Figures
- FIGURE 1
Airway epithelium regenerated from COPD basal cells shows an abnormal phenotype. Basal cells from 8 normal and 8 COPD subjects were cultured in Transwells at air–liquid interface (ALI) for 4 weeks. The Transwells were transferred to new receiver plates containing fresh medium and incubated for 24 h prior to harvesting. The cultures in the Transwells were fixed in 10% buffered formalin, embedded in paraffin, and the paraffin sections were stained with a) haematoxylin and eosin (H & E) or b) periodic acid Schiff (PAS). Arrows in panel (a) indicate cilia and arrows in panel (b) indicate goblet cells. Images in (a and b) are representative of eight COPD and eight normal regenerated airway epithelial cell cultures. c) From identical cultures, cells were dissociated and subjected to flow cytometry to determine different cell population and data represent mean±sd. d) Transepithelial resistance (TER) was measured after culturing basal cells at ALI for 2 weeks. Data represent median and range. The statistical significance was either determined by unpaired t-test (c) or by Mann–Whitney nonparametric test (d). Scale bars=20 μm.
- FIGURE 2
Differentially regulated genes in basal COPD and normal cells. a) Volcano plot showing differential gene expression between basal cells from 8 COPD and 8 normal subjects: red, downregulated genes; green, upregulated genes. Total RNA isolated from the basal cells was subjected to real-time quantitative PCR for b) HOXA1, c) HOXB2, d) IVL, e) ELF5, f) VGLL1, g) KRTDAP, h) STC2 and i) ADAMTSL3. Data represent median with range from 8 COPD and 8 normal subjects and statistical significance was determined by Mann–Whitney U-test.
- FIGURE 3
Expression of HOXA1 and HOXB2 in basal cells correlates with transepithelial resistance (TER) and not differentiation markers. Basal cells from 8 normal and 8 COPD subjects were cultured at air–liquid interface (ALI) for 2 weeks (to assess TER) or 4 weeks (to assess MUC5AC and FOXJ1). Total RNA was isolated from basal cells and cells cultured at ALI for 4 weeks. mRNA expression of HOXA1 and HOXB2 in basal cells, and MUC5AC and FOXJ1 were determined by probe-based qPCR. The data were normalised to G3PDH. TER measured in cells cultured at ALI for 2 weeks correlates with the expression of HOXA1 and HOXB2 (a and d). The expression of either HOXA1 or HOXB2 does not show strong correlation with differentiation markers MUC5AC or FOXJ1 (b, c, e and f). Red and black dots in all the panels represent data from normal and COPD cells, respectively.
- FIGURE 4
COPD cells show reduced expression of E-cadherin and occludin proteins. a) Total protein isolated from polarised cells was subjected to Western blot analysis to determine the protein expression of E-cadherin and occludin. Image is representative of 8 normal and 8 COPD subjects. The density of bands were determined and expressed as fold change over β-actin (b and c). The data represent mean±sd calculated from 8 normal and 8 COPD basal cells from 3 independent experiments and the significance was assessed by unpaired t-test.
- FIGURE 5
HOXB2 expression shows dynamic changes during culturing. Total RNA from basal cells and basal cells cultured at air–liquid interface (ALI) for 1, 2, and 4 weeks was isolated, cDNA was synthesised and subjected to probe-based qPCR to determine the expression of a) HOXA1 and b) HOXB2. The data represent median with range. Statistical significance was determined by ANOVA on ranks with Kruskal–Wallace post hoc test. Numbers in bold represent difference in the expression of genes between normal and COPD cells at each time point. HOXB2 expression significantly changed with time of culturing but not in COPD cells.
- FIGURE 6
HOXB2 contributes to epithelial cell polarisation. a) The 16HBE14o− cells cultured in Transwells (90% confluent) were transfected with nontargeting (NT) or HOXB2 short hairpin (sh)RNA and incubated for another 2 days to promote polarisation and then transepithelial resistance (TER) was measured. Cells were fixed to assess the localisation of b) occludin and c) E-cadherin by immunofluorescence microscopy. d) From identical cultures, total protein was isolated and expression of occludin and E-cadherin was determined by Western blot analysis. Density of the bands was determined and expressed as fold change over β-actin (e and f). The data represent mean±sd calculated from 4 independent experiments and unpaired t-test was used to calculate the p-value. g) HOXB2 knockdown was confirmed by Western blot analysis. Images in (b, c, d and g) are representative of 4 independent experiments. Scale bars=20 μm.
