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

Liu, Tao; Hecker, Julian; Liu, Siqi; Rui, Xianliang; Boyer, Nathan; Wang, Jennifer; Yu, Yuzhen; Zhang, Yihan; Mou, Hongmei; Gomez-Escobar, Luis  Guillermo; Choi, Augustine  M.K.; Raby, Benjamin  A.; Weiss, Scott  T.; Zhou, Xiaobo

doi:10.35534/jrbtm.2024.10005

Journal of Respiratory Biology and Translational Medicine

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

Tao Liu ¹

Julian Hecker ¹ Siqi Liu ¹

Xianliang Rui ¹

Nathan Boyer ¹

Jennifer Wang ¹ Yuzhen Yu ¹ Yihan Zhang ² Hongmei Mou ²

Luis Guillermo Gomez-Escobar ³ Augustine M.K. Choi ³ Benjamin A. Raby ⁴ Scott T. Weiss ¹

Xiaobo Zhou ¹^,*

Author Information

Channing Division of Network Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA

The Mucosal Immunology and Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02115, USA

Weil Cornell Medical School, Joan and Sanford I. Weill Department of Medicine, New York, NY 10065, USA

⁴

Division of Pulmonary Medicine, Department of Pediatrics, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA

Authors to whom correspondence should be addressed.

PMCID:PMC11086750

PMID:38737375

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Journal of Respiratory Biology and Translational Medicine 2024, 1 (1), 10005; https://doi.org/10.35534/jrbtm.2024.10005

Received: 27 February 2024 Accepted: 22 March 2024 Published: 31 March 2024

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 translocalization 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: cGAS-STING pathway, GSDMB, ISGs, Asthma, Airway inflammation

Graphical Abstract

1. Introduction

Asthma, the most prevalent airway disease in school-age children, imposes a significant economic burden globally [1]. Despite effective treatment to manage asthma symptoms, the disease persists with no cure. Genetic predisposition determines the risk for individuals to develop asthma with single nucleotide polymorphisms (SNPs) at chromosome 17q21 as the most reproducible and significant asthma susceptibility locus associated with early-onset childhood asthma [2]. Risk alleles in these 17q21 SNPs are associated with asthma severity and exacerbation and increased expression of the gasdermin B (GSDMB) gene in airway epithelial cells [3,4,5,6]. Pyroptosis is a form of inflammatory cell death characterized by Gasdermins (GSDMs)-mediated cell lysis and involves the activation of inflammatory caspases [7,8]. GSDMB, unique to humans without mouse homolog, belongs to Gasdermins (GSDMs) family, which induces pyroptosis in tumors or upon bacterial infections [9,10,11,12]. Non-pyroptosis-related function of GSDMB was reported in inflammatory bowel disease (IBD), through regulation of restoration of the intestinal lining [13,14]. Interestingly, over expression of human GSDMB isoform 1 lacking exon 6 in mice led to airway remodeling without profound inflammation [13]. However, whether and how full length GSDMB promotes airway inflammation in patients with asthma remains unclear. Proper activation of cGAS-STING pathway is a vital mechanism responds to aberrant cytosolic DNA, which are majorly originated from DNA virus or dysregulated self-DNA. However, over-activated cGAS-STING pathway promotes inflammation that accelerates a variety of inflammatory diseases, such as amyotrophic lateral sclerosis [15], doxorubicin-induced cardiotoxicity (DIC) [16], or systemic lupus erythematosus [17]. Mechanically, cGAS directly binds to DNA, converting guanosine 5´-triphosphate (GTP) and adenosine 5´-triphosphate (ATP) into the second messenger 2’ 3’ cyclic GMP-AMP (cGAMP), which binds to and activates stimulator of interferon genes (STING) [18]. Subsequently, STING oligomerizes and translocates from the endoplasmic reticulum (ER) to the Golgi apparatus where it recruits TBK1, phosphorylating IRF3 [19,20,21,22,23,24,25,26], leading to the translocation of phosphorylated and dimerized IRF3 into nucleus activating the transcription of type I and type III IFNs, inducing expression of ISGs [27,28,29,30]. The levels of ISGs expression, are elevated in asthma and associated with reduced lung function and ER stress [31,32]. For example, RANTES (also known as CCL5), an important pro-inflammatory ISG with increased expression in asthmatics, contributes to the recruitment of eosinophils in allergic airway inflammation [33,34,35,36]. Also, RANTES contribute to neutrophilic inflammation in asthma, potentially serving as a bridge between type 1 and type 2 inflammation [37]. Moreover, targeting RANTES-responsive cells within the lung prevents chronic fungal asthma [38]. However, how ISGs is dysregulated in asthma remain unclear. In various pulmonary diseases, including coronavirus disease 2019 (COVID-19) [39], idiopathic pulmonary fibrosis (IPF) [40,41], extrapulmonary sarcoidosis [42], as well as asthma [43,44,45,46], aberrant release of mitochondrial DNA (mtDNA) through the BAK/BAX macropores was reported, which is a potent agonist of cGAS-STING pathway, potentiating inflammation in response to stress or injury [47,48,49,50,51,52]. While the genetic analyses have identified the association between GSDMB and interferon response [53], the molecular mechanisms and ensuing biological effects of GSDMB in mtDNA-induced cGAS-STING pathway in the context of asthma remain largely undefined. In this study, we found that GSDMB promotes mtDNA-induced IFN production and subsequent ISGs activation through regulating the cGAS-STING pathway in human bronchial epithelial cells. Mechanically, GSDMB interacts with STING, facilitating its Golgi localization and subsequent TBK1 recruitment, without affecting cGAMP binding or STING oligomerization. Hence, our findings provide novel molecular insights into how the genetic determinant GSDMB regulates lung inflammation in asthma development, suggesting a promising therapeutic strategy for treating airway inflammation in genetically susceptible asthmatics.

