GSDMB induces an asthma phenotype characterized by increased airway responsiveness and remodeling without lung inflammation

Abstract

Gasdermin B (GSDMB) on chromosome 17q21 demonstrates a strong genetic linkage to asthma, but its function in asthma is unknown. Here we identified that GSDMB is highly expressed in lung bronchial epithelium in human asthma. Overexpression of GSDMB in primary human bronchial epithelium increased expression of genes important to both airway remodeling [TGF-β1, 5-lipoxygenase (5-LO)] and airway-hyperresponsiveness (AHR) (5-LO). Interestingly, hGSDMB^Zp3-Cre mice expressing increased levels of the human GSDMB transgene showed a significant spontaneous increase in AHR and a significant spontaneous increase in airway remodeling, with increased smooth muscle mass and increased fibrosis in the absence of airway inflammation. In addition, hGSDMB^Zp3-Cre mice showed increases in the same remodeling and AHR mediators (TGF-β1, 5-LO) observed in vitro in GSDMB-overexpressing epithelial cells. GSDMB induces TGF-β1 expression via induction of 5-LO, because knockdown of 5-LO in epithelial cells overexpressing GSDMB inhibited TGF-β1 expression. These studies demonstrate that GSDMB, a gene highly linked to asthma but whose function in asthma is previously unknown, regulates AHR and airway remodeling without airway inflammation through a previously unrecognized pathway in which GSDMB induces 5-LO to induce TGF-β1 in bronchial epithelium.

Keywords: GSDMB; airway-hyperresponsiveness; asthma; inflammation; remodeling.

Fig. 1.

GSDMB is highly expressed in human bronchial epithelial cells in asthma. Human lungs from (A) healthy controls and (B) asthmatic patients were examined by immunohistochemistry with an anti-GSDMB Ab (n = 7 subjects per group). (Magnification: A and B, 200×; Insets, 400×.) (C) The number of peribronchial GSDMB⁺ cells were quantitated in each group by light microscopy (LM) and image analysis. (D) GSDMB⁺ cells in bronchial biopsies from healthy controls, mild asthmatics, and severe asthmatics were quantitated by LM (n = 7 subjects per group). (E) Relative quantification of GSDMB-1 mRNA in primary human bronchial epithelial cells (BEC), alveolar epithelial cells (AEC), macrophages (Mac), smooth muscle cells (SMC), and fibroblasts (Fib). (F) RNA from bronchial epithelial cells was examined using primers specific to GSDMB-1, -2, -3, and -4 isoforms (Table S1). GAPDH mRNA was used as normalization control. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. Fold-change is expressed as mean ± SEM and results are from three independent experiments.

Fig. S1.

GSDMB-1 overexpression in primary human bronchial epithelium cells activates HSPs, CXC chemokines, and CC chemokines. (A) Schematic representation of the exon–intron structure and the alternative splicing isoforms of the human GSDMB gene, as predicted by the National Center for Biotechnology Information (NCBI) database. A black arrow marks the translational start site of GSDMB in exon 2 (e2). Blue boxes represent the 12 coding exons (e1–e12), gray boxes show untranslated regions, and the introns are indicated by solid lines. The alternative processing of exons 6 and 7 (e6 and e7) in the GSDMB isoforms 1, 2, and 4 are represented by dotted lines. The nuclear localization signal is depicted as NLS. (B–D) Primary bronchial epithelial cells were transfected with either empty, GSDMB-1, or GSDMB-1 vector lacking nuclear localization signal (GSDMB-1 ΔNLS) for 72 h. qPCR was performed to measure the mRNA levels of (B) HSPs, HSP60 and HSP70; (C) CXC chemokines, CXCL6 and CXCL17; and (D) CC chemokines, CCL11, CCL26, and CCL28. β-Actin mRNA was used as normalization control and fold-change is expressed as mean ± SEM from four independent experiments. *P < 0.05; NS, not significant.

Fig. 2.

