Assessing Mitochondrial DNA Release into the Cytosol and Subsequent Activation of Innate Immune-related Pathways in Mammalian Cells

被引:43
作者
Bryant, Joshua D. [1 ]
Lei, Yuanjiu [1 ]
VanPortfliet, Jordyn J. [1 ]
Winters, Ashley D. [1 ]
West, A. Phillip [1 ]
机构
[1] Texas A&M Univ, Dept Microbial Pathogenesis & Immunol, Bryan, TX 77843 USA
来源
CURRENT PROTOCOLS | 2022年 / 2卷 / 02期
关键词
cGAS; innate immunity; mitochondria; mitochondrial DNA; STING; GMP-AMP SYNTHASE; TRANSCRIPTION FACTOR; CYCLIC DINUCLEOTIDE; NUCLEOSIDE ANALOGS; CGAS; RESPONSES; TOXICITY; SENSOR; DEATH; MAINTENANCE;
D O I
10.1002/cpz1.372
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Mitochondria have emerged as key drivers of mammalian innate immune responses, functioning as signaling hubs to trigger inflammation and orchestrating metabolic switches required for phagocyte activation. Mitochondria also contain damage-associated molecular patterns (DAMPs), molecules that share similarity with pathogen-associated molecular patterns (PAMPs) and can engage innate immune sensors to drive inflammation. The aberrant release of mitochondrial DAMPs during cellular stress and injury is an increasingly recognized trigger of inflammatory responses in human diseases. Mitochondrial DNA (mtDNA) is a particularly potent DAMP that engages multiple innate immune sensors, although mounting evidence suggests that cytosolic mtDNA is primarily detected via the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. cGAS and STING are widely expressed in mammalian cells and serve as key regulators of type I interferon and cytokine expression in both infectious and inflammatory diseases. Despite growing roles for the mtDNA-cGAS-STING axis in human disease, assays to quantify mtDNA release into the cytosol and approaches to link mtDNA to cGAS-STING signaling are not standardized, which increases the possibility for experimental artifacts and misinterpretation of data. Here, we present a series of protocols for assaying the release of mtDNA into the cytosol and subsequent activation of innate immune signaling in mammalian cells. We highlight genetic and pharmacological approaches to induce and inhibit mtDNA release from mitochondria. We also describe immunofluorescence microscopy and cellular fractionation assays to visualize morphological changes in mtDNA and quantify mtDNA accumulation in the cytosol. Finally, we include protocols to examine mtDNA-dependent cGAS-STING activation by RT-qPCR and western blotting. These methods can be performed with standard laboratory equipment and are highly adaptable to a wide range of mammalian cell types. They will permit researchers working across the spectrum of biological and biomedical sciences to accurately and reproducibly measure cytosolic mtDNA release and resulting innate immune responses. (c) 2022 Wiley Periodicals LLC. Basic Protocol 1: siRNA-mediated knockdown of TFAM to induce mtDNA instability, cytosolic release, and activation of the cGAS-STING pathway Alternate Protocol: Pharmacological induction of mtDNA release and cGAS-STING activation using ABT-737 and Q-VD-OPH Basic Protocol 2: Isolation and quantitation of DNA from cytosolic, mitochondrial, and nuclear fractions Basic Protocol 3: Pharmacological inhibition of mtDNA replication and release
引用
收藏
页数:31
相关论文
共 78 条
  • [71] Recognition and signaling by toll-like receptors
    West, A. Phillip
    Koblansky, Anna Alicia
    Ghosh, Sankar
    [J]. ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, 2006, 22 : 409 - 437
  • [72] Mitochondrial dysfunction as a trigger of innate immune responses and inflammation
    West, A. Phillip
    [J]. TOXICOLOGY, 2017, 391 : 54 - 63
  • [73] Mitochondrial DNA in innate immune responses and inflammatory pathology
    West, A. Phillip
    Shadel, Gerald S.
    [J]. NATURE REVIEWS IMMUNOLOGY, 2017, 17 (06) : 363 - 375
  • [74] Mitochondrial DNA stress primes the antiviral innate immune response
    West, A. Phillip
    Khoury-Hanold, William
    Staron, Matthew
    Tal, Michal C.
    Pineda, Cristiana M.
    Lang, Sabine M.
    Bestwick, Megan
    Duguay, Brett A.
    Raimundo, Nuno
    MacDuff, Donna A.
    Kaech, Susan M.
    Smiley, James R.
    Means, Robert E.
    Iwasaki, Akiko
    Shadel, Gerald S.
    [J]. NATURE, 2015, 520 (7548) : 553 - +
  • [75] Xu Jianming, 2005, Curr Protoc Mol Biol, VChapter 28, DOI 10.1002/0471142727.mb2801s70
  • [76] MITOCHONDRIAL DNA IS RELEASED BY SHOCK AND ACTIVATES NEUTROPHILS VIA P38 MAP KINASE
    Zhang, Qin
    Itagaki, Kiyoshi
    Hauser, Carl J.
    [J]. SHOCK, 2010, 34 (01): : 55 - 59
  • [77] Circulating mitochondrial DAMPs cause inflammatory responses to injury
    Zhang, Qin
    Raoof, Mustafa
    Chen, Yu
    Sumi, Yuka
    Sursal, Tolga
    Junger, Wolfgang
    Brohi, Karim
    Itagaki, Kiyoshi
    Hauser, Carl J.
    [J]. NATURE, 2010, 464 (7285) : 104 - U115
  • [78] Mitochondrial DNA-LL-37 Complex Promotes Atherosclerosis by Escaping from Autophagic Recognition
    Zhang, Zhiye
    Meng, Ping
    Han, Yajun
    Shen, Chuanbin
    Li, Bowen
    Hakim, Md Abdul
    Zhang, Xuguang
    Lu, Qiumin
    Rong, Mingqiang
    Lai, Ren
    [J]. IMMUNITY, 2015, 43 (06) : 1137 - 1147