The Asthma Risk Gene, GSDMB, Promotes Mitochondrial DNA-induced ISGs Expression

Abstract

Released mitochondrial DNA (mtDNA) in cells activates cGAS-STING pathway, which induces expression of interferon-stimulated genes (ISGs) and thereby promotes inflammation, as frequently seen in asthmatic airways. However, whether the genetic determinant, Gasdermin B (GSDMB), the most replicated asthma risk gene, regulates this pathway remains unknown. We set out to determine whether and how GSDMB regulates mtDNA-activated cGAS-STING pathway and subsequent ISGs induction in human airway epithelial cells. Using qPCR, ELISA, native polyacrylamide gel electrophoresis, co-immunoprecipitation and immunofluorescence assays, we evaluated the regulation of GSDMB on cGAS-STING pathway in both BEAS-2B cells and primary normal human bronchial epithelial cells (nHBEs). mtDNA was extracted in plasma samples from human asthmatics and the correlation between mtDNA levels and eosinophil counts was analyzed. GSDMB is significantly associated with RANTES expression in asthmatic nasal epithelial brushing samples from the Genes-environments and Admixture in Latino Americans (GALA) II study. Over-expression of GSDMB promotes DNA-induced IFN and ISGs expression in bronchial epithelial BEAS-2B cells and nHBEs. Conversely, knockout of GSDMB led to weakened induction of interferon (IFNs) and ISGs in BEAS-2B cells. Mechanistically, GSDMB interacts with the C-terminus of STING, promoting the translocation of STING to Golgi, leading to the phosphorylation of IRF3 and induction of IFNs and ISGs. mtDNA copy number in serum from asthmatics was significantly correlated with blood eosinophil counts especially in male subjects. GSDMB promotes the activation of mtDNA and poly (dA:dT)-induced activation of cGAS-STING pathway in airway epithelial cells, leading to enhanced induction of ISGs.

Keywords: Airway inflammation; Asthma; GSDMB; ISGs; cGAS-STING pathway.

Figure 1.

GSDMB enhances the induction of IFNs and ISGs by dsDNA. (A,B) The mRNA (A) or protein levels of RANTES (B) were evaluated in BEAS-2B cells transfected with poly (dA:dT) for 6 hours (h). Both GFP-transfected control cells and stable GSDMB-overexpressing cells were used. (C,D) Expression of IFNs, including IFNβ, IFNλ1, IFNλ2/3 (C) and protein levels of IFNβ, IFNλ1/3 (D) were measured in BEAS-2B cells with overexpression of GSDMB and transfection of poly (dA:dT) for 6 h. (E,F) The mRNA (E) or protein levels of RANTES (F) were assessed in nHBEs with stable overexpression of GSDMB post transfection of poly (dA:dT) for 6 h. (G,H) Expression of IFNs, including IFNβ, IFNλ1, IFNλ2/3 (G) and protein levels of IFNλ 1/3 (H) were assessed in nHBEs transfected with poly (dA:dT) for 6 h with or without stable overexpression of GSDMB. (I) The levels of IRF3 and TBK1 phosphorylation were determined by immunoblotting in GFP- or GSDMB-overexpressing BEAS-2B cells post-transfection with poly (dA:dT) for 6 h. (J) IRF3 dimerization was examined in BEAS-2B cells overexpressing GSDMB and transfected with poly (dA:dT) for 0, 3, and 6 hours (h) by resolving protein extracts using native PAGE analysis. Cells expressing GFP were used as a control. Data in A-H is presented as means ± SEM from four independent biological replicates. *p < 0.05; **p < 0.01 (two-way ANOVA). The immunoblot data shown are representatives from two independent biological experiments.

Figure 2.

Deficiency of GSDMB attenuates dsDNA-induced ISGs expression. (A,B) Expression of RANTES and OASL (A) or protein levels of RANTES (B) were measured in empty vector (EV) or GSDMB knockout (KO) BEAS-2B cells transfected with poly (dA:dT) for 6 hours (h). (C,D) BEAS-2B cells with or without GSDMB expression were transfected with poly (dA:dT) for 6 h, and mRNA levels of IFNβ, IFNλ1 and IFNλ2/3 (C) or IFNβ and IFNλ protein levels (D) were measured by RT-PCR or ELISA respectively. (E) The phosphorylation of IRF3 and TBK1 was detected in empty vector (EV)- or GSDMB KO cells transfected with poly (dA:dT) for 6 h. (F) The IRF3 dimerization was examined in BEAS-2B cells with or without GSDMB expression after transfection with poly (dA:dT) at indicated time point. Cells expressing empty vector (EV) were used as a control. Means ± SEM shown in A-D were from three independent biological replicates. *p < 0.05; **p < 0.01 (two-way ANOVA). The immunoblot data shown in E-F were representative repeats from two independent biological experiments. KO#1 and KO#2 are two individual clones generated for GSDMB knockout after CRISPR/Cas-9 editing.

