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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20

Alleviation of Dermatophagoides microceras-induced allergy by an immunomodulatory protein, FIP-fve, from Flammulina velutipes in mice a

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Yu-Chi Chang , Yi-Min Hsiao , Shao-Chi Hung , Ya-Wen Chen , Chu-Chyn Ou , Wei-Ting fg

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Chang , Ko-Huang Lue a

& Jiunn-Liang Ko

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Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan

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Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan c

School of Medicine, Chung Shan Medical University, Taichung, Taiwan

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School of Nutrition, Chung Shan Medical University, Taichung, Taiwan

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Division of Allergy, Department of Pediatrics, Chung-Shan Medical University Hospital, Taichung, Taiwan f

Department of Food and Nutrition, Providence University, Taichung, Taiwan

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Institute of Agriculture Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Published online: 11 Sep 2014.

To cite this article: Yu-Chi Chang, Yi-Min Hsiao, Shao-Chi Hung, Ya-Wen Chen, Chu-Chyn Ou, Wei-Ting Chang, Ko-Huang Lue & Jiunn-Liang Ko (2015) Alleviation of Dermatophagoides microceras-induced allergy by an immunomodulatory protein, FIP-fve, from Flammulina velutipes in mice, Bioscience, Biotechnology, and Biochemistry, 79:1, 88-96, DOI: 10.1080/09168451.2014.956682 To link to this article: http://dx.doi.org/10.1080/09168451.2014.956682

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Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 1, 88–96

Alleviation of Dermatophagoides microceras-induced allergy by an immunomodulatory protein, FIP-fve, from Flammulina velutipes in mice Yu-Chi Chang1, Yi-Min Hsiao2, Shao-Chi Hung3, Ya-Wen Chen3, Chu-Chyn Ou4, Wei-Ting Chang6,7, Ko-Huang Lue3,5,* and Jiunn-Liang Ko1,5,* 1

Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan; 2Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan; 3School of Medicine, Chung Shan Medical University, Taichung, Taiwan; 4School of Nutrition, Chung Shan Medical University, Taichung, Taiwan; 5Division of Allergy, Department of Pediatrics, Chung-Shan Medical University Hospital, Taichung, Taiwan; 6Department of Food and Nutrition, Providence University, Taichung, Taiwan; 7Institute of Agriculture Biotechnology Research Center, Academia Sinica, Taipei, Taiwan

Received April 22, 2014; accepted July 21, 2014

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http://dx.doi.org/10.1080/09168451.2014.956682

Asthma is a major public health concern. Its greatest risk factor is house dust mite (HDM). Dermatophagoides microceras (Der m) is a type of HDM, and in central Taiwan, there is approximately 80% prevalence of sensitization to Der m. FIP-fve is a fungal immunomodulatory protein (FIP) isolated from the fungus Flammulina velutipes, and exhibits anti-inflammatory properties. To investigate whether FIP-fve affects Der m-induced asthma and inflammation, we evaluated hyper-responsiveness (AHR), pathological changes, and cytokines in mice. We demonstrated that oral FIP-fve decreased Der minduced airway AHR, airway inflammation, cell infiltration, and expression of cytokines in the bronchoalveolar lavage fluid of Balb/c mice. The results of this study suggest that FIP-fve suppresses asthma, inflammation, and respiratory pathogenesis stimulated by Der m. FIP-fve is able to maintain immunomodulatory activity even in simulated gastric fluid and intestinal fluid. FIP-fve could be a safe and stable agent for suppression of allergic asthma. Key words:

immunomodulatory protein; FIP-fve (fungal immunomodulatory protein-fve); house dust mite; Der m (Dermatophagoides microceras); asthma

Asthma is a major public health concern affecting millions of people each year.1) Its most important risk factor is house dust mite. Perennial HDM allergen

exposure induces inflammatory diseases, which are manifested as asthma, atopic dermatitis (AD), or allergic rhinitis.2) It has been suggested that approximately 20% of the population in industrialized countries develop a sensitization to HDM allergens.3) Allergens of HDMs, including Dermatophagoides pteronyssinus (Der p), Dermatophagoides farinae (Der f), Der m, and Blomia tropicalis (Blo t), are important inducers of symptoms.4) Der p 1 (cysteine protease of Der p) and Der f 1 (cysteine protease of Der f) are two of the most well-known allergens. They cause the production of pro-inflammatory cytokines and allergic rhinitis.5) Der p 1 increases the permeability of epithelial cells in vivo, presumably through degradation of tight junction proteins, and induces a more specific response. The interaction of epithelial cells and Der p 1 can induce cytokine production, such as interleukin 6 (IL-6) and interleukin 8 (IL-8), or induce fluid secretion from submucosal glands.6) Der f induces acute AD skin lesions and causes epidermal and dermal thickening, dermal infiltration of CD4+ T cells, eosinophils, and Th2 (Type 2 T helper) cytokine expression.7,8) However, a relatively higher proportion of co-sensitization of Der m compared with the other three antigens – Der p, Der f, and Blo t – has been found in Taiwanese allergic children,4) while Der m has rarely been reported except in atopic children in Western countries.4) In the previous study, we focused on Der m from local glycyphagid mites in central Taiwan.4) Allergen exposure induces immune responses that result in naive CD4+T-cell differentiation into Th2 cells

