Skip to main content
SpringerLink
Log in

Characterization of Innate Immune Signalings Stimulated by Ligands for Pattern Recognition Receptors

  • Protocol
  • First Online:
Arthritis Research

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1142))

Abstract

The innate immunity is an essential step as the front line of host defense, and its aberrant activation particularly in response to nucleic acids is closely related to the pathogenesis of autoimmune and inflammatory diseases. Characterization of the innate immune signalings may provide a pathophysiological insight for better understanding of human diseases. Nucleic acid-mediated activation of pattern recognition receptors triggers the activation of two major intracellular signaling pathways, which are dependent on NF-κB and interferon regulatory factors, transcriptional factors. This leads to the subsequent induction of inflammatory cytokines and type I and III interferons. In this chapter, we first overview the representative families of nucleic acid sensors and their ligands and then show the fundamental techniques for extracellular or intracellular stimulation with these nucleic acid ligands and for detection of innate immune response, that is, IFN and proinflammatory cytokine induction, as assessed by luciferase assay, quantitative RT-PCR (qRT-PCR), and enzyme-linked immunosorbent assay.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Takaoka A, Yanai H (2006) Interferon signalling network in innate defence. Cell Microbiol 8:907–922

    Article  CAS  PubMed  Google Scholar 

  2. Mogensen TH (2009) Pathogen recognition and inflammatory signaling in innate immune defenses. Clin Microbiol Rev 22:240–273

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140: 805–820

    Article  CAS  PubMed  Google Scholar 

  4. Vladimer GI, Marty-Roix R, Ghosh S, Weng D, Lien E (2013) Inflammasomes and host defenses against bacterial infections. Curr Opin Microbiol 16:23–31

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Kono H, Rock KL (2008) How dying cells alert the immune system to danger. Nat Cell Biol 8:279–289

    CAS  Google Scholar 

  6. Jounai N, Kobiyama K, Takeshita F, Ishii KJ (2012) Recognition of damage-associated molecular patterns related to nucleic acids during inflammation and vaccination. Front Cell Infect Microbiol 2:168. doi:10.3389/fcimb.2012.00168

    PubMed Central  PubMed  Google Scholar 

  7. Barrat FJ, Meeker T, Gregorio J, Chan JH, Uematsu S, Akira S et al (2005) Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J Exp Med 202:1131–1139

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Barbalat R, Ewald SE, Mouchess ML, Barton GM (2011) Nucleic acid recognition by the innate immune system. Annu Rev Immunol 29:185–214

    Article  CAS  PubMed  Google Scholar 

  9. Takaoka A, Taniguchi T (2008) Cytosolic DNA recognition for triggering innate immune responses. Adv Drug Deliv Rev 60: 847–857

    Article  CAS  PubMed  Google Scholar 

  10. Oldenburg M, Krüger A, Ferstl R, Kaufmann A, Nees G, Sigmund A et al (2012) TLR13 recognizes bacterial 23S rRNA devoid of erythromycin resistance-forming modification. Science 337:1111–1115

    Article  CAS  PubMed  Google Scholar 

  11. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H et al (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408:740–745

    Article  CAS  PubMed  Google Scholar 

  12. Onoguchi K, Yoneyama M, Fujita T (2011) Retinoic acid-inducible gene-I-like receptors. J Interferon Cytokine Res 31:27–31

    Article  CAS  PubMed  Google Scholar 

  13. Yoneyama M, Fujita T (2009) RNA recognition and signal transduction by RIG-I-like receptors. Immunol Rev 227:54–65

    Article  CAS  PubMed  Google Scholar 

  14. Rehwinkel J, Reis e Sousa C (2010) RIGorous detection: exposing virus through RNA sensing. Science 327:284–286

    Google Scholar 

  15. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M et al (2004) The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 5:730–737