2. Materials and Methods

2.1. Cells HEK293, HEK293FT and BEAS-2B cells were grown in DMEM (Life Technologies; Carlsbad, CA, USA) with 10% (v/v) fetal bovine serum (Thermo Fisher;Pittsburgh, PA, USA), 1% Penicillin-Streptomycin-Glutamine (Thermo Fisher). As previously outlined [54,55], normal human bronchial epithelial cells (nHBEs) were cultured in Small Airway Epithelial Cell Medium (Promocell; Heidelberg, Germany) with the addition of 1.0 µM A8301(Tocris Bioscience; Bristol, UK), 0.5 µM CHIR99021(Sigma-Aldrich, St Louis, MO, USA) and 5 µM Y27632 (Sigma-Aldrich) on plates pre-coated with laminin-enriched 804G-conditioned medium. 2.2. GSDMB Stable Overexpression Stable cell lines with GSDMB-overexpression were established by infecting BEAS-2B cells or nHBEs with Gasdermin like (GSDMB) Human Tagged ORF Clone Lentiviral Particle (Cat. RC219044L1V, Origene; Rockville, MD, USA). The expression of GSDMB in the cells was measured by Western blot analysis or qPCR. All details for antibodies are listed in Table S1. 2.3. Generation of Mitochondrial DNA Deficient (ρ0) Cells To deplete mtDNA from cells, BEAS-2B cells were treated with EtBr (100 ng/mL, Sigma-Aldrich) in medium supplemented with pyruvate (100 μg/mL, Sigma-Aldrich) and uridine (50 μg/mL, Sigma-Aldrich) for 7 days at 37 °C with 5% CO₂ in an incubator. The total DNA of indicated cells was obtained by using QIAamp DNA Micro Kit (QIAGEN; Venlo, The Netherlands) per the manufacturer's instructions. The depletion of mitochondrial DNA was quantified by RT-PCR with mtDNA (D-LOOP) primer as previously described [43] (Table S2). 2.4. mtDNA Extraction from Cultured Cells Mitochondria were extracted from BEAS-2B cells or HEK 293 cells using the mitochondrial isolation kit (Abcam, # ab110170; Waltham, MA, USA) based on manufacture’s protocol. The freshly isolated mitochondria were then purified with the QIAamp DSP DNA Blood Mini Kit (Qiagen, Cat. No. 61104) according to the manufacturer's instructions. 2.5. Gene Expression Correlation Analysis in Nasal Epithelial Cells RNA-sequencing data in nasal epithelium was obtained from the Genes-environments & Admixture in Latino Americans (GALA) II study (n = 695, GSE152004) [56]. This dataset contains normalized expression data for 441 asthmatics and 254 healthy controls. We extracted the expression data for the 12 genes including RANTES, CCL8, CXCL9, CXCL10, ISG15, ISG20, GBP1, GBP2, IRF1, IRF7, IFNγ and IFNλ2. We then computed the log2 transformed values of gene expression for correlation analysis between GSDMB and other genes based on the log2 transformed values. To determine whether co-expression differs between cases and controls, we applied a permutation-based approach where the observed absolute difference in co-expressions between cases and controls (measured as the Pearson correlation of the log2 transformed data) is compared to the corresponding absolute difference after shuffling the case/control status randomly. Based on 10,000 permutations, we computed empirical p-values for this comparison. 2.6. Correlation Analysis between mtDNA and Various Clinical Phenotypes in Asthmatics mtDNA was extracted and quantified from plasma samples from asthmatic subjects enrolled in the Costa Rican Asthma cohort. The study of this cohort was approved by the Institutional Review Boards of the Brigham and Women’s Hospital (Boston, MA) entitled “the Genetic Epidemiology of Asthma in Costa Rica”: Protocol #:2000P001130. To analyze the correlation of mtDNA copy number with various clinical characteristic in asthma patients (n = 375), we first filtered out samples with extremely high mitochondrial DNA (mtDNA) measurements (> 10,000 copies/µL plasma). For final analysis, 357 samples were kept for the association analysis between mtDNA levels and various asthma-related outcomes. We also performed an association analysis between mtDNA and eosinophil counts in asthmatics. Both analyses adjusted for age, sex, and height.

3. Results

3.1. GSDMB Elicits dsDNA-induced ISGs Expression in Human Airway Epithelium Increased ISGs expression and hyper-inflammation in airways are closely linked to the decline of lung function in asthma [32,57]. To determine whether GSDMB regulates ISGs expression, we engineered a GSDMB-over-expressing bronchial epithelial BEAS-2B cell line. As compared to the control group, upon transfection of poly (dA:dT), GSDMB-over-expressing cells showed exuberant induction of ISGs including RANTES and 2'-5'-Oligoadenylate Synthetase Like (OASL) (Figure 1A,B and Figure S1A). Moreover, expression of RANTES was more significantly correlated with GSDMB in asthmatic nasal epithelial cells compared to control subjects in the Genes-environments and Admixture in Latino Americans (GALA) II study (n = 254 controls and 441 with asthma) [56,58] (Table S3). In addition to ISGs, overexpression of GSDMB also resulted in the remarkable induction of IFNs (IFNβ, IFNλ1 and IFNλ2/3) (Figure 1C,D). Furthermore, overexpression of GSDMB (Figure S1B) promotes induction of RANTES and IFNs in nHBEs (Figure 1E,F and 1G,H). Consistently, the phosphorylated TBK1/IRF3 and subsequent dimerization of IRF3, important markers to indicate IFN response [59], are significantly upregulated in GSDMB-overexpressing cells (Figure 1I,J), suggesting that GSDMB promotes induction of IFNs and ISGs by poly (dA:dT) through activating the 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.

