Skip to main content

Toll-Like Receptors: Ligands, Cell-Based Models, and Readouts for Receptor Action

  • Protocol
  • First Online:
Toll-Like Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1390))

Abstract

This chapter details Toll-like receptors (TLRs) and the tools available to study their biology in vitro. Key parameters to consider before exploring TLR action such as receptor localization, signaling pathways, nature of ligands and cellular expression are introduced. Cellular models (i.e., host cells and readouts) based on the use of cell lines, primary cells, or whole blood are presented. The use of modified TLRs to circumvent some technical problems is also discussed.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Takeda K, Akira S (2005) Toll-like receptors in innate immunity. Int Immunol 17:1–14

    Article  CAS  PubMed  Google Scholar 

  2. Gay NJ, Keith FJ (1991) Drosophila Toll and IL-1 receptor. Nature 351:355–356

    Article  CAS  PubMed  Google Scholar 

  3. Brikos C, O'Neill LA (2008) Signalling of Toll-like receptors. Handb Exp Pharmacol 183:21–50

    Article  CAS  PubMed  Google Scholar 

  4. Gay NJ, Gangloff M, Weber AN (2006) Toll-like receptors as molecular switches. Nat Rev Immunol 6:693–698

    Article  CAS  PubMed  Google Scholar 

  5. Kawai T, Akira S (2006) TLR signaling. Cell Death Differ 13:816–825

    Article  CAS  PubMed  Google Scholar 

  6. Boehme KW, Compton T (2004) Innate sensing of viruses by toll-like receptors. J Virol 78:7867–7873

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Akira S, Takeda K (2004) Toll-like receptor signalling. Nat Rev Immunol 4:499–511

    Article  CAS  PubMed  Google Scholar 

  8. Barton GM, Kagan JC, Medzhitov R (2006) Intracellular localization of Toll-like receptor 9 prevents recognition of self DNA but facilitates access to viral DNA. Nat Immunol 7:49–56

    Article  CAS  PubMed  Google Scholar 

  9. Saitoh S, Akashi S, Yamada T, Tanimura N, Kobayashi M, Konno K, Matsumoto F, Fukase K, Kusumoto S, Nagai Y, Kusumoto Y, Kosugi A, Miyake K (2004) Lipid A antagonist, lipid IVa, is distinct from lipid A in interaction with Toll-like receptor 4 (TLR4)-MD-2 and ligand-induced TLR4 oligomerization. Int Immunol 16:961–969

    Article  CAS  PubMed  Google Scholar 

  10. Botos I, Liu L, Wang Y, Segal DM, Davies DR (2009) The toll-like receptor 3:dsRNA signaling complex. Biochim Biophys Acta 1789:667–674

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Wang Y, Liu L, Davies DR, Segal DM (2010) Dimerization of Toll-like receptor 3 (TLR3) is required for ligand binding. J Biol Chem 285:36836–36841

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Liu L, Botos I, Wang Y, Leonard JN, Shiloach J, Segal DM, Davies DR (2008) Structural basis of toll-like receptor 3 signaling with double-stranded RNA. Science 320:379–381

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, Lee H, Lee JO (2007) Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130:1071–1082

    Article  CAS  PubMed  Google Scholar 

  14. Kim HM, Park BS, Kim JI, Kim SE, Lee J, Oh SC, Enkhbayar P, Matsushima N, Lee H, Yoo OJ, Lee JO (2007) Crystal structure of the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. Cell 130:906–917

    Article  CAS  PubMed  Google Scholar 

  15. Yoon SI, Kurnasov O, Natarajan V, Hong M, Gudkov AV, Osterman AL, Wilson IA (2012) Structural basis of TLR5-flagellin recognition and signaling. Science 335:859–864

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Tanji H, Ohto U, Shibata T, Miyake K, Shimizu T (2013) Structural reorganization of the Toll-like receptor 8 dimer induced by agonistic ligands. Science 339:1426–1429

    Article  CAS  PubMed  Google Scholar 

  17. Nyman T, Stenmark P, Flodin S, Johansson I, Hammarstrom M, Nordlund P (2008) The crystal structure of the human toll-like receptor 10 cytoplasmic domain reveals a putative signaling dimer. J Biol Chem 283:11861–11865

    Article  CAS  PubMed  Google Scholar 

  18. Gangloff M, Weber AN, Gay NJ (2005) Conserved mechanisms of signal transduction by Toll and Toll-like receptors. J Endotoxin Res 11:294–298

    Article  CAS  PubMed  Google Scholar 

  19. Nunez Miguel R, Wong J, Westoll JF, Brooks HJ, O'Neill LA, Gay NJ, Bryant CE, Monie TP (2007) A dimer of the Toll-like receptor 4 cytoplasmic domain provides a specific scaffold for the recruitment of signalling adaptor proteins. PLoS One 2, e788