Nuclear localization of GSDMB activates TGF-β1, 5-LO, and MMP9 in human bronchial epithelial cells transfected with GSDMB. Primary bronchial epithelial cells were transfected with either empty, GSDMB-1, or GSDMB-1 vector lacking nuclear localization signal (GSDMB-1 ΔNLS) for 72 h and qPCR was performed to measure (A) TGF-β1, (B) 5-LO, and (C) MMP9 mRNA. β-Actin mRNA was used as normalization control. Fold-change is expressed as mean ± SEM from four independent experiments. ELISA was performed to measure (D) active TGF-β1, (E) 5-LO, and (F) MMP9 levels in cells transfected with GSDMB-1 or empty vector. (G) Quantification of cellular localization of GSDMB in bronchial epithelial cells by Western blot. β-Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear extracts, respectively. The purity of these extracts was checked using HSP90, a cytoplasmic marker, and p53, a nuclear marker. Western blot image is a representative of three independent experiments. (H) Bronchial epithelial cells were transfected with RFP-tagged GSDMB-1, GSDMB-1 ΔNLS, or empty vector (red) and cells were mounted with DAPI (blue). (Magnification: 20×.) (I–L) Bronchial epithelial cells overexpressing GSDMB-1 or empty vector were transfected with scrambled (scr.) or 5-LO siRNA. (I) Efficiency of 5-LO mRNA knockdown was assessed by qPCR. β-Actin mRNA was used as normalization control. Protein levels of (J) active TGF-β1, (K) MMP9, and (L) 5-LO were measured by ELISA. Results are expressed as mean ± SEM from three independent experiments. *P < 0.05; **P < 0.01; NS, not significant.

Fig. S2.

Overexpression of 5-LO in primary human bronchial epithelium cells induces TGF-β1 expression. Primary human bronchial epithelial cells were transfected with either empty vector or 5-LO–overexpressing vector for 72 h. Transfected cells were examined to assess the levels of (A) TGF-β1 mRNA by qPCR and β-actin mRNA was used as the normalization control. (B) Active TGF-β1 protein levels were measured by ELISA. *P < 0.05 and results are expressed as mean ± SEM from two independent experiments.

Fig. S3.

Generation of mice expressing human GSDMB transgene (hGSDMB^Zp3-Cre). (A–D) Generation of hGSDMB^Zp3-Cre mice. (A) The human GSDMB Tg construct contains a CAG promoter for universal overexpression (blue), H2B-RFP (red), followed by a transcriptional stop site (yellow), the human GSDMB ORF (green), and rabbit β-globin polyadenylation sequence (purple). The H2B-RFP and transcriptional stop sites are flanked by LoxP sites (red triangles). Cells containing this pCAGEN Lox RFP-H2B STOP Lox hGSDMB construct will express RFP and not hGSDMB, as the transcriptional stop codon (yellow) prevents transcription of hGSDMB. (B) Expression of the hGSDMB gene is Cre recombinase-dependent because the transcriptional stop codon preventing hGSDMB expression and H2B-RFP are excised by Cre recombinase via the LoxP sites. This results in overexpression of hGSDMB only in those cells that express Cre recombinase. (C) Primer sets (F1, R; F2, R) used to detect progeny of RFP-Stop^FLhGSDMB-Tg mice crossed with Zp3-Cre mouse and predicted sizes of transgene DNA as assessed by PCR. (D) PCR products run on 1.5% agarose gel, showing a PCR product band for hGSDMB^Zp3-Cre mice. (E) WT mice (Left) and hGSDMB^Zp3-Cre mice (Right) aged 12 wk appeared morphologically similar. (F) The gross appearance of lungs of WT mice (Left) and hGSDMB^Zp3-Cre mice (Right) aged 12 wk appeared morphologically similar. (G) Lung extracts from WT and hGSDMB^Zp3-Cre mice were examined by Western blot to quantitate the expression of human GSDMB protein. Mouse GAPDH was used as the loading control. Western blot image is a representative of two independent experiments.

