P2X7 Receptor-NOX2 Axis Mediates Tryptophan Oxidation in Murine and Human Nonalcoholic Fatty Liver Disease

Abstract

Tryptophan oxidative metabolite kynurenine is known to be proinflammatory in neurodegeneration, autoimmune disease and sepsis. NAFLD is a proinflammatory condition where resident liver macrophages play a significant role in progression of the disease. With NAFLD being increasingly considered an epidemic in the western world and no suitable therapy available, it is important that novel approaches be studied for possible therapy. The present study shows that P2X7r causes the oxidation of tryptophan via NOX2 induced oxidative stress signaling. Further it may do so by the involvement of JAK/STAT pathway in the diseased liver macrophages. Results in both mouse (MCD Diet) and human liver biopsies showed an increase in tryptophan oxidation product n-formyl kynurenine (NFK) when compared to relevant controls. P2X7r KO mice or use of p47 phox deleted mice significantly decreased NOX2 activation and NFK immunoreactivity. Mechanistically, experiments conducted in immortalized Kupffer cells showed a significant upregulation of STAT3 phosphorylation in LPS-treated and P2X7r-Agonist-treated groups while use of P2X7r antagonist or Apocynin (NOX inhibitor) significantly decreased it. Concomitant use of apocynin and P2X7r agonist significantly decreased STAT3 phosphorylation suggesting that NOX2 acts downstream to P2X7r in these cells. Kynurenine levels in Kupffer cells decreased significantly in groups incubated with P2X7r antagonist, Apocynin or Ruxolitinib (JAK/STAT inhibitor). NASH Histology scores were significantly higher in wildtype MCD fed mice with a normal P2X7r phenotype while p47 phox KO or a P2X7r KO showed a decreased disease pathology. In conclusion, tryptophan metabolite NFK can be a strong indicator of NAFLD inflammation and can predict the stage of the disease based on the evidence in this study.