M. atichisonii used in the present study was provided by the Jeollanam-do Wando Arboretum (Wando, Korea). The fresh fruiting bodies were hot air-dried and ground into a powder. M. atichisonii extract was prepared by boiling 1 kg of mycelium powder in 10 l filtered sterile water for 8 h at 50°C. The supernatant was saved and the pellet was re-boiled with 10 l water for 8 h. Insoluble material was removed by filtration and a two-fold volume of cold ethanol was added to the filtrate to precipitate the polysaccharide. Following standing the mixture at 4°C overnight, the precipitate was collected by centrifugation (4,000 × g, 30 min at room temperature). The precipitate was washed with ethanol and resuspended in water. The resuspended solution was then freeze-dried.

Six-week-old female BALB/c mice (n=80; body weight, 22±2 g) were purchased from Samtako Bio Co. (Osan, Korea), fed with an ad libitum diet and water, and housed in a controlled environment (22±3°C, 12 h light/dark cycle). They were then divided into six treatment groups: (i) Control, (ii) sterilized tap water with ovalbumin (OVA) induction, (iii) 1 mg/kg/day dexamethasone (DEX; Sam Nam Pharm, Chungcheongnam, Korea) with OVA induction, (iv) 10 mg/kg/day M. atichisonii with OVA induction, (v) 100 mg/kg/day M. atichisonii with OVA induction and (vi) 1,000 mg/kg/day M. atichisonii with OVA induction. On days 1 and 8, all mice were sensitized via intraperitoneal injection of 20 µg OVA (cat no. A5503-50G; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 1 mg aluminum hydroxide hydrate (Prod #77161; Thermo Fisher Scientific, Inc. Waltham, MA, USA) in 500 µl saline. From days 21 to 25, all mice, except for those used as control, were challenged once daily with 5% OVA for 30 min using a nebulizer (3 ml/min, NE-U17; Omron Corporation, Kyoto, Japan). During the same 5 day period, the treatment groups were also administered once daily with oral doses of sterilized tap water, DEX, 10, 100 or 1,000 mg/kg/day M. atichisonii 1 h prior to the OVA challenge.

All experiments were approved by the Institutional Animal Care and Use Committee at Dongshin University (Naju, Korea; approval no. 2014-08-04).

A total of one day following the final treatment, the mice were anesthetized with intraperitoneal injections of 50 mg/kg Zoletil (Virbac, Carros, France), and their tracheas were cannulated with disposable animal feeding needles. Lavages were performed with three 0.4 ml aliquots of cold phosphate-buffered saline (PBS; cat no. 17-516F; Lonza, Walkersville, MD, USA). BALF samples were collected and immediately centrifuged at 900 × g for 5 min at room temperature (Sorvall Legend Micro 17R; Thermo Fisher Scientific, Inc.). The cell pellets were resuspended in PBS for total and differential cell counts. The numbers of total and differential cells were counted using the Hemavet Multispecies Hematology system (n=8 per group; Drew Scientific Inc., Miami Lakes, FL, USA). Levels of IgE in the BALF were measured using a specific mouse IgE enzyme-linked immunosorbent assay kit (cat no. 555248; BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol (n=8 per group).

Lung tissue was fixed in 10% (v/v) formaldehyde solution for 10 days at room temperature, dehydrated in a graded ethanol series (99.9, 90, 80 and 70%), and embedded in paraffin. Paraffin-embedded lung tissue was then sectioned (4 µm) longitudinally and stained with hematoxylin and eosin. In addition, the sections were stained with Periodic Acid-Schiff (PAS; periodic acid; cat no. P7875; Sigma-Aldrich; Merck KGaA; Schiff's reagent; cat no HX54780633; EMD Millipore, Billerica, MA, USA) for the semi-quantitative analysis of glycoproteins.

Deparaffinized tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min to remove endogenous peroxidase. Antigen retrieval was carried out with sodium citrate buffer (0.1 M) using a microwave. The slides were incubated with normal serum to block non-specific binding and then incubated overnight at 4°C with the following primary antibodies (diluted 1:100 to 1:200): Rabbit anti-mouse CD3 polyclonal (cat no. ab5690; 1:100; Abcam, Cambridge, MA, USA), rat anti-mouse CD4 monoclonal (cat no. 14-9766; 1:200; eBioscience, Inc., San Diego, CA, USA), rat anti-mouse CD8 monoclonal (cat no. sc-18913; 1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit anti-mouse CD19 polyclonal (cat no. 250585; 1:100; Abbiotec, San Diego, CA, USA), rat anti-mouse MHC class II monoclonal (cat no. sc-59318; 1:100; Santa Cruz Biotechnology, Inc.), rabbit anti-mouse Tbx21/T-bet polyclonal (cat no. bs-3599R; 1:100; BIOSS, Beijing, China), goat anti-mouse GATA-3 (cat no. TA305795; 1:100; OriGene Technologies, Inc., Rockville, MD, USA), rat anti-mouse IFN-γ monoclonal (cat no. sc-74104; 1:100), goat anti-mouse IL-12p35 polyclonal (cat no. sc-9350; 1:100), rat anti-mouse IL-12p40 monoclonal (cat no. sc-57258; 1:100) (all from Santa Cruz Biotechnology, Inc.), rabbit anti-mouse TNF-α polyclonal (cat no. 3053R-100; 1:200; BioVision Milpitas, CA, USA), rat anti-mouse IL-4 monoclonal (cat no. sc-73318; 1:100), rabbit anti-mouse IL-5 polyclonal (cat no. sc-7887; 1:100), goat anti-mouse IL-6 polyclonal (cat no. sc-1265; 1:100), and goat anti-mouse IL-13 polyclonal (cat no. sc-1776; 1:100) (all from Santa Cruz Biotechnology, Inc.). The slides were incubated for 10 min with a biotinylated secondary antibody (cat no. PK-7800; Vector Laboratories, Inc., Burlingame, CA, USA) and horseradish-peroxidase conjugated streptavidin. Signals were detected using a 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution (cat no. SK-4105; Vector Laboratories), and cells were counterstained with Mayer's hematoxylin. To determine the number of positively stained cells, five random, non-overlapping fields of view were selected, and cells were counted (magnification, ×200) from three separately immunostained lung sections per animal (n=8 per group).

