Therapeutic effect of Bacillus Calmette–Guerin polysaccharide nucleic acid on mast cell at the transcriptional level

Patient enrollment

Patients with CSU who were treated with nonsedating H1 antihistamine with or without BCG–PSN during September 2013–December 2013 from Xiangya Hospital were recruited. This cross-sectional study was approved by the Ethics Committee of Xiangya Hospital (201311392). All patients enrolled in our study signed the written informed consent. Each patient was treated with antihistamine monotherapy using conventional doses including desloratadine 5 mg/d and levocetirizine 5 mg/d, with or without BCG–PSN. The enrollment of CSU patients was according to a previous study (Yan et al., 2014; Guo et al., 2015), while patients with severe allergic symptoms, acute disorders, infectious diseases, or glucocorticoid treatment were excluded. The disease severity was assessed by weekly urticaria activity score (UAS7), and when the UAS7 score decreased to 0, the patient was considered as complete relief.

Cell culture

RBL-2H3 cells (ATCC, Manassas, VA, USA) were cultured in 1640 medium (Biological Industries, Israel) with 15% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 °C with 5% CO₂ in a humidified atmosphere.

β-Hexosaminidase (β-HEX) assay

RBL-2H3 cells at 90% confluence were inoculated into a six-well plate at a density of 2 × 10⁵ cells/ml. After overnight sensitization by anti-DNP-IgE (100 ng/ml), cells were pretreated with different concentrations (0, 20, 40, 80, or 100 µl/ml) of BCG–PSN (Jiuzhitang Co., Ltd) at different time points (1, 2, 4, or 6 h) with mizolastine or mizolastine alone. After washing with Tyrode’s buffer twice, the cells were incubated with DNP–HSA (1 µg/ml, Alpha Diagnostic Inc.) for 30 min at 37 °C. The supernatant of each cell culture was withdrawn, and cells were lysed with NP-40 (3 mM) of the same volume. β-HEX solution (50 µl, 3 mM N-acetyl-β-D-glucosaminide, Sigma) was added to 50 µl of the supernatant, which was then mixed and incubated for 90 min at 37 °C. Subsequently, the reaction was suspended by the addition of 150 µl NaHCO₃/Na₂CO₃ solution (pH = 10.5), and the absorbance of the supernatant was detected at 405 nm. The β-HEX release rate was evaluated as described previously (Li et al., 2017).

Cell viability assay

RBL-2H3 cells were seeded into a 96-well culture plate at a cell density of 4000 cells/well. Different concentrations (0, 20, 40, 80, or 100 µl/ml) of BCG–PSN were added to each well. At estimated time points, PMS solution premixed with MTS (1:40) was added into each well and reacted for 2 h at 37 °C, and the absorbance of the supernatant was evaluated at 450 nm. The experiments were conducted at least in triplicate, and readings were normalized to cells treated with control.

LncRNA sequencing

RBL-2H3 cells were seeded into a six-well plate and sensitized with anti-DNP-IgE (100 ng/ml) overnight , then mixed with the appropriate concentration of BCG–PSN for 1 h. After the incubation, DNP-HSA (one µg/ml) was used to stimulate cells for 30 min. Cells were collected subsequently, and total RNA was extracted according to the manufacturer’s instructions. LncRNA sequencing was conducted with an Illumina HiSeq 3000 system. Differential expression was tested between the BCG–PSN drug group and the negative control using the unpaired t-test. LncRNAs and mRNAs with a fold change of more than 2.0 and a corrected p-value less than 0.05 were assessed as differentially expressed.

Real-time PCR (RT-PCR)

RT-PCR was used to verify the differential expression of candidate genes. RNA was extracted from sensitized RBL-2H3 cells pretreated or not with the drug at the appropriate concentrations. Then, two µg of the total RNA was reverse-transcribed to cDNA via the PrimeScript™ Reverse Transcriptase kit (Takara Bio Inc.) according to the manufacturer’s instructions. RT-PCR was performed using an Applied Biosystems 7500 real-time PCR system (ABI, USA). All expression levels were presented as fold changes and were analyzed by the 2^−ΔΔCT method with GAPDH as the internal reference (Table S1).

Statistical analysis

Continuous variables were presented as the mean ± standard deviation (SD). Student’s t-test was used to compare differences between patients receiving and not receiving BCG–PSN and the transcriptional levels of different genes. Categorical variables were expressed as count (%) and compared using the chi-square test. Data analysis was performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). A p-value less than 0.05 was considered statistically significant. The significance level for multiple comparisons among genes was adjusted by Bonferroni correction.