    Article  CAS  PubMed  Google Scholar 

  16. Fujita T (2009) A nonself RNA pattern: tri-p to panhandle. Immunity 31:4–5

    Article  CAS  PubMed  Google Scholar 

  17. Takeuchi O, Akira S (2009) Innate immunity to virus infection. Immunol Rev 227:75–86

    Article  CAS  PubMed  Google Scholar 

  18. Kato H, Takeuchi O, Mikamo-Satoh E, Hirai R, Kawai T, Matsushita K et al (2008) Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5. J Exp Med 205:1601–1610

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Ablasser A, Bauernfeind F, Hartmann G, Latz E, Fitzgerald KA, Hornung V (2009) RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat Immunol 10:1065–1072

    Article  CAS  PubMed  Google Scholar 

  20. Chiu Y-H, MacMillan JB, Chen ZJ (2009) RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 138:576–591

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Sabbah A, Chang TH, Harnack R, Frohlich V, Tominaga K, Dube PH et al (2009) Activation of innate immune antiviral responses by Nod2. Nat Immunol 10:1073–1080

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T et al (2007) DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 448:501–505

    Article  CAS  PubMed  Google Scholar 

  23. Unterholzner L, Keating SE, Baran M, Horan KA, Jensen SB, Sharma S et al (2010) IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol 11:997–1004

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Zhang Z, Yuan B, Bao M, Lu N, Kim T, Liu Y-J (2011) The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat Immunol 12:959–965

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Stetson DB, Medzhitov R (2006) Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity 24:93–103

    Article  CAS  PubMed  Google Scholar 

  26. Burdette DL, Monroe KM, Sotelo-Troha K, Iwig JS, Eckert B, Hyodo M et al (2011) STING is a direct innate immune sensor of cyclic di-GMP. Nature 478:515–518

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  28. Wu J, Sun L, Chen X, Du F, Shi H, Chen C et al (2013) Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339:826–830

    Article  CAS  PubMed  Google Scholar 

  29. Ablasser A, Goldeck M, Cavlar T, Deimling T, Witte G, Röhl I et al (2013) cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING. Nature 498(7454): 380–384

    Article  CAS  PubMed  Google Scholar 

  30. Sadler AJ, Williams BRG (2008) Interferon-inducible antiviral effectors. Nat Cell Biol 8:559–568

    CAS  Google Scholar 

  31. Lamkanfi M, Dixit VM (2012) Inflammasomes and their roles in health and disease. Annu Rev Cell Dev Biol 28:137–161

    Article  CAS  PubMed  Google Scholar 

  32. Hornung V, Ablasser A, Charrel-Dennis M, Bauernfeind F, Horvath G, Caffrey DR et al (2009) AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458:514–518

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Rathinam VAK, Vanaja SK, Fitzgerald KA (2012) Regulation of inflammasome signaling. Nat Immunol 13:333–342

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Yoneyama M, Suhara W, Fukuhara Y, Sato M, Ozato K, Fujita T (1996) Autocrine amplification of type I interferon gene expression mediated by interferon stimulated gene factor 3 (ISGF3). J Biochem 120:160–169

    Article  CAS  PubMed  Google Scholar 

  35. Krieg AM (2002) CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 20:709–760

    Article  CAS  PubMed  Google Scholar 

  36. Schmidt A, Schwerd T, Hamm W, Hellmuth JC, Cui S, Wenzel M et al (2009) 5′-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I. Proc Natl Acad Sci U S A 106:12067–12072

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Hokkaido University, 15-7 Kitaku, Sapporo, 060-0815, Japan

    Takeshi Kameyama & Akinori Takaoka

Authors
  1. Takeshi Kameyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Akinori Takaoka
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Akinori Takaoka .

Editor information

Editors and Affiliations

  1. Department of Rheumatology, Kyushu University Beppu Hospital, Beppu, Japan

    Shunichi Shiozawa

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Kameyama, T., Takaoka, A. (2014). Characterization of Innate Immune Signalings Stimulated by Ligands for Pattern Recognition Receptors. In: Shiozawa, S. (eds) Arthritis Research. Methods in Molecular Biology, vol 1142. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0404-4_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-0404-4_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0403-7

  • Online ISBN: 978-1-4939-0404-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation