Article pubs.acs.org/JAFC

Molecular Cloning and Function Characterization of a New Macrophage-Activating Protein from Tremella fuciformis Chih-Liang Hung,† An-Ju Chang,† Xhao-Kai Kuo,† and Fuu Sheu*,†,‡ †

Department of Horticulture and Landscape Architecture and ‡Center for Biotechnology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10673, Taiwan, R.O.C. S Supporting Information *

ABSTRACT: Silver ear mushroom (Tremella fuciformis) is an edible fungus with health benefits. In this study, we purified a new T. fuciformis protein (TFP) and demonstrated its ability to activate primary murine macrophages. The isolation procedure involved ammonium sulfate fractionation and ion exchange chromatography. TFP naturally formed a 24 kDa homodimeric protein and did not contain glycan residues. The TFP gene was cloned using the rapid amplification of cDNA ends method, and the cDNA sequence of TFP was composed of 408 nucleotides with a 336 nucleotide open reading frame encoding a 112 amino acid protein. TFP was capable of stimulating TNF-α, IL-1β, IL-1ra, and IL-12 production in addition to CD86/MHC class II expression, mRNA expression of M1-type chemokines, and nuclear NF-κB accumulation in murine peritoneal macrophage cells. Furthermore, TFP failed to stimulate TLR4-neutralized and TLR4-knockout macrophages, suggesting that TLR4 is a required receptor for TFP signaling on macrophages. Taken together, these results indicate that TFP may be an important bioactive compound from T. fuciformis that induces M1-polarized activation through a TLR4-dependent NF-κB signaling pathway. KEYWORDS: macrophage-activating protein, Tremella f uciformis, macrophage activation, TLR4, molecular cloning

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and possess strong microbicidal and tumoricidal activities.8 Classically activated M1 macrophages are also considered to be cells that tend toward T helper 1 (Th1) responses, resulting in the production of proinflammatory cytokines such as TNF-α, IL-1, IL-6, and IL-12 and the expression of CXCL10, CCL5, and CXCL9 (M1 chemokines).8,9 In contrast, M2 macrophages undergo alternative M2 activation and have immunoregulatory abilities with anti-inflammatory cytokines such as IL-1ra, IL-10, and IL-1decoyR.8 Unlike M1 macrophages, alternatively activated M2 macrophages produce M2 chemokines (CCL2, CCL17, CCL18, and CXCL4).8 M1 and M2 macrophages are distinct in their polarization mechanisms and expression profiles on secretion of cytokines and chemokines. Toll-like receptors (TLRs) are a critical family of membrane proteins that trigger innate immune responses and are patternrecognition receptors (PRRs) that play critical roles in sensing endocytic vesicles or intracellular organelles.9 The structural character of the ligand recognition by TLRs determines whether there is a response to a specific pathogen infection, and the ligand interaction can induce different signaling pathways through distinct TLRs.9 TLRs bind ligands directly or with accessory molecules, and the intracellular signaling transductions are mediated through the activation of cytoplasmic Toll/interleukin-1 (IL-1) receptor (TIR) domains. The activation of signaling through the TIR finally results in the activation and translocation of NF-κB to the nucleus, which induces proinflammatory cytokine and immune-related gene expression associated with macrophage polarization.8,10 Among

INTRODUCTION Silver ear mushroom (Tremella fuciformis Berk.) is one of the most popular edible fungi in Asia, and its possible healthcare effects have attracted considerable interest. The extracts from T. fuciformis have demonstrated a variety of biological and pharmaceutical properties, including hypocholestolemic,1,2 hypoglycemic,3 antitumor,4 and immunomodulatory5−7 effects. Recently, a growing number of studies have focused on the immune activating and regulatory functions of T. fuciformis extracts. Alkaline and aqueous extract fractions from T. fuciformis were able to inhibit the growth of sarcoma 180 in a mouse model.4 The oral administration of T. fuciformis extracts can induce significantly specific systemic and cecum mucosal antibody production, the antigen-specific proliferation of splenocytes, and erythrocyte rosette-forming cells and erythrocyte-antibody-complement complexes against avian coccidiosis.7 In addition, T. fuciformis extracts have demonstrated the ability to promote cytokine production by human monocytes and mouse splenocytes.5,6 Although multiple immunomodulatory effects for T. fuciformis extracts have been reported, neither the bioactive compounds from T. fuciformis that mediate these effects nor their mechanisms are clear. Macrophages are well-known as important immune cells that remove exogenous pathogens to protect the host. These cells are activated by different stimuli to regulate immune responses.8 On the basis of their different types of activation and biochemical properties, macrophages have been classified into two types. Classically activated macrophages are designated as M1 macrophages, and alternatively activated macrophages are designated as M2 macrophages. M1-type macrophage polarization is defined by macrophages that express high levels of proinflammatory cytokines, produce a large number of reactive nitrogen and oxygen intermediates, © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1526

September 25, 2013 January 6, 2014 January 8, 2014 January 8, 2014 dx.doi.org/10.1021/jf403835c | J. Agric. Food Chem. 2014, 62, 1526−1535