3.2. GSDMB Promotes cGAS-STING Signaling Independent of Pyroptosis, without Affecting ER Stress Given that GSDMB has been previously documented to facilitate inflammasome activation [11,60,61,62,63], leading to pyroptosis and may consequently amplify subsequent ISGs expression [62,64], we thus evaluated whether the regulation of the cGAS/STING pathway by GSDMB depends on pyroptosis. Surprisingly, no evidence showed that GSDMB promotes poly (dA:dT) induced pyroptosis, indicated by intake of propidium iodide (PI) (Figure S2A), rupture of the cell membrane (Figure S2A), release of lactate dehydrogenase (LDH) (Figure S2B) and secretion of interleukin-1β (IL-1β) (Figure S2C). In line with this, the caspase-1 inhibitor YVAD failed to inhibit the induction of RANTES by poly (dA:dT) (Figure S2D). This observation indicates that inflammasome activation is likely dispensable for GSDMB's regulation on poly (dA:dT)-induced activation of cGAS/STING pathway in BEAS-2B cells. Endoplasmic reticulum (ER) stress is a hall-marker of inflammation and exacerbates tissue damage in a wide spectrum of human diseases [65]. In asthma, particularly among patients with mild asthma, ER stress is associated with both type 2 inflammation and ISG expression [32]. As ER stress could be triggered by an overexpression system [66], which can further regulate the interferon (IFN) response, we conducted an assessment of ER stress in the presence or absence of GSDMB. However, comparable expression levels of the ER stress marker, splicing of X-Box binding protein 1 (XBP1) observed in both control and GSDMB-overexpressing cells, indicating no effects of GSDMB on the activation of ER stress (Figure S3). 3.3. Deficiency of GSDMB Suppresses IFN Response and ISGs Activation Additionally, stable knockout (KO) of GSDMB by CRISPR-Cas9 editing dampened induction of ISGs as well as IFNs in BEAS-2B cells transfected with poly (dA:dT) (Figure 2A–D). Furthermore, the phosphorylated TBK1/IRF3 and phosphorylation-dependent dimerization of IRF3 were also subdued in GSDMB KO cells (Figure 2E,F), indicating that GSDMB is indeed required by the double stranded DNA such as poly (dA:dT)-induced activation of cGAS-STING pathway, as well as subsequent induction of IFNs and ISGs.

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.

3.4. GSDMB Facilitates mtDNA-induced IFN Response and ISGs Activation Cell free mtDNA, one major source of double stranded DNA that cells may expose to, has been detected in plasma samples or bronchoalveolar lavage fluid (BALF) from patients with asthma [45,67,68]. We, therefore, extracted and quantified the cell free mtDNA in asthmatic plasma samples (n = 357) from the Costa Rico asthma cohort (GACRS) [69], a relatively mild asthmatic cohort (Table S4). We then compared the correlation of mtDNA levels with multiple clinical characteristics in asthmatics. With limited sample size, we found that mtDNA copy number significantly correlates with eosinophil counts (Figure 3A), mainly in the male, but not female asthmatic samples (Figure 3B and Figure S4A). We therefore set out to determine whether GSDMB regulates mtDNA-induced IFN response and ISGs expression [44]. Firstly, we extracted mtDNA from the bronchial epithelial BEAS-2B cell line and transfected them into GSDMB-overexpressing cells. Similar as poly (dA:dT), transfected BEAS-2B mtDNA induced robust ISGs and IFNs production, which was enhanced by overexpression of GSDMB in both BEAS-2B cells (Figure 3C–E and Figure S4B ) and nHBEs cells (Figure 3F,I). Furthermore, mtDNA extracted from HEK293 cells also activated significant induction of ISGs and IFNs, especially in GSDMB-overexpressing cells (Figure 3J and Figure S4C). Conversely, knockout of GSDMB alleviated mtDNA-induced IFN production and ISGs expression in BEAS-2B cells (Figure 4A–D and Figure S5A–C).

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).

In addition to exogenously transfected mtDNA, we also attempted to induce release of cellular mtDNA by various stimulation. We first employed IL-1β, a pleiotropic inflammatory cytokine that was reported to promote mtDNA release in cancer cell line and immune cells [70]. However, no remarkable mtDNA release was detected in BEAS-2B cells treated with IL-1β (Figure S6). As an alternative approach, we treated cells with QVD-OPh, S63845 and ABT-737 by combined inhibition on pan-caspase, myeloid leukemia 1 (MCL-1) and B-cell lymphoma xL (BCL-xL)/BCL-2/BCL-w respectively, as previously reported for inducing mitochondrial apoptosis and subsequent mtDNA release in cancer cells and primary mouse embryonic fibroblasts (MEFs) [50,51,52,71,72]. Indeed, we observe significant mtDNA release in bronchial epithelial BEAS-2B cells in response to the combined treatment of QVD-OPh, S63845, and ABT-737 (Figure S6). The mtDNA release results in RANTES induction in a cGAS-STING pathway-dependent manner (Figure S7). Furthermore, deficiency of GSDMB significantly impaired induction of RANTES by released endogenous mtDNA (Figure 4E) in KO cells despite comparable amount of released mtDNA in both WT and KO (Figure 4F). Most importantly, depletion of mtDNA by prolonged treatment with ethidium bromide (EtBr) results in significant reduction of RANTES induction in both WT and GSDMB KO cells with combined treatment of QVD-OPh/S63845/ABT-737 in (Figure 4F,G), indicating that induction of RANTES by QVD-OPh/S63845/ABT-737 depends on mtDNA release. Taken together, these findings suggest that GSDMB promotes mtDNA-induced IFNs production and ISGs expression. 3.5. GSDMB Promotes dsDNA-induced ISGs Expression and IFN Production via the cGAS-STING Pathway To gain insight into the mechanisms of how GSDMB regulates dsDNA-induced ISGs expression, we treated cells with IFNα, which leads to comparable induction of ISGs in GSDMB KO cells and control WT cells, indicating that GSDMB likely functions upstream instead of downstream of IFNs (Figure 5A). TBK1 is a central hub for inducing IFN production. BX795, a synthetic inhibitor of TBK1 [73], effectively blocked IFN production and ISGs expression in both control and GSDMB-overexpressing cells in response to transfection of poly (dA:dT) (Figure 5B–E). Similarly, G140 and H151, two potent and selective inhibitors of cyclic GMP-AMP synthase (cGAS) and STING, respectively [74,75], significantly mitigated GSDMB-enhanced ISGs expression and IFN response upon poly (dA:dT) stimulation (Figure 5B–E). These data indicate that GSDMB functions via the cGAS-STING-TBK1 pathway in promoting ISGs expression and IFN production induced by dsDNA.

