Effects of adenosine A₂A receptor activation and alanyl-glutamine in Clostridium difficile toxin-induced ileitis in rabbits and cecitis in mice

Abstract

Background: Severe Clostridium difficile toxin-induced enteritis is characterized by exuberant intestinal tissue inflammation, epithelial disruption and diarrhea. Adenosine, through its action on the adenosine A2A receptor, prevents neutrophillic adhesion and oxidative burst and inhibits inflammatory cytokine production. Alanyl-glutamine enhances intestinal mucosal repair and decreases apoptosis of enterocytes. This study investigates the protection from enteritis by combination therapy with ATL 370, an adenosine A2A receptor agonist, and alanyl-glutamine in a rabbit and murine intestinal loop models of C. difficile toxin A-induced epithelial injury.

Methods: Toxin A with or without alanyl-glutamine was administered intraluminally to rabbit ileal or murine cecal loops. Animals were also given either PBS or ATL 370 parenterally. Ileal tissues were examined for secretion, histopathology, apoptosis, Cxcl1/KC and IL-10.

Results: ATL 370 decreased ileal secretion and histopathologic changes in loops treated with Toxin A. These effects were reversed by the A2A receptor antagonist, SCH 58261, in a dose-dependent manner. The combination of ATL 370 and alanyl-glutamine significantly further decreased ileal secretion, mucosal injury and apoptosis more than loops treated with either drug alone. ATL 370 and alanyl-glutamine also decreased intestinal tissue KC and IL-10.

Conclusions: Combination therapy with an adenosine A2A receptor agonist and alanyl-glutamine is effective in reversing C. difficile toxin A-induced epithelial injury, inflammation, secretion and apoptosis in animals and has therapeutic potential for the management of C. difficile infection.

Figure 1

Effects of ATL 370 alone on C. difficile toxin A-induced secretion and epithelial injury. Increasing doses of intravenous ATL 370 (adenosine A_2A receptor agonist; ng) decreased toxin A (10 μg)-induced intraluminal fluid accumulation (A) and histopathology scores (B) in rabbit ileal loops. Panel 1A: a- P < 0.0001 (one-way ANOVA), and b(PBS vs. Toxin A)- P < 0.001, c(Toxin A vs ATL150)- P < 0.01 (Bonferroni's). Panel 1B: a- P < 0.0001 (one-way ANOVA), b(PBS vs. Toxin A) - P < 0.001 (Bonferroni's), c (Toxin A vs ATL15)- P = 0.04 (Unpaired t-test with Welch's). Results were data pooled from 8 to 28 loops from 2 to 5 rabbits per group from 5 separate experiments.

Figure 2

Effects of SCH-58261 treatment on ATL 370-treated C. difficile toxin A-challenged loops. Increasing doses of adenosine A_2A receptor blocker, SCH 58261 (given IV), reversed the effects of ATL 370 (1.5 ng IV) on secretion (A) and epithelial injury (B) in a dose-dependent manner in rabbit ileal loops. 3.5 ng of SCH 58261 is equimolar to 1.5 ng of ATL370. Panel 2A: a- P < 0.0001 (one-way ANOVA), and b(PBS vs. Toxin A)- P < 0.001, c(Toxin A vs ATL + SCH)- P < 0.01 (Bonferroni's). Panel 2B: a- P < 0.0001 (one-way ANOVA), b(PBS vs. Toxin A)- P < 0.001 (Bonferroni's). Results were data pooled from 4 to 28 loops from 1 to 6 rabbits per group from 5 separate experiments.

Figure 3

Effects of low dose ATL 370 and alanyl-glutamine on C. difficile toxin A-induced secretion and epithelial injury. Varying doses of alanyl-glutamine (AQ; mM) in the lumen when given in addition to intravenous ATL 370 (1.5 ng) showed decrease in toxin A (10 ug)-induced intraluminal fluid accumulation (A) and epithelial injury (B) in rabbit ileal loops. Panel 3A: a- P = 0.08 (one-way ANOVA), and b (PBS vs. Toxin A)- P = 0.0002, c (Toxin A vs ATL/AQ10)- P = 0.02, d (Toxin A vs ATL/AQ30)- P = 0.01, e (Toxin A vs ATL/AQ100)- P = 0.0004 (Unpaired t-test with Welch's). Panel 3B: a- P < 0.0001 (one-way ANOVA), and b(PBS vs. Toxin A)- P < 0.001, c (Toxin A vs ATL/AQ10)- P < 0.01, d (Toxin A vs ATL/AQ30)- P < 0.01, e (Toxin A vs ATL/AQ100)- P < 0.001 (Bonferroni's). Results were data pooled from 4 to 16 loops from 2 rabbits per group from 2 separate experiments.

Figure 4

Effects of ATL 370 and alanyl-glutamine on C. difficile toxin A-induced ileal histopathology. Toxin A (10 μg, B) caused mucosal disruption, increased cellularity and vascular congestion compared with PBS (A) in rabbit ileal tissues. Treatment with intravenous ATL 370 (1.5 ng, D) or intraluminal alanyl-glutamine (100 mM, C) reduced some epithelial injury. Marked preservation of mucosal architecture was noted in tissues treated with the combination of ATL and alanyl-glutamine (E)

Figure 5

Effects of ATL 370 and alanyl-glutamine on cytokine expression. After four hours of incubation, alanyl-glutamine (AQ; 100 mM in loops) or ATL 370 (3 ng/g s.c.) significantly decreased C. difficile toxin A-induced elevation of KC secretion in mouse cecal tissues (A). Combination treatment with ATL 370 and AQ significantly decreased KC secretion further. Likewise, IL-10 secretion was increased by toxin A although not significantly reduced by either AQ or ATL 370 alone (B). Only combination treatment with ATL 370 and alanyl-glutamine significantly decreased IL-10 secretion (B). C and D show corresponding KC and IL-10 mRNA expressions expressed as fold-change from PBS control. Number of mice per group: Panels A&B- 5-6; Panels C&D- 3-5. Panel 5A: a- P < 0.0001 (one-way ANOVA), and b (Toxin A vs AQ)- P < 0.01, c (Toxin A vs AQ)- P < 0.001, d (Toxin A vs ATL)- P < 0.01, e (Toxin A vs ATL/AQ)- P < 0.001 (Bonferroni's). Panel 5B: a- P < 0.0001 (one-way ANOVA), and b (Toxin A vs PBS)- P < 0.01, c (Toxin A vs ATL/AQ)- P < 0.01 (Bonferroni's). Panel 5C: a- P = 0.04 (one-way ANOVA), and b (Toxin A vs ATL/AQ)- P < 0.05 (Bonferroni's). Panel 5D: a- P = 0.056 (one-way ANOVA)

Figure 6

Effects of ATL 370 and alanyl-glutamine on C. difficile toxin A-induced apoptosis. Toxin A (10 μg, B) caused significant apoptosis in the rabbit ileal tissues compared with PBS (A). Treatment with ATL 370 (1.5 ng/rabbit IV, D) or alanyl-glutamine (AQ;100 mM in loops, C) caused some reduction of apoptotic cells but only significant difference was noted with treatment with AQ. A marked decrease in apoptotic cells was noted in tissues treated with the combination of ATL and alanyl-glutamine (E). a- P < 0.0001 (one-way ANOVA), and b (PBS vs. Toxin A)- P < 0.001, c (Toxin A vs ATL/AQ10)- P < 0.01, d (Toxin A vs ATL/AQ100)- P < 0.001 (Bonferroni's). Apoptotic cells were counted in 12 high power fields (3 hpfs/quadrant) per slide from 3 to 4 ileal specimens per group.