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Rapid identification of human mast cell degranulation regulators using functional genomics coupled to high-resolution confocal microscopy

Nature Protocols volume 15, pages 1285–1310 (2020)Cite this article

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Abstract

Targeted functional genomics represents a powerful approach for studying gene function in vivo and in vitro. However, its application to gene expression studies in human mast cells has been hampered by low yields of human mast cell cultures and their poor transfection efficiency. We developed an imaging system in which mast cell degranulation can be visualized in single cells subjected to shRNA knockdown or CRISPR–Cas9 gene editing. By using high-resolution confocal microscopy and a fluorochrome-labeled avidin probe, one can directly assess the alteration of functional responses, i.e., degranulation, in single human mast cells (10–12 weeks old). The elimination of a drug or marker selection step avoids the use of potentially toxic treatment procedures, and the brief hands-on time of the functional analysis step enables high-throughput screening of shRNA or CRISPR–Cas9 constructs to identify genes that regulate human mast cell degranulation. The ability to analyze single cells substantially reduces the total number of cells required and enables the parallel visualization of the degranulation profiles of both edited and non-edited mast cells, offering a consistent internal control not found in other protocols. Moreover, our protocol offers a flexible choice between RNA interference (RNAi) and CRISPR–Cas9 genome editing for perturbation of gene expression using our human mast cell single-cell imaging system. Perturbation of gene expression, acquisition of microscopy data and image analysis can be completed within 5 d, requiring only standard laboratory equipment and expertise.

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Fig. 1: Overview of human mast cell culture, functional genomics and high-resolution confocal microscopy procedures.
Fig. 2: Flowchart illustrating three major components of our protocol: human mast cell culture, functional genomics and high-resolution confocal microscopy.
Fig. 3: Primary human mast cell cultures generated with optimal culture conditions.
Fig. 4: Representative data from selected CRISPR–Cas9-mediated gene knockout studies in primary human mast cells.
Fig. 5: Data analysis and control experiments for validation of regulators of human mast cell degranulation.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author(s) upon reasonable request; an example dataset is provided as Supplementary Data.

Code availability

The custom Fiji macros described in the main text of this study can be accessed and used by readers without restriction. The code in this manuscript has been peer-reviewed.

References

  1. Galli, S. J. & Tsai, M. IgE and mast cells in allergic disease. Nat. Med 18, 693–704 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Galli, S. J. et al. Approaches for analyzing the roles of mast cells and their proteases in vivo. Adv. Immunol. 126, 45–127 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Johnzon, C.-F., Rönnberg, E. & Pejler, G. The role of mast cells in bacterial infection. Am. J. Pathol. 186, 4–14 (2016).

    CAS  PubMed  Google Scholar 

  4. Reber, L. L. et al. Imaging protective mast cells in living mice during severe contact hypersensitivity. JCI Insight 2, 6930 (2017).

    Google Scholar 

  5. Gaudenzio, N. et al. Different activation signals induce distinct mast cell degranulation strategies. J. Clin. Invest 126, 3981–3998 (2016).

    PubMed  PubMed Central  Google Scholar 

  6. Tharp, M. D., Seelig, L. L., Tigelaar, R. E. & Bergstresser, P. R. Conjugated avidin binds to mast cell granules. J. Histochem. Cytochem. 33, 27–32 (1985).

    CAS  PubMed  Google Scholar 

  7. Gaudenzio, N., Laurent, C., Valitutti, S. & Espinosa, E. Human mast cells drive memory CD4+ T cells toward an inflammatory IL-22+ phenotype. J. Allergy Clin. Immunol. 131, 1400–7.e11 (2013).

    CAS  PubMed  Google Scholar 

  8. Joulia, R. et al. Mast cells form antibody-dependent degranulatory synapse for dedicated secretion and defence. Nat. Commun. 6, 6174 (2015).

    CAS  PubMed  Google Scholar 

  9. Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J.-S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

    CAS  PubMed  Google Scholar 

  13. Fu, B. X. H., Hansen, L. L., Artiles, K. L., Nonet, M. L. & Fire, A. Z. Landscape of target: guide homology effects on Cas9-mediated cleavage. Nucleic Acids Res. 42, 13778–13787 (2014).

    CAS  PubMed  Google Scholar 

  14. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Evers, B. et al. CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes. Nat. Biotechnol. 34, 631–633 (2016).