*Corresponding authors. Email: [email protected] (J.-L. Ko); [email protected] (K.-H. Lue) Abbreviations: AD, atopic dermatitis; Blo t, Blomia tropicalis; BALF, bronchoalveolar lavage fluid; CEBPA, CCAAT/enhancer-binding protein (C/EBP); Der f, Dermatophagoides farina; Der m, Dermatophagoides microceras; Der p, Dermatophagoides pteronyssinus; FIP, fungal immunomodulatory protein; G-CSF, granulocyte colony-stimulating factor; H&E, hematoxylin and eosin; HDM, house dust mite; hPBMCs, human peripheral blood mononuclear cells; AHR, hyper-responsiveness; AHR, hyper-responsiveness; IFN-γ, interferon-gamma; IL-13, interleukin 13; IL-4, interleukin 4; IL-5, interleukin 5; IL-6, interleukin 6; IL-8, interleukin 8; MIP-1α, macrophage inflammatory protein 1 alpha; OVA, ovalbumin; PEP, peak expiratory pressure; PIP, peak inspiratory pressure; PHD 1, prolyl hydroxylases 1; PHD 3, prolyl hydroxylases 3; SD, standard deviation; CXCL1, the chemokine (C-X-C motif) ligand 1; TGF-β, transforming growth factor beta; TNF-α, tumor necrosis factor alpha; Th1, type 1 t helper; Th2, type 2 t helper; VEGF-A, vascular endothelial growth factor A. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

FIP-fve inhibits Der m-induced inflammation

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and establish CD4 T-cell memory. Th2 cytokines, such as interleukin 4 (IL-4), interleukin 5 (IL-5), and interleukin 13 (IL-13), are essential in allergic disorders.10) Th2 cells and their cytokines can trigger the development of allergic asthma, and the obstruction of these cytokines is an effective method for alleviating symptoms of asthma both in experimental animal models and in human subjects.1) Fungal immunomodulatory protein (FIP-fve) is an immunomodulatory protein isolated and purified from the edible golden needle mushroom, Flammulina velutipes. It has a molecular mass of ~13 kDa and comprises 114 amino acid residues.11) FIP-fve is a glycoprotein and presents a 1.7 Å X-ray structure by single anomalous diffraction12) in which a potential N-glycosylation site is present at position 54.13) The hinge for the activity of FIPs is stabilized predominantly by hydrophobic interactions within the N-terminal helices.14) Furthermore, FIP-fve has a high degree of amino acid sequence homology with LZ-8, and with 70 invariant amino acid residues.11) FIP-fve has also been found to stimulate IFN-γ (interferon-gamma) production in human peripheral blood mononuclear cells (hPBMCs) via the modulation of Ca2+ release.15,16) IFN-γ is secreted from such local memory Th1 (Type 1 T helper) cells to provide protection against secondary challenge.17) Mice receiving oral FIP-fve treatment during sensitization to OVA demonstrated a Th1-skewing effect with the development of the allergen-specific immune response and showed protection from systemic anaphylaxis-like symptoms.18) Vaccination with Th1directing adjuvants inhibited the development of allergen-induced Th2-type responses via activated CD4+T cells.19) Der m is one of the major allergens in central Taiwan, but it was rarely reported. FIP-fve has the ability of stimulating Th1 cytokine production. We purposed that orally administrated FIP-fve may able to switch the immune response towards Th1 while Der m challenge. To evaluate the hypothesis, we fed Balb/c mice with FIP- fve orally and sensitized with Der m extract.