Fig. 3.

hGSDMB^Zp3-Cre mice exhibit increased AHR, with an increase in peribronchial smooth muscle and collagen deposition without airway inflammation. (A) AHR to MCh and (B) BAL inflammatory cells were assessed in intubated and ventilated WT and hGSDMB^Zp3-Cre mice. (C) Levels of peribronchial smooth muscle were quantitated by immunohistochemistry using an anti–α-smooth muscle actin Ab and (D) image analysis. Results are expressed as the α-smooth muscle actin-stained area (μm²) per circumference (μm) of basement membrane of bronchioles, 150- to 250-μm internal diameter in WT and hGSDMB^Zp3-Cre mice. (E) Levels of peribronchial trichrome staining were imaged using LM and (F) similarly quantitated by image analysis. (G) Levels of mucin were examined by PAS staining and (H) quantitated by image analysis. (Magnification: C, E, and G, 200×; Insets, 400×.) **P < 0.01; ***P < 0.001; NS, not significant. n = 9–12 mice per group. Results are expressed as mean ± SEM from four independent experiments.

Fig. S4.

Multiple hGSDMB^Zp3-Cre mouse lines does not show any significant differences in pathways associated with airway remodeling in asthma. (A) Lungs of WT and hGSDMB^Zp3-Cre mice were examined to assess the levels of GSDM family genes, Gsdma, Gsdmc, and Gsdmd by qPCR. β-Actin mRNA was used as the normalization control. (B–D) To exclude the random genomic integration or copy number contributing to the observed phenotype in hGSDMB^Zp3-Cre mice, three lines of GSDMB transgenic animals (lines 1, 2, and 3) were examined to assess the protein levels of (B) serum 5-LO, (C) lung MMP9, and (D) lung TGF-β1 by ELISA. Results are expressed as mean ± SEM from four independent experiments. *P < 0.05; NS, not significant; n = 9–12 mice per group.

Fig. S5.

Lungs of hGSDMB^Zp3-Cre mice have increased expression of pathways associated with airway remodeling in asthma: TGF-β1, MMP9, 5-LO, and cysteinyl leukotrienes. WT and hGSDMB^Zp3-Cre mice were examined to assess the levels of (A) lung TGF-β1 mRNA by qPCR. ELISA was used to measure the levels of (B) lung TGF-β1 and (C) serum TGF-β1. Levels of (D) lung MMP9 mRNA were measured by qPCR, and the protein levels of (E) lung MMP9 and (F) serum MMP9 were measured by ELISA. (G) Levels of lung 5-LO mRNA in mouse lungs were measured by qPCR. (H) Levels of 5-LO and downstream pathway mediators, (I) cysteinyl leukotrienes (LTC4/D4/E4), and (J) LTB4 were measured in serum samples from WT and hGSDMB^Zp3-Cre mice by ELISA. Levels of (K) lung IL-13 mRNA were measured by qPCR and (L) lung IL-13 protein levels were measured by ELISA. β-Actin mRNA was used as normalization control for qPCR analyses and results are expressed as mean ± SEM from four independent experiments. *P < 0.05; **P < 0.01; NS, not significant; n = 9–12 mice per group.

Fig. S6.

hGSDMB^Zp3-Cre mice have increased levels of serum IgE, lung HSP Hspd1, CXC chemokines Cxcl5 and Cxcl17, and CC chemokine Ccl28. (A) Levels of total IgE in WT and hGSDMB^Zp3-Cre mice were measured in serum samples by ELISA. (B–D) Lungs of WT and hGSDMB^Zp3-Cre mice were examined by qPCR to assess the levels of (B) mouse HSPs, Hspd1 (mouse ortholog of human HSP60) and Hspa2 (mouse ortholog of human HSP70), (C) mouse CXC chemokines Cxcl5 (mouse ortholog of human CXCL6) and Cxcl17, and (D) mouse CC chemokines Ccl11 (mouse ortholog of human eotaxin-1), Ccl26 (mouse ortholog of human eotaxin-3), and Ccl28. β-Actin mRNA was used as the normalization control for qPCR analyses. Lung protein extracts from WT and hGSDMB^Zp3-Cre mice were examined by ELISA to assess the protein levels of (E) mouse Cxcl5 and Cxcl17, and (F) mouse Ccl28. *P < 0.05; **P < 0.01; NS, not significant; n = 9–12 mice per group. Results are expressed as mean ± SEM from four independent experiments.

