Article pubs.acs.org/JAFC
Molecular Cloning of a Poria cocos Protein That Activates Th1 Immune Response and Allays Th2 Cytokine and IgE Production in a Murine Atopic Dermatitis Model Ya-Ting Lu,†,∥ Yen-Chou Kuan,†,∥,‡ Hui-Hsin Chang,†,∥ and Fuu Sheu*,†,‡ †
Department of Horticulture and ‡Center for Biotechnology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10673, Taiwan S Supporting Information *
ABSTRACT: Edible fungus Poria cocos (Schw.) Wolf is a cooking material that has myriad health benefits. However, its active constituents have not been well-defined. We previously purified an immunomodulatory protein, PCP, from P. cocos and described its biochemical features and its ability to activate primary macrophage via TLR4. In this study, we cloned the gene of PCP and demonstrated its ability to activate Th1 response in cell cultures and in mice. The complete cDNA sequence of PCP consisted of 807 bp, which included a 579 bp coding sequence that encoded 194 amino acids. With the addition of co-stimulatory CD3/ CD28 signals, PCP significantly increased the surface expression of CD44 and CD69 on effector T cells. PCP could also upregulate T-bet and STAT4 expressions and IFN-γ and IL-2 secretions. Oral administration of PCP suppressed the production of both total and OVA-specific IgG1 in serum and enhanced the amounts of serum and OVA-specific IgG2a and Th1-related cytokine production in BALB/c splenocytes. In addition, oral administration of PCP significantly reduced IL-4 and IgE expressions in a murine model of atopic dermatitis. In conclusion, these results provide evidence that PCP could regulate mammalian immune cells and reveal their pharmaceutical potential in developing therapeutic strategies against Th2-mediated immune disorders. KEYWORDS: atopic dermatitis, fungal immunomodulatory protein, molecular cloning, Poria cocos, Th1 immune response
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INTRODUCTION Poria cocos (Schw.) Wolf is a fungus widely used as a culinary material. Previous studies have highlighted the health benefits of P. cocos.1,2 The P. cocos-derived extract exhibited an antirejection effect in rats after cardiac allograft implantation3 and two-way regulation on free calcium in cytosol of nerve cells in the brain.4 The P. cocos-derived extract also enhances IL-1β and IL-6 secretion of human peripheral blood monocytes.4 In addition, the P. cocos-derived extract had shown antiinflammatory activity.5 In our previous study, we demonstrated that PCP could activate M1 polarization of murine peritoneal macrophage through a TLR4-MyD88 pathway.6 T cells are principal effector cells in antigen-specific immune response. Mature CD4+ helper T cells and CD8+ cytotoxicity T cells reside in secondary lymphoid organs and are activated by recognizing antigen that is presented on the major histocompatibility complex (MHC) of APCs.7 Stimulation by antigen led to T cell proliferation and Th1 and Th2 effector differentiation.7−9 Th1 cells can produce IL-2 and IFN-γ to mediate cellular immunity, whereas Th2 cells secrete IL-4 and IL-5 to mediate humoral immunity.10 In addition, the Th1 and Th2 response is reciprocally regulated by cytokines such as IFN-γ, IL-4, and IL-10.11,12 In this study, we cloned the gene of PCP, evaluated the activity of PCP to activate murine T cell, and studied the mechanism of PCP-induced T cell differentiation. Additionally, we discovered that PCP can induce a Th1 response in cell culture and in mice and examined its effect in a murine model of atopic dermatitis. © 2014 American Chemical Society
MATERIALS AND METHODS
Preparing PCP from P. cocos. The PCP sample was obtained as previously described.6 In brief, the dried P. cocos sclerotium was homogenized with a Waring blender and then centrifuged. The proteins in the supernatant were salted out by applying ammonium sulfate. PCP was then purified from the proteins by two-step anionexchange chromatography using a self-packed DEAE-cellulose (Whatman, Maidstone, UK) column and a Superdex 75 10/300 GL gel filtration column (GE Healthcare, Little Chalfont, UK). Purified PCP was examined by SDS-PAGE and showed a pattern identical to that of the PCP we reported previously.6 The yield of PCP was generally 175 ± 25 mg of PCP/kg of dried sclerotia