Flammulina velutipes was provided by Shanghai Senyuan biology, China. Losmapimod (purity ≥ 98%) was obtained from Shanghai Beyotime Biotechnology (China). Gossypetin (purity ≥ 99%) was purchased from Shanghai ZhenZhun Biotechnology (China). Acetic acid, ammonium sulfate, NaCl, acetonitrile, Tris, and HCl were provided by Sinopharm Chemical Reagent (China). Coomassie Brilliant Blue R250 was obtained from Shanghai yuanye Bio-Technology (China). Cell lysis buffer (N8031) was obtained from Solarbio (China). DMSO, phytohemagglutinin (PHA), protease inhibitor cocktail (P8340), phosphatase inhibitor cocktail 2 (P5726), and phosphatase inhibitor cocktail 3 (Sigma-Aldrich) were obtained from Sigma (USA).

FIP-fve extraction and purification were performed according to a reported method (1). Briefly, 500 g of the fruiting bodies of Flammulina velutipes were homogenized with 500 ml ice-cold 5% acetic acid solution with an ultrasonic processor (FS-N, Shanghai Ultrasonic Instrument, China), followed by sonication for 90 min (600 W, 3 S, 6 S; 90 min in total) in an ice bath. The soluble proteins in the supernatant were precipitated by the addition of ammonium sulfate to 95% saturated. After centrifugation at 2,000 rpm for 5 min, the precipitate was re-suspended in 10 m Mof Tris-HCl buffer (pH 8.0) and dialyzed against 1 L of Tris-HCl buffer at 4°C for 72 h with five times change of dialysis solution (total of 5 L of buffer). After that, the dialysate was applied to a HiTrapQ-HP column (GE, USA), which was previously equilibrated with 10 mM Tris/HCl at pH 8.0. The column was eluted and purified with a gradient of 0–40% 10 mM Tris/HCl at pH 8.0 and 1 M NaCl (on the chromatograph). The active fractions were collected according to the ultraviolet value and further purified on a Superdex75 column (GE, USA). The Superdex 75 column was previously equilibrated with 20 mM Tris/HCl at pH 8.0 containing 150 mM NaCl. Elution was performed with 20 mM Tris-HCl at pH 8.0 containing 150 mM NaCl. The collected fractions were checked by sodium dodecyl sulfate polyacrylamide Gel Electrophoresis (SDS-PAGE) and a BCA assay. Similar fractions were combined. Then a total of 19.3 mg of purified FIP-fve was obtained (2.14 mg/ml). The molecular weight of purified FIP-fve was measured by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (5800 MALDI-TOF/TOF, AB Sciex, USA) in a positive ion mode, with the calibration ranges of the linear medium molecular weight calibration materials being 5737.609 + 50, 12,362 + 50, and 16,952 + 50 (Sigma, USA).

The purification of FIP-fve was performed according to a previous study (20). Briefly, cellular proteins (15 μg) from each sample were separated by 12% SDS-PAGE (Mini-Protean TGX™ Gels, 10-wellcomb/Bio-Rad). The separation gel was then stained with Coomassie Brilliant Blue R250 for 12 h. After staining, the gel was washed with a destaining solution(10% acetic acid and 25% anhydrous ethanol)until the bands were visualized. Finally, the stained gel was scanned by Image Scanner (GE Healthcare, USA) at a resolution of 300 dpi (full-color mode).

Jurkat E6-1 cells were obtained from the American Type Culture Collection (ATCC). The cells were cultured in RPMI 1640 medium (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, USA) at 37°C under a 5% CO₂. Cell viability was examined using the Cell Counting Kit (CCK-8) (Dojindo, Japan) according to previous studies (21–23). Briefly, cells with a density of 1.0 × 10⁴ cells per well were seeded in 96-well microtiter plates overnight. After incubated with vehicle (PBS) or FIP-fve at concentrations (25, 50, 100, or 200 μg/ml), the culture medium was discarded to remove the FIP-fve. After that, the cells were incubated with 100 μl of CCK-8 solution (10%) in a culture medium for about 1 h. PHA (5 μg/ml) was used as a positive control. The absorbance of the mixture culture medium was measured at 450 nm using a microplate reader (Biotek, USA).

Jurkat E6-1 cells with a density of 1.0 × 10⁴ cells per well were seeded in 96-well microtiter plates overnight. The cells were then treated with vehicle (PBS), FIP-fve at various concentrations (25, 50, 100, or 200 μg/ml), or PHA (1.5 or 5.0 μg/ml, positive control) for 6 h. After that, the supernatant was collected, and the amount of IL-2 in the supernatant was measured using an ELISA kit for human IL-2 (USCN Life Science Inc, China). Absorbance was measured at 450 nm with a Bio-Rad 3550 Microplate Reader (Hercules, CA, USA).

