Twenty-five patients with mild asthma (MA) and 15 patients with SA at the Department of Pulmonary Medicine, The First Affiliated Hospital of Shenzhen University (Shenzhen, China) were recruited into this study. The diagnosis of asthma and the assessment of its severity were carried out according to the Global Initiative for Asthma (GINA) and ERS/ATS guidelines. All participants were subjected to forced expiratory volume in the first second (FEV₁) test and total IgE and HDM-specific IgE measurements. Patients with MA were collected at the time of diagnosis in outpatient, before administration of asthma medication. Patients with SA were hospitalized patients who showed exacerbation in symptoms such as wheezing, breathlessness and chest tightness 24 hours prior to admission to the emergency department and received only rescue medication. Blood samples from these patients were obtained after admission to the emergency department but before the initiation of inhaled corticosteroids (ICSs)/long-acting β2-agonists (LABAs) treatment. Then, these patients were treated with therapy (medications for GINA steps 4–5 asthma). Fifteen healthy donors, with normal pulmonary function and negative allergy tests, were selected as normal controls (NCs). The study was approved by the Research Ethics Board of School of Medicine, Shenzhen University (Shenzhen, China). Written informed consent was obtained from all individuals enrolled in the study.

Heparinized peripheral venous blood (4–5 mL) was collected from each participant. Peripheral blood mononuclear cells (PBMCs) were separated immediately using the Ficoll-Hypaque gradient centrifugation method. CD4⁺ T cells were isolated from PBMCs by human CD4⁺ Micro Beads (Miltenyi Biotec, Bergisch Gladbach, Germany).

Female BALB/c mice (6–8 weeks) were purchased from the Animal Center of Guangdong Province. The mice were maintained in a specific pathogen-free facility at the Experimental Animal Center of Shenzhen University. All animal care and experimental protocols were carried out in accordance with the Institutional Guidelines for Care and Use of Laboratory Animals at Shenzhen University.

The antigen sensitization and challenge in the murine model of allergic asthma were performed as previously described,1718 and the scheme of the study is shown in Fig. 1A. Briefly, BALB/c mice received immunization with 100 µg of HDM extract in 100 µL of PBS with 2% aluminum hydroxide by intraperitoneal injection on days 0, 3, and 7. From days 15 to 21, the mice were challenged intranasally with 50 µg of HDM in 50 µL of PBS. The PBS control group was treated with PBS in all procedures. Twenty-four hours after the final challenge, airway hyperreactivity (AHR) was assayed and on day 23 all the mice were sacrificed. Purified mouse CD4⁺ T cells of both the HDM and PBS groups were isolated from spleens by mouse CD4⁺ Micro Beads (Miltenyi Biotec).

AHR to methacholine (Mch) aerosol was evaluated using unrestrained whole-body plethysmography with a 4-chamber system (Buxco Research Systems, Wilmington, NC, USA). Firstly, the baseline response was recorded for 5 minutes at steady respiration after 10-minute acclimation. Then, the mice were subjected to Mch inhalation at increasing concentrations (6.25, 12.5, 25, and 50 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) or PBS. Tests were temporally separated to allow the respiratory intensity to drop back to baseline. The percentage curves for Penh values at different Mch doses were plotted.

BAL was performed as previously described.18 The supernatant was collected for cytokine determination by enzyme-linked immunosorbent assay (ELISA). Pellets were resuspended and counted with hemocytometer. Cytospin preparations were stained manually with Wright-Giemsa stain. The percentages of lymphocytes, eosinophils, neutrophils and macrophages were determined by counting at least 200 leukocytes in randomly selected areas under a light microscope.

Splenocytes isolated from spleens of BALB/c mice were first challenged with HDM (30 µg/mL) overnight. Then, the cells were divided into 4 groups (1 × 10⁷ cells/group) and treated as follows: 1) pI2 (pIRES2-EGFP vector control), 2) pBcl11b (pIRES2-EGFP-Bcl11b recombinant plasmid group), 3) TGF-β1 (TGF-β1 stimulation group), and 4) pBcl11b+TGF-β1 (pBcl11b + TGF-β1 treatment group). To up-regulate Bcl11b expression in vitro, cells were transfected with 4 µg of mouse Bcl11b plasmid (GeneChem, Shanghai, China) using Amaxa Nucleofector Kit (Lonza, Basel, Switzerland). For TGF-β1 treatment, recombinant TGF-β1 (Peprotech, Rocky Hill, NJ, USA) was prepared as a stock solution at a concentration of 0.1 mg/mL in 10 mM citric acid. Cells were cultured with 5 µM TGF-β1 in the presence (pBcl11b + TGF-β1 group) or absence (TGF-β1 group) of pBcl11b. After 3 days of culture, the levels of cytokines in the supernatant were measured by ELISA and the cells were collected to detect gene expression by quantitative real-time (qRT)-polymerase chain reaction (PCR) and Western blotting.