of P. cocos. Nevertheless, contamination of bacterial LPS in PCP was examined and excluded by carrying out the ToxinSensor chromogenic LAL endotoxin Assay (GenScript Inc., Piscataway, NJ, USA). Molecular Cloning of the Complete cDNA Sequence of the PCP Gene. To clone the gene of PCP, Edman degradation analysis and rapid amplification of cDNA ends (RACE) were performed as previously described.13 In brief, the PCP N-terminal sequence was determined by Edman degradation. Then, the gene encoding PCP was cloned using a SMARTer RACE cDNA Amplification Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer’s manual. In brief, 3′-Ready cDNA was prepared using a 3′-RACE CDS Primer A (5′-AAGCAGTGGTATCAACGCAGAGTAC(T)30VN-3′), and 5′ Ready cDNA was prepared using a 5′RACE CDS Primer A (5′-(T)25VN-3′) plus SMARTer II A Oligonucleotide (5′-AAGCAGTGGTATCAACGCAGAGTACXXReceived: Revised: Accepted: Published: 2861
December 9, 2013 March 6, 2014 March 10, 2014 March 13, 2014 dx.doi.org/10.1021/jf405507e | J. Agric. Food Chem. 2014, 62, 2861−2871
Journal of Agricultural and Food Chemistry
Article
Cell Proliferation Analysis by BrdU Incorporation Assay. Cell proliferation was measured using a cell proliferation ELISA BrdU (colorimetric) kit (Roche, Basel, Switzerland) according to the user’s manual. Data acquisition was carried out by measuring A450 on a BioRad microplate reader (Bio-Rad Laboratories). Cytokines and Igs Quantification by ELISA. The level of cytokines IFN-γ, IL-2, IL4, IL-5, and TNF-α presented in the cell-free culture supernatants were measured by sandwich ELISA using OptEIA murine IFN-γ, IL-2, IL-4, IL-5, and TNF-α ELISA kits (eBioscience, San Diego, CA, USA), respectively, following the manufacturer’s protocols. The levels of the total Igs and the OVA-specific Igs presented in mouse serum were measured by sandwich ELISA using a Mouse MonoAb ID Kit (Invitrogen) and indirect ELISA, respectively, according to the user’s manual. Data acquisition was carried out by measuring A450 on a Bio-Rad microplate reader. The results were expressed as ELISA units (EU), which were calculated by comparison with mouse IgG standards. Cytokine Gene Expression Analysis by Quantitative RealTime PCR. Complementary DNA samples were obtained from T cells treated with or without PCP (100 μg/mL). Gene expression was assessed by quantitative real-time PCR according to the manufacturer’s protocol. The results were normalized to endogenous genes (β-actin or GAPDH). The expression level of each target gene was calculated by cycle threshold (Ct) analysis. The sense and antisense primer pairs are β-actin, 5′-GTGGGCCGCTCTAGGCACC-3′ and 5′-CTCTTTGATGTCACGCACGA-3′; G3PDH, 5′-ATGAATACGGCTACAGCA-3′ and 5′-TGGAAATTGTGAGGGAGAT-3′; T-bet, 5′-GGAGAACTTTGAGTCCAT-3′ and 5′-GTAGAAACGGCTGGGAA-3′; GATA3, 5′-CTGCGGACTCTACCATA-3′ and 5′-TTAGCGTTCCTCCTCCA-3′; ROR γ T, 5′-TGAGATTGCCCTCTACAC-3′ and 5′-GCAGAGATGATGATGGAAA-3′; and Foxp3, 5′-TGAGTTTCGCAAGAAGAG-3′ and 5′-GGTTTCCATAGGTACACG-3′. Detection of Cell Surface Marker and Intracellular IFN-γ by Flow Cytometry. Mouse splenocytes and MACS-purified cells were treated with the indicated samples for 72 h and then collected and suspended in FACS buffer and stained with FITC- or PE-conjugated antibodies specific for CD3, CD4, CD8 (BD Biosciences), CD44, and CD69 (eBioscience) at 4 °C for 0.5 h. Flow cytometry data were acquired on a FACScan (Becton Dickinson and Co., Franklin Lakes, NJ, USA) and analyzed by CellQuest software. To estimate intracellular production of IFN-γ and granzyme B in T cells by PCP, CD4+ and CD8+ cells isolated from splenocytes by the MACS purification system were incubated with the indicated stimulants or medium alone in the presence of anti-CD3/CD28 antibodies for 72 h. After incubation, brefeldin A (10 μg/mL; eBioscience) was added to block cytokine release. After an additional 6 h, cells were washed, fixed in Fixation Solution (eBioscience), and incubated in the dark at room temperature for 30 min. Then, the cells were permeabilized using the Permeabilization Buffer (eBioscience), stained with PE-conjugated mAbs specific for mouse IFN-γ and granzyme B (eBioscience), and analyzed by flow cytometry. Immunoprecipitation of STAT4 and STAT6. Isolated CD90+ cells were stimulated with 100 μg per mL of