Fig. 4.

Mice expressing human GSDMB transgene exhibit increased AHR, airway inflammation, Th2 cytokines, and IgE upon dust mite allergen challenge. (A) Primary human bronchial epithelial cells were incubated with either HDM (25 μg/mL) or LPS (100 ng/mL) for 48 h and GSDMB-1 mRNA levels were measured by qPCR. Cells treated with media alone served as experimental control. β-Actin mRNA was used as normalization control and fold-change is expressed as mean ± SEM. (B) WT and hGSDMB^Zp3-Cre mice were administered either HDM extract (100 μg) or PBS via intranasal challenges on days 0, 7, 14, and 21. AHR to MCh was assessed on day 24 in intubated and ventilated mice. Lungs of WT and hGSDMB^Zp3-Cre mice challenged with or without HDM were examined for the number of lung (C) MBP⁺ peribronchial eosinophils, (D) CD4⁺ T cells, and (E) NE⁺ neutrophils per bronchiole of 150- to 250-μm internal diameter by immunohistochemistry and quantitated by image analysis. Levels of (F) lung IL-4, (G) lung IL-13, and (H) total serum IgE were quantitated by ELISA. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. n = 9–12 mice per group. Results are expressed as mean ± SEM from four independent experiments.

Fig. S7.

hGSDMB^Zp3-Cre mice exhibit increased infiltration of inflammatory cells upon dust mite allergen challenge. (A) Schematic representation of mouse model of HDM-induced asthma. WT and hGSDMB^Zp3-Cre mice aged 8–9 wk were administered either HDM extract (100 μg) or PBS via intranasal (i.n.) challenges on days 0, 7, 14, and 21. AHR to MCh was assessed on day 24 in intubated and ventilated mice (flexiVent ventilator, Scireq). The number of (B) eosinophils, (C) lymphocytes, and (D) neutrophils in BAL was assessed by Giemsa staining. Differential cell counts were determined by light microscopy. (E) Levels of lung IL-5 in WT and hGSDMB^Zp3-Cre mice were quantitated by ELISA. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant, n = 9–12 mice per group. Results are expressed as mean ± SEM from four independent experiments.

Fig. S8.

Lungs of hGSDMB^Zp3-Cre mice did not show a significant IFN-γ response with or without HDM allergen challenge. Lungs of WT and hGSDMB^Zp3-Cre mice were examined to assess the levels of IFN-γ. (A) Lung IFN-γ protein levels were measured by ELISA in mice aged 8, 12, and 24 wk of age without HDM-challenge. (B) Lung IFN-γ levels were measured in HDM-challenged mice at 12 wk of age by ELISA. NS, not significant; n = 9–12 mice per group. Results are expressed as mean ± SEM from four independent experiments.

Fig. S9.

HDM challenge in hGSDMB^Zp3-Cre mice induced airway inflammation, and further increased peribronchial smooth muscle and collagen deposition. Lungs of WT and hGSDMB^Zp3-Cre mice challenged with or without HDM were examined for (A) the levels of mucin by PAS staining and (B) quantitated by image analysis. (C) Levels of peribronchial smooth muscle were quantitated by immunohistochemistry using an anti–α-smooth muscle actin Ab and (D) image analysis. Results are expressed as the α-smooth muscle actin-stained area (μm²) per circumference (μm) of basement membrane of bronchioles, 150- to 250-μm internal diameter in WT and hGSDMB^Zp3-Cre mice. (E) Levels of peribronchial trichrome staining were imaged using LM and (F) similarly quantitated by image analysis. (Magnification: A, C, and E, 200×.) *P < 0.05; **P < 0.01; ***P < 0.001; n = 9–12 mice per group. Results are expressed as mean ± SEM from four independent experiments.