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 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

References

[1] 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.

[2] 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.

[3] 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.

[4] 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.

[5] 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.

[6] 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.

[7] 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.

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

[9] 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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

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

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

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

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

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

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

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

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

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

[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.

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

[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.