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PAGE using a Bio-Rad mini protein III gel apparatus (Bio-Rad Laboratories, Hercules, CA). The gels were stained with Coomassie brilliant blue R250 or periodic acid-Schiff reagent (Sigma). The molecular mass of the protein was compared with those of standard proteins in the range of 10−170 kDa (Fermentas Life Sciences, Glen Burnie, MD). The molecular weight of TFP was further estimated using an FPLC system with a Superdex gel filtration column (GE Healthcare), equilibrated, and eluted with 50 mM phosphate buffer (pH 7.0) containing 0.15 M NaCl. The column was calibrated using the same parameters with protein standards (Amersham Biosciences, Uppsala, Sweden) consisting of ovalbumin, carbonic anhydrase, trypsin inhibitor, and lysozyme (Supplemental Figure 1 in the Supporting Information). The TFP sample was separated by SDSPAGE and electrotransferred onto a poly(vinylidene difluoride) (PVDF) Immobilon P membrane (Millipore, Billerica, MA) using a Trans-Blot cell system (Bio-Rad) with transfer buffer. The TFP protein band was cut out from the membrane, and its N-terminal peptide sequence was determined using automated Edman degradation and sequence analysis (Mission, Taipei, Taiwan). Molecular Cloning of the Gene and cDNA Encoding TFP. The total RNA of T. fuciformis fruiting bodies was extracted using an RNeasy Plant Mini Kit (Qiagen, Germantown, MD) according to the manufacturer’s protocol. First-strand cDNA was synthesized from total RNA by reverse-transcription polymerase chain reaction (RT-PCR) using a Thermoscript RT-PCR system (Invitrogen, Carlsbad, CA) with a 3′-SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA). The 3′-RACE TFP primer and the TFP-specific forward degenerate primer 3T36, which was designed on the basis of the partial N-terminal amino acid sequence with the lowest degeneracy of the genetic code, were paired for PCR amplification of the tf p gene. The resulting 600 bp PCR product (3T36-600) was ligated into the pGEM-T easy vector (Promega, Madison, WI), transformed into Escherichia coli, and sequenced. The 5′ end of TFP was obtained by using a 5′-SMART RACE cDNA Amplification Kit (Clontech). The 5′-RACE TFP primer was paired with the reverse primer Fru5T1, which is specific for the 3T36-600 fragment, for PCR amplification. A 400 bp PCR product (Fru5T1-400 fragment) was sequenced by the same method. The full-length sequence of TFP cDNA was obtained by alignment of the 3T36-600 and Fru5T1-400 fragments. After synthesis of cDNA from the total RNA of T. fuciformis fruiting bodies as the template, the sequence was confirmed via PCR amplification with gene-specific primer pairs that were designed on the basis of the full-length sequence of TFP. Expression and Purification of Recombinant TFP. The cDNA synthesized from the total RNA of T. fuciformis fruiting bodies was used to amplify the TFP gene for expression. The primers used for PCR amplification of TFP were 5′-CCATGGTCGTCCAGAGGGATGACACA-3′ and 5′-CTCGAGATCAAACGCCTAGTCGAACTC3′. After purification, the PCR products were digested with NcoI and XhoI (New England Biolabs, Beverly, MA). Double-digested PCR products were ligated into pET30a(+) vector and transformed into E. coli BL-21 competent cells (Bioman, Taipei, Taiwan) for protein expression. A single BL-21 colony containing the pET30-TFP vector was inoculated into LB/ampicillin medium overnight and then induced with 1 mM IPTG for 3 h at 37 °C. The cultures were centrifuged at 6500 rpm for 30 min and rinsed twice with PBS. After collection, the bacteria were sonicated for 30 min and then centrifuged at 6500 rpm for 30 min. The supernatants were collected for FPLC purification with a HiTrap chelating HP column (GE Healthcare). Before loading of the supernatants, the 1 mL Hitrap chelating column was prepared according to the manufacturer’s instructions and equilibrated with binding buffer (0.02 M sodium phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4) by washing with five column volumes. The column was loaded with supernatants containing His-tagged TFP protein and washed with five column volumes of binding buffer to remove nonbinding protein. His-tagged TFP protein was eluted with elution buffer (0.02 M sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4) in the final fractions. The purities of the fractions containing His-tagged TFP protein were identified by SDS-PAGE and Western blotting analysis with monoclonal antibodies (mAbs) specific

the TLRs, TLR2 is known to recognize several molecules from Gram-positive bacteria and mycobacteria, such as lipoteichoic acid, lipopeptides, and peptidoglycan,11 whereas TLR4 is a transmembrane protein that plays an essential role in the recognition of LPS, which is the main component of the outer membrane of Gram-negative bacteria.12 Both of these receptors have been demonstrated to play important roles in recognition of mushroom compounds that possess bioactive properties and active TLR-mediated signaling pathways that enhance the immune response.13,14 In the present study, a novel immune-activating protein, T. fuciformis immunomodulatory protein (TFP), which was isolated from the dried fruiting bodies of T. fuciformis, was demonstrated to induce macrophage activation. We demonstrated that TFP induces macrophage activation through NFκB. Furthermore, our data revealed that TFP enhances proinflammatory gene expression and induces M1-type polarization of macrophages through a TLR4-dependent pathway.