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).

3.6. GSDMB Interacts with STING and Accelerates STING’s Translocation from ER to Golgi Having positioned GSDMB in the cGAS-STING pathway, we next sought to explore the target of GSDMB in this pathway. Firstly, treatment of the WT and GSDMB KO BEAS-2B cells with cGAMP, the second messenger synthesized by cGAS and recognized by STING [18,76] failed to restore the induction of RANTES in GSDMB KO cells, indicating that GSDMB potentially works downstream of cGAS (Figure 6A). Secondly, co-immunoprecipitation and immunoblot analyses showed that GSDMB interacts with STING, possibly through the C-terminal domain of STING (Figure 6B–D). Importantly, GSDMB KO cells demonstrated reduced interaction between STING and TBK1, suggesting that GSDMB likely facilitates the interaction of STING with TBK1 without influencing the dimerization of STING (Figure 6E,F). Additionally, as the interaction of STING and TBK1 occurs in Golgi, we next examined the location of STING under GSDMB deficiency [77,78,79,80]. Indeed, GSDMB KO led to reduced localization of STING into the Golgi (Figure 6G,H). Overall, GSDMB promotes mtDNA- and poly (dA:dT)-induced activation of cGAS-STING pathway through interacting and promoting the translocation of STING to Golgi and subsequent association with TBK1, activating IFN production and induction of ISGs.

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.

4. Discussion

In our study, we found that GSDMB, the asthma risk gene located in the 17q21, promotes the activation of mtDNA-induced cGAS-STING pathway via facilitating the translocation of STING into Golgi and subsequent interaction with TBK1. Consistent with this, overexpression of GSDMB led to increased, while deficiency of GSDMB led to impaired ISGs expression in normal human bronchial epithelial cells or bronchial epithelial cell lines induced by either transfected mtDNA or endogenously released mtDNA. In addition, chemical inhibition of cGAS by G140, STING by H151 treatment, or TBK1 by BX795 effectively attenuated induction of IFNs and ISGs, suggesting that targeting these signaling molecules may provide a novel therapeutic strategy for airway inflammation in asthmatics, especially for patients who carry the risk alleles at 17q21. Mitochondria, an important organelle, have been linked to the development of asthma with increased levels of ATP content, mitochondrial respiration, mitochondrial mass, reactive oxygen species (ROS), and mitochondrial arginine metabolism detected in asthmatic patients [81,82,83,84]. Additionally, mtDNA has attracted increasing attention in asthma research recently [85]. Alternaria extract and house dust mite extract robustly induced mtDNA release from airway epithelial cells, amplifying type 2 immune response in airways in both cellular and mouse models [46,86]. Perfluoroalkyl substances (PFAS), a commonly used chemical compound in manufacturing processes, promote mtDNA release and enhance subsequent inflammation through AIM2-mediated inflammasome activation, ultimately resulting in aggravated inflammation in mouse models. [43]. These findings support an important role of mtDNA in asthma development. However, roles of mtDNA in human asthmatic samples have been controversial. We found a positive correlation between mtDNA and eosinophil counts in asthmatic plasma samples, consistent with previously reported higher mtDNA levels in asthma samples/models [43,45,67,68,86]. However, asthmatic patients with high fractional exhaled nitric oxide (FeNO) were reported having lower mtDNA levels in bronchoalveolar lavage fluid [45]. Such discrepancy likely results from various endotypes of asthma included in each study given that distinct inflammatory pathways related to eosinophil counts and FeNO are weakly correlated and have different outcomes in large clinical trials in asthma [87,88]. In addition, disease severity, genotypes and genders of asthmatic subjects also need to be considered for data interpretation in each study, given that we observed stronger correlation between mtDNA and eosinophil counts only in the male group. Thus, further investigation on mtDNA levels in a larger cohort with well-characterized clinical endo-types of asthma is necessary to determine roles of mtDNA in asthma, especially the mechanism underlying gender difference. GSDMB regulates inflammasome-induced pyroptosis, similar to its paralogs such as gasdermins A and D [11,60]. The inflammasome, a multi-protein complex responsible for inflammation and cell death, plays a significant role in the onset of various respiratory diseases, including chronic rhinosinusitis (CRS) [64], chronic obstructive pulmonary disease (COPD) [89] and asthma [43]. While the function of GSDMB in anti-tumor immunity through pyroptosis is well characterized [11,60], GSDMB was also reported to have pyroptosis-independent function such as its regulation of PDGF-A-mediated FAK phosphorylation [14], MAVS-TBK1 signaling [90] and 5-lipoxygenase (5-LO)-mediated TGF-β1 expression [13]. Here, we reported another pyroptosis-independent function of GSDMB in human airway epithelial cells by positively regulating the cGAS-STING pathway, leading to a downstream ISGs induction as well as inflammation. Therefore, such pyroptosis-independent function is exclusive to GSDMB, in contrast to GSDMD. Limitations of the study Admittedly, there are several limitations in this study. First, while we observed significant correlation between mtDNA and eosinophil counts only in male asthmatic subjects, which is consistent with the higher prevalence and more severity of asthma in boys compared to girls. However, the underlying mechanism for gender-specific effects requires further studies. Secondly, the in vivo significance of regulation of GSDMB on mtDNA-induced activation of the cGAS-STING pathway needs additional research in the future. Lastly, future investigation is warranted to determine how GSDMB promotes the translocation of STING from ER to Golgi.

5. Conclusions

In summary, we discovered a novel role for GSDMB, a genetic risk factor for asthma associated with 17q21 locus in regulating mtDNA-induced innate immune response in respiratory epithelial cells. Specifically, we found that GSDMB promotes the induction of interferons and subsequent activation downstream ISGs via the cGAS-STING pathway in response to mtDNA. Thus, these findings suggest that GSDMB may have the therapeutic potential for treating asthma especially in genetically susceptible subjects.

Supplementary Materials

The following supporting information can be found at: https://www.sciepublish.com/article/pii/157.

