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Eur J NutrDOI 10.1007/s00394-015-0832-2

ORIGINAL CONTRIBUTION

Active Hexose Correlated Compound (AHCC) promotes an intestinal immune response in BALB/c mice and in primary intestinal epithelial cell culture involving toll‑like receptors TLR‑2 and TLR‑4

Jean‑François Mallet · Émilie Graham · Barry W. Ritz · Kohei Homma · Chantal Matar

Received: 12 September 2014 / Accepted: 8 January 2015 © Springer-Verlag Berlin Heidelberg 2015

Results Feeding AHCC resulted in increased IgA+ cells in the intestine and increased sIgA, IL-10, and IFN-γ in the intestinal fluid. In IECs, contact with AHCC increased IL-6 production but not to the pro-inflammatory level of positive controls, LPS and E. coli. Blocking TLR-2 and TLR-4 reduced the induction of IL-6 by AHCC, suggesting that these innate receptors are involved in generating the immune response of IECs to AHCC.Conclusions AHCC may play a role in the orchestra-tion of immune response and the maintenance of immune homeostasis in part by priming the TLR-2 and TLR-4 gate at the intestinal epithelium. Such a response is likely due to the recognition of non-pathogenic food-associated molecu-lar patterns (FAMPs) such as those found associated with other mushroom or yeast-derived compounds.

Keywords AHCC · Toll-like receptor · E. coli · Infection · Mushroom · Innate · FAMP

Introduction

The use of natural compounds, such as medicinal mush-rooms, to promote general health and prevent and allevi-ate symptoms of disease has long been promoted by vari-ous types of traditional medicine. As part of a trend toward more natural, preventive interventions, the use of medicinal mushrooms and other such products is gaining attention by Western medical practitioners and consumers but requires further investigation. One such mushroom-derived com-pound is Active Hexose Correlated Compound (AHCC), which has been reported to yield a number of therapeutic, primarily immunomodulatory effects. Studies in mouse models have shown that AHCC stimulates the immune sys-tem, particularly natural killer (NK) cell cytotoxic function,

Abstract Purpose Active Hexose Correlated Compound (AHCC®) is a cultured mushroom extract that is commercially avail-able and promoted for immune support. Available data suggest that AHCC supplementation affects immune cell populations and immune outcomes, including natural killer cell response to infection. The mechanism by which AHCC exerts its effects is not well understood. The present work aimed to characterize the immunomodulatory activity of AHCC in the gut and to study the effects of AHCC on toll-like receptor (TLR) signaling in intestinal epithelial cells (IECs).Methods BALB/c mice were fed AHCC by gavage. In vivo activities were assessed by immunohistochemistry and cytokine production. The effects of AHCC on ex vivo pri-mary cell culture from IECs were examined after challenge with LPS or E. coli alone or in the presence of anti-TLR-2 and TLR-4 blocking antibodies.

J.-F. Mallet Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada

É. Graham · C. Matar (*) Nutrition Sciences Program, Faculty of Health Sciences, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canadae-mail: chantal.matar@uottawa.ca

B. W. Ritz Nutrition Sciences Department, College of Nursing and Health Professions, Drexel University, 245 N. 15th Street, Mail Stop 1030, Philadelphia, PA 19102, USA

K. Homma Amino Up Chemical Company, Ltd., 363-32 Shin-ei, Kiyota-ku, Sapporo 004-0839, Japan

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modulating the response against pathogens and increasing survival following infections [1–3].

AHCC is a cultured extract of the mycelia of shiitake mushroom (Lentinula edodes) that is primarily composed of carbohydrates (approximately 70 %), proteins (13 %), minerals (9 %), fats (2 %), and fiber (2 %). A significant portion of the carbohydrates (approximately 20 %) is com-posed of α-1,4-glucans [4–6]. β-glucans, such as from yeasts, are known to alter immune responses by influenc-ing toll-like receptor TLR-2 and TLR-4 activation through interaction with Dectin-1 [7–9]. The principal active com-ponent of AHCC is not known but has been proposed to be the acylated α-1,4-glucan [10]. The quantity of α-glucan to β-glucan in AHCC is almost 30:1, and the relatively low molecular weight of α-glucan (5,000 dalton) may help in its absorption or local activity in the gut [11]. It is still unknown if the presence of α-glucan or its absorption are necessary for the activity of AHCC.