After being treated with FIP-fve for 24 h, Jurkat E6-1 cells were lysed in cell lysis buffer (Solarbio, China) supplemented with protease inhibitor cocktail (Sigma, USA), phosphatase inhibitor cocktail 2 (Sigma, USA), and phosphatase inhibitor cocktail 3 (Sigma, USA) by ultrasonication on an ice bath for 3 min. The protein concentration was measured using the BCA Protein Assay kit (Shanghai EpiZyme Biotechnology, China). The protein was enzymolyzed and labeled with a TMT reagent according to FASP (24) before being sent to Shanghai Luming Biotechnology Co., Ltd. China for Tandem Mass Tag™ (TMT™) proteomic analysis.

Reverse-phase (RP) separation was performed on an 1100 HPLC System (Agilent, USA) equipped with an Agilent Zorbax Extend RP column (5 μm, 150 × 2.1 mm). The mobile phase was composed of 2% acetonitrile in water (solvent A) and 98% acetonitrile in water (solvent B). The flow rate was 0.3 mL/min in the following gradients: 8 min, 98%A; 8.01 min, 95%A; 28 min, 75%A; 50 min, 60%A; 50.01 min, 10%A; 60 min, 10%A; 60.01 min, 98%A; and 65 min, 98%A. The injection volume was 1 μl. PDA was set at 210 and 280 nm. The samples were harvested from 8 to 50 min, and elution buffer was collected in 10 centrifuge tubes every minute and was numbered. The separated peptides were lyophilized for MS detection.

All analyses were performed by a Q Exactive mass spectrometer (Thermo, USA) equipped with a Nanospray Flex source (Thermo, USA). The samples were loaded and separated by a C-18 column (75 μm × 150 nm, 2 μm, 100 A, Dionex, USA) on an EASYnLC™1200 system (Thermo Fisher Scientific, United States). The flow rate was 300 nl/min, and the linear gradient lasted 90 min (0–55 min, 8%B; 55–79 min, 30%B; 79–80 min, 50% B; 80–90 min, 100% B; mobile phase A = 0.1% FA in water; mobile phase B = 80% ACN/0.1% FA in water). Full MS scans were acquired in the mass range of 300–1,600 m/z with a mass resolution of 70,000, and the AGC target value was set at 1e6. The ten most intense MS peaks were fragmented with higher-energy collisional dissociation (HCD) with an NCE of 32. MS/MS spectra were obtained with a resolution of 17,500 with an AGC target of 2e5 and a max infection time of 8 ms. The Q-E dynamic exclusion was set at 30.0 s and run under positive mode.

Database Search Proteome Discoverer (v 2.2) was used to thoroughly search all of the Q Exactive raw data against the sample protein database. Database searches were performed with trypsin digestion specificity. Cysteine alkylation was considered a fixed modification in the database search. For the protein quantification method, TMT6-plex was selected. The global false discovery rate (FDR) was set to 0.01, and the protein groups considered for quantification required at least 2 peptides.

Xcalibur 2.1 data processing software was used for FIP-fve characteristic mass spectrometry identification. Proteome Discoverer TM 2.2 software was used for cell proteomics experimental data analysis, and the UniProt human database was used. The false-positive rate of peptide identification was less than 1%.

All experimental data were the average of at least three independent experiments. Differences between experimental groups were calculated using the Student’s paired t-test. A value of p < 0.05 was considered statistically significant.

The crude extract yielded two distinct protein bands with apparent molecular masses of 13 and 30 kDa in SDS/PAGE (Supplementary Figure 1A). Then, the crude extract was purified with a HiTrap Q-HP column, and the active FIP-fve (13 kDa) (1) obtained from the crude extract was further purified with a Superdex 75 column. The purified FIP-fve identified by an LC, yielded a single band with a molecular mass of 13 kDa (Supplementary Figures 1B,C) as determined by the SDS/PAGE assay. A total of 19.3 mg of purified FIP-fve with a concentration of 2.14 mg/ml was obtained as determined by the BCA method. Moreover, the molecular weight of the purified FIP-fve was 12,737.28 Da as determined by the 5800 MALDI-TOF/TOF (Figure 1).

To evaluate the immunomodulation activity of FIP-fve, we next determined the effect of FIP-fve on cell proliferation and IL-2 release in Jurkat E6-1 cells as the binding of IL-2 to its receptor initiates a series of events that result in cell-cycle progression and T cell proliferation (25). Moreover, the Jurkat E6-1 cells activated by exogenous lectin (such as PHA) can produce a large amount of IL-2. And PHA was used as a positive control (5). The proliferation of Jurkat E6-1 cells was evaluated by CCK-8 kit after 12 and 24 h treatment with control, PHA (5 μg/ml), and FIP-fve (100 μg/ml). Although weaker than PHA, FIP-fve was found to stimulate Jurkat E6-1 cells proliferation significantly (Figure 2). The results were consistent with a previous study (5) where FIP-fve was found to be capable of promoting PBMCs proliferation through enhancing the G₁ to S phase progression of lymhocytes.

