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Ursodeoxycholic acid inhibits colonic mucosal cytokine release and prevents colitis in a mouse model of disease Joseph BJ Ward1, Orlaith Kelly1,2, Siobhan Smith3, Joan Ní Gabhann3, Murtaza Tambuwala4, Frank Murray2, Caroline Jefferies3, Cormac Taylor4 and Stephen Keely1. 1RCSI, Beaumont Hospital. 2 Dept. of Gastroenterology, Beaumont Hospital. 3 RCSI, St. Stephens Green, 4 UCD. 8 * ** 0 *** 0 1 2 3 4 5 6 Time (days) B C UDCA attenuates TLR-3 driven cytokine release from colonic epithelial cells Colon length (mm) Vehicle UDCA (30 mg/kg) DSS (2.5%) + Vehicle 120 DSS (2.5%) + UDCA (30 mg/kg) *** 80 * 60 40 20 0 *** Vehicle + UDCA Vehicle + UDCA (30 mg/kg) (30 mg/kg) 80 ** # 60 n = 6 - 12, ANOVA + Tukey * compared to DSS + Veh ## D 40 Untreated DSS Untreated 1.0 0.8 0.6 0.4 *** - 1 50 100 200 + + - + + - 10 Control PBS DSS (2.5 %) DSS + 6MUDCA (30 mg/kg) 8 6 4 2 0 0 + + 1 2 3 4 5 6 Time (days) n=3-9 UDCA metabolites are elevated in a mouse model of disease + DSS (2.5 %) 40 DSS [UDCA] ( M) n = 6, + - 12 Figure 5. 6MUDCA attenuates in vitro cytokine release, but is not protective in a murine model of colitis. A) The TLR3 agonist, Poly I:C (25 μg/ml), significantly increased TNF-a release compared to control. This response was reduced by cotreatment with either UDCA (200 mM) or a stable analogue, 6MUDCA (n = 5 p 0.001). B) However, in contrast to UDCA, intraperitoneal administration of 6MUDCA (30mg/kg) to male C57 BL/6 mice receiving DSS in their drinking water, did not reduce the DAI (n = 3 - 9). PBS Control P.IC *** 0.2 20 0 B 1.2 0.0 PI:C UDCA 6MUDCA *UDCA compared to DSS RESULTS [TNF- ] (% Poly I:C) *** n = 6-12 Statistical analyses were performed using ANOVA and SNK or Tukey post hoc test. 100 *** 4 METHODS A A 6 2 6MUDCA Disease activity index Here we sought to investigate a potential role for UDCA in regulating colonic epithelial cytokine release and to investigate the therapeutic potential of UDCA in a mouse model of disease. 10 Vehicle UDCA (30 mg/kg) UDCA (100 mg/kg) DSS (2.5 %) DSS + UDCA (30mg/kg) DSS + UDCA (100mg/kg) [bile acid] ( M) A 12 Metabolically stable analogues of UDCA are ineffective against colitis in a mouse model of disease TNF release (fold change over PI:C) Toll-like receptors (TLRs) play a critical role in innate immune responses to intestinal pathogens. In inflammatory bowel disease (Ulcerative Colitis and Crohn’s Disease), epithelial TLR expression is increased. Activation of these TLRs is likely to play an integral role in the pro-inflammatory cytokine release responsible for the large-scale epithelial damage that is typical of active colitis. Ursodeoxycholic acid (UDCA), a bile acid, is a well-established therapy for inflammatory diseases of the liver, where it is known to act at least partly, through inhibition of cytokine release. However, the role of UDCA in regulating intestinal epithelial cytokine secretion is unknown. UDCA ameliorates colitis in a mouse model of disease Disease Activity Index INTRODUCTION * compared to control UDCA CDCA LCA 3- 7-oxo *** 30 *** *** 20 ** 10 # compared to Poly I:C *** n = 4, ANOVA + SNK 1200 * compared to control # pg/ml 20 10 800 40 400 200 UDCA PIC 0 - + - D + + + *** * compared to control 200 + + + n = 4, ANOVA + SNK *** * compared to control # # compared to PIC ** pg/ml pg/ml 2.0 ** 300 100 UDCA PIC + - IL-1 2.5 n = 4, ANOVA + SNK 0 - E IL-6 400 0 UDCA PIC *** 30 20 * compared to 0 mg/kg vehicle # compared to 0 mg/kg DSS 10 0 30 Vehicle + UDCA (mg/kg) 1.0 # *** 0 1.5 0 30 DSS (2.5%) + UDCA (mg/kg) 0.5 - + - + 0.0 UDCA PIC + + - + - + + + Figure 1. UDCA attenuates TLR-3 driven cytokine release from T84 cells. A) The TLR3 agonist, Poly I:C (25 μg/ml), significantly increased TNF-a release compared to control, which was significantly reduced in a concentration-dependent manner by co-treatment with UDCA (0-200 mM). Poly