Reduced MBD2 expression enhances airway inflammation in bronchial epithelium in COPD

Figures & data

Table 1 Characteristics of study subjects

Characteristics	COPD (n=10)	Non-COPD smokers (n=12)	Non-smokers (n=12)
Age (years)	55.60±8.63	54.08±9.24	49.15±10.32
Pack-years smoked	48.10±26.71	37.75±20.98	0*
FEV1 (L)	1.18±0.39	1.88±0.52*	1.95±0.59*
FEV1% of predicted	70.02±19.97	94.87±21.48*	98.24±21.43*
FEV1/FVC %	56.20±13.61	75.65±7.21*	81.51±7.22*

Notes: Data are presented as mean ± SD.

* P<0.05 versus COPD group.

Abbreviations: COPD, chronic obstructive pulmonary disease; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity.

Figure 1 Decreased expression of MBD2 in patients with COPD.

Notes: Representative immunohistochemical images of human lung sections are shown. Relative to the non-smoker (A) and non-COPD smoker (B) groups, MBD2 immunostaining in bronchial epithelium was markedly decreased in patients with COPD (C). Isotype control was stained with rabbit IgG Ab (D), scale bars =100 μM; magnification = ×100. The red-boxed area indicates a region of higher magnification. Semiquantitative analysis of MBD2 expression in airway epithelium using Image-Pro Plus (E). Representative MBD2 expression in whole-lung tissue homogenates by Western blot (F). The band intensity for MBD2 was quantified using ImageJ (G). Data are displayed as mean ± SD, n=10 for COPD, n=12 for non-COPD smokers, and n=12 for non-smokers. P-values were calculated using one-way ANOVA followed by Newman–Keuls test, *P<0.05 and **P<0.01 represent significant differences, compared to the non-smoker group.
Abbreviations: ANOVA, analysis of variance; COPD, chronic obstructive pulmonary disease; MBD2, methyl-CpG-binding domain protein 2; IgG Ab, immunoglobulin G antibody.

Figure 2 Lung function and histological measurements in controls and CS-exposed mice.

Notes: Compared with controls, FEV_0.1s, FEV_0.1s/FVC and C_dyn were significantly decreased in CS-exposed mice (A–C), but R_L was elevated (D). Representative lung tissue section stained with hematoxylin–eosin of controls and CS-exposed mice (E), scale bars =100 μM; magnification = ×40. Compared with controls, MLI measurement was significantly increased in CS-exposed mice (F). Data in C and F panels are presented as mean ± SD; data of other panels are presented as median with range, n=8 for controls and n=8 for CS-exposed mice. **P<0.01 and ***P<0.001 represent significant differences compared to controls.

Abbreviations: CS, cigarette smoke; FEV_0.1s, forced expiratory volume in 0.1s; FEV_0.1s/FVC, ratio of FEV_0.1s to forced vital capacity; C_dyn, dynamic lung compliance; R_L, lung resistance; MLI, mean linear intercept.

Figure 3 Downregulated MBD2 expression in CS-exposed mice.

Notes: Immunohistochemical analysis of MBD2 in mice lung sections are shown (A–C). Compared to controls (A), MBD2 expression was significantly reduced in CS-exposed mice (B), and isotype control was stained with rabbit IgG Ab (C), scale bars =100 μM; magnification = ×100. The red-boxed area indicates a region of higher magnification. Semiquantitative assessment of MBD2 expression using Image-Pro Plus (D). Representative image of Western blots (E). MBD2 band intensity was quantified using ImageJ (F). Data are presented as mean ± SD, n=8 for controls and n=8 for CS-exposed mice. P-values were calculated using Student’s t-test; *P<0.05 and **P<0.01 represents significant differences compared to controls; IgG Ab, immunoglobulin G antibody.
Abbreviations: CS, cigarette smoke; MBD2, methyl-CpG-binding domain protein 2; IgG Ab, immunoglobulin G antibody.

Figure 4 CSE attenuated MBD2 expression in HBE cells in vitro.

Notes: The effects of CSE on HBE cell activity (A). Representative images of Western blots are shown. MBD2 protein expression was reduced in HBE exposed to different CSE concentrations for 48 h (B) or exposed to 10% CSE for various time periods (C). Band intensity was quantified by ImageJ (D and E). Data are displayed as mean ± SD of four independent experiments. P-values were calculated using one-way ANOVA followed by Newman–Keuls test; *P<0.05, **P<0.01, and ***P<0.001 represent significant differences compared to control.
Abbreviations: ANOVA, analysis of variance; CSE, cigarette smoke extract; HBE, human bronchial epithelial; MBD2, methyl-CpG-binding domain protein 2.

Figure 5 MBD2 knockdown increased IL-6 and IL-8 expression in HBE.

Notes: Relative MBD2 mRNA (A) and protein (B) expression was markedly lower in MBD2-siRNA-transfected HBE. Relative IL-6 (C) and IL-8 (D) mRNA expression was higher in HBE cells 24 and 48 h after transfection with MBD2-siRNA. Changes in IL-6 (E) and IL-8 (F) protein expression were consistent with mRNA expression. Data are displayed as mean ± SD of four independent experiments. P-values were calculated using Student’s t-test, *P<0.05, **P<0.01, and ***P<0.001 represent significant differences compared to the corresponding control.
Abbreviations: Con-siRNA, negative control siRNA; HBE, human bronchial epithelial; IL, interleukin; MBD2, methyl-CpG-binding domain protein 2.

Figure 6 MBD2 knockdown enhanced ERK1/2 signal pathway-dependent IL-6 and IL-8 secretion by HBE.

Notes: The increased expression of IL-6 (A, C) and IL-8 (B, D) in HBE after MBD2-siRNA transfection was abrogated by ERK1/2 inhibitors, with or without a CSE milieu. Effects of CSE on HBE cell activity after incubation for 72 h (E). HBE were preincubated with U0126 (10 μM) or DMSO for 1 h, followed by stimulation with medium or 10% CSE for 1 h (F). Data are displayed as mean ± SD of four independent experiments. P-values were calculated using the Student’s t-test; *P<0.05, **P<0.01, and ***P<0.001 represent significant differences compared to the corresponding control.
Abbreviations: Con-siRNA, negative control siRNA; CSE, cigarette smoke extract; DMSO, dimethyl sulfoxide; HBE, human bronchial epithelial; IL, interleukin; MBD2, methyl-CpG-binding domain protein 2.

Figure S1 Representative immunohistochemical image of cells in non-smokers’ lung tissues.

Notes: The red-boxed area indicates a region of higher magnification. The arrows indicate inflammatory cells and asterisks points to alveolar mesenchymal cells.

Figure S2 Levels of IL-6 and CXCL1 (KC) in BALF in controls and CS-exposed mice.

Notes: Compared with controls, levels of IL-6 (A) and CXCL1 (KC) (B) in BALF were increased in CS-exposed mice. Data are presented as means ± SD, n=8 for controls and n=8 for CS-exposed mice. P-values were calculated using Student’s t-test; **P<0.01 and ***P<0.001 represent significant differences compared to controls.

Figure S3 MBD2 protein expression in bronchial epithelium correlated with cytokines in BALF of mice.

Notes: MBD2 expression in bronchial epithelium were negatively correlated with and IL-6 (A) and CXCL1 (KC) (B) level in BALF of mice. Correlations were assessed by using Pearson analysis.

Figure S4 MBD2 mRNA in lung tissues and HBE.

Notes: No significant difference in MBD2 mRNA levels between the CS-exposed samples and controls was observed (A). There were no changes in MBD2 mRNA expression in HBE treated with different concentrations of CSE for different time periods (B).