The in vitro immunomodulatory activity of FIP-fve was investigated by examining IL-2 release in Jurkat E6-1 cells. Jurkat E6-1 cells were cultured with different concentrations of FIP-fve ranging from 35.32 to 766.02 pg/ml for 6 h, and IL-2 production was measured by a human IL-2 ELISA kit. As shown in Figure 3, FIP-fve enhanced IL-2 release in a dose-dependent manner. For example, the levels of IL-2 released in the culture medium of cells treated with FIP-fve at a concentration of 25, 50, 100, and 200 μg/ml were 35.32, 106.88, 321.59, and 766.02 pg/ml, respectively. Moreover, the levels of IL-2 released from the FIP-fve (200 μg/ml) treated cells were almost reachedthat of PHA (5 μg/ml) treated cells (766.02 vs. 798.97 pg/ml). These results indicated that FIP-fve could induce T cell immune activation in Jurkat E6-1 cells.

The above observations confirmed that FIP-fve stimulates cell proliferation and enhances IL-2 release in Jurkat E6-1 cells. Furthermore, unbiased high-throughput proteomics was conducted to explore the mechanism of action of FIP-fve in Jurkat E6-1 cells. As shown in Figure 4A, the principal component analysis (PCA) score chart could distinguish the FIP-fve group from the control group, indicating that there were differences between the two groups. The samples labeled by enzymatic hydrolysis were analyzed by liquid chromatography-mass spectrometry, and the database was searched. After the blank value was removed, a total of 5,668 credible proteins were detected according to the criteria of score sequence HT > 0 and unique peptide ≥ 1, in which 290 differentially expressed proteins were identified according to FC > 1.5 or FC < 2/3 (fold change, FC) and p < 0.05 by the t-test (Figure 4B). Based on 290 differentially expressed proteins data, Gene Ontology (GO) molecular function (MF) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted by Proteome Discoverer TM 2.2 software. As shown in Figure 4C, the main upregulated GO MF were related to protein binding, poly (A) RNA binding, and threonine-type peptidase activity, indicating that FIP-fve treatment increased proteins and nucleic acid anabolism in Jurkat E6-1 cells. Moreover, KEGG analysis showed that FIP-fve treatment upregulated signal pathways involved in amino acid biosynthesis, carbon metabolism, proteasome, and glycolysis/glycogenesis (Figure 4D). Glycolysis, providing ATP for cell protein and nucleic acid anabolism, is considered to be the main metabolic pathway required for T cell activation. Amino acid synthesis is necessary for protein synthesis, nucleic acid synthesis, regulation of the mtroc1 pathway, and T cell stress pathway. Collectively, GO and KEGG pathway analysis indicated that FIP-fve treatment activated pathways related to cell proliferation, further confirming that FIP-fve stimulates cell proliferation in Jurkat E6-1 cells.

Among the 290 differentially expressed proteins, 27 proteins were unregulated, including 4 T cell immune activation markers (ZAP-70, CD69, CD82, and KIF23) (10–12, 26–30) (Table 1). In combination with the enhanced IL-2 release, FIP-fve was further confirmed to induce T cell immune activation. Moreover, the unregulated 27 proteins include 13 MAPK-pathway-related signaling mediators (Table 1). As shown in Table 1, the expression of MAP2K3 in Jurkat E6-1 cells treated with FIP-fve was 1.5 times than that of control. The expression of p38α/MAPK14 was significantly unregulated. The expression of ELK1, AATF, FOS, and JUN-B, which were involved in the nuclear transcription process of the p38 kinase pathway, was also unregulated. Since the MAPK pathway has been reported to be one of the important pathways for T cell activation in the immune response and p38 kinase, the downstream effector of MAPK is considered to be an important kinase for the activation and differentiation of T cells (31), FIP-fve might induce immune activation through MAP2K3/p38α (MAPK14) signaling pathway in Jurkat E6-1 cells. In addition, it was also found that the expression of 5 JAK-STAT-pathway related signaling mediators (JAK3, STAT1, STAT3, STAT5B, and STAT6) was unregulated in response to FIP-fve (50 μg/ml) treatment (Table 1). As JAK/STAT pathways are conventionally involved in the MAPK pathway in the regulation of T cells, JAK3/STAT signaling pathway may be also involved in the immune regulation activity of FIP-fve in Jurkat E6-1 cells.

Gossypetin (a MAP2K3 inhibitor) and losmapimod (a p38α inhibitor) were then used to study the involvement of the MAP2K3/p38α pathway in FIP-fve-induced immune activation in Jurkat E6-1 cells. As shown in Figure 5, 0.1 μM losmapimod and 40 μM gossypetin reduced cells growth stimulated by FIP-fve in Jurkat E6-1 cells by 64 and 71%, respectively (Figure 5A). Moreover, losmapimod and gossypetin attenuated FIP-fve-enhanced IL-2 released in Jurkat E6-1 cells by 83 and 90%, respectively (Figure 5B). These data in combination with results obtained from proteomics analysis indicate that FIP-fve may promote cell proliferation and IL-2 release by activating MAP2K3/p38α signaling pathway in Jurkat E6-1cells.