeutic strategy for pulmonary fibrosis. Funding: Supported by NIH K08HL163406 (to C.H.). This abstract is funded by: NIH K08HL163406
He et al. (Fri,) studied this question.