Compound 48/80 (C48/80) was purchased from Sigma-Aldrich (St. Louis, MO, United States). The Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, United States). Recombinant TCTP proteins r-dTCTP and r-mTCTP were isolated from the BL21 (DE3) PLysS strain that was transformed with PRSET A vector of His-tagged dTCTP and mTCTP. r-dTCTP and r-mTCTP were used for cell treatment after purification using Ni-NTA agarose (QIAGEN, Hilden, Germany) and anion Exchange Chromatography with HiTrap Q Column (GE Healthcare Bio-Sciences Corp, Piscataway, NJ, United States), followed by endotoxin removal. dTBP2 peptide was synthesized at the peptide synthesizer facility PepTron Inc. (Daejeon, Korea) and the purity of the synthetic peptide was confirmed to be 98%.

Bone marrow cell suspensions were isolated by flushing femurs and tibias from C57BL/6J mice (male, 8 weeks old), and cultured in RPMI-1640 supplemented with 10% FBS, 1% penicillin-streptomycin, 50 μM 2-mercaptoethanol, and recombinant murine IL-3 (rmIL-3, 10 ng/ml, BioLegend, San Diego, CA, United States) for 4 weeks (Liao et al., 2020). The purity of BMMCs assessed by staining and flow cytometry analysis with anti-FcεRI (Invitrogen, Carlsbad, CA, United States) and anti-c-kit antibody (eBioscience, San Diego, CA, United States) confirmed that they are 95% pure. The BMMCs were cultivated between days 29 and 49 post-isolation and were used for all functional assays.

Untreated BMMCs or those pre-treated with vehicle, r-dTCTP, and r-mTCTP for 24 h were centrifuges and the cell pellets were resuspended in extracellular buffer (10 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.4 mM Na₂HPO4.7H2O, 1.4 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose, and 0.04% BSA), and treated with 50 μg/ml C48/80 for either 5 min or indicated time. Untreated sample were prepared to use as controls. The cell supernatant was harvested and subjected to β-hexosaminidase release assay using 4-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside in 0.1 M citrate buffer (pH 4.2) (PNAG, 3.4 mg/ml, TCI Chemicals, Japan). Reaction was stopped with 0.4 M glycine buffer (pH 10.8), and optical density was measured at 405 nm.

BMMCs were incubated with LPS (1 µg/ml) for 24 h and treated with C48/80 (50 µg/ml) for 5 min before harvest. Cells were fixed with 4% paraformaldehyde for 10 min at RT, permeabilized in 0.1% Triton X-100/PBS for 10 min, and incubated with antibody against lysosomal membrane-associated protein 1 (LAMP1, Santa Cruz Biotech Inc., Santa Cruz, CA, United States) and TCTP (Abcam, Cambridge, MA, United States), followed by staining with Alexa Fluor 488- or Alexa Fluor 555-coupled secondary antibodies (Invitrogen). Nuclei were stained with DAPI (1 µg/ml). Cells were observed under a confocal laser scanning microscope (ZEISS LSM 880 with Airyscan, Oberkochen, Germany). For LysoTracker staining, cells were pre-treated with dTCTP (10 µg/ml) and/or dTBP2 (100 µg/ml) and incubated with LysoTracker™ (75 nM, Thermo Fischer Scientific) for 1 h at 37°C in dark. Cells were fixed with 4% PFA and observed under a confocal microscope equipped at Drug Development Research Core Center.

BMMCs (1 × 10⁶ cells/ml) were incubated with the vehicle, r-dTCTP, or r-mTCTP in the presence or absence of dTBP2 for 24 h. The culture supernatants were collected and analyzed with commercial ELISA kits according to the manufacturer’s instructions. Histamine level in serum was measured with histamine EIA kit (Oxford biomedical research, Rochester Hills, MI, United States) and serum TNF-α and IL-6 level was measured with murine TNF-α and IL-6 ELISA kit (BioLegend).

BMMCs were incubated with vehicle, r-dTCTP, r-mTCTP in the presence or absence of dTBP2 (100 μg/ml) for 24 h, and centrifuged (Cytospin 4, Thermo Fisher Scientific) and subjected to Giemsa staining (Diff-Quik staining kit, Sysmex Co., Kobe, Japan). BMMCs were also incubated with either vehicle or dTBP2 (100 μg/ml) in the presence of C48/80 (20 μg/ml) and then applied on slide glass with Cytospin 4, and stained with Diff Quik Solution.