    CAS  PubMed  Google Scholar 

  17. Zsebo, K. M. et al. Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63, 213–224 (1990).

    CAS  PubMed  Google Scholar 

  18. Valent, P. et al. Induction of differentiation of human mast cells from bone marrow and peripheral blood mononuclear cells by recombinant human stem cell factor/kit-ligand in long-term culture. Blood 80, 2237–2245 (1992).

    CAS  PubMed  Google Scholar 

  19. Costa, J. J. et al. Recombinant human stem cell factor (kit ligand) promotes human mast cell and melanocyte hyperplasia and functional activation in vivo. J. Exp. Med 183, 2681–2686 (1996).

    CAS  PubMed  Google Scholar 

  20. Dahlin, J. S. et al. KIT signaling is dispensable for human mast cell progenitor development. Blood 130, 1785–1794 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Shimizu, Y. et al. Characterization of ‘adult-type’ mast cells derived from human bone marrow CD34+ cells cultured in the presence of stem cell factor and interleukin-6. Interleukin-4 is not required for constitutive expression of CD54, Fc epsilon RI alpha and chymase, and CD13 expression is reduced during differentiation. Clin. Exp. Allergy 32, 872–880 (2002).

    CAS  PubMed  Google Scholar 

  22. Shimizu, Y., Irani, A. M., Brown, E. J., Ashman, L. K. & Schwartz, L. B. Human mast cells derived from fetal liver cells cultured with stem cell factor express a functional CD51/CD61 (alpha v beta 3) integrin. Blood 86, 930–939 (1995).

    CAS  PubMed  Google Scholar 

  23. Andersen, H. B. et al. Comparison of short term in vitro cultured human mast cells from different progenitors—peripheral blood-derived progenitors generate highly mature and functional mast cells. J. Immunol. Methods 336, 166–174 (2008).

    CAS  PubMed  Google Scholar 

  24. Mitsui, H. et al. Development of human mast cells from umbilical cord blood cells by recombinant human and murine c-kit ligand. Proc. Natl Acad. Sci. 90, 735–739 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Saito, H., Kato, A., Matsumoto, K. & Okayama, Y. Culture of human mast cells from peripheral blood progenitors. Nat. Protoc. 1, 2178–2183 (2006).

    CAS  PubMed  Google Scholar 

  26. Schmetzer, O. et al. A novel method to generate and culture human mast cells: peripheral CD34+ stem cell-derived mast cells (PSCMCs). J. Immunol. Methods 413, 62–68 (2014).

    CAS  PubMed  Google Scholar 

  27. Tam, I. Y. S., Ng, C. W., Tam, S.-Y. & Lau, H. Y. A. Novel six-week protocol for generating functional human connective tissue-type (MCTC) mast cells from buffy coats. Inflamm. Res. 66, 25–37 (2017).

  28. Yin, Y., Bai, Y., Olivera, A., Desai, A. & Metcalfe, D. D. An optimized protocol for the generation and functional analysis of human mast cells from CD34+ enriched cell populations. J. Immunol. Methods 448, 105–111 (2017).

  29. Navinés-Ferrer, A. et al. MRGPRX2-mediated mast cell response to drugs used in perioperative procedures and anaesthesia. Sci. Rep. 8, 11628 (2018).

    PubMed  PubMed Central  Google Scholar 

  30. McNeil, B. D. et al. Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions. Nature 519, 237–241 (2014).

    PubMed  PubMed Central  Google Scholar 

  31. Hamm, A., Krott, N., Breibach, I., Blindt, R. & Bosserhoff, A. K. Efficient transfection method for primary cells. Tissue Eng. 8, 235–245 (2002).

    CAS  PubMed  Google Scholar 

  32. Joung, J. et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat. Protoc. 12, 828–863 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Agrotis, A. & Ketteler, R. A new age in functional genomics using CRISPR/Cas9 in arrayed library screening. Front. Genet. 6, 300 (2015).

    PubMed  PubMed Central  Google Scholar 

  36. de Groot, R., Lüthi, J., Lindsay, H., Holtackers, R. & Pelkmans, L. Large-scale image-based profiling of single-cell phenotypes in arrayed CRISPR-Cas9 gene perturbation screens. Mol. Syst. Biol. 14, e8064 (2018).

    PubMed  PubMed Central  Google Scholar 

  37. Canver, M. C. et al. Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments. Nat. Protoc. 13, 946–986 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

    PubMed  PubMed Central  Google Scholar 

  41. Bischoff, S. C. Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine data. Nat. Rev. Immunol. 7, 93–104 (2007).

    CAS  PubMed  Google Scholar 

  42. Nilsson, G. et al. Phenotypic characterization of the human mast-cell line HMC-1. Scand. J. Immunol. 39, 489–498 (1994).