Acknowledgments

We thank Dr. Yousang Gwack (University of California, Los Angeles) for providing the constructs for the STING deletion mutants. We thank all subjects for their participation in this study.

Author Contributions

T.L, and X.Z. conceived and designed the project. T.L. designed and performed experiments. S.L., N.B., J.W., Y.Z. Y.Y. and X.R. helped with cellular experiments. J.H., helped with data analysis. H.M., L.G.G.-E, B.L., A.M.K.C., B.A.R. and S.T.W. provided comments and suggestions with manuscript preparation. X.Z. supervised the whole project. T.L., and X.Z. wrote the manuscript with input from all authors.

Ethics Statement

All animal experiments were reviewed and approved by the Animal Care and Use Committee (IACUC) at the Brigham and Women’s Hospital (#2016N000391). The study of asthma cohort was approved by the Institutional Review Boards of the Brigham and Women’s Hospital (Boston, MA) entitled “the Genetic Epidemiology of Asthma in Costa Rica”: Protocol #:2000P001130.

Informed Consent Statement

Funding

This work was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health grants NIH (R01HL127200, R01HL148667, R01HL147148 and R01HL162783 to X.Z and R01HL123546 to B.A.R).

Declaration of Competing Interest

A.M.K.C. is a cofounder and consultant, and equity stock holder for Proterris, which develops therapeutic uses for carbon monoxide. A.M.K.C. is a consultant and equity stock holder for SPEXIS. A.M.K.C is a member for Medforth Board of Directors. A.M.K.C. has a use patent on CO. Additionally, A.M.K.C. has a patent in COPD. Other authors declare that they have no relevant conflict of interest.

References

Diseases GBD, Injuries C. Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet 2020, 396, 1204–1222. [Google Scholar]

Ferreira MAR, Mathur R, Vonk JM, Szwajda A, Brumpton B, Granell R, et al. Genetic Architectures of Childhood- and Adult-Onset Asthma Are Partly Distinct. Am. J. Hum. Genet. 2019, 104, 665–684. [Google Scholar]

Stein MM, Thompson EE, Schoettler N, Helling BA, Magnaye KM, Stanhope C, et al. A decade of research on the 17q12-21 asthma locus: Piecing together the puzzle. J. Allergy Clin. Immunol. 2018, 142, 749–764 e3. [Google Scholar]

Smit LA, Bouzigon E, Pin I, Siroux V, Monier F, Aschard H, et al. 17q21 variants modify the association between early respiratory infections and asthma. Eur. Respir. J. 2010, 36, 57–64. [Google Scholar]

Gui H, Levin AM, Hu D, Sleiman P, Xiao S, Mak ACY, et al. Mapping the 17q12-21.1 Locus for Variants Associated with Early-Onset Asthma in African Americans. Am. J. Respir. Crit. Care Med. 2021, 203, 424–436. [Google Scholar]

Moffatt MF, Gut IG, Demenais F, Strachan DP, Bouzigon E, Heath S, et al. A large-scale, consortium-based genomewide association study of asthma. N. Engl. J. Med. 2010, 363, 1211–1221. [Google Scholar]

Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H, et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 2015, 526, 660–665. [Google Scholar]

Shi J, Gao W, Shao F. Pyroptosis: Gasdermin-Mediated Programmed Necrotic Cell Death. Trends Biochem. Sci. 2017, 42, 245–254. [Google Scholar]

Liu X, Zhang Z, Ruan J, Pan Y, Magupalli VG, Wu H, et al. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 2016, 535, 153–158. [Google Scholar]

10.

Zhang Z, Zhang Y, Xia S, Kong Q, Li S, Liu X, et al. Gasdermin E suppresses tumour growth by activating anti-tumour immunity. Nature 2020, 579, 415–420. [Google Scholar]

11.

Panganiban RA, Sun M, Dahlin A, Park HR, Kan M, Himes BE, et al. A functional splice variant associated with decreased asthma risk abolishes the ability of gasdermin B to induce epithelial cell pyroptosis. J. Allergy Clin. Immunol. 2018, , 142, , 1469–1478 e2. [Google Scholar]

12.

Ivanov AI, Rana N, Privitera G, Pizarro TT. The enigmatic roles of epithelial gasdermin B: Recent discoveries and controversies. Trends Cell Biol. 2023, 33, 48–59. [Google Scholar]

13.

Das S, Miller M, Beppu AK, Mueller J, McGeough MD, Vuong C, et al. GSDMB induces an asthma phenotype characterized by increased airway responsiveness and remodeling without lung inflammation. Proc. Natl. Acad. Sci. USA 2016, 113, 13132–13137. [Google Scholar]

14.

Rana N, Privitera G, Kondolf HC, Bulek K, Lechuga S, De Salvo C, et al. GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis. Cell 2022, 185, 283–298 e17. [Google Scholar]

15.

Yu CH, Davidson S, Harapas CR, Hilton JB, Mlodzianoski MJ, Laohamonthonkul P, et al. TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS. Cell 2020, 183, 636–649 e18. [Google Scholar]

16.

Luo W, Zou X, Wang Y, Dong Z, Weng X, Pei Z, et al. Critical Role of the cGAS-STING Pathway in Doxorubicin-Induced Cardiotoxicity. Circ. Res. 2023, 132, e223–e242. [Google Scholar]

17.

Decout A, Katz JD, Venkatraman S, Ablasser A. The cGAS-STING pathway as a therapeutic target in inflammatory diseases. Nat. Rev. Immunol. 2021, 21, 548–569. [Google Scholar]

18.

Sun L, Wu J, Du F, Chen X, Chen ZJ. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 2013, 339, 786–791. [Google Scholar]

19.

Ishikawa H, Ma Z, Barber GN. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 2009, 461, 788–792. [Google Scholar]

20.

Ishikawa H, Barber GN. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 2008, 455, 674–678. [Google Scholar]

21.

Zhong B, Yang Y, Li S, Wang YY, Li Y, Diao F, et al. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity 2008, 29, 538–550. [Google Scholar]

22.