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  20. Akira S, Yamamoto M, Takeda K (2003) Role of adapters in Toll-like receptor signalling. Biochem Soc Trans 31:637–642

    Article  CAS  PubMed  Google Scholar 

  21. Lu YC, Yeh WC, Ohashi PS (2008) LPS/TLR4 signal transduction pathway. Cytokine 42:145–151

    Article  CAS  PubMed  Google Scholar 

  22. Kaisho T, Akira S (2006) Toll-like receptor function and signaling. J Allergy Clin Immunol 117:979–987, quiz 988

    Article  CAS  PubMed  Google Scholar 

  23. Yamamoto M, Sato S, Hemmi H, Sanjo H, Uematsu S, Kaisho T, Hoshino K, Takeuchi O, Kobayashi M, Fujita T, Takeda K, Akira S (2002) Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4. Nature 420:324–329

    Article  CAS  PubMed  Google Scholar 

  24. Takeda K, Akira S (2004) TLR signaling pathways. Semin Immunol 16:3–9

    Article  CAS  PubMed  Google Scholar 

  25. Kawai T, Adachi O, Ogawa T, Takeda K, Akira S (1999) Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11:115–122

    Article  CAS  PubMed  Google Scholar 

  26. Stack J, Doyle SL, Connolly DJ, Reinert LS, O'Keeffe KM, McLoughlin RM, Paludan SR, Bowie AG (2014) TRAM is required for TLR2 endosomal signaling to type I IFN induction. J Immunol 193(12):6090–6102

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Volpi C, Fallarino F, Pallotta MT, Bianchi R, Vacca C, Belladonna ML, Orabona C, De Luca A, Boon L, Romani L, Grohmann U, Puccetti P (2013) High doses of CpG oligodeoxynucleotides stimulate a tolerogenic TLR9-TRIF pathway. Nat Commun 4:1852

    Article  PubMed  CAS  Google Scholar 

  28. Hennessy EJ, Parker AE, O'Neill LA (2010) Targeting Toll-like receptors: emerging therapeutics? Nat Rev Drug Discov 9:293–307

    Article  CAS  PubMed  Google Scholar 

  29. Gay NJ, Gangloff M (2007) Structure and function of Toll receptors and their ligands. Annu Rev Biochem 76:141–165

    Article  CAS  PubMed  Google Scholar 

  30. Rutz M, Metzger J, Gellert T, Luppa P, Lipford GB, Wagner H, Bauer S (2004) Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur J Immunol 34:2541–2550

    Article  CAS  PubMed  Google Scholar 

  31. Oldenburg M, Kruger A, Ferstl R, Kaufmann A, Nees G, Sigmund A, Bathke B, Lauterbach H, Suter M, Dreher S, Koedel U, Akira S, Kawai T, Buer J, Wagner H, Bauer S, Hochrein H, Kirschning CJ (2012) TLR13 recognizes bacterial 23S rRNA devoid of erythromycin resistance-forming modification. Science 337:1111–1115

    Article  CAS  PubMed  Google Scholar 

  32. Takeuchi O, Sato S, Horiuchi T, Hoshino K, Takeda K, Dong Z, Modlin RL, Akira S (2002) Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol 169:10–14

    Article  CAS  PubMed  Google Scholar 

  33. Hasan UA, Dollet S, Vlach J (2004) Differential induction of gene promoter constructs by constitutively active human TLRs. Biochem Biophys Res Commun 321:124–131

    Article  CAS  PubMed  Google Scholar 

  34. Akira S, Uematsu S, Takeuchi O (2006) Pathogen recognition and innate immunity. Cell 124:783–801

    Article  CAS  PubMed  Google Scholar 

  35. Schwandner R, Dziarski R, Wesche H, Rothe M, Kirschning CJ (1999) Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem 274:17406–17409

    Article  CAS  PubMed  Google Scholar 

  36. Sandor F, Latz E, Re F, Mandell L, Repik G, Golenbock DT, Espevik T, Kurt-Jones EA, Finberg RW (2003) Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling. J Cell Biol 162:1099–1110

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Park JS, Gamboni-Robertson F, He Q, Svetkauskaite D, Kim JY, Strassheim D, Sohn JW, Yamada S, Maruyama I, Banerjee A, Ishizaka A, Abraham E (2006) High mobility group box 1 protein interacts with multiple Toll-like receptors. Am J Physiol Cell Physiol 290:C917–C924

    Article  CAS  PubMed  Google Scholar 

  38. Flo TH, Halaas O, Lien E, Ryan L, Teti G, Golenbock DT, Sundan A, Espevik T (2000) Human toll-like receptor 2 mediates monocyte activation by Listeria monocytogenes but not by group B streptococci or lipopolysaccharide. J Immunol 164(4):2064–2069

    Article  CAS  PubMed  Google Scholar 

  39. Ozinsky A, Underhill DM, Fontenot JD, Hajjar AM, Smith KD, Wilson CB, Schroeder L, Aderem A (2000) The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc Natl Acad Sci U S A 97:13766–13771

