Advertisement

Immunophenotyping of Human B Lymphocytes in Blood and in Adipose Tissue

  • Alain Diaz
  • Maria Romero
  • Daniela Frasca
  • Bonnie B. BlombergEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2032)

Abstract

The human obese subcutaneous adipose tissue (SAT) contributes to systemic and B cell intrinsic inflammation, reduced B cell responses, and increased secretion of autoimmune antibodies. Immune cells are recruited to the SAT by chemokines released by both adipocytes and infiltrating immune cells. We describe here the characterization of B lymphocytes from the SAT and blood (control) of obese females undergoing weight reduction surgeries (breast reduction or panniculectomy). We show how to isolate the immune cells from the blood and SAT, how to characterize B cells and their subsets, and how to measure markers of activation and/or transcription factors in SAT-derived B cells and B cell subsets. We also show how to evaluate other immune cell types infiltrating the SAT, including T cells, NK cells, monocyte/macrophages, in order to measure relative proportions of these cell types as compared to the blood.

Key words

Adipose tissue (AT) Body mass index (BMI) Cardiovascular (CV) Dulbecco’s modified Eagle’s medium (DMEM) Fetal calf serum (FCS) Free fatty acids (FFAs) Germinal center (GC) Hanks’ balanced salt solution (HBSS) Insulin resistance (IR) Insulin sensitivity (IS) Peripheral blood mononuclear cells (PBMC) Red blood cells (RBC) Reactive oxygen species (ROS) Room temperature (RT) Subcutaneous adipose tissue (SAT) Stromal vascular fraction (SVF) Type-2 diabetes (T2D) Toll-like receptor (TLR) 

Notes

Acknowledgments

This study was supported by NIH R56 AG32576 (DF/BBB), R21 AG042826 (DF/BBB), R21 AI096446 (BBB/DF), R56 AG059719 [4].

