W. A. Julliard1, J. Fechner1, T. De Wolfe3, N. Safdar2, J. Mezrich1 1University Of Wisconsin,Department Of Surgery, Division Of Transplant Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Medicine, Division Of Infectious Diseases,Madison, WI, USA 3University Of Wisconsin,Department Of Food Science,Madison, WI, USA
Introduction: Clostridium difficile infection is a major epidemic in the United States responsible for over 14,000 deaths a year and greater than $1 billion a year in healthcare costs. The relationship between gut immunity and C. difficile infection is poorly understood. The aryl hydrocarbon receptor (AHR) is a receptor for endogenous and exogenous ligands, and is important in maintenance of gut immunity, generation of FoxP3+ Tregs, and the protective cytokine IL-22. AHR ligands are found in cruciferous vegetables and are made by normal bacteria and probiotics, and may be reduced after certain antibiotic treatment. We hypothesize that dietary administration of the AHR ligand indole-3-carbonyl (I3C) will alter the host immune system and protect against C. difficile infection.
Methods: Male B6 mice were placed on one of three diets for two weeks; standard mouse chow (“standard”), a diet deficient of all AHR ligands (“base diet”), and the same base diet supplemented with 0.1% I3C (“I3C diet”). After two weeks on diet, mice were started on a C. difficile model consisting of antibiotic administration followed by C. difficile inoculation. Mice were then monitored for signs of disease and overall survival. At various time points throughout the experiment, tissue was collected from the cecum to be analyzed by qRT-PCR and histopathology.
Results: Pre-inoculation, mice fed the I3C diet had extremely high levels of mRNA expression of the AHR dependent enzyme Cyp1A1 in their cecum compared to mice on the base diet. I3C mice also had higher levels of FoxP3 mRNA in their cecum compared to base diet mice. After inoculation with C. difficile, standard chow mice had a typical disease response with severe weight loss and 50% mortality by day three. Base diet mice had delayed disease onset, but by day four demonstrated severe disease and overall 87.5% mortality. In stark contrast, I3C diet mice had minimal weight loss and an overall mortality of only 25%. Post-inoculation analysis demonstrated high levels of cecal Cyp1A1, FoxP3, and IL-22 mRNA expression in I3C mice compared to base diet mice. Furthermore, histology of base diet mice demonstrated increased levels of neutrophil margination, tissue edema, and inflammation compared to I3C mice.
Conclusions: Our study demonstrates that the dietary supplementation of the AHR ligand I3C significantly reduces C. difficile associated disease in a well-accepted mouse model. The mechanism for disease prevention appears to be through activation of the AHR as indicated by elevated levels of Cyp1A1 in I3C diet mice. These results suggest a potential therapeutic option for preventing C. difficile infection and could represent a major breakthrough in C. difficile treatment.