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jehanno M, Viala J, Tedin K, Taha MK, Labigne A, Zahringer U, Coyle AJ, DiStefano PS, Bertin J, Sansonetti PJ, Philpott DJ (2003) Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300:1584–1587

    Article  CAS  PubMed  Google Scholar 

  41. de Bouteiller O, Merck E, Hasan UA, Hubac S, Benguigui B, Trinchieri G, Bates EE, Caux C (2005) Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH. J Biol Chem 280(46):38133–38145

    Article  PubMed  CAS  Google Scholar 

  42. Marshall-Clarke S, Downes JE, Haga IR, Bowie AG, Borrow P, Pennock JE, Grencis RK, Rothwell P (2007) Polyinosinic acid is a ligand for toll-like receptor 3. J Biol Chem 282(34):24759–24766

    Article  CAS  PubMed  Google Scholar 

  43. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T, Koh CS, Reis e Sousa C, Matsuura Y, Fujita C, Akira S (2006) Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441:101–105

    Article  CAS  PubMed  Google Scholar 

  44. Zhou Y, Guo M, Wang X, Li J, Wang Y, Ye L, Dai M, Zhou L, Persidsky Y, Ho W (2013) TLR3 activation efficiency by high or low molecular mass poly I:C. Innate Immun 19:184–192

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  45. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr (1997) A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394–397

    Article  CAS  PubMed  Google Scholar 

  46. Caroff M, Karibian D (2003) Structure of bacterial lipopolysaccharides. Carbohydr Res 338:2431–2447

    Article  CAS  PubMed  Google Scholar 

  47. Coats SR, Reife RA, Bainbridge BW, Pham TT, Darveau RP (2003) Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at toll-like receptor 4 in human endothelial cells. Infect Immun 71:6799–6807

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Huber M, Kalis C, Keck S, Jiang ZF, Georgel P, Du X, Shamel L, Sovath S, Mudd S, Beutler B, Galanos C, Freudenberg MA (2006) R-form LPS, the master key to the activation of TLR4/MD-2-positive cells. Eur J Immunol 36:701–711

    Article  CAS  PubMed  Google Scholar 

  49. Mata-Haro V, Cekic C, Martin M, Chilton PM, Casella CR, Mitchell TC (2007) The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science 316:1628–1632

    Article  CAS  PubMed  Google Scholar 

  50. Hirschfeld M, Ma Y, Weis JH, Vogel SN, Weis JJ (2000) Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2. J Immunol 165:618–622

    Article  CAS  PubMed  Google Scholar 

  51. Kawasaki K, Akashi S, Shimazu R, Yoshida T, Miyake K, Nishijima M (2001) Involvement of TLR4/MD-2 complex in species-specific lipopolysaccharide-mimetic signal transduction by Taxol. J Endotoxin Res 7:232–236

    Article  CAS  PubMed  Google Scholar 

  52. Figueiredo RT, Fernandez PL, Mourao-Sa DS, Porto BN, Dutra FF, Alves LS, Oliveira MF, Oliveira PL, Graca-Souza AV, Bozza MT (2007) Characterization of heme as activator of toll-like receptor 4. J Biol Chem 282:20221–20229

    Article  CAS  PubMed  Google Scholar 

  53. Marshak-Rothstein A (2006) Toll-like receptors in systemic autoimmune disease. Nat Rev Immunol 6:823–835

    Article  CAS  PubMed  Google Scholar 

  54. Matzinger P (2002) The danger model: a renewed sense of self. Science 296:301–305

    Article  CAS  PubMed  Google Scholar 

  55. Gao B, Tsan MF (2003) Recombinant human heat shock protein 60 does not induce the release of tumor necrosis factor alpha from murine macrophages. J Biol Chem 278:22523–22529

    Article  CAS  PubMed  Google Scholar 

  56. Ohashi K, Burkart V, Flohe S, Kolb H (2000) Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol 164:558–561

    Article  CAS  PubMed  Google Scholar 

  57. Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA, Barrett SL, Cookson BT, Aderem A (2003) Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat Immunol 4:1247–1253

    Article  CAS  PubMed  Google Scholar 

  58. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A (2001) The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410:1099–1103

    Article  CAS  PubMed  Google Scholar 

  59. Okusawa T, Fujita M, Nakamura J, Into T, Yasuda M, Yoshimura A, Hara Y, Hasebe A, Golenbock DT, Morita M, Kuroki Y, Ogawa T, Shibata K (2004) Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6. Infect Immun 72:1657–1665

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  60. Triantafilou M, Gamper FG, Haston RM, Mouratis MA, Morath S, Hartung T, Triantafilou K (2006) Membrane sorting of toll-like receptor (TLR) -2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic associations with CD36 and intracellular targeting. J Biol Chem 281:31002–31011

