L-cysteine supplementation attenuates local inflammation and restores gut homeostasis in a porcine model of colitis

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Abstract

Background

Inflammatory bowel disease (IBD), a chronic inflammation of the gastrointestinal tract, is characterized by a deregulation of the mucosal immune system and resistance of activated T cells to apoptosis. Current therapeutics show limited efficacy and potential toxicity; therefore there is a need for novel approaches for the treatment of IBD. L-cysteine was examined for its ability to reduce colitis symptoms and modulate local gene expression in a DSS-induced porcine model of colitis.

Methods

Colitis was induced via intra-gastric infusion of dextran sodium sulfate (DSS), followed by the administration of L-cysteine or saline. Clinical signs, morphological measurements, histology and gut permeability were assessed for the prognosis of colitis. Local tissue production of cytokines and gene expression in the colon were analyzed by ELISA and real-time RT-PCR.

Results

L-cysteine supplementation attenuated DSS-induced weight loss and intestinal permeability, reduced local chemokine expression and neutrophil influx, and markedly improved colon histology. Furthermore, cysteine significantly reduced the expression of pro-inflammatory cytokines, including TNF-α, IL-6, IL-12p40, IL-1β, and resulted in increased expression of the apoptosis initiator caspase-8 and decreased expression of the pro-survival genes cFLIP and Bcl-xL.

Conclusions and general significance

These results suggest that L-cysteine administration aids in restoring gut immune homeostasis by attenuating inflammatory responses and restoring susceptibility of activated immune cells to apoptosis, and that cysteine supplementation may be a novel therapeutic strategy for the treatment of IBD.

Introduction

Inflammatory bowel disease (IBD) is a chronically relapsing inflammation of the gastrointestinal tract that manifests itself in one of two main forms, Crohn's disease (CD) and ulcerative colitis (UC). While the etiology of IBD is still not fully understood, it has been suggested that genetic predisposition, environmental factors, intestinal microflora, and impairment of local tolerance leading to an ongoing activation of the intestinal immune system all play a role [1], [2]. Major contributors to IBD pathogenesis include an abundance of pro-inflammatory cytokines, activated lymphocytes, and reactive oxygen species (ROS) in the local inflamed tissue [3], [4]. T lymphocytes in particular may play a key role in the pathogenesis of IBD, and the down-regulation of activated T cells via apoptosis is critical in maintaining immune homeostasis. Recent evidence has suggested that mucosal T cell apoptosis may be inhibited in IBD patients, leading to the accumulation of activated T cells and chronic inflammation [5], [6], [7].

At present, the pharmacological treatments available include 5-aminosalicylate-based compounds, corticosteroids and immunosuppressive and immuno-regulatory agents, however they have limited therapeutic efficacy and are not suitable for long-term use, and have demonstrated toxic side effects [3], [8], [9]. As a result, alternative treatment methods have gained increasing attention, especially nutrition and functional foods. Cysteine, a non-essential amino acid, plays a number of roles in protein metabolism. Similar to other amino acids, cysteine serves as a substrate for protein synthesis; however it encompasses diverse non-nutritional functions. Cysteine is the rate-limiting substrate for the synthesis of glutathione (GSH), which is the most important intracellular antioxidant [10]. Furthermore, cysteine is able to spare methionine by 50–80% [11] and is involved in the synthesis of taurine, both of which are essential for the host defense against oxidative stress [12], [13], [14]. Oxidative stress is an important contributor to the disease pathogenesis of IBD; impairment of GSH synthesis, increased oxidative stress, and anti-oxidant supplementation findings support this claim [3], [15], [16], and DSS-treated animals have been shown to develop acute colitis accompanied by oxidative stress [15]. It has been established that cysteine has potent anti-oxidative roles via GSH synthesis and is able to suppress oxidative stress found in IBD [10]. Moreover, cysteine has been reported to stimulate the synthesis of colonic mucin in rats with colitis, which aids in intestinal epithelial protection [17], and cysteine-containing compounds have been shown to possess anti-inflammatory properties, and to decrease the production of inflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β in murine models of colitis [10], [15]. T lymphocytes have a strong transport activity for cysteine, however circulating cysteine concentrations are typically low (around 10 μM). Plasma concentrations of cystine are much higher, but the membrane transport activity for cystine in human lymphocytes is > 10-fold lower than that for cysteine [18]. Therefore, cysteine may be a promising candidate as a novel therapeutic agent for IBD, and its therapeutic mechanism(s) must be further examined.

Here, the therapeutic efficacy of dietary cysteine supplementation in a dextran sodium sulfate (DSS)-induced porcine model of colitis was evaluated. DSS has been shown in a number of rodent models to induce symptoms of acute colitis, including inflammation in the colon [19], [20], [21]. More recently, porcine models of DSS-induced colitis have been described. Pigs share a similar gastrointestinal morphology and physiology with humans [22] and therefore may represent a more appropriate animal model for the study of IBD therapeutics. Free L-cysteine was used since its metabolism and absorption presents a minimal amount of cellular stress, and does not require digestion or ATP expenditure prior to being absorbed. Thus, cysteine would be rapidly absorbed and not exert additional strain on the already compromised cells [23]. In the present study, we examined the ability of L-cysteine to reduce DSS-induced colitis symptoms and pathology, and evaluated the effect of cysteine on local gene expression, in order to further elucidate the effects of cysteine on intestinal inflammation.

Section snippets

Animals and experimental design

Five-day-old Yorkshire piglets were obtained from the Arkell Swine Research Station (University of Guelph, Guelph, ON). Animals were housed individually in metal floor pens with rubberized floors in a 26 °C temperature controlled 12-hour light/dark cycle room. Piglets were fed three times a day with a commercial milk replacement formula (Soweena® Litter Life; Merrick's Inc. WI) at amounts similar to their ad libitum intake level. All animal work was carried out in accordance with the Canadian

Effect of Cys supplementation on symptomatic parameters in DSS-induced colitis

In order to determine the effect of Cys on the DSS-induced colitis symptoms in the piglets, BW, stool consistency, presence of blood, and feed intake were recorded daily. The majority of the animals had loose stool prior to DSS infusion, as commonly seen during diet adjustment to liquid formula [33]. Following DSS administration, animals in the Pos and Cys groups had bloody and severe diarrhea, which was resolved following cysteine administration. Initial and final BW, daily BW gain and average

Discussion

Current IBD therapeutic agents have demonstrated limited efficacy and potential long-term toxicity. Therefore, there is a need for the development of novel strategies for the treatment of IBD. The purpose of the present study was to evaluate the therapeutic effects of supplementation with L-cysteine using a porcine model of DSS-induced colitis. Nutrition is an important factor for IBD patients due to reduced appetite and compromised intestinal absorption. Furthermore, controlling oxidative

Acknowledgements

The authors would like to thank Dr. M.A. Hayes, Department of Pathobiology, University of Guelph, for histological analyses, and Maggie Lee and Anil Puttaswamy for their technical assistance. This work was funded by Advanced Foods and Materials Network (AFMNet), Networks of Centres of Excellence, Canada.

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