Figure 3.

GSDMB promotes mtDNA-induced ISG expression. (A) Correlation analysis between mtDNA copy number in plasma samples and eosinophil counts in plasma samples from asthmatic individuals from Costa Rico asthma cohort (GACRS). (B) Male subjects were included for the correlation analysis in A. (C–E) The mRNA levels of RANTES (C), IFNβ, IFNλ1 and IFNλ2/3 (D) as well as the protein level of IFNλ (E) were measured in BEAS-2B cells with overexpression of GSDMB and transfection of mitochondrial DNA (mtDNA) extracted from BEAS-2B cells for 12 h. (F–I) The mRNA (F) or protein levels of IFNs (G), as well as the mRNA (H) or protein levels (I) of RANTES were assessed in nHBEs with stable overexpression of GSDMB and transfected with mtDNA extracted from BEAS-2B cells for 12 h. (J) Expression of RANTES was measured in BEAS-2B cells with overexpression of GSDMB and transfection of HEK 293 cells-derived mtDNA for 12 h. Means ± SEM shown in C-J were from three independent biological replicates. *p < 0.05; **p < 0.01 (two-way ANOVA).

Figure 4.

GSDMB deficiency impairs mtDNA-induced ISGs expression. (A–C) BEAS-2B cells with or without knockout (KO) of GSDMB were transfected with mtDNA extracted from BEAS-2B for 12 h, and the mRNA levels of RANTES (A), IFNβ, IFNλ1 and IFNλ2/3 (B) or IFNλ protein levels (C) were then determined. (D) The mRNA levels of RANTES or protein levels of IFNλ were measured in empty vector (EV)-transfected or GSDMB KO BEAS-2B cells which were transfected with 293 mtDNA for 12 h. (E) Expression of RANTES was measured in EV or GSDMB KO BEAS-2B cells treated with ABT-737 (20 μM), S63845 (20 μM) and QVD-OPh (20 μM) for 3 h. (F) The mtDNA amount in total DNA extracts measured by qPCR in EV or GSDMB KO BEAS-2B cells, treated with or without ethidium bromide (EtBr) for 7 days. (G) The expression of RANTES in EV or GSDMB KO BEAS-2B cells depleted of mtDNA followed by treatment with ABT-737 (20 μM), S63845 (20 μM) and QVD-OPh (20 μM) for 3 h. Means ± SEM shown are from three independent biological replicates. *, p < 0.05; **, p < 0.01 (two-way ANOVA).

Figure 5.

GSDMB promotes dsDNA-induced ISGs expression and IFN production through the cGAS-STING pathway. (A) Expression of RANTES and OASL in empty vector (EV)-transfected or GSDMB KO BEAS-2B cells treated with IFNα (500 U/mL) for 24 h. (B) Secreted RANTEs protein was measured in the supernatant from GFP- or GSDMB-overexpressing BEAS-2B cells pretreated with various inhibitors for the cGAS/STING pathway: G140 (40 μM), H151 (20 μM) or BX795 (10 μM) for 1 h followed by transfection with poly (dA:dT) for 6 h. (C) Expression of RANTES and OASL measured in cells as described in B. (D) Expression of IFNβ, IFNλ1, IFNλ2/3 measured in cells as described in B. (E) Protein levels of IFNλ were measured in supernatant from cells described in B. Data are presented as means ± SEM from three independent biological replicates. *p < 0.05; **p < 0.01 (two-way ANOVA).

Figure 6.

GSDMB interacts with STING and promotes its translocation into Golgi. (A) Expression of RANTES in empty vector (EV)-transfected or GSDMB KO BEAS-2B treated with cGAMP. (B–D) Immunoprecipitation assay (IP) using anti-FLAG beads and immunoblotting with HA antibody in HEK293 cells transfected with FLAG-GSDMB and HA-STING (B) or FLAG-STING and HA-GSDMB (C) or FLAG-STING at various lengths (i.e., deletion mutants of STING) and HA-GSDMB (D), respectively. (E) IP assay using anti-FLAG beads in EV or GSDMB KO BEAS-2B cells transfected with Flag-STING and the proteins were further analyzed by immunoblotting with anti-TBK1 antibody. (F) Immunoprecipitation assay (IP) using anti-FLAG beads followed by immunoblotting using anti-GFP antibody in HEK293 cells co-transfected with Flag-STING, HA-GSDMB and GFP-STING to evaluate the dimerization of STING. (G–H) Quantification (G) on immunofluorescence staining (H) of Flag-tagged STING (green) and Golgi tracker Golgi-97 (Cell Signaling Technology) (red) in EV or GSDMB KO BEAS-2B cells transfected with Flag-STING for 48 h followed by transfection with poly (dA:dT) for 6 h. (G). The immunoblotting data shown are representative of two independent biological experiments. Each dot in G represents one independent view from total more than 12 independent views from four repeats in BEAS-2B cells.