- FIGURE 7
Immunolocalisation of HOXB2 in lung tissues. Paraffin sections of COPD and normal lungs were deparaffinised, incubated for 10 min in boiling 10 mM citrate buffer (pH 6.0), allowed to cool down to room temperature. The sections were then treated with 3% hydrogen peroxide to quench endogenous peroxidase activity, blocked with 5% normal horse serum containing 0.3% Triton X-100 for 1 h. Slides were washed and incubated with primary antibody for 16 h at 4°C. The slides were washed, incubated with tyramide conjugated with Alexa Fluor 488 and then counterstained with 4′,6-diamidino-2-phenylindole. Red, HOXB2 in the left panels, blue, nuclei in the centre panels and magenta, colocalisation of HOXB2 with nuclei in right panels. a) Normal and b) COPD lung section respectively stained with HOXB2 antibody. c) Normal lung section stained with isotype control IgG. The images are representative of lung sections from 6 normal and 6 COPD subjects. Scale bars=20 μm.
Tables
- TABLE 1
Expression data for top up- and downregulated genes in COPD compared to normal basal cells and genes involved in epithelial/epidermal differentiation and tissue development
Gene symbol COPD Normal Fold change p-value Top-20 downregulated genes in COPD HOXA1# 1.51±0.70 3.67±0.50 −4.47 1.8×10−6 TENM2 6.98±0.45 8.13±0.28 −2.21 3.3×10−6 SLC2A12 2.93±0.57 4.61±0.65 −3.19 2.0×10−5 VGLL1# 5.06±0.61 6.67±0.57 −3.05 4.7×10−5 ARHGAP40 1.75±0.51 3.21±0.72 −2.76 1.0×10−4 CLIC3 5.23±0.59 6.48±0.66 −2.38 2.0×10−4 HOXB2# 1.89±0.31 3.29±0.93 −2.64 2.0×10−4 IL36RN 2.76±0.45 3.76±0.69 −2.00 3.0×10−4 PADI1 3.31±0.63 4.76±0.76 −2.73 5.0×10−4 MFAP5 4.49±0.68 6.31±0.85 −3.53 5.0×10−4 ATP12A 5.06±1.26 7.23±1.40 −4.48 8.0×10−4 FAM46C 2.21±0.40 3.23±0.60 −2.02 9.0×10−4 MAL 4.20±0.73 6.31±1.75 −4.32 1.0×10−3 KLK5¶ 4.47±0.77 5.90±0.84 −2.68 1.1×10−3 HEPHL1 1.25±0.31 2.59±1.16 −2.53 1.1×10−3 LGALS9B 4.86±0.62 6.43±0.79 −2.97 1.2×10−3 ELF5¶ 3.84±0.57 4.92±0.93 −2.12 2.2×10−3 KLK7¶ 7.42±0.50 8.45±0.93 −2.05 2.4×10−3 SCNN1G 3.45±0.72 4.65±0.60 −2.31 2.7×10−3 UPK1A¶ 1.96±0.55 3.94±1.93 −3.96 2.8×10−3 IVL¶ 3.92±1.03 5.1±1.1 −2.27 0.0093 KRTDAP# 2.47±0.6 3.59±1.57 −2.17 0.0114 S100A7¶ 1.82±0.89 3.17±1.18 −2.55 0.0186 KRT24¶ 1.66±0.94 3.28±1.15 −3.06 0.0241 DSG1¶ 2.64±1.65 4.32±1.96 −3.21 0.0411 Top-10 upregulated genes in COPD HIST3H2BB 4.92±0.41 3.78±0.38 2.21 2.1×10−6 CLGN 3.83±0.80 2.19±0.50 3.12 3.0×10−4 HIST1H3J 5.33±0.44 4.08±0.58 2.39 3.0×10−4 HIST1H2AB 6.02±0.61 4.51±0.51 2.84 4.0×10−4 ANXA10 6.24±1.33 4.24±1.16 3.99 4.0×10−3 HIST1H3H 2.70±0.26 1.62±0.83 2.13 6.0×10−3 CHAC1 6.73±1.32 4.83±0.45 3.72 1.2×10−2 CCNE2 5.67±0.75 4.62±0.29 2.07 1.6×10−2 GTF2H2 1.75±1.09 0.65±0.62 2.15 3.3×10−2 ADAMTSL3 4.25±0.95 3.15±0.84 2.14 3.6×10−2 Control genes G3PDH 4.11±2.11 4.21±0.12 −1.07 0.2273 KRT5 10.13±0.12 10.36±0.17 −1.10 0.0155 Data are presented as log2mean±sd unless otherwise stated. #: involved in tissue development; ¶: involved in epithelial/epidermal differentiation.
Supplementary Material
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Supplementary material 00656-2020.SUPPLEMENT
