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A1.07 Reprogramming of metabolic pathways inhibits TLR2-induced inflammation in ra
  1. T McGarry,
  2. M Biniecka,
  3. W Gao,
  4. DJ Veale,
  5. U Fearon
  1. Centre for Arthritis and Rheumatic Diseases, St. Vincent’s University Hospital, Dublin, Ireland

Abstract

Background and objectives Recent studies have emphasised a tight link between the metabolic state and phenotype of immune/inflammatory cells. Metabolic turnover of the inflamed synovium may induce acellular switch in energy production, characterised by a glycolytic flux, suppression of mitochondrial function andpotentiation of inflammation. The role of Toll-like receptors (TLRs) in immune responses is well documented, however emerging evidence suggests they also play a role in metabolism. In this study, we examine mitochondrial function, cellular metabolism and synovial inflammation in response to TLR2 activation.

Methods RA synovial explant cultures and RA synovial fibroblasts (RASFC) were cultured with TLR2-ligand Pam3CSK4 (1 µg/ml). Mitochondria DNA (mtDNA) mutations were assessed using a Random Mutation Capture assay andreactive oxygen species (ROS), mitochondrial membrane potential (MMP) and ATP levelswere quantified using fluorescent probes. Mitochondrial and glucose metabolism gene arrays were quantified by RT-PCR and the mitochondrial ultrastructure assessed by transmission electron microscopy (TEM). Surrogate markers of glycolysis PKM2, GLUT1, ATP5B and GAPDH were quantified in RA/OA synovium by immunohistochemistry. PKM2 was alsoassessed by western blotting and immunofluorescence. Finally, we examined the effect of glycolytic inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) (20 µM) on TLR2–induced cytokine production, invasion/migration and signalling components NFĸBp65, Notch-1IC and p-STAT3.

Results Pam3CSK4 significantly induced mtDNA mutation frequency, ROS, and inhibited the MMP. In parallel, Pam3CSK4 differentially regulated key mitochondrial genes SOD2, BNIP3 and BBC3 (all p < 0.05) and markedly altered the ultrastructure of mitochondria. Consistent with this, we demonstrated altered metabolic activity in RASFC, with induced PKM2 nuclear translocation, increased ATP levels and differential regulation of metabolic genes FBP1/2, PGAM2 and ACo2 (all p < 0.05), resulting in metabolic shift in favour of glycolysis. This change in metabolic activity was mirrored in-vivo where expression of PKM2, GLUT1 and ATP5B were significantly higher in RA synovium compared to OA (p < 0.05). Finally, reprogramming of metabolism using 3PO significantly inhibited TLR2-induced cytokine production, cell migration/invasion and transcriptional regulation of NFĸBp65 and p-STAT3.

Conclusion Reprogramming of metabolic activity in RA resulted in resolution of inflammation. These results show close interactions between innate immunity, metabolism and inflammation, further elucidation of these interactions may provide new insights to new therapeutic approaches in RA.

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