Sun W, Li Y, Chen L, Chen H, You F, Zhou X, et al. ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization. Proc. Natl. Acad. Sci. USA 2009, 106, 8653–8658. [Google Scholar]

23.

Dobbs N, Burnaevskiy N, Chen D, Gonugunta VK, Alto NM, Yan N. STING Activation by Translocation from the ER Is Associated with Infection and Autoinflammatory Disease. Cell Host Microbe 2015, 18, 157–168. [Google Scholar]

24.

Zhang C, Shang G, Gui X, Zhang X, Bai XC, Chen ZJ. Structural basis of STING binding with and phosphorylation by TBK1. Nature 2019, 567, 394–398. [Google Scholar]

25.

Shang G, Zhang C, Chen ZJ, Bai XC, Zhang X. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP. Nature 2019, 567, 389–393. [Google Scholar]

26.

Liu S, Cai X, Wu J, Cong Q, Chen X, Li T, et al. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation. Science 2015, 347, aaa2630. [Google Scholar]

27.

Motwani M, Pesiridis S, Fitzgerald KA. DNA sensing by the cGAS-STING pathway in health and disease. Nat. Rev. Genet. 2019, 20, 657–674. [Google Scholar]

28.

Jain U, Ver Heul AM, Xiong S, Gregory MH, Demers EG, Kern JT, et al. Debaryomyces is enriched in Crohn's disease intestinal tissue and impairs healing in mice. Science 2021, 371, 1154–1159. [Google Scholar]

29.

Yum S, Li M, Fang Y, Chen ZJ. TBK1 recruitment to STING activates both IRF3 and NF-kappaB that mediate immune defense against tumors and viral infections. Proc. Natl. Acad. Sci. USA 2021, 118, e2100225118. [Google Scholar]

30.

Ablasser A, Hur S. Regulation of cGAS- and RLR-mediated immunity to nucleic acids. Nat. Immunol. 2020, 21, 17–29. [Google Scholar]

31.

da Silva J, Hilzendeger C, Moermans C, Schleich F, Henket M, Kebadze T, et al. Raised interferon-beta, type 3 interferon and interferon-stimulated genes - evidence of innate immune activation in neutrophilic asthma. Clin. Exp. Allergy 2017, 47, 313–323. [Google Scholar]

32.

Bhakta NR, Christenson SA, Nerella S, Solberg OD, Nguyen CP, Choy DF, et al. IFN-stimulated Gene Expression, Type 2 Inflammation, and Endoplasmic Reticulum Stress in Asthma. Am. J Respir. Crit. Care Med. 2018, 197, 313–324. [Google Scholar]

33.

Fu Y, Wang J, Zhou B, Pajulas A, Gao H, Ramdas B, et al. An IL-9-pulmonary macrophage axis defines the allergic lung inflammatory environment. Sci. Immunol. 2022, 7, eabi9768. [Google Scholar]

34.

Tillie-Leblond I, Hammad H, Desurmont S, Pugin J, Wallaert B, Tonnel AB, et al. CC chemokines and interleukin-5 in bronchial lavage fluid from patients with status asthmaticus. Potential implication in eosinophil recruitment. Am. J. Respir. Crit. Care Med. 2000, 162, 586–592. [Google Scholar]

35.

Gauthier M, Kale SL, Oriss TB, Scholl K, Das S, Yuan H, et al. Dual role for CXCR3 and CCR5 in asthmatic type 1 inflammation. J. Allergy Clin. Immunol. 2022, 149, 113–124 e7. [Google Scholar]

36.

Rey-Jurado E, Espinosa Y, Astudillo C, Jimena Cortes L, Hormazabal J, Noguera LP, et al. Deep immunophenotyping reveals biomarkers of multisystemic inflammatory syndrome in children in a Latin American cohort. J. Allergy Clin. Immunol. 2022, 150, 1074–1085 e11. [Google Scholar]

37.

Gauthier M, Kale SL, Oriss TB, Gorry M, Ramonell RP, Dalton K, et al. CCL5 is a potential bridge between type 1 and type 2 inflammation in asthma. J. Allergy Clin. Immunol. 2023, 152, 94–106.e12. [Google Scholar]

38.

Schuh JM, Blease K, Bruhl H, Mack M, Hogaboam CM. Intrapulmonary targeting of RANTES/CCL5-responsive cells prevents chronic fungal asthma. Eur. J. Immunol. 2003, 33, 3080–3090. [Google Scholar]

39.

Domizio JD, Gulen MF, Saidoune F, Thacker VV, Yatim A, Sharma K, et al. The cGAS-STING pathway drives type I IFN immunopathology in COVID-19. Nature 2022, 603, 145–151. [Google Scholar]

40.

Sakamoto K, Furukawa T, Yamano Y, Kataoka K, Teramachi R, Walia A, et al. Serum mitochondrial DNA predicts the risk of acute exacerbation and progression of idiopathic pulmonary fibrosis. Eur. Respir. J. 2021, 57, 2001346. [Google Scholar]

41.

Ryu C, Sun H, Gulati M, Herazo-Maya JD, Chen Y, Osafo-Addo A, et al. Extracellular Mitochondrial DNA Is Generated by Fibroblasts and Predicts Death in Idiopathic Pulmonary Fibrosis. Am. J. Respir. Crit. Care Med. 2017, 196, 1571–181. [Google Scholar]

42.

Ryu C, Brandsdorfer C, Adams T, Hu B, Kelleher DW, Yaggi M, et al. Plasma mitochondrial DNA is associated with extrapulmonary sarcoidosis. Eur. Respir. J. 2019, 54, 1801762. [Google Scholar]

43.

Wang LQ, Liu T, Yang S, Sun L, Zhao ZY, Li LY, et al. Perfluoroalkyl substance pollutants activate the innate immune system through the AIM2 inflammasome. Nat. Commun. 2021, 12, 2915. [Google Scholar]

44.

Song MA, Kim JY, Gorr MW, Miller RA, Karpurapu M, Nguyen J, et al. Sex-specific lung inflammation and mitochondrial damage in a model of electronic cigarette exposure in asthma. Am. J. Physiol. Lung Cell Mol. Physiol. 2023, 325, L568–L579. [Google Scholar]

45.