The TLRs function as pattern recognition receptors, a group of receptors expressed on the surface of epithelial cells, as well as intra- and extracellular surfaces of certain immune cells that recognize molecular moieties typically associated with pathogens and not present in the vertebrate host (pathogen-associated molecular patterns, PAMPs). As such, TLR activation contributes to first-line, innate self and non-self discrimination and the subsequent immune response. They play a fundamental role in prompting the innate immune system in defending the host against a wide selection of pathogens [12]. TLR-2 binds a large array of ligands, including lipoteichoic acid, peptidoglycan, and bacterial lipoproteins present in the cell wall of gram-pos-itive and gram-negative bacteria [13]. Lipopolysaccharide (LPS), the most studied TLR ligand, is found on the surface of bacteria and is recognized by TLR-4, inducing a strong, pro-inflammatory immune response through NF-κB [14]. Escherichia coli are gram-negative, facultative anaerobic bacteria found in the healthy flora of the intestines of mam-mals and birds [15, 16]. Most E. coli are non-pathogenic in their normal host, but some strains (e.g., O157:H7) can cause foodborne illness and may result in severe outcomes like hemorrhagic diarrhea and kidney failure [17, 18].

Intestinal epithelial cells express TLR-2 and TLR-4 on their apical surface [19]. Both TLR-2 and TLR-4 induce the production and release of IL-6 through the activation of transcription factor NF-κB. These TLRs may participate in immune priming and homeostasis while remaining capable of recognizing pathogens and inducing an effector response when necessary [20, 21].

As a complex food product that contains glucans, it has been proposed that the effects of AHCC on immune response might be mediated by TLRs [4]. In this experi-ment, we first established the immunomodulatory effects of AHCC in murine intestinal epithelium. We then blocked

TLR-2 and TLR-4 in a primary culture of intestinal epi-thelial cells (IECs) ex vivo to better understand the role of pattern recognition in the response of IECs in contact with AHCC. We also evaluated the influence of AHCC on the response to E. coli infection of IECs ex vivo. To our knowl-edge, this is the first time the effects of AHCC and its inter-action with TLRs have been evaluated in a primary culture of IECs.

Materials and methods

Animals and feeding procedures

Six- to eight-week-old female BALB/c mice weighing 18–20 g were obtained from Charles River (Montreal, QC, Canada). The mice were treated in accordance with the guidelines of the Canadian Council on Animal Care, and the experimental design was approved by the Animal Care Committee of the University of Ottawa under proto-col ME-260. Mice were housed together in plastic micro-isolator cages in a controlled atmosphere (temperature: 22 ± 2 °C; humidity: 55 ± 2 %) on a 12-h light/dark cycle and fed a conventional balanced diet (2018 Teklad Global 18 % Protein Rodent diet, Harlan Laboratories Inc, Madi-son, Wisconsin, USA) and water ad libitum. An aqueous solution of AHCC was administered by gavage (0.1, 0.5, or 1.0 g/kg bw) to the animals for 7 consecutive days. The control group was given water instead of the AHCC suspension.

At the end of each feeding period, the animals were anesthetized with intraperitoneal injections of ketamine (100 mg/ml), xylazine (20 mg/ml), and acepromazine (10 mg/ml) and killed by cervical dislocation to obtain the various tissues (intestinal epithelium, serum, intestinal fluid, and pulmonary wash) to be used in the immunologi-cal assays.

Identification of IgA+ and IgG+ cell populations

The small intestines were removed for histological exami-nation as previously described [22]. Briefly, the tissue was prepared using the Sainte-Marie technique for paraffin inclusion, and the paraffin blocks were cut into 4-µm sec-tions. The numbers of IgA-producing (IgA+) and IgG+ cells on histological sections of the samples from the ileum region near Peyer’s patch were determined by direct immu-nofluorescence. The immunofluorescence tests were per-formed using FITC-conjugated goat (α-chain specific) pol-yclonal anti-mouse IgA or FITC-conjugated goat (γ-chain specific) polyclonal anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA). The histological sections were depar-affinized and rehydrated in a graded series of ethanol (from

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95 to 40 %). The deparaffinized histological samples were examined using a fluorescent light microscope. The results were expressed as the number of IgA+ or IgG+ cells (pos-itive: fluorescent cell) per 10 fields (magnification 1,000×). The data represent the mean of three histological sections from each animal for each feeding period.

Determination of in vivo cytokine production

Blood was collected from anesthetized mice by cardiac puncture. The blood was coagulated by allowing it to rest for 30 min at room temperature and then was centrifuged for 10 min at 1,000g. The serum was stored at −20 °C until it was analyzed. The small intestine was flushed with 3 ml of ice cold PBS, and the lung was perfused with 1 ml of ice cold PBS. Resulting liquids were spun at 1,000g for 5 min. Cytokines (IL-6, IL-10, IL-12, and IFN-γ) were quanti-fied by ELISA (eBioscience, Inc., San Diego, CA, USA). Secretory IgA (sIgA) was analyzed by DAS-ELISA using affinity-purified goat anti-mouse IgA antibodies (Sigma Chemical Co., St Louis, MI, USA).

