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Bone Marrow-Derived Macrophage Immortalization of LXR Nuclear Receptor-Deficient Cells

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Lipid-Activated Nuclear Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1951))

Abstract

Macrophages are professional phagocytic cells that play key roles in innate and adaptive immunity, metabolism, and tissue homeostasis. Lipid metabolism is tightly controlled at the transcriptional level, and one of the key players of this regulation in macrophages and other cell types is the LXR subfamily of nuclear receptors (LXRα and LXRβ). The use of LXR double knockout (LXR-DKO) macrophages in vitro has yielded extensive benefits in metabolism research, but this technique is hindered by primary macrophage cell expansion capability, which diminishes along terminal cell differentiation process. Here we detail a method to immortalize LXR double knockout bone marrow-derived macrophage cells at an early stage of differentiation, using a retroviral delivery of a combination of murine v-myc and v-raf oncogenes. This methodology enables the generation of autonomous self-renewing macrophages bearing an LXR-DKO genetic background, as a valuable tool for research in lipid metabolism and other LXR receptor-mediated effects.

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References

  1. Svensson S, Ostberg T, Jacobsson M, Norstrom C, Stefansson K, Hallen D, Johansson IC, Zachrisson K, Ogg D, Jendeberg L (2003) Crystal structure of the heterodimeric complex of LXRalpha and RXRbeta ligand-binding domains in a fully agonistic conformation. EMBO J 22(18):4625–4633. https://doi.org/10.1093/emboj/cdg456

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Peet DJ, Turley SD, Ma W, Janowski BA, Lobaccaro JM, Hammer RE, Mangelsdorf DJ (1998) Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR alpha. Cell 93(5):693–704. https://doi.org/10.1016/S0092-8674(00)81432-4

    Article  CAS  PubMed  Google Scholar 

  3. Castrillo A, Tontonoz P (2004) Nuclear receptors in macrophage biology: at the crossroads of lipid metabolism and inflammation. Annu Rev Cell Dev Biol 20:455–480. https://doi.org/10.1146/annurev.cellbio.20.012103.134432

    Article  CAS  PubMed  Google Scholar 

  4. Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H, Mangelsdorf DJ (2002) Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver X receptors alpha and beta. J Biol Chem 277(21):18793–18800. https://doi.org/10.1074/jbc.M109927200

    Article  CAS  PubMed  Google Scholar 

  5. Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P (2003) Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat Med 9(2):213–219. https://doi.org/10.1038/nm820

    Article  CAS  PubMed  Google Scholar 

  6. N AG, Bensinger SJ, Hong C, Beceiro S, Bradley MN, Zelcer N, Deniz J, Ramirez C, Diaz M, Gallardo G, de Galarreta CR, Salazar J, Lopez F, Edwards P, Parks J, Andujar M, Tontonoz P, Castrillo A (2009) Apoptotic cells promote their own clearance and immune tolerance through activation of the nuclear receptor LXR. Immunity 31(2):245–258. https://doi.org/10.1016/j.immuni.2009.06.018

    Article  CAS  Google Scholar 

  7. Bensinger SJ, Bradley MN, Joseph SB, Zelcer N, Janssen EM, Hausner MA, Shih R, Parks JS, Edwards PA, Jamieson BD, Tontonoz P (2008) LXR signaling couples sterol metabolism to proliferation in the acquired immune response. Cell 134(1):97–111. https://doi.org/10.1016/j.cell.2008.04.052

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Joseph SB, Bradley MN, Castrillo A, Bruhn KW, Mak PA, Pei L, Hogenesch J, O'Connell RM, Cheng G, Saez E, Miller JF, Tontonoz P (2004) LXR-dependent gene expression is important for macrophage survival and the innate immune response. Cell 119(2):299–309. https://doi.org/10.1016/j.cell.2004.09.032

    Article  CAS  PubMed  Google Scholar 

  9. Valledor AF, Hsu LC, Ogawa S, Sawka-Verhelle D, Karin M, Glass CK (2004) Activation of liver X receptors and retinoid X receptors prevents bacterial-induced macrophage apoptosis. Proc Natl Acad Sci U S A 101(51):17813–17818. https://doi.org/10.1073/pnas.0407749101

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. N AG, Guillen JA, Gallardo G, Diaz M, de la Rosa JV, Hernandez IH, Casanova-Acebes M, Lopez F, Tabraue C, Beceiro S, Hong C, Lara PC, Andujar M, Arai S, Miyazaki T, Li S, Corbi AL, Tontonoz P, Hidalgo A, Castrillo A (2013) The nuclear receptor LXRalpha controls the functional specialization of splenic macrophages. Nat Immunol 14(8):831–839. https://doi.org/10.1038/ni.2622

    Article  CAS  Google Scholar 

  11. Frenoux JM, Vernet P, Volle DH, Britan A, Saez F, Kocer A, Henry-Berger J, Mangelsdorf DJ, Lobaccaro JM, Drevet JR (2004) Nuclear oxysterol receptors, LXRs, are involved in the maintenance of mouse caput epididymidis structure and functions. J Mol Endocrinol 33(2):361–375. https://doi.org/10.1677/jme.1.01515

    Article  CAS  PubMed  Google Scholar 

  12. Blasi E, Mathieson BJ, Varesio L, Cleveland JL, Borchert PA, Rapp UR (1985) Selective immortalization of murine macrophages from fresh bone marrow by a raf/myc recombinant murine retrovirus. Nature 318(6047):667–670. https://doi.org/10.1038/318667a0

