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

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.

mtDNA Extraction and Quantification from Plasma Plasma samples (n = 375) from asthmatic subjects were thawed on ice and mixed briefly using a vortex mixer.Next, 50 µL of plasma was combined with 170 µL PBS and mixed thoroughly.The diluted sample was then centrifuged for 5 min at 4 degrees at 700 g.After centrifugation, 200 µL of supernatant was collected and further centrifuged for 15 min at 4 degrees at 18,000 g.The collected supernatants were used for DNA extraction and quantification as previously described [1], using the QIAamp DSP DNA Blood Mini Kit (Qiagen, Cat.61104).The quantity of mtDNA was then determined by qPCR, using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific).The primer sequences were provided in Table S2.The linear dsDNA standard was synthesized from IDT to measure copy number of human mitochondrially encoded NADH dehydrogenase 1 (MTND1).The sequence is: 5'-AACAACATACCCATGGCCAACCTCCTACTCCTCATTGTACCCATTCTAATCGCAATGGCATTCCTAATGC TTACCGAACGAAAAATTCTAGGCTATATACAACTACGCAAAGGCCCCAACGTTGT -3'.

Quantitative RT-PCR
Total RNA was extracted using the Direct-zol™ RNA MiniPrep with TRI-Reagent® (Zymo Research) following the manufacturer's instructions.To perform the RT-PCR analysis, cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit with RNase and then gene expression was measured by either Applied Biosystems Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) or TaqMan™ Fast Advanced Master Mix (LIFE TECHNOLOGIES CORPORATION).The expression levels of all genes were normalized to GAPDH.human GAPDH (Hs.PT.39a.22214836) and human XBP1 (Hs.PT.58.1903847) probes are purchased from Integrated DNA Technologies IDT.Primer sequences were provided in Table S2.

Immunoprecipitation and Immunoblot Analysis
For immunoprecipitation, total cellular protein was extracted from cells transfected with plasmids or poly (dA:dT).The extracts were then lysed in low-salt lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100), and incubated overnight with anti-FLAG beads (Sigma-Aldrich).The beads were washed three to five times with low-salt lysis buffer, and the immunoprecipitates were washed then loaded with 4x Laemmli Sample Buffer (BioRad) and separated by pre-cast 5-12% SDS-PAGE gel (Bio-Rad, #4561093 and #4561096).The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad) and further probed with the relevant antibodies listed in Table S1.Proteins was detected by Thermo Scientific SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific) or Thermo Scientific Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and images were captured using the G:Box system (Syngene).

Native Polyacrylamide Gel Electrophoresis (PAGE) Analysis of Oligomers
IRF3 oligomers and dimers were detected using native PAGE in BEASE-2B cells with stable overexpression or knockout of GSDMB.Briefly, cellular protein extracts were obtained from BEAS-2B cells after transfection with poly (dA:dT) at designated time points using a low-salt lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2 , 10% glycerol, 1% Triton X-100).The samples were then mixed with Native Sample Buffer (Bio-Rad) and separated on a pre-cast 4-20% Mini-PROTEAN TGX Gel (Bio-Rad), using TGS buffer (Thermo Fisher Scientific) for 30 min before the proteins were transferred to PVDF membranes (Bio-Rad) and detected with the corresponding antibodies including IRF3, STING, GFP and TBK1 (Table S1) by Western Blotting as described above.
Table S3.Differential correlation analysis of GSDMB with various interferon stimulated genes (ISGs) in asthmatic and control samples from Genes-environments and Admixture in Latino Americans (GALA) II study.

Cytotoxicity Assay
After cells were transfected with poly (dA:dT) for 6 h, supernatants were collected and the level of cell death was then determined by measurements of the lactate dehydrogenase (LDH) release using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) based on the manufacture's protocol.

Figure S1 .
Figure S1.GSDMB promotes dsDNA-induced ISG expression.(A) The mRNA level of OASL was measured in BEAS-2B cells transfected with poly (dA:dT) for 6 h.Both control cells stably transfected with GFP and GSDMB-overexpressing cells were used.(B) Expression of GSDMB in nHBEs with stable overexpression of GSDMB as assessed by qPCR.Mean ± SEMs shown from at least three independent biological replicates.*p < 0.05; **p < 0.01 (two-way ANOVA).

Figure S2 .
Figure S2.Overexpression of GSDMB showed no impacts on dsDNA-induced cell death.(A) Phase contrast images of BEAS-2B cells with or without overexpression of GSDMB after transfection with poly (dA:dT) (2 µg/mL) for 6 h.Cells in red color have positive staining with propidium iodide (PI) indicative of dead cells.(B) Cell death measurements by lactate dehydrogenase (LDH) release culture supernatant in BEAS-2B cells transfected with poly (dA:dT) for 6 h.(C) Measurements of IL-1β in supernatant from BEAS-2B cells with or without GSDMB overexpression transfected with poly (dA:dT) (2 µg/mL) for 6 h.THP-1 cells stimulated with phorbol 12-myristate 13-acetate (PMA) (100 ng/mL) overnight followed by subsequent transfection of poly (dA:dT) (2 µg/mL) for 6 h were used as a positive control for IL-1β secretion.(D) Expression of RANTES (Left) and OASL (Right) in BEAS-2B cells, with or without overexpression of GSDMB and transfected with poly (dA:dT) (2 µg/mL) for 6 h with or without pretreatment of YVAD (50 µM) for 1 h.Error bars represent standard errors of mean(SEM) from four independent biological replicates.*p < 0.05; **p < 0.01 (two-way ANOVA).

Figure S4 .
Figure S4.GSDMB elicits mtDNA-induced interferon response.(A) Female subjects were included for the 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) Expression of OASL was measured in BEAS-2B cells with overexpression of GSDMB after transfection of mtDNA derived from BEAS-2B cells for 12 h.(C) Expression of IFNλ was measured in BEAS-2B cells with overexpression of GSDMB after transfection of mtDNA extracted from HEK 293 cells for 12 h.

Figure S7 .
Figure S7.Mitochondrial apoptosis-induced IFN response depends on cGAS-STING pathway.Measurement of RANTES expression in BEAS-2B cells pretreated with 40 M G140, 20 M H151 or 10 M BX795 for 1 h followed by additional treatment of 20 M ABT-737, 20 M S63845 and 20 M QVD-OPh for 3 h.Mean expression of RANTES from three independent experiments was shown with standard errors for the mean.*p < 0.05 (Student's t-test),

Table S1 .
Antibodies used in Western Blotting.

Table S2 .
Primers used in RT-PCR assays.

Table S4 .
Demographics (mean±SDs) of subjects in Costa Rica asthma cohort.