Bovine tumor necrosis factor-alpha Increases IL-6, IL-8, and PGE2 in bovine fibroblast-like synoviocytes by metabolic reprogramming

Lameness is a common condition in dairy cattle caused by infectious or noninfectious agents. Joint lesions are the second most common cause of lameness and can be diagnosed in association with the presentation of digit injuries. Fibroblast-like synoviocyte (FLS) are predominant cells of synovia and play a key role in the pathophysiology of joint diseases, thus increasing the expression of proinflammatory mediators. Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine involved in cyclooxygenase 2 (COX-2) and proinflammatory cytokine expression in FLS. Previously, TNF-alpha was demonstrated to increase hypoxia-inducible Factor 1 (HIF-1), a transcription factor that rewires cellular metabolism and increases the expression of interleukin (IL)-6 in bovine FLS (bFLS). Despite this, the proinflammatory effects of TNF-alpha in bFLS on metabolic reprogramming have been poorly studied. We hypothesized that TNF-alpha increases glycolysis and in this way controls the expression of IL-6, IL-8, and COX-2 in bFLS. Results first, gas chromatography/mass spectrometry (GC/MS)-based untargeted metabolomics revealed that bTNF-alpha altered the metabolism of bFLS, increasing glucose, isoleucine, leucine, methionine, valine, tyrosine, and lysine and decreasing malate, fumarate, alpha-ketoglutarate, stearate, palmitate, laurate, aspartate, and alanine. In addition, metabolic flux analysis using D-glucose-C-13(6) demonstrated an increase of pyruvate and a reduction in malate and citrate levels, suggesting a decreased flux toward the tricarboxylic acid cycle after bTNF-alpha stimulation. However, bTNF-alpha increased lactate dehydrogenase subunit A (LDHA), IL-6, IL-8, IL-1 beta and COX-2 expression, which was dependent on glycolysis and the PI3K/Akt pathway. The use of FX11 and dichloroacetate (DCA), an inhibitor of LDHA and pyruvate dehydrogenase kinase (PDK) respectively, partially reduced the expression of IL-6. Our results suggest that bTNF-alpha induces metabolic reprogramming that favors glycolysis in bFLS and increases IL-6, IL-8, IL-1 beta and COX-2/PGE2.