PCP for 4, 8, 16, and 24 h, respectively, in the presence or absence of anti-CD3/CD28 antibodies. Then, the cells were homogenized in RIPA buffer. The homogenate was centrifuged, and the supernatant was collected for total protein detection. The supernatant was immunoprecipitated using the mouse monoclonal antibodies against STAT4 or STAT6 (Santa Cruz Biotechnology, Dallas, TX, USA) overnight with gentle mixing at 4 °C. After incubation with STAT4 or STAT6 antibodies, the lysate was incubated with Protein G plus/Protein A agarose for 3 h with gentle mixing at 4 °C. The solution was then centrifuged, and the pellet was suspended in 20 μL of SDS sample buffer and subjected to SDS-PAGE and Western blot analysis. Blots were incubated with mouse antiphosphotyrosine antibodies (Merck KGaA) and subsequently detected using a HRP-conjugated anti-mouse IgG (KPL, Gaithersburg, MD, USA). Membranes were washed extensively with Tris-buffered saline with Tween 20. Proteins were detected using the Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Waltham, MA, USA) and subjected to X-ray autoradiography. In addition, antibodies were
XXX-3′; this is an adaptor sequence that attaches to the 5′ end of the mRNA template; X stands for undisclosed nucleotides protected by the manufacturer). The RACE ready cDNA was synthesized by a SMARTScribe reverse transcriptase. Then, 3′-RACE PCR was performed using the 3′-RACE Ready cDNA as a template, a Universal Primer A Mix (long, 5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′; short, 5′-CTAATACGACTCACTATAGGGC-3′), and a gene-specific degenerate primer p320 (5′GGTTACGGTCAATGTATGTGAT-3′) obtained from the PCP Nterminal amino acid sequence. 5′-RACE PCR was performed using the 5′-RACE Ready cDNA as a template, a Universal Primer A Mix, and a gene-specific primer p5-320-2 (5′-ATTGTATGTGAGTTGCATCATGTC-3′) designed on the basis of the sequence of 3′-RACE products. Finally, the complete cDNA sequence of PCP was revealed by combining the product sequences of 3′-RACE PCR and the 5′RACE PCR. The accession number of the PCP cDNA sequence is JN571084.1. Primary Cell Acquisition and Cell Cultures. Primary lymphocytes were acquired from murine spleens as previously described.14 In brief, the total splenocytes were obtained from spleens excised from euthanized mice, and the splenic T cells were either positively or negatively purified using a MACS system with microbeads of anti-CD90, -CD4, -CD8, or anti-B220 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively. The purity of the purified T cells used in this study was >95%. The cells were seeded in 96-well flat-bottom plates (Corning, Lowell, MA, USA) at 4 × 106 cells/mL and were stimulated with or without PCP at indicated concentrations, ConA (5 μg/mL) or LPS (1 μg/mL), for 72 h. In some experiments, the purified T cells were seeded in rat anti-mouse CD3/CD28 antibody-coated 96-well plates. All cells were grown in a CO2 incubator under humidified atmosphere of 5% CO2 at 37 °C. Mice and Animal Experiments. Female 6−8-week-old BALB/c were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and used for splenic cell acquisition and in vivo studies. To assess the in vivo effect of PCP, mice were ip injected with 50 μg/100 μL OVA adsorbed on aluminum hydroxide adjuvant or with adjuvant i alone as a nonimmunized vehicle. The mice were randomly grouped into four (n = 6 each). The first two groups were orally administered by gavage 0.5 mL of PCP solution (2 mg/mL in PBS) at 50 mg/kg body weight (1 mg/mouse) once daily and immunized with OVA or with saline vehicle, respectively. The two other groups were given an equal volume of PBS and immunized with OVA or with saline vehicle, respectively. OVA-immunized mice were immunized on days 0 and 14. Blood samples (100 μL per mouse) were collected from tail veins at various time points (days 0, 10, and 24) after immunization. To evaluate the potential of PCP to attenuate Th2-dominat allergic response, an OVA-induced atopic dermatitis (AD) murine model, which was developed by Spergel and his collegues,15 was performed. In brief, female BALB/c mice aged 6−8 weeks were randomly divided into three groups, each consisting of four mice (n = 4). The back skin of all the mice was shaved and tape stripped six times