Food Chemistry 136 (2013) 1288–1295 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem The nutritional supplement Active Hexose Correlated Compound (AHCC) has direct immunomodulatory actions on intestinal epithelial cells and macrophages involving TLR/MyD88 and NF-jB/MAPK activation Abdelali Daddaoua a, Enrique Martínez-Plata b, Mercedes Ortega-González c, Borja Ocón d, Carlos J. Aranda c, Antonio Zarzuelo d, María D. Suárez c, Fermín Sánchez de Medina d, ⇑ Olga Martínez-Augustin c, a Department of Environmental Protection, Consejo Superior de Investigaciones Científicas, C/ Profesor Albareda 1, E-18008 Granada, Spain b Pharmacy Department, Hospital de Poniente, El Ejido, Almería, Spain c Department of Biochemistry and Molecular Biology II, CIBERehd, School of Pharmacy, University of Granada, Granada, Spain d Department of Pharmacology, CIBERehd, School of Pharmacy, University of Granada, Granada, Spain article info abstract Article history: Active Hexose Correlated Compound (AHCC) is an immunostimulatory nutritional supplement. AHCC Received 29 May 2012 effects and mechanism of action on intestinal epithelial cells or monocytes are poorly described. AHCC Received in revised form 31 July 2012 was added to the culture medium of intestinal epithelial cells (IEC18 and HT29 cells) and monocytes Accepted 5 September 2012 (THP-1 cells) and assessed the secretion of proinflammatory cytokines by ELISA. Inhibitors of NFjB Available online 23 September 2012 and MAPKs were used to study signal transduction pathways while TLR4 and MyD88 were silenced in IEC18 cells using shRNA. It was found that AHCC induced GROa and MCP1 secretion in IEC18 and IL-8 Keywords: in HT29 cells. These effects depended on NFjB activation, and partly on MAPKs activation and on the AHCC presence of MyD88 and TLR4. In THP-1 cells AHCC evoked IL-8, IL-1b and TNF- secretion. The induction Enterocyte a Macrophage immunomodulatory of IL-8 depended on JNK and NFjB activation. Therefore, AHCC exerts immunostimulatory effects on NFjB intestinal epithelial cells and monocytes involving TLR4/MyD88 and NFjB/MAPK signal transduction TLR4 pathways. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction subject of multiple in vitro, in vivo and clinical studies in the last few decades (Aviles, Belay, Vance, Sun, & Sonnenfeld, 2004; Dad- Active Hexose Correlated Compound (AHCC) is a nutritional daoua et al., 2007; Gao et al., 2006; Turner & Chaudhary, 2009; supplement used in Japan and other Asian countries, as well as Ye, Wakame, Ichimura, & Matsuzaki, 2004; Yin, Fujii, & Walshe, in the United States, as a dietary supplement to boost immune 2010). Studies in mouse models have shown that AHCC stimulates function (Matsushita et al., 1998; Yagita, Maruyama, Wakasugi, & the immune system modulating the response against pathogens Sukegawa, 2002). It is a mixture of polysaccharides, amino acids and increasing the survival following infections in mice (Ritz, and lipids enriched in a-1,4-linked glucans (Kidd, 2000; Matsui 2008). These studies include a variety of infectious agents like et al., 2002; Matsushita et al., 1998) and it is derived from the influenza, Pseudomonas aeruginosa or meticillin-resistant Staphylo- mycelia of species of Basidiomycetes mushrooms: Shintake (Lenti- coccus aureus (Ritz, 2008). An intestinal anti-inflammatory effect of nus edodes) and Shimeji (Lyophyllum shimeji). AHCC has been the AHCC dependent probably on its prebiotic effect has also been de- scribed in rats (Daddaoua et al., 2007). AHCC has been used widely Abbreviations: AHCC, Active Hexose Correlated Compound; ELISA, enzyme- in immunocompromised patients, especially those with cancer, in linked immunosorbent assay; GROa, growth regulated oncognene a; IL, interleukin; order to hasten patient recovery from antineoplastic therapy (Kidd, IECs, intestinal epithelial cells; IRAK, IL-1 receptor-associated kinase; IFN-c, 2000; Matsui et al., 2002; Matsushita et al., 1998; Shah et al., 2011; interferon-c; LPS, lipopolysaccharide; MAPK, mitogen activated protein kinase; MCP-1, monocyte chemotactic protein-1; NFjB, nuclear factor jB; TLR, Toll-like Sumiyoshi et al., 2010; Turner & Chaudhary, 2009). Recent research receptor; TNF-a, tumour necrosis factor-a; TRAF6, tumour necrosis factor receptor indicates that AHCC enhances the immune response by multiple associated factor 6. mechanisms, including augmented macrophage and natural killer ⇑ Corresponding author. Address: Department of Biochemistry and Molecular cell proliferation (Matsushita et al., 1998; Nishioka, Akao, & Biology II, CIBERehd, School of Pharmacy, Campus de Cartuja s/n, 18071 Granada, Spain. Tel.: +34 958 241305; fax: +34 958 248960. Wakame, 2009; Ritz, Nogusa, Ackerman, & Gardner, 