GC-MS (Agilent 5975C MSD and 7890A GC; Agilent Technologies, Inc., Santa Clara, CA, USA) was tuned by perfluorotribuylamine (cat no. 442747; Sigma-Aldrich; Merck KGaA) using three mass fragments (m/z) of 69.0, 219.0 and 502.0 in the condition of electron ionization. A 5MS GC column (DB-5 cross-linked 5% phenylmethyl silicone; Agilent Technologies, Inc.) was used for the analysis as this column has low bleed that improves sensitivity for constituent identification. The GC oven was heated using the following program: Isothermal at 65°C for 10 min and increasing by 10°C every min to 300°C with He as the carrier gas. The summarized operation parameters for the GC-MS are presented in Table I. In order to analyze the quality of extract in M. aitchisonii, the certain amount of dried samples by dehydrofreezing procedure was prepared. The samples were extracted using 10 min sonication at room temperature, twice with dichloromethane, followed by sonication twice with acetone using a sonicator (Bransonic^® CPXH; Branson Ultrasonics Corporation, Dallas, TX, USA). The composited extract was evaporated by high volume nitrogen blowdown (TurboVap II; Caliper Life Sciences, Hopkinton, MA, USA) to reach 5–10 ml and was further evaporated to a final volume of 100 µl using low volume nitrogen blowdown (MGS-2200; Tokyo Rikakikai Co., Ltd., Tokyo, Japan). A final extract volume of 100 µl was sialylated with N,O-bis(trimethylsilyl) trifluoroacetamide (Supelco; Sigma-Aldrich; Merck KGaA) to derivatize the constituents to their trimethylsilyl-derivatives (TMS-derivatives) prior to GC-MS analysis.

Data are presented as the mean ± standard deviation. Group differences were evaluated by one-way analysis of variance followed by Dunnett's multiple comparison tests. Statistical significance was set at P<0.05 or P<0.01.

In the OVA-induced asthma group, M. atichisonii dramatically increased WBC count, when compared with the level of WBCs in the control group. However, in the DEX treated group, WBC number decreased by a similar amount in the control group (Fig. 1A; P<0.05 or P<0.01). M. atichisonii suppressed WBC proliferation induced by OVA in a dose-dependent manner. The number of WBCs in the 1,000 mg/kg M. atichisonii treatment group was similar to that in the DEX treatment.

Eosinophil number was dramatically was more affected by M. atichisonii treatment than WBC number (Fig. 1B; P<0.05 or P<0.01). M. atichisonii dose-dependently suppressed eosinophil proliferation and the levels of eosinophils in the 1,000 mg/kg/day M. atichisonii treatment group, and this was similar to that in DEX treatment.

IgE is related to the hyperresponsiveness of asthma and serves an important role in asthma occurrence (28). M. atichisonii effectively decreased the level of IgE in a dose-dependent manner, which was increased by OVA treatment, when compared with that the in OVA treatment group (8.2±0.69 ng/ml). In addition, the quantity of IgE in the 1,000 mg/kg treatment group (2.7±1.02 ng/ml) was similar to that in the DEX treatment group (2.4±0.54 ng/ml; Fig. 2; P<0.05 or P<0.01).

To analyze the morphological changes in the lung that were induced by OVA treatment, hematoxylin and eosin staining was used, in addition to PAS staining (Fig. 3). In the lungs of mice with OVA-induced asthma, typical histopathological changes were observed such as airway remodeling, mucous hyper-secretion, epithelial hyperplasia and eosinophil infiltration (Fig. 3A-b). In particular, PAS stained mucous hyper-secretion in the bronchioles was clearly observed using the specific staining method (Fig. 3B-b). DEX, which is typically used as a representative anti-asthmatic drug, significantly suppressed the morphological changes in the lung (Fig. 3A-c) and completely prevented mucous secretion (Fig. 3B-c). M. atichisonii not only recovered the asthmatic alterations (Fig. 3A-d-f) but also suppressed mucous secretion (Fig. 3B-d-f). These results suggested that M. atichisonii may ameliorate airway obstruction.

