DOP21 Role of P2X7 in intestinal fibrosis and inflammasome activation: Relevance in Crohn´s Disease
Lis-López, L.(1);Bauset, C.(1);Ortiz-Masia, D.(1);Gisbert-Ferrándiz, L.(1);Coll, S.(1);Calatayud, S.(1);Barrachina, M.D.(1);Cosín-Roger, J.(2)
(1)Faculty of Medicine- University of Valencia, Department of Pharmacology, Valencia, Spain;(2)Hospital Dr. Peset, Department of Endocrinology and Metabolism, Valencia, Spain
Background
Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract whose etiology is unknown. CD is associated with complications such as fibrosis or fistula, which cannot be pharmacologically reversed, requiring repeated surgery. Although a profibrotic effect of the P2X7 receptor has been described in some scenarios such as lung, heart and liver, its role in intestinal fibrosis has not been analysed yet. Given the crosstalk between fibrosis and inflammasome, we aim to analyze the relevance of P2X7 in intestinal fibrosis and inflammasome activation.
Methods
Surgical intestinal resections of CD patients and healthy ileum of carcinoma patients were obtained. Murine chronic colitis was induced by 4 cycles of DSS in wild-type (WT) or P2X7-/- (KO) mice. HT29 cells were treated 24 hours with an inflammasome activator cocktail (LPS, TNF-α and IFN-γ) and the P2X7 antagonist A-80. Gene and protein expression of P2X7, inflammasome markers (NLRP3, ASC, CASPASE1, IL1β and IL18) and alternative inflammasome pathways (APIs) (NLRP1, NLRC4 and AIM2) were analysed by qPCR and Western Blot. The collagen layer was analysed by Sirius Red Staining. Results are expressed by mean±SEM. Statistical analysis was performed with one-way ANOVA and correlations were analysed with Spearman coefficient.
Results
In CD patients, the expression of P2X7 (2.97±0.50), Nlrp3 (2.53±0.41), Asc (5.61±0.76), Caspase1 (6.90±1.41), IL18 (4.17±0.89) and APIs Nlrp1 (3.07±0.40), Nlrc4 (6.99±1.19) is significantly increased vs non-IBD patients. Moreover, P2X7 expression positively and significantly correlates with the expression of the inflammasome markers NLRP3 (r=0.51), ASC (r=0.38), CASPASE1 (r=0.46), IL18 (r=0.36) and API such as NLRP1 (r=0.73), NRLC4 (r=0.67) and AIM2 (r=0.51) in CD patients (n≥45). The chronic murine model of DSS revealed that: a) KO-DSS showed more aggravated colitis with lower survival and greater weight loss compared with WT-DSS; b) the expression of NLRP3, IL18, IL1β and NLRP1 were significantly increased in KO-DSS (101.00±16.33, 3.28±1.49, 327.50±113.90, 4.92±1.00 respectively) vs WT-DSS; c) the thickness of the collagen layer in KO-DSS was increased vs WT-DSS. As expected, HT29 cells treated with the inflammasome cocktail increased protein expression of caspase-1 and the treatment with the P2X7 antagonist A-80 impaired the inflammasome activation since it significantly reduced the protein expression of caspase-1.
Conclusion
An increased expression of P2X7 receptor, the inflammasome and its APIs is detected in CD patients. Lack of P2X7 worsens chronic colitis associated with an increased activation of the inflammasome. Additional studies are needed in order to elucidate this dual role of P2X7 in intestinal fibrosis.