T-5224 (PubChem CID: 23626877) was purchased from TargetMol (Shanghai, China). Monoclonal DNP-specific IgE, DNP-HSA, and 4-nitrophenyl N-acetyl-β-D-glucosaminide were obtained from Sigma-Aldrich (St. Louis, MO). Aluminum adjuvant was purchased from Thermo Scientific (Waltham, MA). Evans blue, ketotifen fumarate (PubChem CID: 5282408), formamide and toluidine blue were purchased from Meilun Biotechnology Co., Ltd (Dalian, China).
Female Balb/c mouse pups were used in accordance with ethical protocols. Female Balb/c mice (6–8 wks, 18–20 g) from Guangdong Medical Laboratory Animal Center (Benxi, Liaoning, China) were bred in an aseptic-specified pathogen-free environment with controlled humidity (55 ± 10%) and temperature (24 ± 1°C). Animal experiments were carried out according to protocols approved by our University’s Animal Care and Use Committee and in compliance with the Guidelines on Animal Welfare of the School of Medicine of Shenzhen University. Mouse health was monitored twice daily, and early humane euthanasia was conducted in cases of weight loss > 15% body weight, lethargy, inability to stand, or anorexia.
Rat basophilic leukemia cells (RBLs; RBL-2H3 cell line from Cellcook Biotechnology, Guangzhou, China) were cultured in complete Dulbecco’s modified Eagle medium with 4.0 mM L-glutamine, sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, non-essential amino acid (Solarbio, Beijing, China) and 10% fetal bovine serum (Gibco, Grand Island, NY) in a humidified incubator at 37°C, 5% CO2.
Preparation of BMMCs
BMMCs were isolated from 6–8-week-old female Balb/c mouse femurs. The animals were euthanized with CO2 and submerged in 70% ethanol; then intact femurs were removed. Sterile endotoxin-free medium was flushed through bone shafts repeatedly with a syringe and needle. A suspension of collected bone marrow cells was centrifuged (300 ×g, 5 min). The spun-down cells were cultured (0.5 × 106 nucleated cells/ml) in RPMI 1640 media supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), sodium pyruvate (10 mM), l-glutamine (2 mM), 4-(2-hydroxyethyl)-piperazine ethane sulfonic acid (10 mM), and recombinant stem cell factor and IL-3 (10 ng/ml each). After ~ 5 weeks in culture, BMMC purity was ~ 95%, as determined by flow cytometry of cell-surface CD117 and FcεRI.
Cell viability was evaluated with a Cell Counting Kit 8 assay kit (MedChem Express, Monmouth Junction, NJ). RBLs (2 × 103 /well) and BMMCs (1 × 104 /well) were each cultured in 96-well plates and incubated with T-5224 for 24 h. Kit-provided solution was added wells and incubated at 37°C for 1 h. Absorbance at 450 nm was detected by a multi-well plate reader (Bio-Rad, Hercules, CA). Viability was calculated as relative absorbance expressed as a percentage of control values.
β-hexosaminidase release served as a MC degranulation index. Dinitrophenol (DNP)-targeting IgE (50 ng/mL)-sensitized RBLs or BMMCs were pretreated with T-5224 for 1 h. After two phosphate buffered saline (PBS) washes, cells were stimulated with DNP-HSA (100 ng/ml) for 30 min at 37°C. Supernatant aliquots (50 µL) were transferred to 96-well plates and incubated with an equal volume of substrate solution (1 mM 4-nitrophenyl-N-acetyl-β-d-glucosaminide) for 90 min at 37°C. To obtain total β-hexosaminidase release, cells in control wells were lysed with 0.1% Triton X-100 before plate reading. The reaction was terminated with 150 µl of stop solution (0.1 M Na2CO3 and NaHCO3). Absorbance at 405 nm was detected by a multi-well plate reader (Bio-Rad, Hercules, CA).
RNA-seq was performed with a BGISEQ-500 sequencer (Beijing Genomics Institute, Beijing, China). Total RNA was extracted from RBLs with Trizol. The sequencing data were filtered with SOAPnuke (v1.5.2) by removing reads that contained the sequencing adapter or had a low-quality (< 5) base ratio > 20%. The remaining clean reads were stored in FASTQ format and mapped to the reference genome using HISAT2 (v2.0.4). The reads were aligned in RSEM (v.1.2.12) with Bowtie (v2.2.5). Differential expression was analyzed with DESeq2 (v1.4.5), and gene expression heatmaps were drawn in pheatmap (v1.0.8). Our RNA-seq data were deposited in GEO (accession GSE161317).