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MATERIALS AND METHODS

Mice. BALB/c and C57BL6 mice that were 8−10 weeks of age were purchased from the National Laboratory Animal Center, Taipei, Taiwan. TLR4-deficient mice (TLR4−/−, strain C57BL/10ScN) were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in our animal facility under specific pathogen-free conditions. All mice were fed a standard mice diet (LabDiet, Richmond, IN). All experimental procedures were approved by the Institutional Animal Care and Use Committee of National Taiwan University (NTUIACUC-95-139). Cell Preparation. Peritoneal macrophages were obtained from BABL/c, C57BL/6J, and TLR4−/− mice 3 days after i.p. injection of 1.8 mL of sterile 3% thioglycollate (Sigma, St. Louis, MO). Macrophages were harvested by sacrificing mice and flushing ∼6 mL of cold phosphate-buffered saline (PBS) into the mouse peritoneal cavity twice. The fluid containing the macrophages was centrifuged and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT) with 10% (v/v) fetal bovine serum (FBS) (Life Technologies, Grand Island, NY). The cells were counted, plated in 24-well (2 × 106 cells/well) or 12-well (4 × 106 cells/well) flat-bottom culture plates (Costar, Cambridge, MA), and incubated for 2 h in a humidified atmosphere of 5% CO2 at 37 °C. After the incubation, the supernatants were removed, and the cells were washed with warm PBS to remove nonadherent cells. The cells were further incubated in DMEM containing 10% FBS before and during all experiments. Purification of TFP. One kilogram of weighed dried fruiting bodies of T. fuciformis that were obtained from a local market were soaked in water for 2 h for rehydration and then soaked in a 5% (v/v) acetic acid solution containing 50 mM 2-mercaptoethanol at 4 °C overnight. After soaking, the fruiting bodies were homogenized and centrifuged at 8500g for 50 min. The soluble proteins in the supernatant were precipitated by adding ammonium sulfate from 60% saturation and centrifuging at 8500g for 50 min to collect the precipitates. The precipitates were dialyzed in 20 mM Tris-HCl buffer (pH 8.2) for 96 h with at least eight changes of the dialysis buffer. The main active fractions in the dialysate, which was capable of increasing TNF-α production by RAW 264.7 macrophages, were fractionated and purified using a fast protein liquid chromatography (FPLC) system with a Resource-Q anion exchange column (GE Healthcare, Uppsala, Sweden) in 10 mM Tris-HCl buffer containing 0.5 M NaCl (pH 8.0). Each fraction was analyzed with denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the fraction containing a single 24 kDa protein was chosen for activity analysis. The 24 kDa protein that could induce RAW264.7 macrophages to produce TNF-α was our target protein, TFP. The fraction containing TFP was further dialyzed and concentrated optimally for the subsequent analysis and in vitro experiments. Molecular Weight Determination and Amino Acid Sequencing. The purity and molecular weight of TFP were analyzed by SDS1527

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Figure 1. Chromatographic purification and electrophoretic analysis of native TFP. (A) The elution profile of the ammonium sulfate precipitates of a T. fuciformis dried fruiting body from FPLC using a Resource Q column displayed a single peak of TFP. (B) SDS-PAGE analysis of the crude protein from T. fuciformis (lane 1), purified TFP (lane 2), and purified TFP with periodic acid/Schiff’s staining (lane 3) indicated that TFP had a mobility similar to that of a 24 kDa protein that did not contain carbohydrates. (C) Isoelectric focusing analysis (lane 4) indicated that the pI of TFP is 3.45. for TFP and the His tag. The His tag was removed from the recombinant His-tagged TFP protein using EKMax enterokinase (Invitrogen) to yield recombinant TFP. All of the proteins were dialyzed with PBS and further concentrated optimally for the subsequent analysis. Determination of Cytokine Production. The production of the cytokines TNF-α, IL-1β, IL-10, and IL-12p70 in cell-culture supernatants was determined by sandwich enzyme-linked immunosorbent assay (ELISA) using mouse TNF-α, IL-1β, and IL-10 ELISA kits (eBioscience, San Diego, CA) and a DuoSet mouse IL-12p70 ELISA Kit (R&D Systems, Minneapolis, MN) according to each manufacturer’s protocols. Quantitative Real-Time PCR. The cells were lysed using TRIzol reagent (Invitrogen), and the total RNA was extracted from the cell lysate according to the manufacturer’s protocols. The quality and concentration of RNA were determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The total RNA was used as a template to synthesize the first-strand cDNA with a ThermoScript RT-PCR system (Invitrogen) for evaluation of the expression of genes implicated in TFP-induced activation in macrophages. The expression differences of individual genes were measured using real-time PCR. The primers were based on macrophage-specific cytokines, chemokines, and transcription factor sequences from NCBI and designed for SYBR green probes with Probe Design software (Roche, Mannheim, Germany) (Supplemental Table 1). Gene amplifications were normalized to the β-actin housekeeping gene. Real-time PCR was performed using a Bio-Rad MyiQ single-color detection system (Bio-Rad). The expression level of the individual target genes was quantified with the relative levels of the specific mRNA using cycling threshold (Ct) analysis. The relative expression value of significantly different expression in the gene should be higher than 4-fold relative to the 0 h control. Electrophoretic Mobility Shift Assay. The DNA−protein interaction was determined using a LightShift Chemiluminescent EMSA kit (Pierce, Rockford, IL) according to the manufacturer’s protocol. The nuclear protein extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce) and incubated with biotinylated double-stranded NF-κB probes. The DNA−protein complexes were analyzed using a native 6% polyacrylamide gel and electrotransferred onto nylon membranes. When the transfer was completed, the biotinylated NF-κB−DNA complexes were detected using a chemiluminescent substrate kit (Pierce) with peroxidaseconjugated streptavidin. Fluorescence-Activated Cell Sorting (FACS) Analysis. The expressions of surface CD80, CD86, and MHC-II on mouse peritoneal macrophages that were cultured with TFP for 24 h were detected using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ) with fluorescein isothiocyanate (FITC)-conjugated antibodies specific