2006) and a E-mail address: [email protected] (O. Martínez-Augustin). higher production of various cytokines by macrophages and T 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.09.039 A. Daddaoua et al. / Food Chemistry 136 (2013) 1288–1295 1289 lymphocytes (interferon-c (IFN-c), interleukin (IL)-8, IL-1b, (25 mM glucose) and RPMI-1640 medium, respectively, supple- tumour necrosis factor (TNF-a, IL-2 and IL-12) (Yin et al., 2010). mented with heat-inactivated foetal bovine serum (10% v/v), Intestinal mucosal immunity is thought to be modulated by 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 2.5 lg/ml amphoter- cytokine release from intestinal epithelial cells, but it is unclear icin and 2 mM glutamine. how this crosstalk actually works, especially in relation to the influence of luminal factors, including nutrients and various die- 2.3. AHCC preparation tary compounds. Moreover, luminal bacteria are considered to play a pivotal role in this regard, and the microbiota clearly influences AHCC was provided by Amino Up Chemical (Sapporo, Japan). intestinal epithelial cell (IEC) gene expression. In IECs the nuclear The AHCC manufacturing process has been described elsewhere factor jB (NFjB) is a key player in maintaining intestinal barrier (Miura, Kitadate, Nishioka, & Wakame, 2010). Commercial AHCC integrity since its stimulation is important for defensin production was freshly prepared by dissolving the original powder in complete (Voss et al., 2006). Upon activation by various stimuli, NFjB trans- DMEM at a final concentration of 5 mg/ml. After 10 min of sonica- criptionally regulates many cellular genes involved in early inflam- tion to ensure total dissolution, it was passed through 0.22 lm fil- matory responses, including cytokines, suggesting a possible role ters and used immediately. in inflammatory bowel disease (IBD) pathogenesis. However, this activation may be also important to contain effectively the luminal microorganisms and prevent bacterial translocation. Thus the 2.4. Effect of AHCC on cytokine secretion mucosal immune system acts as a double edged sword in the inter- phase between the lumen and the internal milieu. Cytokine secretion (GROa and MCP-1 for IEC18 cells, IL-8 for Colonic bacteria are probably handled in the intestinal mucosa HT29 cells and IL-1b, TNF-a and IL-8 for THP-1 cells) was measured to a great extent by innate immunity mechanisms, which involve in the 24 h supernatant of cells treated with AHCC (0.05, 0.5 and ligation by bacterial products of receptors devoted to the recogni- 5 mg/ml). In some cases, specific pathway inhibitors were added tion of microbe-associated molecular patterns. These include the prior to AHCC treatment (see below). Cytokines were quantitated Toll-like receptors (TLRs). One of the TLRs that has received most by ELISA (Biosource Europe, Nivelles, Belgium and BD Biosciences, attention is TLR4. In vitro, monocyte stimulation with the TLR4 Erembodegem, Belgium), following the protocols recommended by agonist lipopolysaccharide (LPS) generates a significant change of the manufacturers. gene expression (Frost, Nystrom, & Lang, 2002; Nau et al., 2002; Shoham, Huang, Chen, Golenbock, & Levitz, 2001), including the 2.5. Characterisation of the signal transduction pathways using the production of chemokines and cytokines. After recognition of NFjB and MAPK inhibitors microbial ligands, TLR signalling is initiated by binding of the adap- ter molecule MyD88 to the cytoplasmic Toll/interleukin-1 receptor The cells were treated as above using the concentration of 5 mg/ (IL-1R) domain present in all TLRs. Recruitment of IL-1R-associated ml of AHCC, after the addition of specific inhibitors, including Bay kinases (IRAK4, IRAK1 and tumour necrosis factor (TNF) receptor 11-7082 (IjB-a phosphorylation), PD98059 (ERK1/2 MAPK), associated factor 6 (TRAF6)) results in activation of the mitogen SB203580 (p38 MAPK), SP600152 (JNK MAPK) and wortmannin activated protein kinases (MAPK) and NFjB pathways (Akira & (phosphatidylinositol-3 kinase, PI3K). These inhibitors of intracel- Takeda, 2004). lular signalling were added to the culture 60 min before the addi- The effects of AHCC on intestinal epithelial cells and monocytes/ tion of 5 mg/ml of AHCC to cells. All inhibitors were used at a macrophages have been poorly studied. Some reports indicate im- concentration of 10 lM except wortmannin, which was used at mune enhancing effects on macrophages, but the molecular mech- 1 lM. anism of action involved has been not assessed. Both intestinal epithelial cells and monocytes/macrophages are involved in the intestinal innate immune response. The study of the effects and 2.6. Western blot mechanism of action of AHCC