Efficacy of BCG–PSN in combination with antihistamines

A total of 110 patients were enrolled in this study. Among the participants, 53 were treated with BCG–PSN accompanied by antihistamine monotherapy, while 57 were treated with antihistamine monotherapy. The demographic and clinical characteristics of the patients were shown in Table 1. Age, gender, course, and baseline disease severity were not significantly different between groups. According to Table S2, the rate of treatment effectiveness after 4 weeks was not significantly different between the BCG–PSN group (71.7%) and the monotherapy group (71.9%). However, the average time of complete relief in the BCG–PSN group was significantly shorter than that in the monotherapy group (p = 0.026, Table 1).

β-HEX release rate in sensitized RBL-2H3 cells pretreated with BCG–PSN

After stimulation with DNP-HSA (1 µg/ml) for 30 min, the release rate of β-HEX decreased in RBL-2H3 cells in a manner that was associated with BCG–PSN concentration. The β-HEX release ratio decreased most extensively when cells were treated with 100 µl/ml BCG–PSN (p < 0.001, Fig. 1A). The MTS assay showed that the cell viability was not significantly inhibited between groups treated with varying concentrations of BCG–PSN (Fig. 1B). Therefore, no more than 100 µl/ml BCG–PSN was considered an appropriate concentration for treating RBL-2H3 cells. As BCG-PSN was used for combined therapy with nonsedating antihistamines, we subsequently evaluated the mast cell activation by the BCG-PSN combined with or without the representative antihistamine mizolastine. The degranulation level of the BCG-PSN combined treatment group showed a lower β-HEX release ratio compared with either BCG-PSN or mizolastine (Fig. 1C), which indicated that BCG-PSN inhibited the sensitized mast cell activation and that this effect was enhanced when BCG-PSN was combined with the nonsedating antihistamine mizolastine.

Expression of mRNA and lncRNA via heatmap analysis and mRNA expression validation in sensitized RBL-2H3 cells pretreated with BCG–PSN

As shown above, BCG-PSN decreased the degranulation level of sensitized RBL-2H3 cells with an unknown mechanism. Therefore, lncRNA sequencing was conducted to explore the deeper mechanism at the transcription levels, including mRNA and lncRNA expression levels. During sequencing, we first qualified the sample RNA libraries. Q30% ranged from 94.02% to 94.83%, and all libraries were appropriate for the following analysis. Detailed data are shown in Table S3.

According to the sequencing data analysis, a total of 6368 lncRNAs were detected preliminarily, and after PLEK, CNCI, and CPAT computer-filtered analysis, only 2126 lncRNAs remained. The classifications of all candidate lncRNAs are shown in Fig. S1.

Cluster analysis was performed using a heatmap analysis including mRNA and lncRNAs. All statistics were divided into control (IgE sensitized cells) and treatment groups (IgE sensitized cells pretreated with BCG-PSN) in triplicates. We observed a significant difference in the expression of lncRNAs (Fig. 2) and mRNAs (Fig. 3) between the groups treated with and without BCG-PSN. After setting the filter criteria of log₂(fold change) >1 or <−1 and a p-value <0.05, we observed that 34 lncRNAs and 84 mRNAs were downregulated and 29 lncRNAs and 136 mRNAs were upregulated in the BCG–PSN treatment group compared with the control group (Table S4). Among them, TCR signaling and cytokine release-related genes, including growth factor independent 1B transcriptional repressor (GFI1B) and Erb-B2 receptor tyrosine kinase 4 (ERBB4), were downregulated, while a radical S-adenosyl methionine domain containing 2 (RSAD2) was upregulated (Table S5). Gene ontology (GO) analysis of the differentially expressed genes showed significant enrichment of proteins targeting intracellular organelles, membrane-bounded organelles, and intracellular parts. The upregulation of tyrosine phosphorylation pathway related genes such as Colony Stimulating Factor 2 (CSF2) and ERBB4 also showed significance. To further validate significantly differentially expressed genes, RT-PCR was performed. Validation of the mRNA levels of genes such as GFI1B, discoidin domain receptor tyrosine kinase 1 (DDR1), RSAD2, and ERBB4 showed significant downregulation of ERBB4 and upregulation of RSAD2, while the changes of DDR1 and GFI1B showed no significances (Fig. 4).