for mouse CD80, CD86, and MHC-II (eBioscience). The cells were harvested with PBS, centrifuged at 1500g, and resuspended with PBS containing 2% bovine serum albumin and 0.1% sodium azide (FACS buffer). Antibody was added to the cell suspensions, which were then incubated at 4 °C for 30 min. After the treatments, the cells were washed with FACS buffer to remove the unbound antibodies. For phagocytosis experiments, the cells were incubated with TFP for 24 h, collected, centrifuged, and resuspended in FACS buffer. The phagocytosis ability of macrophages was analyzed using a Vybrant Phagocytosis Assay Kit (Molecular Probes) according to the manufacturer’s protocol. After addition of the fluorescent E. coli BioParticles, cells (106 cells/mL) were incubated at 4 °C as a negative control and for 37 °C for 1 h. Antibody-stained or E. coli-treated cells were analyzed to determine the mean fluorescence intensity (MFI) and percentage of positive fluorescent cells using a FACScan flow cytometer with CellQuest software (BD Biosciences). The peak fluorescence intensities of the control and experiment groups were compared. Statistical Analysis. All data are presented as means ± standard deviations of three independent experiments (n = 3) performed in triplicate. The statistical comparisons were executed by analysis of variance (ANOVA). When the P value was below 0.05 (i.e., P < 0.05), the differences were considered to be statistically significant.

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RESULTS Purification of TFP. To isolate the bioactive protein from T. fuciformis, the crude protein of the dry fruiting body that was obtained from homogenization and ammonium sulfate precipitation was subjected to FPLC using a Resource Q anion exchange column with a linear gradient of sodium chloride (0−0.5 M in 20 mM Tris-HCl buffer, pH 8.2). Measuring absorbance at 280 nm revealed a remarkable peak in the elution profile (Figure 1A). With this procedure, we were able to collect around 61 mg of this purified protein from 100 g of the dry fruiting bodies. The protein of this peak was then collected, and its activity was investigated by coculturing with RAW 264.7 macrophages. Significant TNF-α production was observed in the supernatant of RAW 264.7 cells that were cultured with the peak fraction (Supplemental Figure 2). This active fraction was analyzed with SDS-PAGE and displayed a single protein with an observed molecular mass of approximately 24 kDa (Figure 1B). This bioactive protein was designated TFP. The endotoxin level in the TFP sample which as determined with a ToxinSensor Chromogenic LAL Endotoxin Assay Kit was around 0.14 EU/μg (data not 1528

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Figure 2. Nucleotide and translated amino acid sequence of TFP. The N-terminal amino acid sequence obtained by Edman degradation is underlined, and the signal peptide of TFP is shown in bold. The forward degenerate primer (3T36) based on the TFP N-terminal sequence (GTAEQY) and used in 3′-RACE PCR is indicated above the right-pointing arrow. The DNA sequence of the reverse degenerate primer (Fru5T1) used in 5′-RACE with the 3′-RACE product sequence (VDACCCED) is shown above the left-pointing arrow. The full-length sequence of TFP reported in this paper has been deposited in the GenBank database (accession number EF152774).

