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Measurement of NF-κB Activation in TLR-Activated Macrophages

  • Orna Ernst
  • Sharat J. Vayttaden
  • Iain D. C. FraserEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1714)

Abstract

Nuclear factor kappa-B (NF-κB) is a key transcription factor in the regulation of the innate immune inflammatory response in activated macrophages. NF-κB functions as a homo- or hetero-dimer derived from one or more of the five members of the NF-κB family, and is activated through a well-studied process of stimulus-dependent inhibitor degradation, post-translational modification, nuclear translocation, and chromatin binding. Its activity is subject to multiple levels of feedback control through both inhibitor protein activity and direct regulation of NF-κB components. Many methods have been developed to measure and quantify NF-κB activation. In this chapter, we summarize available methods and present a protocol for image-based measurement of NF-κB activation in macrophages activated with microbial stimuli. Using either a stably expressed GFP-tagged fusion of the RelA NF-κB protein, or direct detection of endogenous RelA by immunocytochemistry, we describe data collection and analysis to quantify NF-κB cytosol to nuclear translocation in single cells using fluorescence microscopy.

Keywords

NF-κB RelA Nuclear translocation Macrophage Transcription factor GFP High-content imaging 

Notes

Acknowledgments

We thank Dr. Jing Sun for helpful discussions and comments on the nuclear translocation protocols. This work was generously supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