Zhao J, Dar HH, Deng Y, St Croix CM, Li Z, Minami Y, et al. PEBP1 acts as a rheostat between prosurvival autophagy and ferroptotic death in asthmatic epithelial cells. Proc. Natl. Acad. Sci. USA 2020, 117, 14376–14385. [Google Scholar]

46.

Han Y, Chen L, Liu H, Jin Z, Wu Y, Wu Y, et al. Airway Epithelial cGAS Is Critical for Induction of Experimental Allergic Airway Inflammation. J. Immunol. 2020, 204, 1437–1447. [Google Scholar]

47.

Huang LS, Hong Z, Wu W, Xiong S, Zhong M, Gao X, et al. mtDNA Activates cGAS Signaling and Suppresses the YAP-Mediated Endothelial Cell Proliferation Program to Promote Inflammatory Injury. Immunity 2020, 52, 475–486 e5. [Google Scholar]

48.

He WR, Cao LB, Yang YL, Hua D, Hu MM, Shu HB. VRK2 is involved in the innate antiviral response by promoting mitostress-induced mtDNA release. Cell. Mol. Immunol. 2021, 18, 1186–1196. [Google Scholar]

49.

Willemsen J, Neuhoff MT, Hoyler T, Noir E, Tessier C, Sarret S, et al. TNF leads to mtDNA release and cGAS/STING-dependent interferon responses that support inflammatory arthritis. Cell Rep. 2021 , 37, 109977. [Google Scholar]

50.

McArthur K, Whitehead LW, Heddleston JM, Li L, Padman BS, Oorschot V, et al. BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis. Science 2018, 359, eaao6047. [Google Scholar]

51.

White MJ, McArthur K, Metcalf D, Lane RM, Cambier JC, Herold MJ, et al. Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production. Cell 2014, 159, 1549–1562. [Google Scholar]

52.

Riley JS, Quarato G, Cloix C, Lopez J, O'Prey J, Pearson M, et al. Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis. EMBO J. 2018, 37, e99238. [Google Scholar]

53.

Li X, Christenson SA, Modena B, Li H, Busse WW, Castro M, et al. Genetic analyses identify GSDMB associated with asthma severity, exacerbations, and antiviral pathways. J. Allergy Clin. Immunol. 2021, 147, 894–909. [Google Scholar]

54.

Levardon H, Yonker LM, Hurley BP, Mou H. Expansion of Airway Basal Cells and Generation of Polarized Epithelium. Bio. Protoc. 2018, 8, e2877. [Google Scholar]

55.

Mou H, Vinarsky V, Tata PR, Brazauskas K, Choi SH, Crooke AK, et al. Dual SMAD Signaling Inhibition Enables Long-Term Expansion of Diverse Epithelial Basal Cells. Cell Stem Cell 2016, 19, 217–231. [Google Scholar]

56.

Jackson ND, Everman JL, Chioccioli M, Feriani L, Goldfarbmuren KC, Sajuthi SP, et al. Single-Cell and Population Transcriptomics Reveal Pan-epithelial Remodeling in Type 2-High Asthma. Cell Rep. 2020, 32, 107872. [Google Scholar]

57.

Ravi A, Koster J, Dijkhuis A, Bal SM, Sabogal Pineros YS, Bonta PI, et al. Interferon-induced epithelial response to rhinovirus 16 in asthma relates to inflammation and FEV(1). J. Allergy Clin. Immunol. 2019, 143, 442–427 e10. [Google Scholar]

58.

Sajuthi SP, DeFord P, Li Y, Jackson ND, Montgomery MT, Everman JL, et al. Type 2 and interferon inflammation regulate SARS-CoV-2 entry factor expression in the airway epithelium. Nat. Commun. 2020, 11, 5139. [Google Scholar]

59.

Jin S, Tian S, Luo M, Xie W, Liu T, Duan T, et al. Tetherin Suppresses Type I Interferon Signaling by Targeting MAVS for NDP52-Mediated Selective Autophagic Degradation in Human Cells. Mol. Cell 2017, 68, 308–322 e4. [Google Scholar]

60.

Zhou Z, He H, Wang K, Shi X, Wang Y, Su Y, et al. Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells. Science 2020, 368, eaaz7548. [Google Scholar]

61.

Ding J, Wang K, Liu W, She Y, Sun Q, Shi J, et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 2016, 535, 111–116. [Google Scholar]

62.

Zheng Y, Liu Q, Wu Y, Ma L, Zhang Z, Liu T, et al. Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis. EMBO J. 2018, 37, e99347. [Google Scholar]

63.

Wang Y, Ning X, Gao P, Wu S, Sha M, Lv M, et al. Inflammasome Activation Triggers Caspase-1-Mediated Cleavage of cGAS to Regulate Responses to DNA Virus Infection. Immunity 2017, 46, 393–404. [Google Scholar]

64.

Liu T, Zhou YT, Wang LQ, Li LY, Bao Q, Tian S, et al. NOD-like receptor family, pyrin domain containing 3 (NLRP3) contributes to inflammation, pyroptosis, and mucin production in human airway epithelium on rhinovirus infection. J. Allergy Clin. Immunol. 2019, 144, 777–787 e9. [Google Scholar]

65.

You K, Wang L, Chou CH, Liu K, Nakata T, Jaiswal A, et al. QRICH1 dictates the outcome of ER stress through transcriptional control of proteostasis. Science 2021, 371, eabb6896. [Google Scholar]

66.

Lin JH, Walter P, Yen TS. Endoplasmic reticulum stress in disease pathogenesis. Annu. Rev. Pathol. 2008, 3, 399–425. [Google Scholar]

67.

Cocco MP, White E, Xiao S, Hu D, Mak A, Sleiman P, et al. Asthma and its relationship to mitochondrial copy number: Results from the Asthma Translational Genomics Collaborative (ATGC) of the Trans-Omics for Precision Medicine (TOPMed) program. PLoS One 2020, 15, e0242364. [Google Scholar]

68.