Primary culture of mouse small intestine epithelial cells

The primary cultures of the enterocytes were prepared as described previously [23], with the slight modifications that are described here. Following the feeding period (AHCC or water), the animals were euthanized as described above. The small intestines were removed and placed in a digestion buffer of Hanks’ balanced salt solution (HBSS) (Sigma-Aldrich, St. Louis, MO, USA) with 2 % glucose (Sigma-Aldrich), 100 U/ml penicillin (Sigma-Aldrich), and 0.1 mg/ml streptomycin (Sigma-Aldrich). The intestines were flushed six times with 10 ml of the digestion buffer. They were then cut into 2- to 3-mm fragments and digested in 20 ml of digestion buffer supplemented with 300 U/ml Type XI collagenase (Sigma-Aldrich) and 0.1 mg/ml dis-pase (Gibco, Grand Island, NY, USA) at 25 °C at 150-rpm for 45 min. Digestion was stopped by adding 20 ml of Dul-becco’s modified Eagle’s medium (DMEM) without phe-nol red (Gibco), supplemented with 10 % heat-inactivated fetal bovine serum (ATCC, Manassas, VA, USA), 10 ng/ml epidermal growth factor (US Biological, Swampscott, MA, USA), insulin–transferrin–selenium-A (2.50, 0.55 µg/ml, and 1.68 pg/ml, respectively) from a 100× ready-to-use solution (Gibco), 100 U/ml penicillin (Sigma-Aldrich), and 0.1 mg/ml streptomycin (Sigma-Aldrich). Large fragments were removed by allowing them to settle for 2 min at the bottom of the flask. The supernatant was centrifuged for 3 min at 300 rpm. The pellet was washed twice with the DMEM solution and then re-suspended in the same culture medium at a concentration of 4 × 105 to 6 × 105 organoids (single cells or IEC clusters) per ml. The IEC suspensions

were then transferred to 96-well cell culture plates (200 µl/well) and incubated for 8 h (37 °C; 5 % CO2). An in vitro toxicology assay kit, a 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT)-based assay (Sigma-Aldrich), and trypan blue (0.4 %) exclusion were used to assess cell viability. As previously reported, the cell sus-pension is composed of 83 % IECs with some cellular debris and immune cells present [24]. All assays were com-pleted within 8 h.

Challenging with test substances and blocking with anti-TLR-2 and anti-TLR-4

The challenge and blocking experiments were performed as previously described with IL-6 production as the response measure [20]. Suspensions of primary IECs were obtained as described above and challenged with AHCC (at the con-centrations indicated), standard lipopolysaccharide from Escherichia coli 0111:B4 (0.01 μg/ml, Sigma), or E. coli MM294 (105 cells/ml). For blocking experiments, IECs were first incubated for 30 min at room temperature in the presence of 40 µg/ml of functional grade purified anti-mouse TLR-2 or TLR-4/MD2 (eBioscience), or DMEM (control). After the blocking period using the anti-TLRs, 0.1 mg/ml of AHCC was added to the supernatant. LPS, a potent inducer of IL-6, was used as a positive control. The IEC suspensions were incubated for 8 h (37 °C; 5 % CO2), and the supernatants were recovered and frozen until IL-6 levels were quantified by ELISA (BD OptEIA, BD Bio-sciences Pharmingen, San Diego, CA, USA).

Statistical analysis

The data were analyzed using a one-way analysis of variance procedure, using SPSS software (IBM, Armonk, NY). The differences among the means were detected with Duncan’s multiple-range test with significance at a p value <0.05.

Results

Supplementation of BALB/c mice with AHCC by gavage for 7 days increased the number of IgA-producing cells at the lamina propria as evaluated by immunohistochemistry (Fig. 1). The increase in the number of IgA+ cells in the lamina propria of the small intestine after the feeding peri-ods was accompanied by an increase in the luminal con-tent of total sIgA, determined by ELISA. Increased IgA production was also confirmed in the serum. No significant increase in IgA was reported in bronchial fluid of mice fed with AHCC. There were no effects on the number of IgG producing cells in the small intestine (Fig. 1) or on IgG lev-els (not shown).