    Article  CAS  PubMed  Google Scholar 

  61. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C (2004) Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303:1529–1531

    Article  CAS  PubMed  Google Scholar 

  62. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H, Bauer S (2004) Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303:1526–1529

    Article  CAS  PubMed  Google Scholar 

  63. Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, Iwasaki A, Flavell RA (2004) Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci U S A 101

    Google Scholar 

  64. Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, Horiuchi T, Tomizawa H, Takeda K, Akira S (2002) Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 3:196–200

    Article  CAS  PubMed  Google Scholar 

  65. Heil F, Ahmad-Nejad P, Hemmi H, Hochrein H, Ampenberger F, Gellert T, Dietrich H, Lipford G, Takeda K, Akira S, Wagner H, Bauer S (2003) The Toll-like receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily. Eur J Immunol 33:2987–2997

    Article  CAS  PubMed  Google Scholar 

  66. Kobold S, Wiedemann G, Rothenfusser S, Endres S (2014) Modes of action of TLR7 agonists in cancer therapy. Immunotherapy 6:1085–1095

    Article  CAS  PubMed  Google Scholar 

  67. Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A, Schlee M, Uematsu S, Noronha A, Manoharan M, Akira S, de Fougerolles A, Endres S, Hartmann G (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med 11:263–270

    Article  CAS  PubMed  Google Scholar 

  68. Savarese E, Chae OW, Trowitzsch S, Weber G, Kastner B, Akira S, Wagner H, Schmid RM, Bauer S, Krug A (2006) U1 small nuclear ribonucleoprotein immune complexes induce type I interferon in plasmacytoid dendritic cells through TLR7. Blood 107:3229–3234

    Article  CAS  PubMed  Google Scholar 

  69. Gorden KK, Qiu XX, Binsfeld CC, Vasilakos JP, Alkan SS (2006) Cutting edge: activation of murine TLR8 by a combination of imidazoquinoline immune response modifiers and polyT oligodeoxynucleotides. J Immunol 177:6584–6587

    Article  CAS  PubMed  Google Scholar 

  70. Krieg AM (2006) Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov 5:471–484

    Article  CAS  PubMed  Google Scholar 

  71. Tian J, Avalos AM, Mao SY, Chen B, Senthil K, Wu H, Parroche P, Drabic S, Golenbock D, Sirois C, Hua J, An LL, Audoly L, La Rosa G et al (2007) Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Nat Immunol 8(5):487–496

    Article  CAS  PubMed  Google Scholar 

  72. Magnusson M, Tobes R, Sancho J, Pareja E (2007) Cutting edge: natural DNA repetitive extragenic sequences from gram-negative pathogens strongly stimulate TLR9. J Immunol 179:31–35

    Article  CAS  PubMed  Google Scholar 

  73. Boule MW, Broughton C, Mackay F, Akira S, Marshak-Rothstein A, Rifkin IR (2004) Toll-like receptor 9-dependent and -independent dendritic cell activation by chromatin-immunoglobulin G complexes. J Exp Med 199:1631–1640

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  74. Latz E, Verma A, Visintin A, Gong M, Sirois CM, Klein DC, Monks BG, McKnight CJ, Lamphier MS, Duprex WP, Espevik T, Golenbock DT (2007) Ligand-induced conformational changes allosterically activate Toll-like receptor 9. Nat Immunol 8:772–779

    Article  CAS  PubMed  Google Scholar 

  75. Lenert P (2005) Inhibitory oligodeoxynucleotides – therapeutic promise for systemic autoimmune diseases? Clin Exp Immunol 140:1–10

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  76. Kalantari P, DeOliveira RB, Chan J, Corbett Y, Rathinam V, Stutz A, Latz E, Gazzinelli RT, Golenbock DT, Fitzgerald KA (2014) Dual engagement of the NLRP3 and AIM2 inflammasomes by plasmodium-derived hemozoin and DNA during malaria. Cell Rep 6:196–210

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  77. Hasan U, Chaffois C, Gaillard C, Saulnier V, Merck E, Tancredi S, Guiet C, Briere F, Vlach J, Lebecque S, Trinchieri G, Bates EE (2005) Human TLR10 is a functional receptor, expressed by B cells and plasmacytoid dendritic cells, which activates gene transcription through {MyD} 88. J Immunol 174:2942–2950

    Article  CAS  PubMed  Google Scholar 

  78. Govindaraj RG, Manavalan B, Lee G, Choi S (2010) Molecular modeling-based evaluation of hTLR10 and identification of potential ligands in Toll-like receptor signaling. PLoS One 5, e12713

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  79. Oosting M, Cheng SC, Bolscher JM, Vestering-Stenger R, Plantinga TS, Verschueren IC, Arts P, Garritsen A, van Eenennaam H, Sturm P, Kullberg BJ, Hoischen A, Adema GJ, van der Meer JW, Netea MG, Joosten LA (2014) Human TLR10 is an anti-inflammatory pattern-recognition receptor. Proc Natl Acad Sci U S A 111:E4478–E4484

