P018 Expression of annexin A1 and NLRP3 inflamassome in Chron’s disease

Abstract

Abstract Background Despite annexin A1 (ANXA1), an anti-inflammatory and pro-resolving protein, regulates the NLRP3 inflammasome activation in inflammatory cells, this relationship has not been explored in inflammatory bowel disease. Then, this study evaluated the expression patterns of ANXA1 and NLRP3 inflammasome in Crohn’s disease (CD). Methods Paraffin-embedded human intestine biopsies, obtained from CD (n=20) and control (n=8) patients were processed for histological and immunohistochemical analyses. The study was approved by the UNIFESP Ethics Committee (CEP 4.491.836/2020). GSE179285 study, which features ileum (Control - n=8, CD non-inflamed - n=48, CD inflamed - n=33) and colon (Control - n=12, CD non-inflamed – n=74, CD inflamed - n=14) transcriptome, was selected from the Gene Expression Omnibus repository. Datasets were analyzed using the GEO2R tool to detect ANXA1, CASP1, NLRP3, and PYCARD expressions. Results Bioinformatics analysis of GSE179285 samples showed a marked increase in the ANXA1 expression in the CD inflamed ileum and colon compared to CD non-inflamed and controls samples (Fig.1A). Regarding the genes associated with the NLRP3 inflammasome activation, transcriptome analysis also revealed a significant increase in NLRP3 and CASP1 in the CD inflamed intestine compared to the other ones (Fig.1B,C). PYCARD expression did not show alterations between the different conditions (Fig.1D). Immunohistochemical analyzes showed intense immunoreactivity for ANXA1 and NLRP3 proteins in the CD ileum and colon, especially in inflammatory infiltrate and epithelium of the mucosal layer (Fig.2C,D,G,H), compared to controls (Fig. 2A,B,E,F). These findings corroborate the transcriptome analyses. Conclusion ANXA1 and NLRP3 expression is increased in CD-inflamed intestines. High levels of ANXA1 may contribute to controlling inflammation and repairing intestinal injuries. High levels of NLRP3 evidence dysfunction in the pro-inflammatory activity of DC, contributing to the release of IL-1β and IL-18. Support: FAPESP (grants 20/03565-2, 21/01541-1 and 21/06059-3). Figure 1. ANXA1, NLRP3, CASP1 and PYCARD expressions. Analysis performed in the healthy or CD terminal ileum and ascending/descending colon of human biopsies from GSE179285 study. Data represent mean ± S.E.M. of the gene expression values. **p<0.01; ***p<0.001; ****p<0.0001 (Kruskal-Wallis, Dunn’s post-test). Figure 2. ANXA1 and NLRP3 expression in human intestine. Representative images of ileum (A, C, E, G) and colon (B, D, F, H) of control (A,B,E,F) and CD patients (C,D,G,H). ANXA1 and NLRP3 expression detected in intestinal epithelium (arrows) and inflammatory infiltrate (asterisks). Counterstaining: Carazzi’s Hematoxylin. Bars: 50 µm.