Total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantification of human Bcl11b mRNA was performed as previously described.16 Bcl11b copies were normalized to 10⁵ copies of the β2m reference gene. Changes in the mRNA relative expression of human and mouse IL-17A and TGF-β1, and mouse Bcl11b, Smad2, Smad3, and Smad4 were determined by SYBR Green based real-time PCR. Prime and probe sequences are detailed in Table 1. PCR amplification was performed in a total volume of 20 µL with 1 µL of cDNA, 0.5 mmol/L of each primer and 9 µL of SYBR Green I Master Mix (Takara, Beijing, China). Relative target gene expression was calculated by normalization to β-2M or β-actin with the 2^−ΔΔCt method and presented as folds of change against controls.

The levels of human and mouse cytokines were determined by ELISA according to the instructions of reagent kits.

Proteins were extracted from cells, separated by SDS-PAGE and transferred to PVDF membranes. After blocking, the membranes were incubated overnight with antibodies against Bcl11b, Smad2, phosphorylated (p)-Smad2, Smad3, p-Smad3, Smad4, and β-actin (Cell Signaling Technology, Danvers, MA, USA). Immune reaction bands were detected by using specific secondary antibodies (Cell Signaling Technology) and enhanced chemiluminescence reagents (EMD Millipore, Billerica, MA, USA), and viewed by Kodak Image Station 4000MM (Eastman Kodak, Rochester, NY, USA). The relative abundance of protein was determined by quantitative densitometry using Image J software (National Institutes of Health, Bethesda, MA, USA), and the data were normalized to β-actin.

Data are expressed as mean ± standard error of the mean of at least 3 independent experiments. Statistical analysis was performed using Prism 7.0 (Graphpad Software, La Jolla, CA, USA). A 2-tailed Student's t-test was used for pairwise comparison. Where appropriate, the ANOVA test and the Bonferroni post hoc test were used to analyze multiple comparisons and the Mann-Whitney U test was used to analyze non-parametric data. The correlations were examined by Spearman's rank correlation test. The P values of < 0.05 were considered significant.

According to the GINA guideline, patients may be divided into 2 groups: MA and SA. No significant differences were identified in terms of age, sex and BMI between the MA, SA and NC groups. We observed significant increases in blood absolute eosinophil/neutrophil count, total IgE and HDM-specific IgE in SA group in comparison to the MA group. FEV₁ (% pred.) was lower in patients with MA or SA than that in NC (Table 2).

To determine whether Th2 cytokines (IL-4, IL-5, and IL-13) and Th17 cytokine (IL-17A) are both implicated in asthmatic patients positive for HDM-specific IgE, we measured the plasma levels of IL-4, IL-5, IL-13, and IL-17A. We found that IL-4, IL-5, and IL-13 expression were increased in the MA and SA groups when compared to NC. The levels of IL-4 and IL-5 were higher in the SA group than in the MA group; however, no significant difference was observed in the levels of IL-13 between these 2 groups (Fig. 2A). Patients in the SA group showed an increased concentration of IL-17A compared with the other groups. The levels of IL-17A in MA and NC were no different (Fig. 2B). In addition, we noted that the levels of TGF-β1 were increased in both the MA and SA groups compared to NC (Fig. 2B).