Materials and methods Purification of fungal immunomodulatory protein (FIP-fve). FIP-fve was purified as previously described.20) In brief, the fruiting bodies of F. velutipes (300 g) were homogenized following soaking in ice-cold 5% acetic acid in the presence of 0.05 M 2-mercaptoethanol. The collected soluble proteins were precipitated by the addition of ammonium sulfate to 80% saturation. The precipitate was dialyzed against the dialysis solution (10 mM sodium acetate, pH 5.2), and the dialyzed proteins were centrifuged, then the supernatant was collected for crude extract. The dialysate equilibrated with 10 mM sodium acetate (pH 5.2) was applied to a CM-52 column (2 × 5 cm). The column was washed with equilibration buffer and then eluted with 200 ml 0–0.5 M NaCl in 10 mM sodium acetate. Finally, approximately 80 mg of purified FIP-fve was collected. To confirm the purification, crude extract and purified FIP-fve were analyzed by 12% SDS-PAGE and stained with Coomassie blue

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(0.26%) for 30 min. The stained gel was destained by destaining buffer (50% methanol and 3% glycerol) overnight. Animals. Female Balb/c mice at 6–8 weeks of age and with body weights of 20–25 g were purchased from the National Laboratory Animal Center in Taiwan. All animal housing requirements and procedures were performed in accordance with the Animal Use Committee of Chung Shan Medical University. Mice were individually housed in rack-mounted stainless steel cages with access to food and water and grouped as follows: (1) the control group had intranasal application of normal saline; (2) Der m group mice fed normal saline and were intranasal with 6.25 mg/kg/day of Der m extract for 10 days; (3) FIP-fve/Der m group mice were intranasal with 6.25 mg/kg/day of Der m extract for 10 days. Each mouse was administered 10 mg/kg FIPfve, 2 days before and up to 10 days, after intranasal application of Der m. Airway hyper-responsiveness and bronchoalveolar lavage fluid collection. The effect of FIP-fve on RSV-induced AHR was measured using a whole-body barometric plethysmography (Model PLY 3211; Buxco Electronic Inc., Sharon, CT). During inspiration and expiration with increasing doses of inhaled methacholine (Sigma-Aldrich, St. Louis, MO), the change in the chamber pressure was calculated and recorded as Penh, which is a dimensionless parameter used to evaluate pulmonary resistance. Each mouse rested in a chamber and received an initial baseline challenge with saline, followed by increasing doses of inhaled methacholine (0, 5, 10, 20, and 40 mg/ml). This was followed by a 3-min spray of nebulized methacholine, after which the breathing frequency was read and recorded for 3 min. The respiratory cycle resulted in box pressure waveforms that constitute the peak expiratory pressure (PEP), the peak inspiratory pressure, and the time of expiration. Penh was calculated by the following formula: Penh = Pause × peak expiratory pressure (PEP)/ peak inspiratory pressure (PIP). Finally, the Penh values were averaged and reported as baseline saline values in percentages.21,22). After measurement of AHR, lungs were lavaged via the trachea with 1 ml of normal saline for collecting bronchoalveolar lavage fluid (BALF). The cellularity of the BALF was investigated using a hemocytometer. The cells were centrifuged onto slides, fixed and then stained with Liu’s staining. The remaining BALF was stored at −70 °C until used in the assay. Mouse cytokine array. A mouse cytokine array (Proteome ProfilerTM Array, R&D system Catalog Number ARY006) was conducted according to the manufacturer’s instructions to analyze the cytokine profiles of the BALF of mice. Briefly, the sample was first mixed with the detection antibody at room temperature for 1 h and was then added to the array membrane. The membrane was incubated at 2–8 °C on a shaker overnight. After incubation, the membrane was washed.

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Horseradish peroxidase-conjugated streptavidin was then added to the membrane and incubated at room temperature on a shaker for 30 min. Following washing, array signals were detected by a chemiluminescence imaging system (MultiGel-21). Finally, the mean pixel densities of spots were analyzed using ImageJ analysis software.

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Cytokine measurement. BALF was collected via the trachea with 1 ml of normal saline lavation. Airway inflammation was evaluated by IL-6 in the BALF and assessed using an ELISA kit (88-7066-88; eBioscience) according to the manufacturer’s protocol. The ELISA plate was read at 450 nm using a Bio-Rad ELISA reader. Samples were compared using a standard curve containing seven dilution points, which were the relevant recombinant cytokines on each assay plate. Histological analysis. To assess pathological changes, the lungs of the mice were soaked in 10% formaldehyde for fixation and embedded in paraffin immediately after sacrificed by CO2. Lung tissue sections were stained with hematoxylin and eosin (H&E) after being cut from the paraffin blocks. Statistical analysis. Kruskal–Wallis H test was used for non-parametric multiple comparisons. Then, Mann– Whitney was used to test the differences between control and Der m subgroups, and then to test the differences between Der m and FIP-fve/Der m subgroups. A p value