References

  1. 1.
    Apovian CM, Gokce N (2012) Obesity and cardiovascular disease. Circulation 125(9):1178–1182.  https://doi.org/10.1161/CIRCULATIONAHA.111.022541CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Hotamisligil GS (2006) Inflammation and metabolic disorders. Nature 444(7121):860–867.  https://doi.org/10.1038/nature05485CrossRefPubMedGoogle Scholar
  3. 3.
    Johnson AM, Olefsky JM (2013) The origins and drivers of insulin resistance. Cell 152(4):673–684.  https://doi.org/10.1016/j.cell.2013.01.041CrossRefPubMedGoogle Scholar
  4. 4.
    Shoelson SE, Lee J, Goldfine AB (2006) Inflammation and insulin resistance. J Clin Invest 116(7):1793–1801.  https://doi.org/10.1172/JCI29069CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Renehan AG, Tyson M, Egger M, Heller RF, Zwahlen M (2008) Body-mass index and incidence of cancer: a systematic review and meta-analysis of prospective observational studies. Lancet 371(9612):569–578.  https://doi.org/10.1016/S0140-6736(08)60269-XCrossRefPubMedGoogle Scholar
  6. 6.
    Setty AR, Curhan G, Choi HK (2007) Obesity, waist circumference, weight change, and the risk of psoriasis in women: nurses’ health study II. Arch Intern Med 167(15):1670–1675.  https://doi.org/10.1001/archinte.167.15.1670CrossRefPubMedGoogle Scholar
  7. 7.
    Casas R, Sacanella E, Estruch R (2014) The immune protective effect of the Mediterranean diet against chronic low-grade inflammatory diseases. Endocr Metab Immune Disord Drug Targets 14(4):245–254CrossRefGoogle Scholar
  8. 8.
    Hass DJ, Brensinger CM, Lewis JD, Lichtenstein GR (2006) The impact of increased body mass index on the clinical course of Crohn's disease. Clin Gastroenterol Hepatol 4(4):482–488.  https://doi.org/10.1016/j.cgh.2005.12.015CrossRefPubMedGoogle Scholar
  9. 9.
    Collaborators GBDO, Afshin A, Forouzanfar MH, Reitsma MB, Sur P, Estep K, Lee A, Marczak L, Mokdad AH, Moradi-Lakeh M, Naghavi M, Salama JS, Vos T, Abate KH, Abbafati C, Ahmed MB, Al-Aly Z, Alkerwi A, Al-Raddadi R, Amare AT, Amberbir A, Amegah AK, Amini E, Amrock SM, Anjana RM, Arnlov J, Asayesh H, Banerjee A, Barac A, Baye E, Bennett DA, Beyene AS, Biadgilign S, Biryukov S, Bjertness E, Boneya DJ, Campos-Nonato I, Carrero JJ, Cecilio P, Cercy K, Ciobanu LG, Cornaby L, Damtew SA, Dandona L, Dandona R, Dharmaratne SD, Duncan BB, Eshrati B, Esteghamati A, Feigin VL, Fernandes JC, Furst T, Gebrehiwot TT, Gold A, Gona PN, Goto A, Habtewold TD, Hadush KT, Hafezi-Nejad N, Hay SI, Horino M, Islami F, Kamal R, Kasaeian A, Katikireddi SV, Kengne AP, Kesavachandran CN, Khader YS, Khang YH, Khubchandani J, Kim D, Kim YJ, Kinfu Y, Kosen S, Ku T, Defo BK, Kumar GA, Larson HJ, Leinsalu M, Liang X, Lim SS, Liu P, Lopez AD, Lozano R, Majeed A, Malekzadeh R, Malta DC, Mazidi M, McAlinden C, McGarvey ST, Mengistu DT, Mensah GA, Mensink GBM, Mezgebe HB, Mirrakhimov EM, Mueller UO, Noubiap JJ, Obermeyer CM, Ogbo FA, Owolabi MO, Patton GC, Pourmalek F, Qorbani M, Rafay A, Rai RK, Ranabhat CL, Reinig N, Safiri S, Salomon JA, Sanabria JR, Santos IS, Sartorius B, Sawhney M, Schmidhuber J, Schutte AE, Schmidt MI, Sepanlou SG, Shamsizadeh M, Sheikhbahaei S, Shin MJ, Shiri R, Shiue I, Roba HS, Silva DAS, Silverberg JI, Singh JA, Stranges S, Swaminathan S, Tabares-Seisdedos R, Tadese F, Tedla BA, Tegegne BS, Terkawi AS, Thakur JS, Tonelli M, Topor-Madry R, Tyrovolas S, Ukwaja KN, Uthman OA, Vaezghasemi M, Vasankari T, Vlassov VV, Vollset SE, Weiderpass E, Werdecker A, Wesana J, Westerman R, Yano Y, Yonemoto N, Yonga G, Zaidi Z, Zenebe ZM, Zipkin B, Murray CJL (2017) Health effects of overweight and obesity in 195 countries over 25 years. N Engl J Med 377(1):13–27.  https://doi.org/10.1056/NEJMoa1614362CrossRefGoogle Scholar
  10. 10.
    Frasca D, Ferracci F, Diaz A, Romero M, Lechner S, Blomberg BB (2016) Obesity decreases B cell responses in young and elderly individuals. Obesity 24(3):615–625.  https://doi.org/10.1002/oby.21383CrossRefPubMedGoogle Scholar
  11. 11.
    Ovsyannikova IG, White SJ, Larrabee BR, Grill DE, Jacobson RM, Poland GA (2014) Leptin and leptin-related gene polymorphisms, obesity, and influenza A/H1N1 vaccine-induced immune responses in older individuals. Vaccine 32(7):881–887.  https://doi.org/10.1016/j.vaccine.2013.12.009CrossRefPubMedGoogle Scholar