    CAS  PubMed  Google Scholar 

  43. Xia, Y. C. et al. Human mast cell line-1 (HMC-1) cells transfected with FcεRIα are sensitive to IgE/antigen-mediated stimulation demonstrating selectivity towards cytokine production. Int. Immunopharmacol. 11, 1002–1011 (2011).

    CAS  PubMed  Google Scholar 

  44. Rådinger, M., Jensen, B. M., Kuehn, H. S., Kirshenbaum, A. & Gilfillan, A. M. Generation, isolation, and maintenance of human mast cells and mast cell lines derived from peripheral blood or cord blood. Curr. Protoc. Immunol. 90, 7.37 (2010).

  45. Guhl, S., Babina, M., Neou, A., Zuberbier, T. & Artuc, M. Mast cell lines HMC-1 and LAD2 in comparison with mature human skin mast cells—drastically reduced levels of tryptase and chymase in mast cell lines. Exp. Dermatol 19, 845–847 (2010).

    CAS  PubMed  Google Scholar 

  46. Laidlaw, T. M. et al. Characterization of a novel human mast cell line that responds to stem cell factor and expresses functional FcεRI. J. Allergy Clin. Immunol. 127, 815–22.e1–5 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Sheen, C. H., Schleimer, R. P. & Kulka, M. Codeine induces human mast cell chemokine and cytokine production: involvement of G-protein activation. Allergy 62, 532–538 (2007).

    CAS  PubMed  Google Scholar 

  48. Guschin, D. Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247–256 (2010).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank C. Liu for technical support and A. van Stigt for helpful discussions regarding MRGPRX2 expression and gene knockout in human mast cell models. We thank P. Dubreuil (Inserm, Marseille, France) for the kind donation of CHO cells. This research was supported by NIH grants U19AI104209, R01 AR067145 and R01 AI32494 (to S.J.G.) and a Marie Skłodowska-Curie Individual Fellowship (H2020-MSCA-IF-2016 no. 749629), the European Research Council (ERC-2018-STG no. 802041), the INSERM ATIP-Avenir program (N.G.) and the Lung Foundation Netherlands (grant 4.1.18.226 to R.W.H). J.F. was supported by a Fulbright Fellowship (financed by the Netherland-America Foundation). R.S. was supported by an NWO Veni Fellowship (grant 91617114) and an Erasmus MC Fellowship.

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Authors and Affiliations

  1. Department of Pathology, Stanford University School of Medicine, Stanford, California, USA

    Jelle Folkerts, See-Ying Tam & Stephen J. Galli

  2. Department of Dermatology and Allergy, Charité–Universitätsmedizin Berlin, Berlin, Germany

    Jelle Folkerts & Marcus Maurer

  3. Department of Pulmonary Medicine, Erasmus MC Rotterdam, Rotterdam, the Netherlands

    Jelle Folkerts, Rudi W. Hendriks & Ralph Stadhouders

  4. Unité de Différenciation Epithéliale et Autoimmunité Rhumatoïde (UDEAR), UMR 1056, INSERM, Université de Toulouse, Toulouse, France

    Nicolas Gaudenzio

  5. Department of Cell Biology, Erasmus MC Rotterdam, Rotterdam, the Netherlands

    Ralph Stadhouders

  6. Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, USA

    Stephen J. Galli

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Contributions

J.F. and N.G. designed the experiments and optimized the protocol. J.F. and N.G. performed the experiments and contributed to the data analysis. M.M., R.W.H., R.S., S.-Y.T. and S.J.G. supervised the projects and participated in experimental design and technical discussions. J.F., N.G, M.M., R.W.H., R.S., S.-Y.T. and S.J.G. contributed to the drafting of the manuscript; J.F. wrote the first draft of the manuscript and J.F., S.-Y.T. and S.J.G. completed the finalized version of the manuscript.

Corresponding authors

Correspondence to See-Ying Tam or Stephen J. Galli.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Hydar Ali and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Gaudenzio, N. et al. J. Clin. Invest. 126, 3981–3998 (2016): https://doi.org/10.1172/JCI85538

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Folkerts, J., Gaudenzio, N., Maurer, M. et al. Rapid identification of human mast cell degranulation regulators using functional genomics coupled to high-resolution confocal microscopy. Nat Protoc 15, 1285–1310 (2020). https://doi.org/10.1038/s41596-019-0288-6

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