Advertisement

B-Cell Commitment to IL-10 Production: The VertX Il10egfp Mouse

Protocol
First Online:
  • 754 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2270)

Abstract

Although the inflammatory cytokine IL-10 is pivotal in regulatory B-cell function, detecting IL-10-producing B cells by intracellular IL-10 staining requires multiple steps and tedious preparation. In contrast, the Il10-eGFP reporter mouse model (VertX), generated in 2009, allows easier and quicker detection of IL-10-producing B cells with the possibility of sorting viable cells without membrane permeabilization and ex vivo activation. Even though detecting IL-10+ cells is simpler, several nuances are important. For example, methanol-containing buffers delete GFP signal, while long-term fixation can maintain GFP intensity but decreases other intracellular signals (FOXP3, etc.). Here, we provide optimized and improved protocols for GFP detection in intestinal B cells and isolation techniques of lamina propria, spleen, mesenteric lymph node, peritoneum, and blood cells from VertX mice.

Key words

Regulatory B cell Interleukin-10 GFP Mucosal immunology Flow cytometry Microbiota Gnotobiotic mice 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

We thank Dr. C.L. Karp for providing VertX mice and Dr. B. Vanhaesebroeck for providing PI3KδD910A mice. The authors are supported by grants from National Institute of Health (P01DK094779; P30DK034987; P40OD010995) and Crohn’s & Colitis Foundation Research Fellowship Award (407007) and Gnotobiotic Facility. The UNC Flow Cytometry Core Facility is supported in part by P30CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center. The flow cytometry research reported in this publication was supported in part by the North Carolina Biotech Center Institutional Support Grant 2012-IDG-1006.