References

  1. 1.
    Sen R, Baltimore D (1986) Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism. Cell 47(6):921–928CrossRefPubMedGoogle Scholar
  2. 2.
    Ghosh S, Hayden MS (2008) New regulators of NF-kappaB in inflammation. Nat Rev Immunol 8(11):837–848. https://doi.org/10.1038/nri2423 CrossRefPubMedGoogle Scholar
  3. 3.
    Ghosh S, May MJ, Kopp EB (1998) NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16:225–260. https://doi.org/10.1146/annurev.immunol.16.1.225 CrossRefPubMedGoogle Scholar
  4. 4.
    Jurjus A, Eid A, Al Kattar S, Zeenny MN, Gerges-Geagea A, Haydar H, Hilal A, Oueidat D, Matar M, Tawilah J, Hussein IH, Schembri-Wismayer P, Cappello F, Tomasello G, Leone A, Jurjus RA (2016) Inflammatory bowel disease, colorectal cancer and type 2 diabetes mellitus: the links. BBA Clin 5:16–24. https://doi.org/10.1016/j.bbacli.2015.11.002 CrossRefPubMedGoogle Scholar
  5. 5.
    Hoffmann A, Baltimore D (2006) Circuitry of nuclear factor kappaB signaling. Immunol Rev 210:171–186. https://doi.org/10.1111/j.0105-2896.2006.00375.x CrossRefPubMedGoogle Scholar
  6. 6.
    Smale ST (2012) Dimer-specific regulatory mechanisms within the NF-kappaB family of transcription factors. Immunol Rev 246(1):193–204. https://doi.org/10.1111/j.1600-065X.2011.01091.x CrossRefPubMedGoogle Scholar
  7. 7.
    Karin M, Ben-Neriah Y (2000) Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol 18:621–663. https://doi.org/10.1146/annurev.immunol.18.1.621 CrossRefPubMedGoogle Scholar
  8. 8.
    Huang B, Yang XD, Lamb A, Chen LF (2010) Posttranslational modifications of NF-kappaB: another layer of regulation for NF-kappaB signaling pathway. Cell Signal 22(9):1282–1290. https://doi.org/10.1016/j.cellsig.2010.03.017 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Perkins ND, Felzien LK, Betts JC, Leung K, Beach DH, Nabel GJ (1997) Regulation of NF-kappaB by cyclin-dependent kinases associated with the p300 coactivator. Science 275(5299):523–527CrossRefPubMedGoogle Scholar
  10. 10.
    Hochrainer K, Racchumi G, Anrather J (2013) Site-specific phosphorylation of the p65 protein subunit mediates selective gene expression by differential NF-kappaB and RNA polymerase II promoter recruitment. J Biol Chem 288(1):285–293. https://doi.org/10.1074/jbc.M112.385625 CrossRefPubMedGoogle Scholar
  11. 11.
    Zhong H, May MJ, Jimi E, Ghosh S (2002) The phosphorylation status of nuclear NF-kappa B determines its association with CBP/p300 or HDAC-1. Mol Cell 9(3):625–636CrossRefPubMedGoogle Scholar
  12. 12.
    Saccani S, Marazzi I, Beg AA, Natoli G (2004) Degradation of promoter-bound p65/RelA is essential for the prompt termination of the nuclear factor kappaB response. J Exp Med 200(1):107–113. https://doi.org/10.1084/jem.20040196 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kunsch C, Ruben SM, Rosen CA (1992) Selection of optimal kappa B/Rel DNA-binding motifs: interaction of both subunits of NF-kappa B with DNA is required for transcriptional activation. Mol Cell Biol 12(10):4412–4421CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Holden NS, Tacon CE (2011) Principles and problems of the electrophoretic mobility shift assay. J Pharmacol Toxicol Methods 63(1):7–14. https://doi.org/10.1016/j.vascn.2010.03.002 CrossRefPubMedGoogle Scholar
  15. 15.
    Renard P, Ernest I, Houbion A, Art M, Le Calvez H, Raes M, Remacle J (2001) Development of a sensitive multi-well colorimetric assay for active NFkappaB. Nucleic Acids Res 29(4):E21CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Colleran A, Collins PE, Carmody RJ (2015) Assessing sites of NF-kappaB DNA binding using chromatin immunoprecipitation. Methods Mol Biol 1280:47–59. https://doi.org/10.1007/978-1-4939-2422-6_4 CrossRefPubMedGoogle Scholar
  17. 17.
    Nowak DE, Tian B, Brasier AR (2005) Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation. BioTechniques 39(5):715–725CrossRefPubMedGoogle Scholar
  18. 18.
    Collins PE, O'Carroll C, Carmody RJ (2015) Measurement of NF-kappaB transcriptional activity and identification of NF-kappaB cis-regulatory elements using luciferase assays. Methods Mol Biol 1280:25–43. https://doi.org/10.1007/978-1-4939-2422-6_3 CrossRefPubMedGoogle Scholar
  19. 19.
    Woods JW, Coffey MJ, Brock TG, Singer II, Peters-Golden M (1995) 5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation. J Clin Invest 95(5):2035–2046. https://doi.org/10.1172/JCI117889 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Nabbi A, Riabowol K (2015) Rapid isolation of nuclei from cells in vitro. Cold Spring Harb Protoc 2015(8):769–772. https://doi.org/10.1101/pdb.prot083733 PubMedGoogle Scholar
  21. 21.
    Luo Y, Hara T, Ishido Y, Yoshihara A, Oda K, Makino M, Ishii N, Hiroi N, Suzuki K (2014) Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays. BMC Immunol 15:586. https://doi.org/10.1186/s12865-014-0062-z CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Takashiba S, Van Dyke TE, Amar S, Murayama Y, Soskolne AW, Shapira L (1999) Differentiation of monocytes to macrophages primes cells for lipopolysaccharide stimulation via accumulation of cytoplasmic nuclear factor kappaB. Infect Immun 67(11):5573–5578PubMedPubMedCentralGoogle Scholar