In the 100 mg/kg/day M. atichisonii treatment and 1,000 mg/kg/day M. atichisonii treatment, expression of CD3+ total T cell (Fig. 4A; P<0.05 or P<0.01) was almost fully diminished. The expression of CD4+ helper T cells was suppressed in a dose-dependent manner (Fig. 4B; P<0.05 or P<0.01). A total of 1,000 mg/kg/day M. atichisonii treatment effectively inhibited the expression of CD4+ helper T cells (Fig. 4B-f; P<0.05 or P<0.01) when compared with the DEX treatment group (Fig. 4B-c; P<0.05 or P<0.01). In the 100 mg/kg/day or more M. atichisonii treatment group, the expression pattern of CD8+ cytotoxic T cells (Fig. 4C; P<0.05 or P<0.01) was the same as that observed for the CD4+ helper T cells, which was that they were almost fully diminished. Although M. atichisonii downregulated the expression of CD19+ B cells, the decreased level of CD19+ B cells was lower compared with other groups including CD3+ total T cell, CD4+ helper T cell, and CD8+ cytotoxic T cell (Fig. 4D; P<0.05 or P<0.01). The expression of MHC class II+ molecule (Fig. 4E; P<0.05 or P<0.01) almost disappeared in the 100 mg/kg/day M. atichisonii treatment and in the 1,000 mg/kg/day M. atichisonii treatment groups. The 1,000 mg/kg/day M. atichisonii treatment effectively inhibited the expression of MHC class II+ molecules (Fig. 4E-f; P<0.05 or P<0.01) more, when compared with the DEX treatment group (Fig. 4E-c; P<0.05 or P<0.01).

Although M. atichisonii appears to control levels of both T cells (CD3+, CD4+, CD8+) and B cells (CD19+), the potency to modulate B cell (CD19+) proliferation may be smaller than that of T cell (CD3+, CD4+, CD8+) proliferation, however it downregulated CD3+ positive cells (total T cell) and CD8+ positive cells (cytotoxic T cell) and dose-dependently regulated CD4+ positive cell (helper T cell).

In order to compare the suppressive effects of the Th1 cell transcription factor, T-bet, and the Th2 cell transcription factor, GATA-3, the changes of expressions on T-bet and GATA-3 were measured via immunohistochemical staining (Fig. 5). The expressions of T-bet and GATA-3 in the OVA treatment group was significantly increased, when compared with the control, however DEX decreased the expressions of T-bet and GATA-3, which was induced by OVA, to a similar level as the control groups (Fig. 5A and B). Although M. atichisonii dose-dependently downregulated the expression of T-bet, there was no significant difference between 100 mg/kg M. atichisonii treatment and 1,000 mg/kg M. atichisonii treatment (Fig. 5C; P<0.05 or P<0.01). Conversely, from treatment with 10 mg/kg M. atichisonii to 1,000 mg/kg, the changes in GATA-3 expression were dependent on the dose-response relationship (Fig. 5C; P<0.05 or P<0.01).

M. atichisonii dose-dependently suppressed the expression of IFN-γ and the effect of M. atichisonii on INF-γ was less than DEX (Fig. 6A). The results of IFN-γ by M. atichisonii were similar to that of T-bet (Fig. 5A). M. atichisonii inhibited the expression of IL-12p35 (Fig. 6B). The effect of 1,000 mg/kg M. atichisonii treatment on IL-12p40 was similar to that of dexamethasone. DEX and M. atichisonii effectively suppressed the expression of IL-12p40 (Fig. 6C). Although there was no significant difference between 10 mg/kg M. atichisonii treatment and 100 mg/kg M. atichisonii treatment (P>0.05), in the 1,000 mg/kg M. atichisonii treatment group, IL-12p35 levels were almost fully diminished (Fig. 6D).

In order to measure the downregulation effect of M. atichisonii on Th2-related cytokines, such as TNF-α (Fig. 7A), IL-4 (Fig. 7B), IL-5 (Fig. 7C), IL-6 (Fig. 7D) and IL-13 (Fig 7E), immunohistochemical analysis was conducted. Although M. atichisonii did not completely prevent the expression of TNF-α, it was similar to that of DEX (Fig. 7A). M. atichisonii dose-dependently suppressed the expression of IL-4, but the suppression was less than DEX (Fig. 7B and F). The expression of IL-5 was controlled by M. atichisonii and the effect was similar to DEX. In particular, IL-6 and IL-13 were dose-dependently suppressed by M. atichisonii treatment more than by DEX (Fig. 7F).

To identify the compounds in M. aitchisonii that may possess anti-asthmatic properties, GC-MS analysis was conducted. Not all compounds in M. aitchisonii were isolated, however nicotinic acid (niacin), oleic acid and linoleic acid were identified as candidate compounds. Fig. 8 presents the identification of silylated fatty acids. Of the identified fatty acids, niacin, linoleic acid and linoleic acid were analyzed at retention times of 18.21, 28.01 and 28.06 min respectively.