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  80. Zhang D, Zhang G, Hayden MS, Greenblatt MB, Bussey C, Flavell RA, Ghosh S (2004) A toll-like receptor that prevents infection by uropathogenic bacteria. Science 303:1522–1526

    Article  CAS  PubMed  Google Scholar 

  81. Yarovinsky F, Zhang D, Andersen JF, Bannenberg GL, Serhan CN, Hayden MS, Hieny S, Sutterwala FS, Flavell RA, Ghosh S, Sher A (2005) TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 308:1626–1629

    Article  CAS  PubMed  Google Scholar 

  82. Roach JC, Glusman G, Rowen L, Kaur A, Purcell MK, Smith KD, Hood LE, Aderem A (2005) The evolution of vertebrate Toll-like receptors. Proc Natl Acad Sci U S A 102:9577–9582

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  83. Andrade WA, Souza Mdo C, Ramos-Martinez E, Nagpal K, Dutra MS, Melo MB, Bartholomeu DC, Ghosh S, Golenbock DT, Gazzinelli RT (2013) Combined action of nucleic acid-sensing Toll-like receptors and TLR11/TLR12 heterodimers imparts resistance to Toxoplasma gondii in mice. Cell Host Microbe 13:42–53

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  84. Mishra BB, Gundra UM, Teale JM (2008) Expression and distribution of Toll-like receptors 11–13 in the brain during murine neurocysticercosis. J Neuroinflammation 5:53

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  85. Latz E, Visintin A, Lien E, Fitzgerald KA, Monks BG, Kurt-Jones EA, Golenbock DT, Espevik T (2002) Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction. J Biol Chem 277:47834–47843

    Article  CAS  PubMed  Google Scholar 

  86. Yang RB, Mark MR, Gray A, Huang A, Xie MH, Zhang M, Goddard A, Wood WI, Gurney AL, Godowski PJ (1998) Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling. Nature 395:284–288

    Article  CAS  PubMed  Google Scholar 

  87. Pridmore AC, Wyllie DH, Abdillahi F, Steeghs L, van der Ley P, Dower SK, Read RC (2001) A lipopolysaccharide-deficient mutant of Neisseria meningitidis elicits attenuated cytokine release by human macrophages and signals via toll-like receptor (TLR) 2 but not via TLR4/MD2. J Infect Dis 183:89–96

    Article  CAS  PubMed  Google Scholar 

  88. Matsuguchi T, Takagi K, Musikacharoen T, Yoshikai Y (1999) Gene expressions of lipopolysaccharide receptors, toll-like receptors 2 and 4, are differently regulated in mouse T lymphocytes. Blood 95(56):1378–1385

    Google Scholar 

  89. Yoshimura A, Lien E, Ingalls RR, Tuomanen E, Dziarski R, Golenbock D (1999) Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2. J Immunol 163:1–5

    CAS  PubMed  Google Scholar 

  90. Hasan UA, Trinchieri G, Vlach J (2005) Toll-like receptor signaling stimulates cell cycle entry and progression in fibroblasts. J Biol Chem 280:20620–20627

    Article  CAS  PubMed  Google Scholar 

  91. Wang J, Shao Y, Bennett TA, Shankar RA, Wightman PD, Reddy LG (2006) The functional effects of physical interactions among Toll-like receptors 7, 8, and 9. J Biol Chem 281:37427–37434

    Article  CAS  PubMed  Google Scholar 

  92. Takeshita F, Leifer CA, Gursel I, Ishii KJ, Takeshita S, Gursel M, Klinman DM (2001) Cutting edge: role of Toll-like receptor 9 in CpG DNA-induced activation of human cells. J Immunol 167:3555–3558

    Article  CAS  PubMed  Google Scholar 

  93. Watanabe N, Narita M, Yamahira A, Nakamura T, Tochiki N, Saitoh A, Kaji M, Hashimoto S, Furukawa T, Toba K, Fuse I, Aizawa Y, Takahashi M (2010) Transformation of dendritic cells from plasmacytoid to myeloid in a leukemic plasmacytoid dendritic cell line (PMDC05). Leuk Res 34:1517–1524

    Article  CAS  PubMed  Google Scholar 

  94. Chaperot L, Blum A, Manches O, Lui G, Angel J, Molens JP, Plumas J (2006) Virus or TLR agonists induce TRAIL-mediated cytotoxic activity of plasmacytoid dendritic cells. J Immunol 176:248–255

    Article  CAS  PubMed  Google Scholar 

  95. Maeda T, Murata K, Fukushima T, Sugahara K, Tsuruda K, Anami M, Onimaru Y, Tsukasaki K, Tomonaga M, Moriuchi R, Hasegawa H, Yamada Y, Kamihira S (2005) A novel plasmacytoid dendritic cell line, CAL-1 established from a patient with blastic natural killer, cell lymphoma. Int J Hematol 81:148–154