Tremella encephala β-tubulin (tub2) gene. We obtained a 600 bp sequence of the 3′-RACE product (3T36-600), and its translated amino acid sequence was highly similar to the Nterminal sequence of TFP. On the basis of this 3′-RACE product, the primer Fru5T1 was then designed and used for the 5′-RACE PCR, which further generated a 400 bp fragment, Fru5T1-400, that encoded the same starting amino residues (DDTIYIGTAEQYTPV) as native TFP. By merging the two open reading frames of 3T36-600 and Fru5T1-400, we obtained the full-length sequence of TFP (GenBank accession number EF152774). A conceptual translation and analysis of the 336 bp open reading frames of the 408 bp TFP transcript suggested that this gene encodes a 112 aa protein with a predicted molecular mass of 11.34337 kDa (Figure 2). Native TFP Exists as a Homodimer. According to the gene sequence of TFP, its predicted molecular mass (11 kDa) is approximately half of the mass of native TFP from the SDSPAGE (24 kDa) (Figure 1B, lane 2). To clarify the conflict in the values of the molecular mass from the prediction and the SDS-PAGE observations, we determined the mobility of

shown). To further analyze the biochemical characteristics of TFP, we used carbohydrate staining and isoelectric focusing electrophoresis to determine that TFP was not a glycoprotein (Figure 1B and Supplemental Figure 3) and that its pI value was 3.45 (Figure 1C), respectively. Cloning of TFP. To clone the full-length cDNA sequence of TFP, we used N-terminal amino acid analysis and RACE by RT-PCR. First, the TFP after electrophoretic analysis with SDS-PAGE was immobilized onto a PVDF membrane and then subjected to N-terminal amino acid analysis. The sequence of the 15 N-terminal amino acid residues, DDTIYIGTAEQYTPV, was obtained from Edman degradation and analysis. This sequence exhibited low similarity to any known protein based on an NCBI database search with BLASTP. Next, we designed degenerate primers for RACE according to the TFP N-terminal amino acid sequence. The full-length DNA sequence was determined by a combination of RT-PCR and 3′/5′-RACE PCR with degenerate oligonucleotide primers. The 3′-RACE primer was designed by selecting the codon with the highest frequency based on the NCBI codon usage table of the 1529

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Figure 3. TFP is a homodimer protein. (A) Western blotting using an mAb against recombinant His-tagged TFP (lane 1), the enterokinase-digested product of His-tagged TFP (lane 2), and native TFP (lane 3). (B) The MALDI-TOF MS spectrum obtained from a purified TFP sample indicated a protein mass/charge ratio of between 10 400 and 12 000. The TFP peak is marked.

Figure 4. Effect of TFP on cytokine production and activation in mouse peritoneal macrophages. (A−C) Peritoneal macrophages freshly prepared from BALB/c mice were cultured in the presence of TFP at the indicated concentrations for 24 h. TFP induced (A) TNF-α, (B) IL-1β, and (C) IL12p70 production. (D−F) Peritoneal macrophages stimulated with TFP (6 μg/mL) or LPS (1 μg/mL) for 24 h were analyzed, and the TFPstimulated cells displayed enhanced (D) phagocytosis activity, (E) CD86 expression, (F) and MHC class II expression. The shaded areas indicate unstained controls, the green dotted lines indicate cells without stimulation, the thin blue solid lines indicate LPS-stimulated cells, and the thick red lines indicate TFP-stimulated cells.

TFP, the protein was analyzed with MALDI-TOF mass spectrometry. As shown in Figure 3B, TFP had a molecular mass of 11 033 Da, which is approximately the same as the predicted molecular mass from the TFP gene analysis. In addition, in the presence of a high percentage of βmercaptoethanol, the mobility of TFP in SDS-PAGE still equaled that of a 24 kDa protein in the SDS-PAGE (Supplemental Figure 4). According to these results, we assume that the TFP dimer results not from disulfide bonds but rather from other protein interactions. Furthermore, we

recombinant TFP (rTFP) and its mass spectrum. To produce rTFP, the cDNA sequence encoding TFP from Asp25 to the stop codon, containing 110 amino acids, was cloned into a pET-30a(+) vector and then transformed into the bacteria host E. coli BL21 with the constructed plasmid. Interestingly, transformant-expressed rTFP exhibited the mobility of a 24 kDa protein even though the inserted gene was predicted to translate an 11 kDa protein (Figure 3A), implying that TFP from the fruiting body of T. fuciformis could natively form a homodimer. Furthermore, to establish the molecular mass of 1530

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Figure 5. Effect of TFP on NF-κB activity in peritoneal macrophages. TFP-induced nuclear NF-κB DNA binding in mouse peritoneal macrophages occurred in a dose- and time-dependent manner. Nuclear NF-κB activity was determined by EMSA after the binding of nuclear extract to the labeled oligonucleotide corresponding to the NF-κB binding site. The biotin-labeled NF-κB DNA binding complex was visualized using chemiluminescence detection. (A) TFP-induced NF-κB DNA binding varied with the treatment concentration. The cells were cultured with the indicated concentration of TFP for 1.5 h, collected for nuclear protein extraction, and analyzed with EMSA. (B) The results of the densitometry analyses were compared with that for untreated cells (0 μg/mL TFP). (C) TFP initiated nuclear NF-κB DNA binding after 1 h of TFP treatment. The cells were treated by TFP (6.25 μg/mL) for the indicated times. (D) The results of the densitometry analyses were compared with that for the untreated time point (0 h).

