P011 Altered Expression and Enzymatic Activity of Xanthine Oxidase in Inflammatory Bowel Disease

Abstract

Abstract Background Xanthine oxidase (XO) is involved in the last steps of purine metabolism; indeed, it catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid (UA) with the production of superoxide anion. The accumulation of UA has been shown to initiate the inflammatory process through NLRP3 inflammasome and the production of reactive oxygen species (ROS) contributes to inflammation-related tissue damage. In murine models of colitis, XO inhibitors, allopurinol or febuxostat, reduced the expression of proinflammatory cytokines supporting the involvement of XO in the intestinal inflammatory process. This study aims to conduct a thorough investigation of the role XO plays in IBD pathogenesis and to explore the potential of XO inhibition as a therapeutic strategy. Methods Biopsies from the colon of patients with ulcerative colitis (UC) and Crohn’s disease (CD) were collected at University Hospital of Cagliari. Disease activity was assessed using Mayo and SES-CD scores. XO expression levels were evaluated using Real-Time PCR, validated by GEO database (GSE11223, GSE117993). XO enzymatic activity was quantified spectrophotometrically in protein extracts from the biopsies. XO and NLRP3 inflammasome activation pathway, including NLRP3, ASC, and CASPASE1, was investigated using immunohistochemistry (IHC). In vitro, Caco2, HT29, SW480 cells were treated with xanthine, XO inhibitors (allopurinol, febuxostat) to assess XO inhibition on NLRP3 activation, analyzed by immunocytochemistry (ICC). Results Biopsies from the sigmoid colon of 15 HC, 20 UC, and 15 CD patients, as well as from the ileum of 10 HC and 10 CD, were analyzed. Increased XO expression was found in UC and CD sigmoid mucosa (Relative Quantification [RQ]: 1.93 vs. 0.99, p=0.004), and CD ileum (RQ: 2.72 vs. 1.26, p=0.004), confirmed by GEO data. XO activity was higher in UC and CD sigmoid (3.86 nmol UA/min/mg in UC, 4.82 in CD vs. 1.03 in HC, p<0.001) and CD ileum (8.41 vs. 1.83 in HC, p<0.001). IHC confirmed that XO and the NLRP3 inflammasome pathway are more highly expressed in IBD patients than HC, predominantly within epithelial cells. In vitro studies on Caco2, HT29, and SW480 cells demonstrated that suppressing XO activity with XO inhibitors leads to the inhibition of the NLRP3 pathway. Conclusion XO expression was higher in IBD pts than in HC individuals. Alongside this increase in XO activity, there was a concurrent hyperactivation of the NLRP3 inflammasome. The inhibition of XO activity in cell lines resulted in reduced activation of the NLRP3 inflammasome pathway. These findings support the hypothesis that XO plays a critical role in the pathogenesis of IBD and highlight its potential as a therapeutic target.