    Article  CAS  PubMed  Google Scholar 

  13. Lutz MB, Granucci F, Winzler C, Marconi G, Paglia P, Foti M, Assmann CU, Cairns L, Rescigno M, Ricciardi-Castagnoli P (1994) Retroviral immortalization of phagocytic and dendritic cell clones as a tool to investigate functional heterogeneity. J Immunol Methods 174(1-2):269–279. https://doi.org/10.1016/0022-1759(94)90031-0

    Article  CAS  PubMed  Google Scholar 

  14. Girolomoni G, Lutz MB, Pastore S, Assmann CU, Cavani A, Ricciardi-Castagnoli P (1995) Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin. Eur J Immunol 25(8):2163–2169. https://doi.org/10.1002/eji.1830250807

    Article  CAS  PubMed  Google Scholar 

  15. Rapp UR, Cleveland JL, Fredrickson TN, Holmes KL, Morse HC, Jansen HW, Patschinsky T, Bister K (1985) Rapid induction of hemopoietic neoplasms in newborn mice by a raf(mil)/myc recombinant murine retrovirus. J Virol 55(1):23–33

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Flaveny CA, Griffett K, El-Gendy Bel D, Kazantzis M, Sengupta M, Amelio AL, Chatterjee A, Walker J, Solt LA, Kamenecka TM, Burris TP (2015) Broad Anti-tumor Activity of a Small Molecule that Selectively Targets the Warburg Effect and Lipogenesis. Cancer Cell 28(1):42–56. https://doi.org/10.1016/j.ccell.2015.05.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Tavazoie MF, Pollack I, Tanqueco R, Ostendorf BN, Reis BS, Gonsalves FC, Kurth I, Andreu-Agullo C, Derbyshire ML, Posada J, Takeda S, Tafreshian KN, Rowinsky E, Szarek M, Waltzman RJ, McMillan EA, Zhao C, Mita M, Mita A, Chmielowski B, Postow MA, Ribas A, Mucida D, Tavazoie SF (2018) LXR/ApoE Activation Restricts Innate Immune Suppression in. Cancer Cell 172(4):825–840.e818. https://doi.org/10.1016/j.cell.2017.12.026

    Article  CAS  Google Scholar 

  18. Palleroni AV, Varesio L, Wright RB, Brunda MJ (1991) Tumoricidal alveolar macrophage and tumor infiltrating macrophage cell lines. Int J Cancer 49(2):296–302. https://doi.org/10.1002/ijc.2910490226

    Article  CAS  PubMed  Google Scholar 

  19. Adami C, Brunda MJ, Palleroni AV (1993) In vivo immortalization of murine peritoneal macrophages: a new rapid and efficient method for obtaining macrophage cell lines. J Leukoc Biol 53(4):475–478. https://doi.org/10.1002/jlb.53.4.475

    Article  CAS  PubMed  Google Scholar 

  20. Gupta D, Shah HP, Malu K, Berliner N, Gaines P (2014) Differentiation and characterization of myeloid cells. Curr Protoc Immunol 104Unit 22F.25. https://doi.org/10.1002/0471142735.im22f05s104

  21. Earle WR, Schilling EL, Stark TH, Straus NP, Brown MF, Shelton E (1943) Production of Malignancy in Vitro. IV. The Mouse Fibroblast Cultures and Changes Seen in the Living Cells. J Natl Cancer Inst 4(2):165–212. https://doi.org/10.1093/jnci/4.2.165

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo Superior de Investigaciones Científicas (CSIC), Centro Mixto CSIC-Universidad Autónoma de Madrid, Madrid, Spain

    Ana Ramón-Vázquez, Juan Vladimir de la Rosa, Carlos Tabraue & Antonio Castrillo

  2. Unidad de Biomedicina IIBM-ULPGC (Unidad Asociada al CSIC), Universidad de Las Palmas de Gran Canaria, Las Palmas, Spain

    Ana Ramón-Vázquez, Juan Vladimir de la Rosa, Carlos Tabraue & Antonio Castrillo

  3. Grupo de Investigación Medio Ambiente y Salud (GIMAS), Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS) de la ULPGC, Las Palmas, Spain

    Ana Ramón-Vázquez, Juan Vladimir de la Rosa, Carlos Tabraue & Antonio Castrillo

Authors
  1. Ana Ramón-Vázquez
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  2. Juan Vladimir de la Rosa
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  3. Carlos Tabraue
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  4. Antonio Castrillo
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Corresponding author

Correspondence to Antonio Castrillo .

Editor information

Editors and Affiliations

  1. Division of Medicine, Centre for Cardiometabolic Medicine, University College London, London, UK

    Matthew C. Gage

  2. Division of Medicine, Centre for Cardiometabolic Medicine, University College London, London, UK

    Inés Pineda-Torra

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Ramón-Vázquez, A., de la Rosa, J.V., Tabraue, C., Castrillo, A. (2019). Bone Marrow-Derived Macrophage Immortalization of LXR Nuclear Receptor-Deficient Cells. In: Gage, M., Pineda-Torra, I. (eds) Lipid-Activated Nuclear Receptors. Methods in Molecular Biology, vol 1951. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9130-3_6

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  • DOI: https://doi.org/10.1007/978-1-4939-9130-3_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9129-7

  • Online ISBN: 978-1-4939-9130-3

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