References

  1. 1.
    Mizoguchi A, Mizoguchi E, Smith RN et al (1997) Suppressive role of B cells in chronic colitis of T cell receptor α mutant mice. J Exp Med 186:1749–1756CrossRefGoogle Scholar
  2. 2.
    Tedder TF (2015) B10 cells: a functionally defined regulatory B cell subset. J Immunol 194:1395–1401CrossRefGoogle Scholar
  3. 3.
    Madan R, Demircik F, Surianarayanan S et al (2009) Nonredundant roles for B cell-derived IL-10 in immune counter-regulation. J Immunol 183:2312–2320CrossRefGoogle Scholar
  4. 4.
    Mishima Y, Liu B, Hansen JJ, Sartor RB (2015) Resident bacteria-stimulated IL-10-secreting B cells ameliorate T cell-mediated colitis by inducing Tr-1 cells that require IL-27-signaling. Cell Mol Gastroenterol Hepatol 1:295–310CrossRefGoogle Scholar
  5. 5.
    Oka A, Ishihara S, Mishima Y et al (2014) Role of regulatory B cells in chronic intestinal inflammation: association with pathogenesis of Crohn’s disease. Inflamm Bowel Dis 20:315–328CrossRefGoogle Scholar
  6. 6.
    Mizoguchi A, Bhan AK (2006) A case for regulatory B cells. J Immunol 176:705–710CrossRefGoogle Scholar
  7. 7.
    Wei B, Velazquez P, Turovskaya O et al (2005) Mesenteric B cells centrally inhibit CD4+ T cell colitis through interaction with regulatory T cell subsets. Proc Natl Acad Sci U S A 102:2010–2015CrossRefGoogle Scholar
  8. 8.
    Mauri C, Menon M (2017) Human regulatory B cells in health and disease: therapeutic potential. J Clin Invest 127:772–779CrossRefGoogle Scholar
  9. 9.
    Mishima Y, Ishihara S, Aziz MM et al (2010) Decreased production of interleukin-10 and transforming growth factor-β in toll-like receptor-activated intestinal B cells in SAMP1/Yit mice. Immunology 131:473–487CrossRefGoogle Scholar
  10. 10.
    Mishima Y, Oka A, Liu B et al (2019) Microbiota maintain colonic homeostasis by activating TLR2/MyD88/PI3K signaling in IL-10-producing regulatory B cells. J Clin Invest 130:3702–3716CrossRefGoogle Scholar
  11. 11.
    Oka A, Mishima Y, Liu B et al (2019) Phosphoinositide 3-kinase P110δ-signaling is critical for microbiota-activated IL-10 production by B cells that regulate intestinal inflammation. Cells 8:1121.  https://doi.org/10.3390/cells8101121CrossRefPubMedCentralGoogle Scholar
  12. 12.
    The Jackson Laboratory (2020) Mouse strain datasheet, B6(Cg)-Il10tm1.1Karp/J, Stock No:014530, Vert-X (or VertX). https://www.jax.org/strain/014530. Accessed 2 Feb 2020
  13. 13.
    The National Gnotobiotic Rodent Resource Center, University of North Carolina at Chapel Hill (2020). Price list Gnotobiotic mice. https://www.med.unc.edu/ngrrc/price-list/. Accessed 2 Feb 2020
  14. 14.
    Okkenhaug K, Bilancio A, Farjot G et al (2002) Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice. Science 297:1031–1034PubMedGoogle Scholar
  15. 15.
    The Jackson Laboratory (2020) Mouse strain datasheet, C57BL/6J, Stock No:000664, Black 6. https://www.jax.org/strain/000664. Accessed 2 Feb 2020
  16. 16.
    Calado DP, Paixão T, Holmberg D, Haury M (2006) Stochastic monoallelic expression of IL-10 in T cells. J Immunol 177:5358–5364CrossRefGoogle Scholar
  17. 17.
    Kamanaka M, Kim ST, Wan YY et al (2006) Expression of Interleukin-10 in intestinal lymphocytes detected by an Interleukin-10 reporter Knockin tiger mouse. Immunity 25:941–952CrossRefGoogle Scholar
  18. 18.
    Maynard CL, Harrington LE, Janowski KM et al (2007) Regulatory T cells expressing interleukin 10 develop from Foxp3+ and Foxp3− precursor cells in the absence of interleukin 10. Nat Immunol 8:931–941CrossRefGoogle Scholar
  19. 19.
    Atarashi K, Tanoue T, Shima T et al (2011) Induction of colonic regulatory T cells by indigenous Clostridium species. Science 331:337–341CrossRefGoogle Scholar
  20. 20.
    Neves P, Lampropoulou V, Calderon-Gomez E et al (2010) Signaling via the MyD88 adaptor protein in B cells suppresses protective immunity during Salmonella typhimurium infection. Immunity 33:777–790CrossRefGoogle Scholar
  21. 21.
    Bouabe H, Liu Y, Moser M et al (2011) Novel highly sensitive IL-10–β-lactamase reporter mouse reveals cells of the innate immune system as a substantial source of IL-10 in vivo. J Immunol 187:3165–3176CrossRefGoogle Scholar
  22. 22.
    Bouabe H (2012) Cytokine reporter mice: the special case of IL-10. Scand J Immunol 75:553–567CrossRefGoogle Scholar
  23. 23.
    Roederer M (2002) Compensation in Flow Cytometry. Curr Protoc Cytom 22:Unit 1.14.1–Unit 1.1420Google Scholar
  24. 24.
    Schwarz MK, Scherbarth A, Sprengel R et al (2015) Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains. PLoS One 10:e0124650.  https://doi.org/10.1371/journal.pone.0124650CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Tighe S, Held MA (2010) Isolation of total RNA from transgenic mouse melanoma subsets using fluorescence-activated cell sorting. Methods Mol Biol 632:27–44CrossRefGoogle Scholar
  26. 26.
    Ballas ZK, Krieg AM, Warren T et al (2001) Divergent therapeutic and immunologic effects of Oligodeoxynucleotides with distinct CpG motifs. J Immunol 167:4878–4886CrossRefGoogle Scholar
  27. 27.
    Lynch L, Michelet X, Zhang S et al (2015) Regulatory iNKT cells lack expression of the transcription factor PLZF and control the homeostasis of T(reg) cells and macrophages in adipose tissue. Nat Immunol 16:85–95CrossRefGoogle Scholar
  28. 28.
    Weigmann B, Tubbe I, Seidel D et al (2007) Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue. Nat Protoc 2:2307–2311CrossRefGoogle Scholar
  29. 29.
    Sefik E, Geva-Zatorsky N, Oh S et al (2015) Individual intestinal symbionts induce a distinct population of RORγ+ regulatory T cells. Science 349:993–997CrossRefGoogle Scholar
  30. 30.
    Ivanov II, McKenzie BS, Zhou L et al (2006) The orphan nuclear receptor RORγt directs the differentiation program of Proinflammatory IL-17+ T helper cells. Cell 126:1121–1133CrossRefGoogle Scholar
  31. 31.
    Mulder WMC, Koenen H, van de Muysenberg AJC et al (1994) Reduced expression of distinct T-cell CD molecules by collagenase/DNase treatment. Cancer Immunol Immunother 38:253–258PubMedGoogle Scholar
  32. 32.
    Abuzakouk M, Feighery C, O’Farrelly C (1996) Collagenase and Dispase enzymes disrupt lymphocyte surface molecules. J Immunol Methods 194:211–216CrossRefGoogle Scholar
  33. 33.
    Grupillo M, Lakomy R, Geng X et al (2011) An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells. BioTechniques 51:417–420CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Center for Gastrointestinal Biology and Disease, Department of Medicine, Division of Gastroenterology and HepatologyUniversity of North Carolina at Chapel HillChapel HillUSA
  2. 2.Department of Internal Medicine IIShimane University Faculty of MedicineIzumoJapan
  3. 3.Department of Microbiology and ImmunologyUniversity of North Carolina at Chapel HillChapel HillUSA
  4. 4.National Gnotobiotic Rodent Resource CenterUniversity of North Carolina at Chapel HillChapel HillUSA

Personalised recommendations

Cite protocol

Buy options