  23. 23.
    Nelson DE, Ihekwaba AE, Elliott M, Johnson JR, Gibney CA, Foreman BE, Nelson G, See V, Horton CA, Spiller DG, Edwards SW, McDowell HP, Unitt JF, Sullivan E, Grimley R, Benson N, Broomhead D, Kell DB, White MR (2004) Oscillations in NF-kappaB signaling control the dynamics of gene expression. Science 306(5696):704–708. https://doi.org/10.1126/science.1099962 CrossRefPubMedGoogle Scholar
  24. 24.
    Ashall L, Horton CA, Nelson DE, Paszek P, Harper CV, Sillitoe K, Ryan S, Spiller DG, Unitt JF, Broomhead DS, Kell DB, Rand DA, See V, White MR (2009) Pulsatile stimulation determines timing and specificity of NF-kappaB-dependent transcription. Science 324(5924):242–246. https://doi.org/10.1126/science.1164860 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Kellogg RA, Tay S (2015) Noise facilitates transcriptional control under dynamic inputs. Cell 160(3):381–392. https://doi.org/10.1016/j.cell.2015.01.013 CrossRefPubMedGoogle Scholar
  26. 26.
    Lee RE, Walker SR, Savery K, Frank DA, Gaudet S (2014) Fold change of nuclear NF-kappaB determines TNF-induced transcription in single cells. Mol Cell 53(6):867–879. https://doi.org/10.1016/j.molcel.2014.01.026 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Sung MH, Salvatore L, De Lorenzi R, Indrawan A, Pasparakis M, Hager GL, Bianchi ME, Agresti A (2009) Sustained oscillations of NF-kappaB produce distinct genome scanning and gene expression profiles. PLoS One 4(9):e7163. https://doi.org/10.1371/journal.pone.0007163 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Tay S, Hughey JJ, Lee TK, Lipniacki T, Quake SR, Covert MW (2010) Single-cell NF-kappaB dynamics reveal digital activation and analogue information processing. Nature 466(7303):267–271. https://doi.org/10.1038/nature09145 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Friedrichsen S, Harper CV, Semprini S, Wilding M, Adamson AD, Spiller DG, Nelson G, Mullins JJ, White MR, Davis JR (2006) Tumor necrosis factor-alpha activates the human prolactin gene promoter via nuclear factor-kappaB signaling. Endocrinology 147(2):773–781. https://doi.org/10.1210/en.2005-0967 CrossRefPubMedGoogle Scholar
  30. 30.
    Hoffmann A, Levchenko A, Scott ML, Baltimore D (2002) The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation. Science 298(5596):1241–1245. https://doi.org/10.1126/science.1071914 CrossRefPubMedGoogle Scholar
  31. 31.
    De Lorenzi R, Gareus R, Fengler S, Pasparakis M (2009) GFP-p65 knock-in mice as a tool to study NF-kappaB dynamics in vivo. Genesis 47(5):323–329. https://doi.org/10.1002/dvg.20468 CrossRefPubMedGoogle Scholar
  32. 32.
    Sung MH, Li N, Lao Q, Gottschalk RA, Hager GL, Fraser ID (2014) Switching of the relative dominance between feedback mechanisms in lipopolysaccharide-induced NF-kappaB signaling. Sci Signal 7(308):ra6. https://doi.org/10.1126/scisignal.2004764 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Li N, Sun J, Benet ZL, Wang Z, Al-Khodor S, John SP, Lin B, Sung MH, Fraser ID (2015) Development of a cell system for siRNA screening of pathogen responses in human and mouse macrophages. Sci Rep 5:9559. https://doi.org/10.1038/srep09559 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Gottschalk RA, Martins AJ, Angermann BR, Dutta B, Ng CE, Uderhardt S, Tsang JS, Fraser ID, Meier-Schellersheim M, Germain RN (2016) Distinct NF-kappaB and MAPK activation thresholds uncouple steady-state microbe sensing from anti-pathogen inflammatory responses. Cell Syst 2(6):378–390. https://doi.org/10.1016/j.cels.2016.04.016 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Miller AH, Vayttaden SJ, Al-Khodor S, Fraser ID (2015) Assay development for image-based quantification of intracellular bacterial replication and analysis of the innate immune response to infection. Assay Drug Dev Technol 13(9):515–528. https://doi.org/10.1089/adt.2015.664 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Lee TK, Denny EM, Sanghvi JC, Gaston JE, Maynard ND, Hughey JJ, Covert MW (2009) A noisy paracrine signal determines the cellular NF-kappaB response to lipopolysaccharide. Sci Signal 2(93):ra65. https://doi.org/10.1126/scisignal.2000599 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Gerner MY, Kastenmuller W, Ifrim I, Kabat J, Germain RN (2012) Histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes. Immunity 37(2):364–376. https://doi.org/10.1016/j.immuni.2012.07.011 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Orna Ernst
    • 1
  • Sharat J. Vayttaden
    • 1
  • Iain D. C. Fraser
    • 1
    Email author
  1. 1.Signaling Systems Unit, Laboratory of Immune System BiologyNational Institute of Allergy and Infectious Diseases, National Institutes of HealthBethesdaUSA

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