Our previous study demonstrated a negative impact of Bcl11b on TGF-β/Smad signaling in naïve T cells using global gene expression analysis. We hypothesize that a reduction of Bcl11b results in enhanced TGF-β/Smad signaling that is essential for Th17 differentiation and IL-17A expression. We examined Bcl11b mRNA levels by TaqMan-based real-time PCR in CD4⁺ T cells, and found that Bcl11b copies were significantly decreased in the MA or SA group compared with NC donors (Fig. 2C). Conversely, IL-17A and TGF-β1 relative mRNA levels were increased (Fig. 2D). However, there were no differences in Bcl11b, IL-17A, and TGF-β1 mRNA expression between the MA and SA groups. We also analyzed the possible correlation between Bcl11b and Th2 cytokines/IL-17A/TGF-β1. In the SA group, the copies of Bcl11b were inversely correlated with IL-17A and TGF-β1 mRNA levels (Fig. 2E and F). However, in the MA group, no significant correlations were found between IL-17A or TGF-β levels and the copies of Bcl11b (data not shown). In addition, there were no differences in the correlations of Th2 inflammation markers (IL-4, IL-5, and IL-13) and Bcl11b between the MA and SA groups (data not shown). Together, these findings suggest that Bcl11b regulates IL-17 production in asthmatic patients positive for HDM-specific IgE.

To explore the role of Bcl11b in the pathogenesis of asthma, we established a HDM-induced allergic airway inflammation model (Fig. 1A). We found that mice immunized and challenged with HDM exhibited increased inflammatory cells (lymphocytes, eosinophils, neutrophils, and macrophages) in bronchoalveolar lavage (Fig. 1B) and AHR (Fig. 1C). Consistently, HDM-specific IgE and IgG1 (Fig. 1D), Th2 cytokines (IL-4, IL-5, and IL-13) (Fig. 1E), IL-17A and TGF-β1 in BALF (Fig. 1F) were all significantly increased in mice immunized and challenged with HDM. These results indicated that HDM-challenged mice appear to have both Th2- and Th17-mediated allergic inflammation.

To determine the mechanism of Bcl11b regulating IL-17 in asthma, we assessed expression of Bcl11b, IL-17A, TGF-β1, Smad2, Smad3, and Smad4 in CD4⁺ T cells using qRT-PCR or western blotting. CD4⁺ T cells isolated from HDM-treated mice showed a 2.56-fold decrease in Bcl11b mRNA, but significantly up-regulated IL-17A and TGF-β1 mRNA compared with control mice (Fig. 3A). Western blot analysis showed similar change in Bcl11b protein expression (Fig. 3B and C). Although the total protein amounts of Smad2, Smad3, and Smad4 did not significantly differ between the 2 groups, the levels of phosphorylated Smad2 and Smad3 were significantly increased in CD4⁺ T cells from HDM-treated mice (Fig. 3B and C). Taken together, our data suggest that down-regulation of Bcl11b in response to HDM treatment may enhance TGF-β/Smad signaling in CD4⁺ T cells to promote IL-17A expression.

Our data demonstrated an inverse relationship between Bcl11b expression and Smad2/3 phosphorylation or IL-17A accumulation. To confirm whether Bcl11b negatively regulates the TGF-β/Smad pathway, we transfected HDM-stimulated mouse splenocytes with mouse Bcl11b expression plasmid in the presence or absence of TGF-β1. Compared with the pI2-empty vector group, the pBcl11b-transfected groups showed increased Bcl11b expression at both mRNA (Fig. 4A) and protein levels (Fig. 4E and F). Ectopic expression of Bcl11b suppressed mRNA expression of IL-17A, TGF-β1, and Smad2, but did not affect Smad3 expression (Fig. 4B and C). Down-regulation of IL-17A by Bcl11b overexpression was confirmed in the culture supernatant by ELISA (Fig. 4D). In addition, Western blot analysis demonstrated that overexpression of Bcl11b decreased Smad2 activation, evidenced by a 1.3-fold reduction in Smad2 phosphorylation (Fig. 4E and F).

We finally investigated whether Bcl11b also suppresses TGF-β1-stimulated Smad activation and IL-17A expression. While TGF-β1 treatment enhanced IL-17A expression at both mRNA and protein levels, such effects were abrogated by ectopic expression of Bcl11b (Fig. 4B and D). Accordingly, TGF-β1 induced Smad2 expression, and activation were suppressed by Bcl11b overexpression (Fig. 4C, E, and F). Surprisingly, the levels of Smad3 and pSmad3 had no obvious change in either group (Fig. 4C, E, and F). These results indicated that overexpression of Bcl11b might restrain IL-17A expression through suppressing the TGFβ/Smad2 signaling pathway.