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Cytokine response was evaluated in intestinal and pul-monary fluids by ELISA. There was a trend for increased intestinal IL-6. IL-6 was significantly increased in bron-chial fluid at a dose of 0.5 g/kg body weight, and there was

a trend for decreased IL-12 in the lungs. AHCC at a dose of 1.0 g/kg body weight resulted in an increase in IL-10, IL-12, and IFN-γ in intestinal fluid (Fig. 2).

To establish the optimal AHCC dosage, primary IECs were isolated from BALB/c mice and challenged in con-tact with different AHCC concentrations. Challenge with AHCC at 0.1 and 1 mg/ml increased IL-6 production when compared to the control (Fig. 3).

Naïve IECs were pre-treated with anti-TLR-2 or anti-TLR-4 and then cocultured in contact with AHCC (0.1 mg/ml) or LPS as a positive control (0.01 μg/ml).

Fig. 1 Number of IgA (a) and IgG (b) positive cells in the small intestines of BALB/c mice fed AHCC for 7 days. Representative pic-ture of IgA+ immunohistochemistry slides of control mice (c) and mice fed AHCC (d). Examples of positive cells are indicated by a white arrow. Concentration of sIgA in serum (e), intestinal fluid (f), and pulmonary wash (g) from mice fed AHCC for 7 days. Values rep-resent the mean ± SEM, n = 3 mice, *p < 0.05 versus control

Fig. 2 Cytokine levels in the intestinal fluid and pulmonary wash of BALB/c mice fed AHCC for 7 days. Values rep-resent the mean ± SEM, n = 3 mice, *p < 0.05 versus control

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AHCC resulted in a fivefold induction in IL-6 compared to untreated control, which was significantly less than the response induced by LPS, as expected (Fig. 4). Both anti-TLR-2 and anti-TLR-4 inhibited IL-6 production by IECs in response to AHCC compared to the untreated control. Blocking either TLR fully eliminated the induction of IL-6 by AHCC. There was no additive effect when both TLRs were blocked in combination (not shown).

To determine if exposure to AHCC would influence the response of IECs to stimuli dependent on TLR-2 and TLR-4 recognition, an ex vivo culture of IECs from mice previously fed AHCC was challenged by E. coli in the presence or absence of AHCC. AHCC eliminated IL-6

production in the response of IECs to E. coli (Fig. 5). This result in culture was only apparent when IECs were derived from mice pre-fed AHCC. No such effect was observed in IECs treated with AHCC and challenged with E. coli, sug-gesting that a priming response was required (not shown).

Discussion

These results confirm that AHCC is an immunomodulatory compound, as previously demonstrated, and such effects are apparent at the site of contact, the intestinal epithelium, as well as systemically. Supplemental AHCC increased the number of IgA+ cells in the intestinal epithelium as well as sIgA production. Production of sIgA appears to be highly influenced by nutritional interventions, and health benefits such as enhancing mucosal defences and reducing infections have been described [25, 26]. Supplementation with AHCC also yielded small but significant influences on inflammatory cytokine production at the epithelium, as well as in the lungs. These responses were indicative of a local and systemic immune response but not of a physi-ologically pro-inflammatory response, as increases were modest and equivocal. Previous reports in mouse models have indicated that AHCC is associated with an enhanced response to infection, reduction of severity of symptoms, and increased survival [2, 3].

The effects of AHCC on an infectious challenge were evaluated ex vivo in the primary IEC model using E. coli. When IECs from mice pre-fed AHCC were treated with AHCC prior to challenge with E. coli, the IL-6 response to E. coli was eliminated to the level of untreated control. Interestingly, this result was only observed when IECs

Fig. 3 IL-6 production ex vivo by small intestinal epithelial cells exposed to different concentrations of AHCC. Production of IL-6 by cultured IECs isolated from naive mice after treatment with com-plete medium (control) and 0.01, 0.1, and 1.0 mg/ml of AHCC. Val-ues represent mean results from three replicates normalized to fold change ± SEM, *p ≤ 0.05 versus control

Fig. 4 AHCC associated IL-6 production mediated by the TLRs. Production of IL-6 by cultured IECs isolated from naive mice after treatment with complete medium (control), LPS (0.01 μg/ml), AHCC (0.1 mg/ml), or AHCC + anti-TLR-2 or anti-TLR-4. Values represent mean results from three replicates normalized to fold change ± SEM. Bars that have no letter in common are significantly different from each other (p ≤ 0.05)

Fig. 5 Inhibition of E. coli-induced IL-6 production by AHCC in an ex vivo culture of IECs from mice. Production of IL-6 by cultured IECs isolated from mice after treatment with complete medium (con-trol), AHCC (0.1 mg/ml), E. coli MM294 (105 cells/ml) or AHCC (0.1 mg/ml) + E. coli MM294 (105 cells/ml). Values represent mean results from two experiments normalized to fold change ±SEM, **p ≤ 0.01