with 3M tape to simulate the skin injury inflicted by scratching in patients with AD. The first group of mice was orally administered daily 200 μL of PBS and sensitized daily in the sensitizing periods with 20 μL of saline vehicle placed on a 1 cm2 patch of sterile gauze, which is secured to the skin with a transparent bio-occlusive dressing to repel licking by the mice. The other group of mice was administered daily 200 μL of PBS and sensitized with 20 μL of skin-patched OVA (100 mg/mL) in the manner described above. The last group of mice was administered daily 200 μL of PCP (5 mg/mL) and sensitized with skin-patched OVA (100 mg/mL) in the manner described above. There were two sensitizing periods in the AD model; the first period began from day 3 to day 8, and the second period began from day 22 to day 27. Oral administration began on day 0 and terminated on day 32, the end point of the AD model. Blood samples (100 μL per mouse) were collected on day 8 via tail veins of the mice. The lymphocytes of the mice were obtained from the axillary nodes, the brachial nodes, and the inguinal nodes and cultured in the presence or absence of OVA (100 mg/mL) for 48 h. The cell-free culture supernatant was collected for cytokine quantification via ELISA. 2862
dx.doi.org/10.1021/jf405507e | J. Agric. Food Chem. 2014, 62, 2861−2871
Journal of Agricultural and Food Chemistry
Article
Figure 1. Complete cDNA sequence of PCP gene cloned via RACE PCR. (A) The full-length cDNA sequence of PCP was generated by combining the 3′- and 5′-RACE products. The N-terminal amino acid sequence is boxed, the signal peptide sequence is indicated by a gray background, and the stop codon is indicated by an asterisk. (B) Multiple alignment of PCP amino acid sequence with sequences of proteins from Polyporaceae fungi was performed by ClustalW omega program on the Web site of EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/clustalo/). Identical, highly similar, and similar residues are marked with asterisks, colons, and dots, respectively. 2863
dx.doi.org/10.1021/jf405507e | J. Agric. Food Chem. 2014, 62, 2861−2871
Journal of Agricultural and Food Chemistry
Article
Table 1. Homology Analysis of PCP Amino Acid Sequence with Proteins from Polyporaceae Fungi species
name
accession no.
length
identity (%)
similarity (%)
gap (%)
P. cocos F. radiculosa T. versicolor D. squalens
PCP predicted protein TRAVEDRAFT_31886 DICSQDRAFT_157655
AEM91639.1 CCL98455.1 EIW52848.1 EJF56964.1
193 190 293 182
49 46 43
65 60 59
1 3 9
Figure 2. PCP activates murine splenocytes. (A) Mouse splenocytes were incubated with PCP at the indicated concentrations or ConA (5 μg/mL) as a positive control for 54 h. Cell proliferation was determined by measuring BrdU incorporation into DNA for an additional 18 h. (B) Mouse splenocytes were incubated with PCP at the indicated concentrations for 72 h. Culture supernatants were collected to determine their IFN-γ and IL5 levels by sandwich ELISA methods. Statistical difference was compared to the level in untreated control cells (∗, P < 0.05). (C, D) Mouse splenocytes were treated with PCP at each time point. RNAs from treated cells were isolated and subjected to RT-PCR using specific primer sets for mouse Th1-associated cytokines (C; IFN-γ and IL-2), Th2-associated cytokines (D; IL-4 and IL-5), and β-actin. The values were normalized by dividing by the amount of β-actin gene expression in real-time PCR analysis. Each bar of the histogram represents a minimum of three experiments, and data are presented as the mean ± SD (∗, P < 0.05 compared to the culture medium control (0 h). removed by 2% SDS, 62.5 mM Tris, pH 6.8, 100 mM βmercaptoethanol buffer. The membrane was reprobed with rabbit anti-mouse STAT4 or STAT6 antibodies (Santa Cruz Technology) and detected with HRP-conjugated anti-rabbit IgG (KPL) as a loading control. Statistical Analysis. All in vitro data are presented as means ± SD of three independent experiments (n = 3). All animal experiment data are presented as means ± SE of at least two independent experiments performed in quadruplicate or hexaplicate. Statistical comparison was made by one-way ANOVA followed by a Duncan multiplecomparisons test. Differences were considered as statistically significant when the P values were
