Abstract
Macrophages have emerged as crucial regulators of tissue homeostasis, inflammation, and tissue regeneration. In vivo bioluminescence imaging could offer a powerful tool to study many poorly understood aspects of macrophage biology. Thus, we recently developed a straightforward method for the production of large numbers of green fluorescent protein (GFP) and firefly luciferase (fLUC)-expressing reporter macrophages for various in vivo bioluminescence imaging applications. Lentivirus vector containing the GFP/fLUC reporter gene is produced and mouse bone marrow macrophages are isolated following established protocols. Macrophages are then exposed to the lentivirus in the presence of 10 μM cyclosporine for 24 h. After a 24-h recovery period, the transduction is repeated. Three days after the second infection the cells are ready to be used in vivo. Following this cyclosporine-mediated double infection strategy up to 60% of the macrophages express GFP in flow cytometry. The macrophages maintain their ability to polarize to M1 and M2 phenotypes and, when injected to the systemic circulation of a mouse model, reporter cells are both easily detectable with BLI and migrate to a local site of inflammation. These GFP/fLUC-expressing reporter macrophages could prove to be useful tools to study the role of macrophages in health and disease.
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References
Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8:958–969
Solinas G, Germano G, Mantovani A et al (2009) Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation. J Leukoc Biol 86:1065–1073
Chawla A, Nguyen KD, Goh YP (2011) Macrophage-mediated inflammation in metabolic disease. Nat Rev Immunol 11:738–749
Murray PJ, Wynn TA (2011) Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11:723–737
Nich C, Takakubo Y, Pajarinen J et al (2013) Macrophages-key cells in the response to wear debris from joint replacements. J Biomed Mater Res A 101:3033–3045
Goodman SB, Gibon E, Pajarinen J et al (2014) Novel biological strategies for treatment of wear particle-induced periprosthetic osteolysis of orthopaedic implants for joint replacement. J R Soc Interface 11:20130962
Brown BN, Sicari BM, Badylak SF (2014) Rethinking regenerative medicine: a macrophage-centered approach. Front Immunol 5:510
Nassiri S, Graney P, Spiller KL (2014) Manipulation of macropahges to enhance bone repair and regeneration. In: Zreiqat H, Dunstan CR, Rosen V (eds) A tissue regeneration approach to bone and cartilage repair. Springer, New York. ISBN: 978-3-319-13265-5
Noser JA, Towers GJ, Sakuma R et al (2006) Cyclosporine increases human immunodeficiency virus type 1 vector transduction of primary mouse cells. J Virol 80(15):7769–7774
Zhang X, Edwards J, Mosser D (2009) The expression of exogenous genes in macrophages: obstacles and opportunities. In: Reiner N (ed) Macrophages and dendritic cells, methods in molecular biology. Springer Protocols, New York, pp 123–143
Cao YA, Wagers AJ, Beilhack A et al (2004) Shifting foci of hematopoiesis during reconstitution from single stem cells. Proc Natl Acad Sci U S A 101:221–226
Cao YA, Bachmann MH, Beilhack A et al (2005) Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation. Transplantation 80:134–139
Pajarinen J, Lin TH, Sato T et al (2015) Establishment of green fluorescent protein and firefly luciferase expressing mouse primary macrophages for bioluminescence imaging. PLoS One 10:e0142736
Zufferey R, Nagy D, Mandel RJ et al (1997) Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15:871–875
Weischenfeldt J, Porse B (2008) Bone marrow-derived macrophages (BMM): isolation and applications. CSH Protoc 2008:pdb.prot5080
Martinez FO, Helming L, Milde R et al (2013) Genetic programs expressed in resting and IL-4 alternatively activated mouse and human macrophages: similarities and differences. Blood 121:e57–e69
Ren PG, Irani A, Huang Z et al (2011) Continuous infusion of UHMWPE particles induces increased bone macrophages and osteolysis. Clin Orthop Relat Res 469:113–122
Acknowledgments
Work was supported by NIH grants 2R01 AR055650 and 1R01AR063717 and the Ellenburg Chair in Surgery in Stanford University. J.P. thanks Jane and Aatos Erkko foundation for postdoctoral fellowship.
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Pajarinen, J., Lin, TH., Goodman, S.B. (2018). Production of GFP and Luciferase-Expressing Reporter Macrophages for In Vivo Bioluminescence Imaging. In: Dubey, P. (eds) Reporter Gene Imaging. Methods in Molecular Biology, vol 1790. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7860-1_8
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DOI: https://doi.org/10.1007/978-1-4939-7860-1_8
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