BMMCs were harvested and extracted with RIPA lysis buffer. Protein extracts and culture supernatants were resolved by SDS-polyacrylamide gel electrophoresis, followed by electrotransfer. Protein blots were incubated with antibody against TCTP (Abcam), Phospho-NF-κB p65, NF-kB p65, Phospho-p38, p38, Phospho-ERK, ERK (Cell signaling technology, Danvers, MA, United States), and β-actin (bs-0061R, 1:5000, Bioss, Woburn, MA, United States), and protein signal was visualized with Image Analyzer (ChemiDoc MP, Bio-Rad, Hercules, CA, United States).

BMMCs were plated into black wall, transparent bottom 96-well plates (BD, Franklin Lakes NJ, United States) and pre-treated with vehicle, LPS, r-dTCTP (10 µg/ml) or r-mTCTP (10 µg/ml) in the presence or absence of dTBP2 (100 µg/ml) for 24 h. The cells were then assayed for calcium with Fluo-4 Direct Calcium Assay Kit (Thermo Fisher Scientific), followed by measurement using Tecan infinite F200 fluorescence microplate reader (Tecan, Männedorf, Switzerland). After 30 min incubation, cells were additionally treated with C48/80 (20 µg/ml) and measured with fluorescence microplate reader for 5 min. The basal fluorescence intensity was subtracted to calculate the relative fluorescence intensity over time.

C57BL/6J mice (male, 8 weeks old) were obtained from OrientBio Inc. (Seongnam, Korea), and were housed at a temperature of 22 ± 1°C and relative humidity of 55 ± 10%. Mice (males, 8 weeks old) were injected intraperitoneally with PBS or C48/80 (8 mg/kg) freshly dissolved in PBS. Low (0.3 mg/kg) and high doses (1 mg/kg) dTBP2 or dexamethasone (DXM, 4 mg/kg) were injected into the animals 15 min before C48/80. Mortality rate was monitored for 1 h after induction of systemic anaphylactic reactions by C48/80. Blood samples were collected through heart puncture to measure the levels of histamine, TNF-α, and IL-6. All animal experiments were conducted in accordance with the international guidelines approved by Ewha Womans University’s Institutional Animal Care and Use Committee (IACUC 18-040).

All data are expressed as the mean ± SEM. Results were analyzed by one-way ANOVA or two-tailed Student’s t-test. p values less than 0.05 were considered to indicate a statistically significant difference.

Since dTBP2 has been identified as a specific inhibitor for dTCTP, which is primarily produced by mononuclear cells and released under allergic conditions, we first asked whether mast cells produce dTCTP and dTCTP modulates mast cell function. Both dTCTP and mTCTP were strongly expressed in mast cells, which were further increased by LPS stimulation (Figure 1A). Interestingly, dTCTP stably expressed as a dimer even under reducing conditions, and intracellular TCTP was rarely detected in the extracellular compartment. But dTCTP secretion was increased by treatment with C48/80 (Figure 1A). Immunofluorescence staining confirmed that intracellular TCTP expression was markedly increased by LPS, mostly co-localized with LAMP1 in the cytoplasm, and rapidly released as secretory lysosomes by C48/80 treatment (Figure 1B). In addition, the release of dTCTP into the extracellular compartment was inhibited by treatment with the lysosomal inhibitor, chloroquine (CQ) (Figure 1C). Furthermore, time-course analysis of dTCTP secretion by C48/80 treatment demonstrated a rapid release of dTCTP within 5 min after C48/80 treatment and persistent release of dTCTP during mast cell degranulation induced by C48/80 (Figure 1D). We thus purified endotoxin-free recombinant proteins r-dTCTP and r-mTCTP and investigated whether extracellular recombinant TCTP proteins affect mast cell degranulation. To prevent overwhelming effect of C48/80, BMMCs were incubated with LPS, r-dTCTP, or r-mTCTP for 24 h and briefly treated with C48/80 to induce mast cell degranulation. C48/80 treatment slightly increased β-hexosaminidase release through mast cell degranulation, and LPS dramatically increased mast cell degranulation. In addition, while r-mTCTP treatment had no effect on β-hexosaminidase release by BMMCs, r-dTCTP significantly increased mast cell degranulation (Figure 1E), indicating that dTCTP is a major mediator of LPS-induced mast cell degranulation. Furthermore, intracellular calcium mobilization triggered by C48/80 was further increased in LPS-stimulated BMMCs than in vehicle-treated cells. Likewise, r-dTCTP treatment enhanced intracellular calcium mobilization in BMMCs, whereas r-mTCTP showed similar levels compared to vehicle-treated BMMCs (Figure 1F). These results suggest that dTCTP is rapidly released by activated mast cells and further potentiates mast cell degranulation via an autocrine activation loop.