Total cellular RNA was isolated with an RNeasy Mini Kit (Qiagen, Duesseldorf, Germany) and reverse transcribed into cDNAs with a TIAN Script II RT Kit (Tiangen Biotech, Beijing, China) and primers (Figure Table 1) according to the manufacturer’s instructions. Real-time qRT-PCRs were performed with TB Green® Premix ExTaq™ (Takara, Tokyo, Japan) in a qTOWER 2.2 system (Analytik Jena, Upland, CA). Expression levels were computed by normalizing relative to GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA levels with the 2−ΔΔCt technique. The primers were shown in Table S1.
Cell lysate preparation and immunoblotting were performed as previously described . Anti-DNP IgE (50 ng/ml)-sensitized RBLs (5 × 105/well in 6-well plates) were pretreated with T-5224 for 1 h and then stimulated with DNP-HSA for 4 h (p–c-Fos FRA1, and EGR1) or 30 min (others). The cells were washed with PBS twice and lysed with 200 µl RIPA buffer containing protease inhibitor cocktail (MedChem Express, Monmouth Junction, NJ). Lysed samples were cooled on ice for 15 min and centrifuged at 12,000 rpm for 10 min at 4°C. Protein concentrations were measured with a BCA kit (Beyotime, Beijing, China). Equal amounts of lysate were separated by 10% sodium dodecyl sulfate–polyacrylamide-gel electrophoresis and transferred to polyvinylidene difluoride membranes.
Membranes were incubated with primary antibodies at 4°C, and then incubated with horse radish peroxidase-conjugated anti-rabbit antibody for 1 h at room temperature. The following rabbit antibodies were purchased from Cell Signaling Technology (Danvers, MA) and applied at 1:1000 dilutions: p-p44/42 MAPK (ERK1/2) (#4370, Thr202/Thr204, monoclonal); p44/42 MAPK (ERK1/2) (#4695, monoclonal); JNK (#9252, polyclonal); p-JNK (Thr183/Tyr185) (#4668, monoclonal); p38 (#8690, monoclonal); p-p38 (#4511, monoclonal). The following rabbit monoclonal antibodies were purchased from Abcam (Cambridge, MA, USA): c-Fos (ab134122, 1:2000), FosB (ab184938, 1:10,000) and EGR1 (ab133695, 1:2000). Mouse anti-GAPDH monoclonal antibody (sc-365062) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA)(1:1000). Second antibodies included anti-rabbit (#7074, 1:5000) and anti-mouse (#7076, 1:5000) IgG-horseradish peroxidase (HRP) were both from Cell Signaling Technologies., Inc. Protein bands were visualized using the enhanced chemiluminescence (Meilun, Dalian, China) and analyzed using ImageJ software (ImageJ 1.80v; National Institutes of Health, Bethesda, MA, USA).
Chromatin immunoprecipitation (ChIP)
RBLs were collected as previously described . Proteins were crosslinked to DNA by adding 270 µl of 37% formaldehyde to 10 ml culture medium (final concentration, 1%). After 10-min incubations at 37°C, reactions were quenched by adding 0.125 M glycine for 5 min at room temperature. After washing with ice-cold PBS twice, fixed cells were lysed with 1 ml RIPA buffer (MedChem Express, Monmouth Junction, NJ) on ice for 10 min. After centrifugation, nuclei were sonicated (30% amplitude, 20 sets of 15-s bursts) for four rounds. Debris was removed by centrifugation at 13,000 rpm at 4°C for 10 min; supernatants were transferred to new tubes. Lysates were precleared with protein A/G beads before being incubated with c-Fos antibodies (Cell Signaling Technology, Danvers, MA) or IgG (Santa Cruz, CA) at 4°C overnight with rotation. The immunocomplex was washed (once each) in low salt, high salt, and LiCl solutions and washed twice in TE buffer. Bead-bound immunocomplexes were eluted with 200 µl elution buffer. To reverse histone-DNA crosslinks, samples were combined with 5 M NaCl and heated at 65°C for 4 h. DNA product was purified in a spin column system (Omega Bio-Tek, Norcross, GA).
Knockdown of c-Fos
We cloned shFos (5′-GCAGACCGAGATTGCCAATTT-3′) and GFP mRNA as a negative control (shNC, 5′-GCAAGCTGACCCTGAAGTTCAT-3′) into pLKO.1-puro vectors (Sigma, St. Louis, MO). Sequences were confirmed by DNA sequencing (Beijing Genomics Institute, Shenzhen, China). Liposomes (Bioinno-Profei Tech, Qingdao, China) were applied to enable cell transfection.
