Abstract
RNA interference (RNAi) offers a promising base for therapeutic knockdown of clinically relevant genes. Local delivery routes as well as targeted delivery to specific cell populations have been shown to circumvent several hurdles of successful siRNA delivery in vivo. To evaluate and quantify the treatment effect in a precise way, next to measuring the downregulation on gene and protein levels, it is equally essential to investigate the influence on downstream factors such as generated cytokines. Here, we describe an expressive method to specifically isolate the desired target cells and determine their levels of intracellular cytokines by flow cytometry using the example of murine lungs after pulmonary in vivo transfection with siRNA.
Therefore, the lungs of treated mice are harvested and processed into single cell suspensions, in which CD4 positive T cells are marked by antibody-coupled magnetic beads and isolated via magnetic separation. These purified target cells are then fixed and permeabilized, making their intracellular interleukins accessible for staining with fluorescently labeled antibodies. Thus, the cytokine levels and hence the precise influence of the siRNA treatment on intracellular conditions can be measured.
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References
Mocellin S, Provenzano M (2004) RNA interference: learning gene knock-down from cell physiology. J Transl Med 2(1):39
Aagaard L, Rossi JJ (2007) RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev 59(2–3):75–86
Merkel OM, Rubinstein I, Kissel T (2014) siRNA delivery to the lung: what’s new? Adv Drug Deliv Rev 75:112–128
Paranjpe M, Muller-Goymann CC (2014) Nanoparticle-mediated pulmonary drug delivery: a review. Int J Mol Sci 15(4):5852–5873
Lam JK, Liang W, Chan HK (2012) Pulmonary delivery of therapeutic siRNA. Adv Drug Deliv Rev 64(1):1–15
Kim NH, Nadithe V, Elsayed M, Merkel OM (2013) Tracking and treating activated T cells. J Drug Deliv Sci Technol 23(1):17–21
Jeffery PK (1992) Pathology of asthma. Br Med Bull 48(1):23–39
Ray A, Cohn L (1999) Th2 cells and GATA-3 in asthma: new insights into the regulation of airway inflammation. J Clin Invest 104(8):985–993
Gwinn WM, Damsker JM, Falahati R, Okwumabua I, Kelly-Welch A, Keegan AD et al (2006) Novel approach to inhibit asthma-mediated lung inflammation using anti-CD147 intervention. J Immunol 177(7):4870–4879
Biedermann T, Schwarzler C, Lametschwandtner G, Thoma G, Carballido-Perrig N, Kund J et al (2002) Targeting CLA/E-selectin interactions prevents CCR4-mediated recruitment of human Th2 memory cells to human skin in vivo. Eur J Immunol 32(11):3171–3180
O'Reilly S, Hugle T, van Laar JM (2012) T cells in systemic sclerosis: a reappraisal. Rheumatology (Oxford) 51(9):1540–1549
Pelaia G, Vatrella A, Maselli R (2012) The potential of biologics for the treatment of asthma. Nat Rev Drug Discov 11(12):958–972
Sel S, Wegmann M, Dicke T, Sel S, Henke W, Yildirim AO et al (2008) Effective prevention and therapy of experimental allergic asthma using a GATA-3-specific DNAzyme. J Allergy Clin Immunol 121(4):910–916 e5
Krug N, Hohlfeld JM, Kirsten AM, Kornmann O, Beeh KM, Kappeler D et al (2015) Allergen-induced asthmatic responses modified by a GATA3-specific DNAzyme. N Engl J Med 372(21):1987–1995
Xie Y, Kim NH, Nadithe V, Schalk D, Thakur A, Kilic A et al (2016) Targeted delivery of siRNA to activated T cells via transferrin-polyethylenimine (Tf-PEI) as a potential therapy of asthma. J Control Release 229:120–129
Miltenyi S, Muller W, Weichel W, Radbruch A (1990) High gradient magnetic cell separation with MACS. Cytometry 11(2):231–238
Jamur MC, Oliver C (2010) Permeabilization of cell membranes. Methods Mol Biol 588:63–66
Acknowledgments
This work was supported by the ERC Starting Grant ERC-2014-StG-637830 “Novel Asthma Therapy.” The authors are grateful to Ayse Kilic for expert support.
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Kandil, R., Feldmann, D., Xie, Y., Merkel, O.M. (2019). Evaluating the Regulation of Cytokine Levels After siRNA Treatment in Antigen-Specific Target Cell Populations via Intracellular Staining. In: Ogris, M., Sami, H. (eds) Nanotechnology for Nucleic Acid Delivery. Methods in Molecular Biology, vol 1943. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9092-4_21
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DOI: https://doi.org/10.1007/978-1-4939-9092-4_21
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