I: C-stimulated B) TNF-a release, C) IL-8 release, D) IL-6 release and E) IL-1b release were all significantly reduced to by co-treatment with UDCA (200 mM). UDCA attenuates TRIF/TBK1 signalling Effect of UDCA on P125 promoter transcription Luciferase activity (fold change over control EV) Figure 6. UDCA administration alters the caecal bile acid pool. Intraperitoneal administration of UDCA (30 mg/kg) to male C57 BL/6 mice receiving DSS in their drinking water, significantly elevated levels of UDCA metabolites, LCA and 7keto LCA. E 600 100 Control UDCA (200 M) * 80 Figure 3. UDCA is protective in a murine model of colitis. A) Intraperitoneal administration of UDCA (30 mg/kg and 100 mg/kg) to male C57 BL/6 mice receiving DSS in their drinking water, significantly reduced the disease activity index (DAI) from 10 ± 0.3 (DSS alone) to 7.2 ± 0.7 (UDCA 30mg/kg) and 5.8 ± 0.5 (UDCA 100mg/kg) (n = 6 - 12, p 0.001). B) Mice treated with DSS alone had shorter colons and a lack of faecal pellet formation. In contrast, mice co-treated with UDCA had longer colons with clear evidence of faecal pellet formation. C) The average length of colons in DSS-treated mice was shorter than in mice cotreated with UDCA (30 mg/kg) (n = 6-12, p < 0.05). D) H and E staining was performed on colonic sections and E) UDCA (30 mg/kg) significantly reduced the inflammation score of the sections from 37.3 ± 0.3 in DSS alone, to 29.0 ± 3.5 (ANOVA, n = 5, p 0.05). Biochemistry of UDCA metabolism ANOVA + SNK, N = 6, p 0.05 60 40 20 0 EV TRIF TBK1 IKKe IRF3 + P125 luciferase Figure 2. UDCA inhibits TLR signalling at TRIF/TBK1. Overexpression analysis of proteins in the TLR signalling pathway revealed that UDCA (200 mM) exerts its inhibitory effect at the TIR-domain-containing adapter-inducing interferonβ (TRIF)/TANK-binding kinase 1 (TBK1) junction (n = 6, p 0.05). DSS DSS Basal + UDCA n = 3, ANOVA + SNK, ** P 0.01, ** P 0.001 *** 1000 UDCA * vs respective basal bile acid # # compared to PIC *** Basal Figure 4. UDCA and its derivatives can be readily interconverted. Liver enzymes along with bacterial enzymes in the colon can readily interconvert UDCA to LCA and 7-keto LCA. The UDCA metabolite, LCA prevents colitis in a mouse model of disease 1.2 1.0 0.8 0.6 0.4 *** 0.2 0.0 Poly I:C 0 - 0 + 0.001 0.1 + + 1 + LCA ( M; 24 hours) n = 7, ANOVA + SNK, p 0.001 compared to Poly I:C 10 + Disease Activity Index 30 1400 *** IL-8 TNF release (fold change over control) n = 4, ANOVA + SNK * compared to control # compared to PIC 0 Inflammation score 40 pg/ml C TNF- UDCA B 12 Control LCA (30 mg/kg) DSS DSS + LCA (30 mg/kg) 10 8 *** 6 *** 4 2 0 0 1 2 3 4 5 Time (days) n = 5, * DSS + LCAcompared to DSS Figure 7. LCA attenuates in vitro cytokine release and is protective in a murine model of colitis. A) The TLR3 agonist, Poly I:C (25 μg/ml), significantly increased TNF-a release compared to control. This response was reduced by co-treatment with LCA (10 mM) (n = 7 p 0.001). B) Intraperitoneal administration of the UDCA metabolite, LCA (30 mg/kg), to male C57 BL/6 mice receiving DSS in their drinking water, significantly reduced the DAI from 11.2 ± 0.6 (DSS alone) to 5.4 ± 0.9 (n = 5, p 0.001). SUMMARY & CONCLUSION These studies reveal a novel role for UDCA in regulating colonic epithelial cytokine secretion. UDCA significantly attenuates TLR-3dependent cytokine release from colonic epithelial cells through a pathway dependent on TRIF/TBK-1. UDCA also ameliorates the effects of colitis in a mouse model. Interestingly, metabolically stable analogues of UDCA do not prevent colitis in a mouse model, whereas the metabolic product of UDCA, LCA, practically abolishes DSSinduced colitis. In conclusion, our data suggest that, by virtue of their effects in preventing TLR-induced proinflammatory cytokine release, UDCA and its metabolic products are good targets for developing new approaches to treat IBD. 6