We next investigated whether dTBP2 directly attenuates potentiated degranulation of BMMCs by prolonged triggering with C48/80 in vitro. β-Hexosaminidase activity, the indication of mast cell degranulation, increased in BMMCs in a C48/80 dose-dependent manner, and pretreatment of BMMCs with dTBP2 significantly decreased mast cell degranulation potentiated by C48/80 (Figure 2A). In addition, dTBP2 dose-dependently decreased β-hexosaminidase release in BMMCs induced by C48/80 (Figure 2B). Giemsa staining also demonstrated that mast cell degranulation was increased by C48/80 but was instantly inhibited by the addition and increase of dTBP2 during mast cell degranulation (Figure 2C). Moreover, in vitro degranulation of BMMCs was time-dependently increased with prolonged incubation with C48/80, and the transient incubation with dTBP2 during mast cell degranulation without pretreatment effectively inhibited mast cell degranulation (Figure 2D). The potent inhibitory activity of dTBP2 on in vitro mast cell degranulation was also confirmed at various C48/80 concentrations (Figure 2E). These results indicate that dTBP2 directly and effectively inhibits antigen-induced degranulation of mast cells.

We next attempted to confirm the dTCTP-dependent inhibitory activity of dTBP2 and investigated its underlying molecular mechanisms. As previously confirmed, dTBP2 substantially inhibited mast cell degranulation potentiated by prolonged treatment or high concentration of C48/80. In addition, the release of β-hexosaminidase was increased by r-dTCTP and significantly decreased by dTBP2 addition in BMMCs, whereas r-mTCTP did not induce mast cell degranulation (Figure 3A). Moreover, histamine release by BMMCs was also significantly increased by r-dTCTP but not r-mTCTP, and dTCTP-induced histamine release was markedly decreased by dTBP2 treatment (Figure 3B). Extracellular r-dTCTP, not r-mTCTP, treatment increased the formation of dense purple granules within BMMCs and instantly induced mast cell degranulation. The dTCTP-induced mast cell granule formation and degranulation was impaired in the presence of dTBP2, which was evidenced by Giemsa staining (Figure 3C). Further analysis confirmed that phosphorylation of p38 MAPK was increased by r-dTCTP but not affected by r-mTCTP. dTBP2 treatment inhibited the r-dTCTP-induced p38 MAPK signaling pathway essential for mast cell degranulation, but did not affect the NF-kB signaling pathway (Figure 3D). In addition, intracellular calcium mobilization increased by r-dTCTP was decreased by treatment with dTBP2, but there was no change in r-mTCTP-treated mast cells (Figure 4E). Interestingly, expansion and acidification of lysosomal compartments essential for anaphylactic degranulation (Pejler et al., 2017) were prominently found in r-dTCTP-treated BMMCs but were consequently decreased by addition of dTBP2 (Figure 3F). Our results indicate that the anti-anaphylactic activity of dTBP2 is due to the specific inhibition of p38 MAPK activation and calcium mobilization in mast cells activated by autocrine dTCTP signaling.

To evaluate the in vivo anti-anaphylactic effect of dTBP2, an animal model for systemic anaphylaxis was established by intraperitoneal injection of C48/80 into mice and additionally administered low-dose and high-dose dTBP2 through intravenous injection. Most mice died within 1 h after C48/80 injection, and mortality reached 80%. Low-dose dTBP2 delayed morality but had no effect on C48/80-induced mortality, whereas high-dose dTBP2 significantly delayed and improved mortality (Figure 4A). DXM injection reduced C48/80-induced mortality, which was similar to the effect of high-dose dTBP2. Histamine release induced by C48/80 injection was decreased in the high-dose dTBP2 injection group as in the DXM-treated group, whereas there was no significant change in histamine release in the low-dose dTBP2 injection group (Figure 4B). Consistently, serum levels of pro-inflammatory cytokines TNF-α and IL-6 were elevated in C48/80-induced anaphylaxis mouse models but were significantly reduced by treatment with DXM. Both low-dose and high-dose dTBP2 also markedly decreased the production of pro-inflammatory cytokines (Figures 4C,D). These results indicate that dTBP2 is effective in inhibiting the C48/80-induced anaphylactic reactions in vivo.