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were first primed with AHCC in vivo. This is in agree-ment with previous observations in infectious models in which AHCC was shown to exhibit an effector response, such as increased NK cell activity, only when the animals were challenged with an infection [1]. Importantly, AHCC alone does not appear to induce a physiologically relevant immune effector response, but does appear to increase the response when superimposed with an infection. Whether the reduction in IL-6 response to E. coli challenge would translate to a positive or negative response to infection in the animal must be considered. Although not evaluated in the current study, AHCC has been shown to enhance bac-terial clearance and consistently increase survival in mice challenged with an acute infection [2, 27]. Therefore, we hypothesize that the observed reduction in IL-6 in response to E. coli represents a less inflammatory, potentially less damaging response that would reduce the severity of the infection but would not impede clearance and recovery. This requires further study.

Importantly, the ability of AHCC in contact with IECs to induce IL-6 production was dependent on interaction with either TLR-2 or TLR-4. Blocking TLR-2 or TLR-4 access fully eliminated the response of IECs to AHCC. Therefore, it is clear that the immunomodulatory activities of AHCC are mediated at least in part through contact with pattern recog-nition receptors present on the intestinal epithelium. Whether other TLRs might also be involved must be determined, but TLR-2 and TLR-4 are reasonable first candidates, since both are known to respond to the types of surface moieties expected to be present in AHCC, such as α- and β-glucans [4, 7]. We have previously demonstrated similar responses to other nutritional bioactives, including dairy peptides [20, 28], and hypothesize that a large family of complex food prod-ucts including not only yeast glucans, mushrooms, and dairy peptides, but also probiotics, polyphenols and their metab-olites, and certain fatty acids may also participate in innate immune recognition as a special category of food-associated (pathogen-like) molecular patterns or FAMPs. These com-mon food patterns serve as non-pathogenic, non-danger sig-nals that instead of eliciting a strong and potentially damag-ing response are involved in keeping the immune response primed, balanced, and tolerant.

β-Glucans have been shown to change the gut microbi-ota and raise the presence of bacteria that are believed to help the host to stay in good health [29]. It is difficult to dissociate the effects of AHCC on the epithelial cells from possible effects on the gut microbiota since they might use the same receptors to induce their effect [20]. Further research is needed to investigate the impact of AHCC on the beneficial bacteria present in the gut.

A strength of this work was the combination of an in vivo measure of immune response in the intestinal epithe-lium with an ex vivo assay using a primary culture of IECs.

Such studies are difficult to conduct, but help to estab-lish mechanism in a controlled assay in primary tissue as opposed to a representative cell culture model. A signifi-cant limitation is that the IEC culture does not replicate the digestion of a complex food product such as AHCC. There-fore, it is still difficult to compare results from IEC chal-lenge with test substances such as AHCC with the in vivo experience. Further removed are potential clinical expe-riences in human subjects. We did not endeavor to deter-mine the specific active component in AHCC responsible for these outcomes. While it follows that glucans may be responsible for at least some of the effects, we hypothesize that AHCC as a complex food product likely interfaces with the immune system through multiple mechanisms, and indeed, that it is this plurality that renders such natu-ral compounds effective in supporting the body’s natural defense and homeostatic mechanisms, as opposed to induc-ing a hyperstimulated, potentially damaging response.

AHCC and other complex natural food compounds offer an exciting new area of research for understanding how the innate immune system interacts with its food environment and how we might capitalize on such advancements for health promotion.

Acknowledgments We would like to thank Mr. Jairo Duarte for his help in the isolation of the intestinal epithelial cells. This work was made possible by a non-restrictive research grant from the manufac-turer of AHCC, Amino Up Chemical Company, Sapporo, Japan.

Conflict of interest Dr. Kohei Homma is employed at Amino Up Chemical Co., Ltd. as a research scientist. Amino Up Chemical is the developer and manufacturer of the AHCC product used in this study. The author has not received personal financial gain from sales of the AHCC product. All findings and views expressed in this paper are those of the authors and do not necessarily reflect the views of Amino Up Chemical. As the vice president of scientific and regulatory affairs for Atrium Innovations, Dr. Barry Ritz is involved in the commer-cialization of many natural products, including AHCC. Currently, two atrium companies, Douglas Laboratories and Pure Encapsulations, commercialize products that contain AHCC as an ingredient. The other authors have no conflict of interest to declare.

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