    Article  PubMed  Google Scholar 

  96. Tapping RI, Orr SL, Lawson EM, Soldau K, Tobias PS (1999) Membrane-anchored forms of lipopolysaccharide (LPS)-binding protein do not mediate cellular responses to LPS independently of CD14. J Immunol 162:5483–5487

    CAS  PubMed  Google Scholar 

  97. InvivoGen (2006) InvivoGen Insight Newsletter: TLR7 and TLR8. http://www.InvivoGen.com/docs/Insight200609.pdf

    Google Scholar 

  98. Park EK, Jung HS, Yang HI, Yoo MC, Kim C, Kim KS (2007) Optimized THP-1 differentiation is required for the detection of responses to weak stimuli. Inflamm Res 56:45–50

    Article  CAS  PubMed  Google Scholar 

  99. Zarember KA, Godowski PJ (2002) Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol 168:554–561

    Article  CAS  PubMed  Google Scholar 

  100. Chanput W, Mes JJ, Wichers HJ (2014) THP-1 cell line: an in vitro cell model for immune modulation approach. Int Immunopharmacol 23:37–45

    Article  CAS  PubMed  Google Scholar 

  101. Hippenstiel S, Opitz B, Schmeck B, Suttorp N (2006) Lung epithelium as a sentinel and effector system in pneumonia—molecular mechanisms of pathogen recognition and signal transduction. Respir Res 7:97

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  102. Jego G, Bataille R, Geffroy-Luseau A, Descamps G, Pellat-Deceunynck C (2006) Pathogen-associated molecular patterns are growth and survival factors for human myeloma cells through Toll-like receptors. Leukemia 20:1130–1137

    Article  CAS  PubMed  Google Scholar 

  103. Sha Q, Truong-Tran AQ, Plitt JR, Beck LA, Schleimer RP (2004) Activation of airway epithelial cells by toll-like receptor agonists. Am J Respir Cell Mol Biol 31:358–364

    Article  PubMed  CAS  Google Scholar 

  104. Ma Y, Li J, Chiu I, Wang Y, Sloane JA, Lu J, Kosaras B, Sidman RL, Volpe JJ, Vartanian T (2006) Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis. J Cell Biol 175:209–215

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  105. Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdorfer B, Giese T, Endres S, Hartmann G (2002) Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 168:4531–4537

    Article  CAS  PubMed  Google Scholar 

  106. Applequist SE, Wallin RP, Ljunggren HG (2002) Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines. Int Immunol 14:1065–1074

    Article  CAS  PubMed  Google Scholar 

  107. Nagase H, Okugawa S, Ota Y, Yamaguchi M, Tomizawa H, Matsushima K, Ohta K, Yamamoto K, Hirai K (2003) Expression and function of Toll-like receptors in eosinophils: activation by Toll-like receptor 7 ligand. J Immunol 171:3977–3982

    Article  CAS  PubMed  Google Scholar 

  108. Komai-Koma M, Jones L, Ogg GS, Xu D, Liew FY (2004) TLR2 is expressed on activated T cells as a costimulatory receptor. Proc Natl Acad Sci U S A 101:3029–3034

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  109. Hemont C, Neel A, Heslan M, Braudeau C, Josien R (2013) Human blood mDC subsets exhibit distinct TLR repertoire and responsiveness. J Leukoc Biol 93:599–609

    Article  CAS  PubMed  Google Scholar 

  110. Siren J, Pirhonen J, Julkunen I, Matikainen S (2005) IFN-alpha regulates TLR-dependent gene expression of IFN-alpha, IFN-beta, IL-28, and IL-29. J Immunol 174:1932

    Article  CAS  PubMed  Google Scholar 

  111. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira S (1999) Cutting edge: Toll-like receptor 4 (TLR4) -deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 162:6686–6687

    Google Scholar 

  112. Kurt-Jones EA, Sandor F, Ortiz Y, Bowen GN, Counter SL, Wang TC, Finberg RW (2004) Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity. J Endotoxin Res 10:419–424

    Article  CAS  PubMed  Google Scholar 

  113. Thurm CW, Halsey JF (2005) Measurement of cytokine production using whole blood. Curr Protoc Immunol Unit 7:18B

    PubMed  Google Scholar 

  114. Blimkie D, Fortuno ES III, Yan H, Cho P, Ho K, Turvey SE, Marchant A, Goriely S, Kollmann TR (2011) Variables to be controlled in the assessment of blood innate immune responses to Toll-like receptor stimulation. J Immunol Methods 366:89–99

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  115. Hermann C, von Aulock S, Dehus O, Keller M, Okigami H, Gantner F, Wendel A, Hartung T (2006) Endogenous cortisol determines the circadian rhythm of lipopolysaccharide—but not lipoteichoic acid—inducible cytokine release. Eur J Immunol 36:371–379

