A CpG-Oligodeoxynucleotide Suppresses Th2/Th17 Inflammation by Inhibiting IL-33/ST2 Signaling in Mice from a Model of Adoptive Dendritic Cell Transfer of Smoke-Induced Asthma

Abstract

Tobacco smoke exposure is a major environmental risk factor that facilitates the development and progression of asthma. Our previous study showed that CpG oligodeoxynucleotide (CpG-ODN) inhibits thymic stromal lymphopoietin (TSLP)-dendritic cells (DCs) to reduce Th2/Th17-related inflammatory response in smoke-related asthma. However, the mechanism underlying CpG-ODN -downregulated TSLP remains unclear. A combined house dust mite (HDM)/cigarette smoke extract (CSE) model was used to assess the effects of CpG-ODN on airway inflammation, Th2/Th17 immune response, and amount of IL-33/ST2 and TSLP in mice with smoke-related asthma induced by adoptive transfer of bone-marrow-derived dendritic cells (BMDCs) and in the cultured human bronchial epithelium (HBE) cells administered anti-ST2, HDM, and/or CSE. In vivo, compared to the HDM alone model, the combined HDM/CSE model had aggravated inflammatory responses, while CpG-ODN attenuated airway inflammation, airway collagen deposition, and goblet cell hyperplasia and reduced the levels of IL-33/ST2, TSLP, and Th2/Th17-cytokines in the combined model. In vitro, IL-33/ST2 pathway activation promoted TSLP production in HBE cells, which could be inhibited by CpG-ODN. CpG-ODN administration alleviated Th2/Th17 inflammatory response, decreased the infiltration of inflammatory cells into the airway, and improved the remodeling of smoke-related asthma. The underlying mechanism may be that CpG-ODN inhibits the TSLP-DCs pathway by downregulating the IL-33/ST2 axis.

Keywords: CpG-ODN; ST2; asthma; dendritic cell; interleukin-33.

Figure 1

CpG-ODN inhibits airway inflammation in mice with smoke-related asthma. (a) Representative hematoxylin and eosin-stained lung tissue sections (×200). The red arrows point to the area shown at a higher magnification in the left lower quadrant. Scale bars: 50 μm. (b) Total number of inflammatory cells in BALF. (c) ELISA of CCL11 and CXCL1 in lung homogenates. All experiments were performed two times independently, with n = 6 mice/experiment and three technical replicates. Shapiro–Wilk tests were used to test for normality, and one-way ANOVA was used for multiple unmatched groups, followed by Sidak’s test for multiple comparisons. Data are mean ± s.e.m. * p < 0.05 and ** p < 0.01 vs. the counterpart group.

Figure 2

CpG-ODN inhibits airway remodeling in mice with smoke-related asthma. (a) Representative micrographs showing airway collagen deposition in Picro Sirius red-stained lung samples (×200). The red arrows point to the area shown at a higher magnification in the left lower quadrant. Scale bars: 50 μm. Proportion of collagen area derived as collagen-positive area/total bronchiole area in bronchioles of similar size using Image-Pro Plus 4.5 (Media Cybernetics, Inc., Rockville, MD, USA). (b) Representative micrographs showing mucin production in Alcian blue (pH 2.5)-stained lung sections (×200). The red arrows point to the area shown at a higher magnification in the left lower quadrant. Scale bars: 50 μm. Proportion of mucin area derived as mucin-positive area/total bronchiole area in bronchioles of similar size using Image-Pro Plus 4.5. (c) Relative Muc5ac and COL1A1 mRNA content was measured by qRT-PCR in lung tissue samples. All experiments were performed two times independently, with n = 6 mice/experiment and three technical replicates. Shapiro–Wilk tests were used to test for normality, and one-way ANOVA was used for multiple unmatched groups, followed by Sidak’s test for multiple comparisons. Data are mean ± s.e.m. * p < 0.05 and ** p < 0.01 vs. the counterpart group.

Figure 3

CpG-ODN treatment regulates TSLP in smoke-related asthma. (a) Representative images of TSLP (green) immunofluorescence of lung samples (×200). Scale bars: 50 μm. Semiquantitative protein expression of TSLP detected by immunofluorescence was determined as integral optical density (IOD) of TSLP (green)/number of cells. (b) TSLP protein level in BMDCs supernatants, assessed by ELISA. (c) TSLP protein content in lung homogenates, assessed by ELISA. (d) TSLP gene expression in lung homogenates, evaluated by qRT-PCR. All experiments were performed two times independently, with n = 6 mice/experiment and three technical replicates. Shapiro–Wilk tests were used to test for normality, and one-way ANOVA was used for multiple unmatched groups, followed by Sidak’s test for multiple comparisons. Data are mean ± s.e.m. * p < 0.05 and ** p < 0.01 vs. the counterpart group.

