Linezolid prevents fibroblast activation and ameliorates tissue fibrosis by inhibition of mitochondrial translation

Background: Beyond its role as a ribosome-targeting antibiotic, linezolid was recently shown to modulate immune responses by inhibiting mitochondrial translation. Since mitochondrial dysfunction is implicated in various fibrotic diseases, including systemic sclerosis (SSc), this study aimed to evaluate the antifibrotic potential of linezolid and delineate its underlying mechanisms in SSc. Methods: The effects of linezolid on fibrotic tissue remodeling were assessed using multiple experimental systems: human dermal fibroblasts, human macrophages, three-dimensional SSc skin equivalents (SScSE), the murine model of sclerodermatous chronic graft-versus-host disease (sclGvHD) and precision-cut skin slices (PCSS) obtained from SSc patients, by RNA sequencing, immunofluorescence, Western Blot and histology. Mitochondrial function was evaluated using Seahorse assays alongside mitochondrial protein synthesis assessments. Results: Linezolid inhibited TGFß-induced fibroblast activation in cultured human fibroblasts, SScSE, sclGvHD mice and SSc PCSS, as demonstrated by reversal of profibrotic gene expression programs, downregulation of TGFß, WNT, and JAK-STAT signaling and by reductions in aSMA expression or stress fiber formation, which led to reduced collagen deposition and ameliorated skin or lung fibrosis in vivo. Mechanistically, linezolid induced metabolic alterations by inhibiting mitochondrial translation in fibroblasts to hamper the oxidative phosphorylation, reduce the NAD?/NADH ratio and downregulate glycolysis, an essential metabolic pathway for fibroblast activation. Conclusions: This study provides first evidence that inhibiting mitochondrial translation with linezolid ameliorates fibrotic tissue remodeling. Since linezolid is already clinically approved as a reserve antibiotic, these findings hold translational promise and support the use of linezolid as a novel treatment for fibrotic disorders after further validation. Overall design: Mice sample: Allogeneic transplanted mice (cGVHD model) and syngeneic transplanted control mice (Syn) received daily oral gavage of 0.4% methylcellulose (vehicle) starting on day 21 after allogeneic or syngeneic bone marrow transplantation (alloBMT). Treatment groups included linezolid monotherapy (4 mg daily, orally) and vancomycin (4 mg daily, orally). All mice were sacrificed seven weeks post-alloBMT. Fibroblast RNA-seq: Primary dermal fibroblasts were stimulated with TGF-ß for 7 days, with or without linezolid, and harvested on day 7 for bulk RNA-seq. Macrophage RNA-seq: iPSC-derived macrophages were exposed to TGF-ß for 48 h, with or without linezolid, then collected for bulk RNA-seq. Precision-cut skin slices (PCSS) RNA-seq. PCSS prepared from patient skin were cultured ex vivo and treated for 48 h with either control medium (positive control) or linezolid.