    Article  CAS  PubMed  Google Scholar 

  116. Petrovsky N, Harrison LC (1995) Cytokine-based human whole blood assay for the detection of antigen-reactive T cells. J Immunol Methods 186:37–46

    Article  CAS  PubMed  Google Scholar 

  117. Schindler S, Asmus S, von Aulock S, Wendel A, Hartung T, Fennrich S (2004) Cryopreservation of human whole blood for pyrogenicity testing. J Immunol Methods 294:89–100

    Article  CAS  PubMed  Google Scholar 

  118. Smolen KK, Cai B, Gelinas L, Fortuno ES III, Larsen M, Speert DP, Chamekh M, Cooper PJ, Esser M, Marchant A, Kollmann TR (2014) Single-cell analysis of innate cytokine responses to pattern recognition receptor stimulation in children across four continents. J Immunol 193:3003–3012

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  119. Burl S, Townend J, Njie-Jobe J, Cox M, Adetifa UJ, Touray E, Philbin VJ, Mancuso C, Kampmann B, Whittle H, Jaye A, Flanagan KL, Levy O (2011) Age-dependent maturation of Toll-like receptor-mediated cytokine responses in Gambian infants. PLoS One 6, e18185

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  120. Levy O, Zarember KA, Roy RM, Cywes C, Godowski PJ, Wessels MR (2004) Selective impairment of TLR-mediated innate immunity in human newborns: neonatal blood plasma reduces monocyte TNF-alpha induction by bacterial lipopeptides, lipopolysaccharide, and imiquimod, but preserves the response to R-848. J Immunol 173:4627–4634

    Article  CAS  PubMed  Google Scholar 

  121. Labuda LA, de Jong SE, Meurs L, Amoah AS, Mbow M, Ateba-Ngoa U, van der Ham AJ, Knulst AC, Yazdanbakhsh M, Adegnika AA (2014) Differences in innate cytokine responses between European and African children. PLoS One 9, e95241

    Article  PubMed Central  PubMed  Google Scholar 

  122. Deitschel SJ, Kerl ME, Chang CH, DeClue AE (2010) Age-associated changes to pathogen-associated molecular pattern-induced inflammatory mediator production in dogs. J Vet Emerg Crit Care (San Antonio) 20:494–502

    Article  Google Scholar 

  123. Figueiredo MD, Moore JN, Vandenplas ML, Sun WC, Murray TF (2008) Effects of the second-generation synthetic lipid A analogue E5564 on responses to endotoxin in [corrected] equine whole blood and monocytes. Am J Vet Res 69:796–803

    Article  CAS  PubMed  Google Scholar 

  124. Karper JC, Ewing MM, de Vries MR, de Jager SC, Peters EA, de Boer HC, van Zonneveld AJ, Kuiper J, Huizinga EG, Brondijk TH, Jukema JW, Quax PH (2013) TLR accessory molecule RP105 (CD180) is involved in post-interventional vascular remodeling and soluble RP105 modulates neointima formation. PLoS One 8, e67923

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  125. Chen YW, Smith ML, Sheets MP, Ballaron SJ, Trevillyan JM, Fey TA, Gauvin DM, Kolano R, Pong MS, Hsieh GC, Bauch J, Marsh K, Carter G, Luly J, Djuric S, Mollison KW (1999) Ex vivo assessment of immunosuppression in undiluted whole blood from pigs dosed with tacrolimus (FK506). Clin Immunol 90:133–140

    Article  CAS  PubMed  Google Scholar 

  126. Dietsch GN, Dipalma CR, Eyre RJ, Pham TQ, Poole KM, Pefaur NB, Welch WD, Trueblood E, Kerns WD, Kanaly ST (2006) Characterization of the inflammatory response to a highly selective PDE4 inhibitor in the rat and the identification of biomarkers that correlate with toxicity. Toxicol Pathol 34:39–51

    Article  CAS  PubMed  Google Scholar 

  127. Duffy D, Rouilly V, Libri V, Hasan M, Beitz B, David M, Urrutia A, Bisiaux A, Labrie ST, Dubois A, Boneca IG, Delval C, Thomas S, Rogge L, Schmolz M, Quintana-Murci L, Albert ML, Milieu Interieur Consortium (2014) Functional analysis via standardized whole-blood stimulation systems defines the boundaries of a healthy immune response to complex stimuli. Immunity 40:436–450

    Article  CAS  Google Scholar 

  128. O'Neill LA, Bowie AG (2007) The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 7:353–364

    Article  PubMed  CAS  Google Scholar 

  129. Hansen MC, Nederby L, Henriksen MO, Hansen M, Nyvold CG (2014) Sensitive ligand-based protein quantification using immuno-PCR: a critical review of single-probe and proximity ligation assays. Biotechniques 56:217–228