used the COTH software,15 a web service for protein complex structure prediction, to identify the complex structure of TFP from its primary amino acid sequence (Supplemental Figure 5). Taken together, these data suggest that the observed TFP is an 11 kDa protein and that it mostly forms natural homodimers. TFP Activates the NF-κB Pathway in Mouse Peritoneal Macrophages. We then investigated the influence of TFP on cytokine production in mouse peritoneal macrophages to explore the activating capability of TFP in innate immune systemic cells. The peritoneal macrophages were cultured with medium that contained TFP at different concentrations ranging from 1.5 to 100 μg/mL. The supernatants were harvested to analyze the production of TNF-α, IL-1β, and IL-12p70. The macrophages cultured in the presence of TFP displayed dosedependent upregulation of TNF-α, IL-1β, and IL-12p70 (Figure 4A−C). In addition, we investigated the effects of TFP on the phagocytic activity and activation marker expression in macrophages with flow cytometry. In the phagocytic activity assay, TFP significantly enhanced the phagocytosis of mouse peritoneal macrophages (Figure 4D). The MFI value of the TFP-induced cells (91.72; red thick line) was significantly higher (P < 0.05) than that of the control cells (51.25; green dotted line). Similarly, the cells cultured in the presence of TFP expressed more CD86 and MHC class II molecules than the control cells (Figure 4E,F). However, TFP treatment did not induce CD80 expression in the macrophages (Supplemental Figure 6). These results suggest that TFP can activate the phagocytic and antigen-presentation capabilities of mouse macrophage cells. NF-κB, one of the most essential transcription factors in the generation of host defense and the innate immune system, has an important role in the induction of TNF-α and IL-12p40 gene expression in macrophages.16 In the present study, we

investigated whether TFP induces the activation of NF-κB, resulting in upregulation of the production of the previously described cytokines. As shown in Figure 5A,B, TFP directly induced an increase of NF-κB in the cell nucleus, and stimulation with different concentrations of TFP could enhance the NF-κB activation. In addition, the activation of NF-κB displayed a time-dependent pattern with the TFP treatment (Figure 5C,D). These results reveal that TFP can induce NFκB activation and further modulate cellular immune responses. TFP Induces M1-Type Polarization in Mouse Peritoneal Macrophages. Polarization is an important method of differentiation of function and phenotype in macrophages and has been broadly classified into types M1 and M2 on the basis of functional property differences.17 Given the high levels of pro-inflammatory cytokine expression in TFP-induced mouse peritoneal macrophages, we examined some of the known markers of activated M1 macrophages.18,19 We performed realtime PCR assays to determine the cytokine gene expression in macrophages that were treated with 6.25 μg/mL TFP for 8 h. As shown in Figure 6, TFP treatment increased the mRNA expression of TNF-α, IL-1β, IL-6, and IL-12p35, all of which are M1-type cytokines, but not IL-10, which is an M2-type cytokine. In addition, we observed that TFP-induced cells displayed enhanced mRNA expression of M1-type chemokines, including CCL3 and CXCL10 (Figure 7A,D). The CCL4 and CCL5 expression levels were unaffected (Figure 7B,C). However, the mRNA expression levels of CCL17 and CCL24, which are M2-type chemokines, were not changed during 8 h of 6.25 μg/mL TFP treatment (Figure 7E,F). These results indicate that TFP might upregulate the expression of M1-type cytokines and chemokine mRNA expression and therefore may induce M1 polarization of mouse macrophages. 1531

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Figure 6. Effect of TFP on cytokine gene expression in peritoneal macrophages. A time course evaluation of cytokine mRNA expression in TFPinduced macrophages is shown. The cells were cultured in the presence of TFP (6.25 μg/mL) for the indicated times and collected for total RNA extraction. The expression of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) IL-12p40, (E) IL-12p35, and (F) IL-10 mRNA was analyzed with quantitative real-time PCR. The mRNA expression level in each case was calculated on the basis of the Ct value and is shown as the ratio of the product to βactin expression level. The relative mRNA expression of each time event is presented as the fold change relative to the 0 h time point. Asterisks indicate that the mRNA expression was 4-fold higher than that of the 0 h control.

TLR4 Is Associated with the Stimulation of TFP in Macrophages. The activation of TLR4 is believed to promote a Th1 response and induce the production of Th1-associated cytokines, specifically IL-12p70.20 Therefore, we investigated whether TFP promotes activation through TLR4 in mouse peritoneal macrophages. Preliminary experiments were performed by using the anti-mouse TLR4 mAb to neutralize the TLR4-related activation in the cells. The TFP-induced TNF-α production was inhibited in the TLR4-neutralized cells (Figure 8A). To further confirm this result, we measured the TNF-α production in TFP-treated macrophages from TLR4-deficient mice (TLR4−/−, C57BL/10ScN). As expected, the TNF-α secretion in wild-type (WT) macrophages was not observed in TLR4−/− cells (Figure 8B). On the basis of these results, we concluded that TLR4 is involved in TFP-mediated activation in mouse peritoneal macrophages.

polypeptide through O- and N-glycosylation according to the predictions using NetNGlyc 1.0 and DictyOGlyc 1.1 software (Supplemental Figures 7 and 8). Moreover, we found that TFP exists as a homodimeric molecule in the experiments. Dimerization of proteins could be related to many biological functions and could be a key process in the control a variety of cellular actions.21 For the previously mentioned proteins, the formation of the homodimer through the N-terminal α-A-helix is necessary for recognition by their receptors on the cell surface and their immunomodulatory activities.22−24 The method of Garnier25 was used to predict the secondary structure of TFP, and the TFP was determined not to contain the N-terminal α-A-helix structure (Supplemental Figure 9), which differed from fungal immunomodulatory proteins (FIPs). Many proteins that have been purified from edible mushrooms have been classified as lectins and FIPs on the basis of their biochemical activities and conserved amino acid sequences.23,24,26,27 Fip-vvo was classed as a lectin because of its hemagglutination and cell proliferation activity.24 Lectins, known as carbohydrate-binding proteins, can agglutinate red blood cells and can recognize a wide variety of pathogens, and they consequently play an important role in innate immunity.9,27 As shown in Supplemental Figure 10, the purified TFP (0.03125−1 mg/mL) lacked the ability to agglutinate mouse red blood cells. Moreover, the amino acid sequence of TFP exhibited low homology to any known FIP that has been classified as lectin (Supplemental Figure 11). Taken together,

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DISCUSSION In this work, we first identified that TFP, an immune-activating protein from T. fuciformis, is capable of enhancing Th1 cytokine secretion, phagocytic activity, co-stimulatory molecule expression, and M1 polarization of macrophages via the TLR4 signaling pathway. The cloned TFP cDNA sequence was dissimilar to those of other fungi or any other species (Supplemental Table 2). After biochemical and genetic analysis, we demonstrated that TFP consists of 112 amino acid residues (Figure 2) and lacks a glycosylation in T. fuciformis (Figure 1). In addition, TFP might lack a carbohydrate moiety linking the 1532

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Figure 7. Time course evaluation of chemokine mRNA expression in TFP-induced macrophages. The cells were cultured in the presence of TFP (6.25 μg/mL) for the indicated times and collected for total RNA extraction. The expression of (A) CCL3, (B) CCL4, (C) CCL5, (D) CXCL10, (E) CCL17, and (F) CCL24 mRNA was analyzed with quantitative real-time PCR. The mRNA expression level in each case was calculated on the basis of the Ct value and is shown as the ratio of the product to the β-actin expression level. The relative mRNA expression of each time event is presented as the fold change relative to the 0 h time point. Asterisks indicate that the mRNA expression was 4-fold higher than that of the 0 h control.

these results show that TFP is different from those FIPs in regard to amino acid composition and biological activity. As shown in Supplemental Table 2, the two known proteins identified in mushroom-forming fungi that are most similar to TFP, according to the NCBI nr database, are the noncatalytic module family EXPN protein (expressed by the wooddegrading fungus Schizophyllum commune)28 and an expansin family protein (secreted by the ectomycorrhizal fungus Laccaria bicolor).29 The noncatalytic module family EXPN proteins with high similarity (max identity >50%) from S. commune and L. bicolor are both classified as expansin superfamily proteins.28,29 Expansins, the plant cell-wall-loosening proteins involved in plant cell growth processes, are not only found in plants but also secreted by mycorrhizal fungi, which develop symbiotic tissues in plant root tissues by using expansins to induce extension in the cell wall of roots.30 As T. fuciformis is a wooddegrading fungus, the role of TFP may be associated with wood digestion and fungi development. As shown in Supplemental Figure 12, one of the TFP-similar proteins (max identity 37%) that is distinct from the expansins was found in L. bicolor, a riboflavin-aldehyde-forming enzyme that functions as an upregulator of the expanding sporophores.31 It is possible that TFP plays an important role in the growth and development of T. fuciformis. Nevertheless, T. fuciformis genomics data remain sparse, and the physiological function of TFP in T. fuciformis remains unclear and requires further investigation. TFP directly activates macrophages accompanying the enhanced macrophage immune functions in the innate immune responses as an immune modulator. It is well-known that

Figure 8. TFP-mediated activation of mouse peritoneal macrophages involves TLR4. (A) WT macrophages were pretreated with control (isotype) or anti-TLR4 mAb 30 min before incubation with 1 μg/mL ultrapurified LPS or 6.25 μg/mL TFP for 18 h. (B) TLR4−/− macrophages were incubated with 1 μg/mL ultrapurified LPS or 6.25 μg/mL TFP for 24 h. The supernatants were collected to determine the TNF-α production by macrophages using ELISA. **, P < 0.01 vs the isotype control.

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In conclusion, TFP is a novel immune-activating protein and may be an important bioactive molecule from T. fuciformis. The current study is the first to provide evidence that TFP activates mouse macrophages (promoting M1-type polarization) via the TLR4-NF-κB pathway and might promote a further Th1 response. This research has revealed the potential for the use of TFP in the development of health foods and pharmaceutical products.

macrophages rely on phagocytosis to eliminate exogenous pathogens and the production of cytokines to further improve the differentiation of T cells in adaptive immunity.32−34 We demonstrated that TFP-activated macrophages display an increased phagocytosis ability and secrete a high level of TNF-α, IL-1β, and IL-12. TNF-α can promote the M1 polarization of macrophages, which leads to the tissue macrophages giving rise to classically activated macrophages. Subsequently, those macrophages produced more IL-12, which enhanced Th1 cell development.32,33 Furthermore, we observed that TFP can induce the expression of M1 chemokines such as CCL3, CCL4, and CXCL10 but not M2 chemokines such as CCL17 and CCL24. These results suggest that TFP-mediated macrophage activation is associated with M1 polarization, which could further upregulate the Th1-response-integrated macrophages that are involved in type I inflammation, resulting in the killing of intracellular microorganisms and tumor resistance.19 The TFP-activated macrophages exposed to TFP increased the expression of CD86, which is a well-known activation marker of macrophages and a considerable T cell costimulatory molecule involved in proliferation, cytokine production, and cytotoxic T lymphocyte generation during adaptive immune responses.35 Therefore, we theorize that TFP might be a potent macrophage activator that drives the activation and M1 polarization of macrophages to promote innate immune responses. TLRs are critical for the recognition of pathogen components and play important roles in eliciting innate immune responses, especially TLR4.16,19 TLR4 has been reported to be capable of recognizing several pathogen molecules, including LPS from bacteria and mycobacteria, glycoinositolphospholipids from parasites, mannan from fungus, and structural proteins from viruses.16 Using an antiTLR4 mAb neutralization experiment and TLR4-deficient mice (TLR4−/−), we demonstrated that TLR4 could be a major TFP-recognition receptor for macrophages and that blocking TLR4 causes macrophages to decrease TNF-α secretion. In addition, it is known that MyD88 and TIR-domain-containing adaptor protein (TIRAP) play necessary roles in the production of inflammatory cytokines such as TNF-α and IL-12p40 after the stimulation of TLR4.10,36 Through the TLR4-mediated MyD88-dependent signaling pathway, recruited IRAK activates TRAF6 to trigger the activation of the IκB kinase (IKK) complex, leading to the translocation of NF-κB and expression of inflammatory cytokines.10,36 We observed that TFPmediated macrophage activation is related to the activation of NF-κB. This observation suggests that TFP regulates macrophage activation via the TLR4-NF-κB signaling pathway. Furthermore, NF-κB regulates the transcriptional program of inflammatory-response-related gene expression as the mediator of innate responses and promotes specific gene expression involving the recruitment and survival of leukocytes as an initiator of adaptive immune responses.37,38 It was demonstrated previously that TFP induces the production of IL-12p70 and the expression of IL-12p35 and IL-12p40, which have been reported as bridges between the innate and adaptive immune responses.37 It has also been reported that IL-12 has the ability to induce the production of IFN-γ, the differentiation of Th1 and NK cells, and the generation of cytotoxic T lymphocytes and LAK cells.38 Taken together, these data suggest that TFP might utilize the TLR4-NF-κB signaling pathway to stimulate macrophages that have the potential to enhance the adaptive immune response.

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ASSOCIATED CONTENT

S Supporting Information *

Gel filtration chromatogram of TFP; effects of TFP on the production of TNF-α in RAW264.7 macrophages; PAS glycosylation analysis of TFP; effects of TFP on the expression of CD80 in mouse peritoneal macrophages; NetNGlyc 1.0 prediction results for TFP; DictyOGlyc 1.1 prediction results for TFP; GOR secondary structure prediction for TFP; hemagglutinating activity of TFP; SDS-PAGE of TFP with different percentages of β-mercaptoethanol in the sample dye; COTH prediction results for TFP; alignment of the amino acid sequences of LZ8, Fip-fve, Fip-vvo, and TFP; alignment of the amino acid sequences of extensin family protein (L. bicolor S238N-H82) and TFP; primers used in this study; and the 26 best results obtained in the NCBI Blast searches with the nr database. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

The nucleotide sequence data reported for TFP from Tremella f uciformis have been submitted to the GenBank Nucleotide Sequence Database under accession number EF152774.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 886-2-33664846. Fax: 886-223673103. Funding

This work was supported by grants from the National Science Council (NSC), Taiwan (99-2628-B-002-003-MY3) to F.S. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED MHC, major histocompatibility complex; LPS, lipopolysaccharide; FPLC, fast protein liquid chromatography; RACE, rapid amplification of cDNA ends; RT-PCR, reverse-transcription polymerase chain reaction; TNF-α, tumor necrosis factor-α; IL, interleukin; EMSA, electrophoretic mobility shift assay; CXCL, chemokine (C-X-C motif) ligand; CCL, chemokine (C−C motif) ligand 1; Th1, T helper 1; TLR, Toll-like receptor.

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REFERENCES

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