Dimasuay KG, Schaunaman N, Martin RJ, Pavelka N, Kolakowski C, Gottlieb RA, et al. Parkin, an E3 ubiquitin ligase, enhances airway mitochondrial DNA release and inflammation. Thorax 2020, 75, 717–724. [Google Scholar]

69.

Sharma S, Murphy AJ, Soto-Quiros ME, Avila L, Klanderman BJ, Sylvia JS, et al. Association of VEGF polymorphisms with childhood asthma, lung function and airway responsiveness. Eur. Respir. J. 2009, 33, 1287–1294. [Google Scholar]

70.

Aarreberg LD, Esser-Nobis K, Driscoll C, Shuvarikov A, Roby JA, Gale M, Jr. Interleukin-1beta Induces mtDNA Release to Activate Innate Immune Signaling via cGAS-STING. Mol. Cell 2019, 74, 801–815 e6. [Google Scholar]

71.

Cosentino K, Hertlein V, Jenner A, Dellmann T, Gojkovic M, Pena-Blanco A, et al. The interplay between BAX and BAK tunes apoptotic pore growth to control mitochondrial-DNA-mediated inflammation. Mol. Cell 2022, 82, 933–949 e9. [Google Scholar]

72.

Rongvaux A, Jackson R, Harman CC, Li T, West AP, de Zoete MR, et al. Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA. Cell 2014, 159, 1563–1577. [Google Scholar]

73.

Clark K, Plater L, Peggie M, Cohen P. Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation. J. Biol. Chem. 2009, 284, 14136–14146. [Google Scholar]

74.

Chen J, Jin Z, Zhang S, Zhang X, Li P, Yang H, et al. Arsenic trioxide elicits prophylactic and therapeutic immune responses against solid tumors by inducing necroptosis and ferroptosis. Cell. Mol. Immunol. 2023, 20, 51–64. [Google Scholar]

75.

Lama L, Adura C, Xie W, Tomita D, Kamei T, Kuryavyi V, et al. Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression. Nat. Commun. 2019, 10, 2261. [Google Scholar]

76.

Ablasser A, Goldeck M, Cavlar T, Deimling T, Witte G, Rohl I, et al. cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING. Nature 2013, 498, 380–384. [Google Scholar]

77.

Mukai K, Konno H, Akiba T, Uemura T, Waguri S, Kobayashi T, et al. Activation of STING requires palmitoylation at the Golgi. Nat. Commun. 2016, 7, 11932. [Google Scholar]

78.

Mukai K, Ogawa E, Uematsu R, Kuchitsu Y, Kiku F, Uemura T, et al. Homeostatic regulation of STING by retrograde membrane traffic to the ER. Nat. Commun. 2021, 12, 61. [Google Scholar]

79.

Zhang BC, Nandakumar R, Reinert LS, Huang J, Laustsen A, Gao ZL, et al. STEEP mediates STING ER exit and activation of signaling. Nat. Immunol. 2020, 21, 868–879. [Google Scholar]

80.

Srikanth S, Woo JS, Wu B, El-Sherbiny YM, Leung J, Chupradit K, et al. The Ca(2+) sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum. Nat. Immunol. 2019, 20, 152–162. [Google Scholar]

81.

Beaufils F, Esteves P, Enaud R, Germande O, Celle A, Marthan R, et al. Mitochondria are involved in bronchial smooth muscle remodeling in severe preschool wheezers. J. Allergy Clin. Immunol. 2021, 148, 645–651 e11. [Google Scholar]

82.

Esteves P, Blanc L, Celle A, Dupin I, Maurat E, Amoedo N, et al. Crucial role of fatty acid oxidation in asthmatic bronchial smooth muscle remodelling. Eur. Respir. J. 2021, 58, 2004252. [Google Scholar]

83.

Trian T, Benard G, Begueret H, Rossignol R, Girodet PO, Ghosh D, et al. Bronchial smooth muscle remodeling involves calcium-dependent enhanced mitochondrial biogenesis in asthma. J. Exp. Med. 2007, 204, 3173–3181. [Google Scholar]

84.

Xu W, Ghosh S, Comhair SA, Asosingh K, Janocha AJ, Mavrakis DA, et al. Increased mitochondrial arginine metabolism supports bioenergetics in asthma. J. Clin. Invest. 2016, 126, 2465–2481. [Google Scholar]

85.

Carpagnano GE, Lacedonia D, Malerba M, Palmiotti GA, Cotugno G, Carone M, et al. Analysis of mitochondrial DNA alteration in new phenotype ACOS. BMC Pulm. Med. 2016, 16, 31. [Google Scholar]

86.

Srisomboon Y, Iijima K, Colwell M, Maniak PJ, Macchietto M, Faulk C, et al. Allergen-induced DNA release by the airway epithelium amplifies type 2 immunity. J. Allergy Clin. Immunol. 2023, 151, 494–508 e6. [Google Scholar]

87.

Colak Y, Afzal S, Nordestgaard BG, Marott JL, Lange P. Combined value of exhaled nitric oxide and blood eosinophils in chronic airway disease: the Copenhagen General Population Study. Eur. Respir. J. 2018, 52, 1800616. [Google Scholar]

88.

Malinovschi A, Fonseca JA, Jacinto T, Alving K, Janson C. Exhaled nitric oxide levels and blood eosinophil counts independently associate with wheeze and asthma events in National Health and Nutrition Examination Survey subjects. J. Allergy Clin. Immunol. 2013, 132, 821–827 e1-5. [Google Scholar]

89.

Liu T, Liu S, Zhou X. Innate Immune Responses and Pulmonary Diseases. Adv. Exp. Med. Biol. 2021, 1304, 53–71. [Google Scholar]

90.

Liu T, Liu S, Rui X, Cao Y, Hecker J, Guo F, et al. Gasdermin B, an asthma-susceptibility gene, promotes MAVS-TBK1 signaling and airway inflammation. Eur. Respir. J. 2024, 21, 2301232. [Google Scholar]

Journal of Respiratory Biology and Translational Medicine