    CAS  PubMed  Google Scholar 

  130. Pranada AL, Metz S, Herrmann A, Heinrich PC, Muller-Newen G (2004) Real time analysis of STAT3 nucleocytoplasmic shuttling. J Biol Chem 279:15114–15123

    Article  CAS  PubMed  Google Scholar 

  131. Schindler U, Baichwal VR (1994) Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression. Mol Cell Biol 14:5820–5831

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  132. Johnson CM, Tapping RI (2007) Microbial products stimulate human Toll-like receptor 2 expression through histone modification surrounding a proximal NF-kappaB-binding site. J Biol Chem 282:31197–31205

    Article  CAS  PubMed  Google Scholar 

  133. Berger J, Hauber J, Hauber R, Geiger R, Cullen BR (1988) Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene 66:1–10

    Article  CAS  PubMed  Google Scholar 

  134. Markova SV, Golz S, Frank LA, Kalthof B, Vysotski ES (2004) Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme. J Biol Chem 279:3212–3217

    Article  CAS  PubMed  Google Scholar 

  135. Zhou S, Cerny AM, Bowen G, Chan M, Knipe DM, Kurt-Jones EA, Finberg RW (2010) Discovery of a novel TLR2 signaling inhibitor with anti-viral activity. Antiviral Res 87:295–306

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  136. Ehrhardt C, Kardinal C, Wurzer WJ, Wolff T, von Eichel-Streiber C, Pleschka S, Planz O, Ludwig S (2004) Rac1 and PAK1 are upstream of IKK-epsilon and TBK-1 in the viral activation of interferon regulatory factor-3. FEBS Lett 567:230–238

    Article  CAS  PubMed  Google Scholar 

  137. Civas A, Genin P, Morin P, Lin R, Hiscott J (2006) Promoter organization of the interferon-A genes differentially affects virus-induced expression and responsiveness to TBK1 and IKKepsilon. J Biol Chem 281:4856–4866

    Article  CAS  PubMed  Google Scholar 

  138. Monteith GR, Bird GS (2005) Techniques: high-throughput measurement of intracellular Ca(2+)—back to basics. Trends Pharmacol Sci 26:218–223

    Article  CAS  PubMed  Google Scholar 

  139. National Center for Biotechnology Information (NCBI) (2015) http://www.ncbi.nlm.nih.gov/pcassay/?term = tlr

    Google Scholar 

  140. Reindl M, Lutterotti A, Ingram J, Schanda K, Gassner C, Deisenhammer F, Berger T, Lorenz E (2003) Mutations in the gene for toll-like receptor 4 and multiple sclerosis. Tissue Antigens 61:85–88

    Article  CAS  PubMed  Google Scholar 

  141. Karima R, Matsumoto S, Higashi H, Matsushima K (1999) The molecular pathogenesis of endotoxic shock and organ failure. Mol Med Today 5:123–132

    Article  CAS  PubMed  Google Scholar 

  142. Mansson A, Adner M, Hockerfelt U, Cardell LO (2006) A distinct Toll-like receptor repertoire in human tonsillar B cells, directly activated by PamCSK R-837 and CpG-2006 stimulation. Immunology 118:539–548

    PubMed Central  PubMed  Google Scholar 

  143. Caron G, Duluc D, Fremaux I, Jeannin P, David C, Gascan H, Delneste Y (2005) Direct stimulation of human T cells via TLR5 and TLR7/8: flagellin and R-848 up-regulate proliferation and IFN-gamma production by memory CD4+ T cells. J Immunol 175:1551–1557

    Article  CAS  PubMed  Google Scholar 

  144. Tabiasco J, Devevre E, Rufer N, Salaun B, Cerottini JC, Speiser D, Romero P (2006) Human effector CD8+ T lymphocytes express TLR3 as a functional coreceptor. J Immunol 177:8708–8713

    Article  CAS  PubMed  Google Scholar 

  145. Kadowaki N, Ho S, Antonenko S, Malefyt RW, Kastelein RA, Bazan F, Liu YJ (2001) Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med 194:863–869

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  146. Hart OM, Athie-Morales V, O'Connor GM, Gardiner CM (2005) TLR7/8-mediated activation of human NK cells results in accessory cell-dependent IFN-gamma production. J Immunol 175:1636–1642

    Article  CAS  PubMed  Google Scholar 

  147. Ito T, Wang YH, Liu YJ (2005) Plasmacytoid dendritic cell precursors/type I interferon-producing cells sense viral infection by Toll-like receptor (TLR) 7 and TLR9. Springer Semin Immunopathol 26:221–229

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jennifer K. Dowling .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Dowling, J.K., Dellacasagrande, J. (2016). Toll-Like Receptors: Ligands, Cell-Based Models, and Readouts for Receptor Action. In: McCoy, C. (eds) Toll-Like Receptors. Methods in Molecular Biology, vol 1390. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3335-8_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3335-8_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3333-4

  • Online ISBN: 978-1-4939-3335-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics