Elsevier

Immunobiology

Volume 219, Issue 10, October 2014, Pages 802-812
Immunobiology

Bacterial β-(1,3)-glucan prevents DSS-induced IBD by restoring the reduced population of regulatory T cells

https://doi.org/10.1016/j.imbio.2014.07.003Get rights and content

Abstract

Bacterial β-(1,3)-glucan has more advantages in terms of cost, yield and efficiency than that derived from mushrooms, plants, yeasts and fungi. We have previously developed a novel and high-yield β-(1,3)-glucan produced by Agrobacterium sp. R259. This study aimed to elucidate the functional mechanism and therapeutic efficacy of bacterial β-(1,3)-glucan in dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD).Mice were orally pretreated with bacterial β-(1,3)-glucan at daily doses of 2.5 or 5 mg/kg for 2 weeks. After 6 days of DSS treatment, clinical assessment of IBD severity and expression of pro-inflammatory cytokines were evaluated. In vivo cell proliferation was examined by immunohistochemistry using Ki-67 and ER-TR7 antibodies. The frequency of regulatory T cells (Tregs) was analyzed by flow cytometry. Natural killer (NK) activity and IgA level were evaluated using NK cytotoxicity assay and ELISA.The deterioration of body weight gain, colonic architecture, disease score and histological score was recovered in DSS-induced IBD mice when pretreated with bacterial β-(1,3)-glucan. The recruitment of macrophages and the gene expression of proinflammatory cytokines, such as IL-1β, IL-6 and IL-17A/F, were markedly decreased in the colon of β-(1,3)-glucan-pretreated mice. β-(1,3)-Glucan induced the recovery of Tregs in terms of their frequency in DSS-induced IBD mice. Intriguingly, β-(1,3)-glucan reversed the functional defects of NK cells and excessive IgA production in DSS-induced IBD mice.We conclude that bacterial β-(1,3)-glucan prevented the progression of DSS-induced IBD by recovering the reduction of Tregs, functional defect of NK cells and excessive IgA production.

Introduction

Inflammatory bowel disease (IBD) is caused by intestinal inflammation or ulceration and is often referred to as Crohn's disease (CD) or ulcerative colitis (UC) (Baumgart and Sandborn, 2007, Hendrickson et al., 2002). While CD causes inflammation in any part of gastrointestinal tract from the mouth to the anus, UC only affects the colon and rectum (Baumgart and Sandborn, 2007, Hendrickson et al., 2002, Podolsky, 2002, Strober et al., 2002, Fiocchi, 1998, Huibregtse et al., 2007). IBD is characterized by pathologic symptoms, such as bloody diarrhea, intestinal motility disorder and colonic shortening (Baumgart and Sandborn, 2007, Hendrickson et al., 2002). Although environmental, genetic and immune factors are likely involved in the development of IBD, its pathogenesis remains unclear (Coombes et al., 2005, Kaser et al., 2010, Allez and Mayer, 2004, Singh et al., 2001). In experimental studies, DSS-induced IBD is a standard model that is widely used to test the efficacy of therapeutic approaches for IBD (Jurjus et al., 2004, Wirtz and Neurath, 2007).

Among cells with the ability to inhibit progression of IBD, Tregs play critical roles in the regulation of inflammatory responses by inhibiting the proliferation of inflammatory cells and the production of proinflammatory cytokines (Groux and Powrie, 1999, Pandolfi et al., 2009). CD8αα+TCRαβ+ Tregs, a newly described subset of CD8αα+TCRαβ+ Tregs, recognize T cell receptor-derived peptide in the context of class Ib major histocompatibility complex (MHC) molecule Qa-1. CD8αα+TCRαβ+ Tregs play an important role in controlling autoimmune disease including IBD by suppressing activated CD4+ and CD8+ T cells (Fanchiang et al., 2012, Smith and Kumar, 2008). In contrast, CD4+Foxp3+ Tregs recognize class II MHC and primarily suppress the priming of naïve or unstimulated CD4+ and CD8+ T cells. CD4+Foxp3+ Tregs were compromised in lympho-proliferative disorder, autoimmune disease and allergy (Fanchiang et al., 2012, Smith and Kumar, 2008). Moreover, Tregs suppress the effector functions of immune cells, such as natural killer (NK) cells and B cells (Terme et al., 2008, Ghiringhelli et al., 2005, Lim et al., 2005). NK cell – mediated lysis of human tumor cells is impaired by Tregs in cancer patients (Yadav et al., 2011). In secondary lymphoid organs, IgA production is reduced by Treg-mediated inhibition of B cell activation and Ig class switch recombination (Manzano et al., 1992). With regard to IBD, studies in vivo and in vitro have demonstrated that the incidence of IBD is accompanied by a reduction of NK cytotoxicity (Yadav et al., 2011, Manzano et al., 1992) and an augmentation of IgA production (Sandborn, 2004, Wang et al., 2011).

Drugs used for the treatment of IBD include antibiotics, immunosuppressive drugs, anti-inflammatory drugs and antibody-based therapeutics (Hendrickson et al., 2002). Although numerous studies have aimed to develop side effect-free treatment for IBD, many of these drugs cause adverse side effects, including osteoporosis, neurotoxicity and gastrointestinal intolerance (Hendrickson et al., 2002). To circumvent these side effects, several studies have attempted to develop an IBD therapy using a variety of natural products (Debnath et al., 2013, Hur et al., 2012, Fan et al., 2013). The curcumin extracted from the rhizome of Curcuma longa decreases the disease activity index, histological colitis score, cellular infiltration and epithelial disruption in DSS-induced colitis mice (Deguchi et al., 2007). Anthocyanins extracted from blueberries inhibit the levels of nitric oxide (NO), IL-12, TNF-α and IFN-γ in trinitrobenzene sulfonic acid (TNBS)-induced IBD mice (Wu et al., 2011). Other natural products, including probiotics, vitamin C/E and n-3 fatty acids, have been reported to improve symptoms of IBD (Haddad et al., 2005). However, these findings are inconsistent, and the underlying mechanisms remain unclear.

β-(1,3)-Glucans are a heterogeneous group of polysaccharides derived from bacteria, fungi, yeast and mushrooms, primarily in the form of β-(1,3)/(1,6)-glucan, and are found in oats and barleys as β-(1,3)/(1,4)-glucan (Mantovani et al., 2008). While β-(1,3)/(1,6)-glucan has immunostimulatory properties and antitumor activities, β-(1,3)/(1,4)-glucan reduces blood sugar and cholesterol levels (Mantovani et al., 2008). The significance of the immunopharmacological activities of β-glucans varies depending on their sources and structures, including molecular weight, degree of branching and conformation (Kim et al., 2006, Brown and Gordon, 2005). Previous studies have reported that β-(1,3)-glucan exhibits various immunomodulatory properties, including humoral and cellular immunity, and thereby protects against tumor development and infection by pathogens (Mantovani et al., 2008, Kim et al., 2006, Brown and Gordon, 2005). β-(1,3)-glucan derived from Geastrum saccatum mushrooms induces anti-inflammatory responses by inhibiting nitric oxide synthase (NOS) and cyclooxygenase (COX) in croton oil-induced ear edema mice (Guerra Dore et al., 2007). Recently, β-(1,3)-glucan extracted from Pleurotus pulmonarius mushrooms revealed anti-inflammatory activity by suppressing expression of pro-inflammatory cytokines, such as IL-1 and TNF-α, in DSS-induced colitis mice (Lavi et al., 2010). In particular, bacterial β-(1,3)-glucan has more advantages in terms of cost, yield and efficiency than that derived from mushrooms, plants, yeasts and fungi (Kim et al., 2003). However, the precise mechanisms by which bacterial β-(1,3)-glucan inhibits inflammatory responses in IBD have not yet been elucidated.

We have previously identified a novel and high-yield β-(1,3)-glucan produced by Agrobacterium sp. R259, which may stimulate immune responses by increasing IFN-γ production in peripheral blood mononuclear cells (Kim et al., 2003). In the present study, to investigate the preventive or therapeutic effects of bacterial β-(1,3)-glucan in DSS-induced IBD, mice were preadministered β-(1,3)-glucan before DSS treatment, and the clinical signs of IBD were evaluated. To date, the mechanisms by which β-(1,3)-glucan is involved in the suppression of DSS-induced IBD through the regulation of Tregs and immune cell homeostasis has not yet been investigated.

Section snippets

Mice

Seven-week-old male C57BL/6 wild-type (WT) mice were purchased from the Damool Animal Breeding company (Daejeon, Korea). All of the mice were maintained under specific pathogen-free conditions, and all of the animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care Committee of Chonnam National University.

Antibodies

Flow cytometry analysis, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were carried out using the following antibodies: horse

Prevention of clinical signs by bacterial β-(1,3)-glucan in DSS-induced IBD mice

The toxicity of bacterial β-(1,3)-glucan has not been reported in both human and rodent studies (Spicer et al., 1999). In our preliminary study, the most potent immunostimulatory effects of β-(1,3)-glucan were observed in the colon of WT mice when orally administered with 2.5 or 5 mg/kg (data not shown). Based on these data, doses of 2.5 and 5 mg/kg β-(1,3)-glucan were chosen for the present study. To determine whether β-(1,3)-glucan prevents DSS-induced IBD, we investigated the clinical signs of

Discussion

This study aimed to elucidate the anti-inflammatory mechanism of bacterial β-(1,3)-glucan in DSS-induced IBD. Here, we provided the first demonstration that β-(1,3)-glucan prevented the progression of DSS-induced IBD through Treg-mediated inhibition of inflammatory responses as well as maintenance of intestinal immune cell homeostasis. Our findings that β-(1,3)-glucan inhibited the expression of inflammatory cytokines and ROS due to the colonic infiltration of macrophages and fibroblasts were

Acknowledgments

This work was supported by Bio-industry Technology Development Program (111052-04-1-SB010, 111052-04-1-SB020 and 111096-03), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. We would like to express our sincere gratitude to Naturence Co., Ltd. (Gongju City, Korea) for the β-(1,3)-glucan used in this study.

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    These authors contributed equally to this work.

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