Basic Science Luminal

Abstract

Introduction:We have previously shown that appendicitis following appendicectomy offered protection against trinitrobenzene sulfonic acid induced colitis in an age and antigen dependent fashion (Ng et al, Gut, 2013 in revision). The protective effect was associated with an expansion of regulatory T cells (Tregs) in the colon following migration from the appendix. It was dependent on the production of the immunoregulatory cytokine interleukin (IL)-10 as neutralisation of IL-10 abrogated the protective effect. The mechanisms underlying the Treg expansion following appendicitis induction remain to be elucidated. Retinoic acid (RA) is an active metabolite of vitamin A that regulates various cellular functions such as proliferation and differentiation and mediates its effects through the retinoic acid receptors (RARs). RA has been shown to play a pivotal role in the development of Tregs in the gastrointestinal system. Based on this, the current study seeks to investigate the role of RA signalling in the appendicitis model with the use of a RAR inhibitor. Methods: The RAR inhibitor LE135 is known to be an effective blocker of RARs in vitro; we used it to block RA signaling in vivo in mice with appendicitis. 5-week-old Balb/c mice were injected with 100 μg of LE135 in dimethyl sulfoxide (DMSO) per 20 g of body weight, daily for 3 days. A separate group of mice were injected with DMSO only at the same dose as a control for RA inhibition. Appendicitis was induced in both groups of mice on day 4 and injections were continued for another 4 days. Both groups of mice were sacrificed 24 hours after the last injection and the liver and colon were harvested and processed for histological analysis to assess toxicity of LE135 and DMSO. To determine the effectiveness of RA signalling inhibition by LE135, RNA was collected from control and treated mouse colons (n = 4) and the changes in the mRNA levels of the putative Treg markers FoxP3 and IL-10 and the surrogate marker for RA inhibition MKP-1 were measured via quantitative real time PCR analysis. Results: Histological examination of the liver and colon from control and LE135-treated mice showed no morphological changes, thus confirming LE135 is safe to use in the appendicitis model. LE135 caused a reduction in MKP-1, FoxP3 and IL-10 mRNA levels in colon tissue compared to mice treated with the vehicle control. These results suggest the current dose of LE135 in vivo is effective in inhibiting the RA pathway in the appendicitis model. Conclusion: LE135 caused a reduction in MKP-1, FoxP3 and IL-10 mRNA levels suggesting that LE135 at the dose of 100 μg per 20 g reduced RA signaling in the colon. In future studies we aim examine the effect of RA inhibition in a colitis model as the regulation of Treg differentiation by RA may be a potential mechanism mediating the appendicitis induced protective effect against colitis. stem cell in the gastrointestinal tract, bone and tumour microenvironment DL WORTHLEY, M CHURCHILL, YL SI, N MANIERI, D LEVIN, Y HAYAKAWA, BW RENZ, XW CHEN, S AS...