Figure 4

CpG-ODN treatment regulates anti-HDM IgE, pro-inflammatory, anti-inflammatory cytokines and Th1/Th2/Th17-cytokines in smoke-related asthma. (a) HDM-specific IgE amounts in BALF, assessed by ELISA. (b) IL-12 and IL-6 protein content in BMDCs supernatants, quantified by ELISA. (c) IFN-γ, IL-13, and IL-17A levels in BALF, as evaluated by ELISA. (d) IL-13 and IL-17A content in lung homogenates, evaluated by ELISA. (e) IL-13 and IL-17A gene expression in lung homogenates, evaluated by qRT-PCR. All experiments were performed two times independently, with n = 6 mice/experiment and three technical replicates. Shapiro–Wilk tests were used to test for normality, and one-way ANOVA was used for multiple unmatched groups, followed by Sidak’s test for multiple comparisons. Data are mean ± s.e.m. * p < 0.05 and ** p < 0.01 vs. the counterpart group.

Figure 5

IL-33 in lung samples is reduced by CpG-ODN treatment. (a) Representative micrographs after immunohistochemical staining of IL-33(×200). The red arrows point to the area shown at a higher magnification in the left lower quadrant. Scale bars: 50 μm. IL-33 signals appeared as brown spots. IOD of IL-33 was calculated by Image-Pro Plus 4.5. The positivity rate of IL-33 was determined as IOD/total bronchiole area. (b) Representative micrographs of IL-33 (red) immunofluorescence in lung sections (×200). Scale bars: 50 μm. IOD of IL-33 was calculated by Image-Pro Plus 4.5. Semiquantitative protein content of IL-33 detected by immunofluorescence, as assessed as IOD of IL-33 (red)/number of cells. All experiments were performed two times independently, with n = 6 mice/experiment and three technical replicates. Shapiro–Wilk tests were used to test for normality, and one-way ANOVA was used for multiple unmatched groups, followed by Sidak’s test for multiple comparisons. Data are mean ± s.e.m. * p < 0.05 vs. the counterpart group.

Figure 6

IL-33/ST2 levels in BMDCs supernatants and lung samples are reduced by CpG-ODN treatment. (a) IL-33 level in BMDCs supernatants, examined by ELISA. (b) IL-33 level in lung homogenates, evaluated by ELISA. (c) IL-33 gene expression in lung homogenates, examined by qRT-PCR. (d) Representative micrographs after immunohistochemical staining of ST2 (×200). The red arrows point to the area shown at a higher magnification in the left lower quadrant. Scale bars: 50 μm. ST2 signals appeared as brown spots. IOD of ST2 was calculated by Image-Pro Plus 4.5. The positivity rate of ST2 was determined as IOD/total bronchiole area. All experiments were performed two times independently, with n = 6 mice/experiment and three technical replicates. Shapiro–Wilk tests were used to test for normality, and one-way ANOVA was used for multiple unmatched groups, followed by Sidak’s test for multiple comparisons. Data are mean ± s.e.m. * p < 0.05 and ** p < 0.01 vs. the counterpart group.

Figure 7

CpG-ODN treatment inhibits IL-33/ST2 upregulation in HBE cells. (a) Western blotting detection of IL-33/ST2 against GAPDH (utilized for normalization). (b) IL-33/GAPDH ratio was determined. (c) ST2/GAPDH ratio was determined. (d) IL-33 levels in HBE cell culture supernatants, examined by ELISA. (e) Relative IL-33 mRNA expression in HBE cells evaluated by qRT-PCR. All experiments were performed two times independently, with n = 3 per experiment and three technical replicates. Shapiro–Wilk tests were used to test for normality, and one-way ANOVA was used for multiple unmatched groups, followed by Sidak’s test for multiple comparisons. Data are mean ± s.e.m. * p < 0.05 vs. the counterpart group.

Figure 8

CpG-ODN treatment decreases TSLP expression in HBE cells. (a) Representative images of TSLP (green) immunofluorescence of HBE cells (×200). Scale bars: 50 μm. Semiquantitative protein expression of TSLP detected by immunofluorescence, determined as IOD of TSLP (green)/number of cells. (b) Western blotting detection of TSLP against GAPDH (utilized for normalization). (c) TSLP/GAPDH ratio was determined. (d) TSLP amounts in HBE cell culture supernatants, examined by ELISA. (e) Relative TSLP mRNA expression in HBE cells, evaluated by qRT-PCR. All experiments were performed two times independently, with n = 3 per experiment and three technical replicates. Shapiro–Wilk tests were used to test for normality, and one-way ANOVA was used for multiple unmatched groups, followed by Sidak’s test for multiple comparisons. Data are mean ± s.e.m. * p < 0.05 vs. the counterpart group.