Dual luciferase promoter-reporter assay
Transient transfection was conducted with liposome transfection reagent. RBLs were seeded in 24-well plates and transfected with the AP-1 luciferase-reporter plasmid pAP1-Luc (Promega, USA), and compared to control plasmid pGL3-basic or pGL3-control. Activities of Firefly luciferase in pGL3-reporters and Renilla luciferase in pRL-TK were determined 48 h later by dual luciferase reporter assays following the manufacturer’s protocol (Promega, Madison, WI). Each transfection was performed in triplicate and each assay was repeated at least three times.
IgE-mediated passive cutaneous anaphylaxis (PCA) mouse model
A PCA mouse model was used to assess T-5224 effects on allergic reactions . In this model, extravasation reflects vessel permeability. Twenty-four mice were randomized into four groups (6/group): PBS; DNP-HSA; DNP-HSA plus T-5224; and DNP-HSA plus ketotifen. Left ears were injected subcutaneously with anti-DNP IgE (500 ng in 0.9% saline). Twenty-four hours later, IgE-sensitized mice were given an intraperitoneal (i.p.) injection of T-5224 (60 mg/kg) or ketotifen (50 mg/kg), and 1 h later challenged with a tail vein injection of 200 µg of DNP-HSA in 1× PBS with 0.5% Evans blue dye. Mice were euthanized 1 h after the challenge; their ears were removed and dissolved in 700 µl formamide overnight at 62°C. Extravasated dye was quantitated with 620-nm spectrophotometry (Bio-Rad, Hercules, CA).
Ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) mouse model
The OVA-induced ASA model is widely used to examine immediate-type hypersensitivity, which is strongly associated with MCs . A cohort of 24 mice was randomized into four groups (6/group): Negative control; OVA sensitization/challenge; OVA sensitization/challenge plus T-5224; and OVA sensitization/challenge plus ketotifen (50 mg/kg). The mice were sensitized on days 0 and 7 with OVA (100 µg i.p. with 2 mg alum adjuvant). From experimental day 9 to 13, mice received T-5224 (60 mg/kg) and ketotifen (50 mg/kg), i.p. on alternating days. On day 14, the mice were challenged with 200 µg OVA (i.p.) and then monitored for 90 min, during which their rectal temperatures were measured every 10 min. Immediately thereafter, orbital venous plexus blood was sampled for serum histamine and IL-4 level measurement by enzyme-linked immunosorbent assay (ELISA).
Oxazolone (OXA)-induced atopic dermatitis model
In the OXA-induced atopic dermatitis model, massive inflammatory cell infiltration leads to skin thickening . To produce the model , the right ears of Balb/c mice were sensitized with 20 µL of 1% OXA (TargetMol, Shanghai, China) in 4:1 (v/v) acetone-olive oil mixture (experimental day 0). Plain acetone/sesame seed oil was injected in the left ears to obtain within-animal controls. On days 7, 9, and 11, the ears were challenged with 20 µL of 1% OXA three times a week for 1 wk. At the same time, T-5224 (60 mg/kg, i.p.) was administered. On day 13, the mice were sacrificed, ear samples were collected for further analysis. Ear swelling change was used as a measure of allergic response.
Cytology and histology
Cell staining methods are described in the Supplemental information. Cells were examined under a light microscope (Carl Zeiss, Goettingen, Germany) to view staining and to detect morphological changes associated with MC degranulation, particularly F-actin cytoskeleton decomposition. Mouse ears were fixed in a 4% paraformaldehyde (Solarbio, Beijing, China) and embedded in paraffin. Sections were cut (thickness, 4 mm), dewaxed, cleared in xylene, hydrated, and stained with hematoxylin and eosin (H&E; Seivicebio, Wuhan, China) to visualize epidermal thickness and eosinophils, and with toluidine blue (Seivicebio, Wuhan, China). Eosinophils and MCs were counted in five randomly selected images.
Histamine and IL4 release
BMMCs were sensitized with anti-DNP IgE (50 ng/ml) overnight, pretreated with T-5224 for 1 h, and then stimulated with DNP-HSA for 30 min (for histamine detection) or 6 h (for IL-4 detection). Cell suspensions were centrifuged (300 ×g, 5 min) to separate cells from media. Supernatant concentrations of histamine and IL4 were determined with enzyme-linked immunosorbent assay (ELISA) kits for mouse histamine (IBL, Hamburg, German) and mouse IL4 (Shanghai Hu Zhen Biological Technology, Shanghai, China).
All clinical and histological evaluations were performed in a blinded manner. Data were analyzed in Prism 8 (GraphPad, La Jolla, CA). For normally distributed quantitative variables, means of triplicate values are reported with standard deviations (SDs). Kruskal–Wallis tests were performed to detect significant differences between treatment groups. One-way analyses of variance (ANOVAs) and Dunnett’s post‑hoc tests were used to find differences between treatment groups in the in vivo experiments. Differences was considered significant at p < 0.05.