  12. 12.
    Sheridan PA, Paich HA, Handy J, Karlsson EA, Hudgens MG, Sammon AB, Holland LA, Weir S, Noah TL, Beck MA (2012) Obesity is associated with impaired immune response to influenza vaccination in humans. Int J Obes 36(8):1072–1077.  https://doi.org/10.1038/ijo.2011.208CrossRefGoogle Scholar
  13. 13.
    George MD, Baker JF (2016) The obesity epidemic and consequences for rheumatoid arthritis care. Curr Rheumatol Rep 18(1):6.  https://doi.org/10.1007/s11926-015-0550-zCrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Ottaviani S, Allanore Y, Tubach F, Forien M, Gardette A, Pasquet B, Palazzo E, Meunier M, Hayem G, Job-Deslandre C, Kahan A, Meyer O, Dieude P (2012) Body mass index influences the response to infliximab in ankylosing spondylitis. Arthritis Res Ther 14(3):R115.  https://doi.org/10.1186/ar3841CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Sandberg ME, Bengtsson C, Kallberg H, Wesley A, Klareskog L, Alfredsson L, Saevarsdottir S (2014) Overweight decreases the chance of achieving good response and low disease activity in early rheumatoid arthritis. Ann Rheum Dis 73(11):2029–2033.  https://doi.org/10.1136/annrheumdis-2013-205094CrossRefPubMedGoogle Scholar
  16. 16.
    Hotamisligil GS (2017) Inflammation, metaflammation and immunometabolic disorders. Nature 542(7640):177–185.  https://doi.org/10.1038/nature21363CrossRefPubMedGoogle Scholar
  17. 17.
    Pearce EL, Pearce EJ (2013) Metabolic pathways in immune cell activation and quiescence. Immunity 38(4):633–643.  https://doi.org/10.1016/j.immuni.2013.04.005CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Grant RW, Dixit VD (2015) Adipose tissue as an immunological organ. Obesity 23(3):512–518.  https://doi.org/10.1002/oby.21003CrossRefPubMedGoogle Scholar
  19. 19.
    Frasca D, Diaz A, Romero M, Thaller S, Blomberg BB (2018) Secretion of autoimmune antibodies in the human subcutaneous adipose tissue. PLoS One 13(5):e0197472.  https://doi.org/10.1371/journal.pone.0197472CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Winer DA, Winer S, Shen L, Wadia PP, Yantha J, Paltser G, Tsui H, Wu P, Davidson MG, Alonso MN, Leong HX, Glassford A, Caimol M, Kenkel JA, Tedder TF, McLaughlin T, Miklos DB, Dosch HM, Engleman EG (2011) B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies. Nat Med 17(5):610–617.  https://doi.org/10.1038/nm.2353CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Gerth AJ, Lin L, Peng SL (2003) T-bet regulates T-independent IgG2a class switching. Int Immunol 15(8):937–944CrossRefGoogle Scholar
  22. 22.
    Naradikian MS, Myles A, Beiting DP, Roberts KJ, Dawson L, Herati RS, Bengsch B, Linderman SL, Stelekati E, Spolski R, Wherry EJ, Hunter C, Hensley SE, Leonard WJ, Cancro MP (2016) Cutting edge: IL-4, IL-21, and IFN-gamma interact to govern T-bet and CD11c expression in TLR-activated B cells. J Immunol 197(4):1023–1028.  https://doi.org/10.4049/jimmunol.1600522CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Peng SL, Szabo SJ, Glimcher LH (2002) T-bet regulates IgG class switching and pathogenic autoantibody production. Proc Natl Acad Sci U S A 99(8):5545–5550.  https://doi.org/10.1073/pnas.082114899CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Santander AM, Lopez-Ocejo O, Casas O, Agostini T, Sanchez L, Lamas-Basulto E, Carrio R, Cleary MP, Gonzalez-Perez RR, Torroella-Kouri M (2015) Paracrine interactions between adipocytes and tumor cells recruit and modify macrophages to the mammary tumor microenvironment: the role of obesity and inflammation in breast adipose tissue. Cancers (Basel) 7(1):143–178.  https://doi.org/10.3390/cancers7010143CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Alain Diaz
    • 1
  • Maria Romero
    • 1
  • Daniela Frasca
    • 1
  • Bonnie B. Blomberg
    • 1
    • 2
    Email author
  1. 1.Department of Microbiology and ImmunologyUniversity of Miami Miller School of MedicineMiamiUSA
  2. 2.Sylvester Comprehensive Cancer CenterUniversity of Miami Miller School of MedicineMiamiUSA

Personalised recommendations