Tumor necrosis factor alpha triggers mitochondrial dysfunction and ROS-driven cytokine amplification in bovine fibroblast-like synoviocytes.

Lameness in dairy cattle, driven by joint inflammation, involves fibroblast-like synoviocytes (FLS) that produce inflammatory mediators and reactive oxygen species (ROS). Tumor necrosis factor alpha (TNF-α) has been proposed as an early systemic inflammatory marker that precedes and accompanies the development of lameness in transition cows. TNF-α signaling promotes ROS production and amplifies the inflammatory response. However, the role of mitochondrial ROS (mtROS) in regulating pro-inflammatory gene expression, particularly in bovine joint inflammation, remains to be elucidated. In this study, we investigated the role of mtROS in TNF-α-stimulated bovine FLS. Using mitochondrial complex inhibitors, complexes I and III were identified as major contributors to mtROS generation. Antioxidants, such as N-acetyl-L-cysteine (NAC) and Mito-Tempo, suppress TNF-α-induced expression of IL-6, IL-8, and IL-1β, with NAC showing broader efficacy in inhibiting COX-2 expression, implicating both cytosolic and mitochondrial ROS in the inflammatory response. TNF-α increased the lactate/pyruvate ratio and the uptake of the fluorescent D-glucose analog 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), suggesting an increase in glucose metabolism and a shift toward aerobic glycolysis. TNF-α-induced NOX2 expression was reduced by NAC and Mito-Tempo, thereby linking cytosolic ROS to inflammatory amplification. Mitochondrial networks dynamically shifted from a "networked" to "fragmented" phenotype after TNF-α exposure, accompanied by transient depolarization. Computational classification (UMAP/K-means) confirmed the morphological alterations. These findings demonstrate that mtROS and metabolic rewiring synergistically drive bovine synovial inflammation, highlighting mitochondrial dysfunction as a potential therapeutic target for joint inflammation-associated lameness.