P0099Investigating Arginase-2 target site blockers as a therapeutic strategy in acute Inflammatory Bowel Disease

Background Macrophage cells, as key meditators of inflammatory processes and their capacity to polarise from a pro-inflammatory (M1) to an anti-inflammatory (M2) phenotype are promising therapeutic targets in inflammatory bowel disease (IBD). Arginase enzymes, Arg1 and Arg2, regulate cellular metabolism, immune responses and proliferation through the urea cycle. These enzymes are broadly considered markers of an M2 macrophage status, particularly Arg1. Our previous work identified that Arg2 is unique in its additional role in regulating cellular oxidative metabolism at mitochondria in inflammatory macrophages1. Subsequently, we developed a small nucleic acid approach, known as Arg2 target site blockers (TSB), to increase Arg2 expression and promote an M2 macrophage phenotype in vivo2. We aimed to confirm Arg2 TSBs as a strategy to modulate IBD via macrophage polarisation. Methods In a preclinical dextran sulfate sodium (DSS)-induced murine model of colitis, a 20mg/kg prophylactic dose of Arg-2 TSB was administered by intraperitoneal injection (i.p.) to wild type (WT) mice. Water (H2O) alone, 2.5% DSS alone and a non-targeting TSB (NT-TSB) + 2.5% DSS group were used as controls. Disease activity was monitored daily using a composite disease activity index (DAI). Histological severity was assessed blindly by an external reviewer using previously published criteria3. Colon lengths were also assessed following animal sacrifice. Inflammatory cytokine concentrations are currently being measured in serum as well colon and liver homogenates using a cytometric bead array (BD Biosciences) and enzyme linked immunosorbent assays (ELISA)(R&D Systems). Results Comparing day 5 to day 0, the NT-TSB + 2.5% DSS and the 2.5% DSS only group both had statistically significant deterioration in DAI scores (Two-way ANOVA with Tukey’s multiple comparisons +3.75 p = 0.043 & +5 p = 0.041 respectively). In contrast, the Arg-2 TSB group, disease activity index reached 3.25 at day 5 compared with baseline and was not statistically significant, indicating a protective effect (p = 0.14). Blinded histological assessment showed trending lower severity scores of inflammation in the Arg2-TSB group, compared to controls (One-way ANOVA p = 0.23). Colon length was preserved in the Arg2-TSB group while the NT-TSB group demonstrated a significant shortening compared with control (Mean difference 1.43cm, p = 0.04). There was no statistically significant differences in both TNF or IL-1β concentrations of liver homogenates (One-way ANOVA p = 0.454 and p = 0.66 respectively). Conclusion These initial studies indicate that prophylactic i.p. administration of 20mg/kg Arg-2 TSB may be protective and ameliorate DAI in the 2.5% DSS murine model of colitis. References: 1.Dowling JK, Afzal R, Gearing LJ, et al. Mitochondrial arginase-2 is essential for IL-10 metabolic reprogramming of inflammatory macrophages. Nat Commun. Mar 5 2021;12(1):1460. doi:10.1038/s41467-021-21617-2 2.De Santi C, Nally FK, Afzal R, et al. Enhancing arginase 2 expression using target site blockers as a strategy to modulate macrophage phenotype. Mol Ther Nucleic Acids. Sep 13 2022;29:643-655. doi:10.1016/j.omtn.2022.08.004 3.Imazu N, Torisu T, Yokote A, et al. Arginase 2 attenuates ulcerative colitis by antioxidant effects of spermidine. J Gastroenterol. Aug 2024;59(8):682-698. doi:10.1007/s00535-024-02104-z Conflict of interest: Dr. Mchale, Ciarán: No conflict of interest Sharma, Shikha: No conflict of interest Agnew, Aidan: No conflict of interest DeSanti, Chiara: No conflict of interest Creagh, Emma: No conflict of interest Corr, Sinead: No conflict of interest Boland, Karen: No conflict of interest Dowling, Jennifer: No conflict of interest