The Optimum Dose of Beta-Glucan for Stimulating Peripheral Blood Mononuclear Cells (PBMCs) to Produce Cytokines: In Vitro Study
Abstract
Background: Beta-glucan has been frequently used in laboratory trials as an immunomodulator in both in vivo and in vitro studies, but the effective dose for measuring its performance has not been established. Like other immunomodulators, researchers must establish the right dose of beta-glucan in their laboratory experiments for the purpose of testing substances as immunomodulators without achieving false-positive or –negative results. This study aimed to determine the optimum dose of beta-glucan to induce cytokine production by peripheral blood mononuclear cells (PBMCs) in vitro.
Subjects and Method: This was a laboratory experimental study. This study measured the production of two cytokines, including interferon gamma (IFN-γ) and interleukin 12 (IL-12), from the isolated PBMCs of healthy subjects. The doses of beta-glucan used as immunomodulator included 1, 5, 10, 20, and 50 μg/ml. Beta-glucan was added to the PBMC culture medium, and the PBMCs were cultured for 6 days. On the sixth day, the supernatant was harvested and the cytokine production was analyzed using sandwich enzyme-linked immunosorbent assay (ELISA). Cytokines were also analyzed using the human IFN-γ ELISA kit and the human IL-12 ELISA kit, and data analysis was performed by one-way ANOVA.
Results: IFN-γ levels were found to be increased in the group treated with 5 μg/ml beta-glucan. The highest IFN-γ levels (70.0 pg/ml) were observed in the group treated with 10 μg/ml beta-glucan. The production of IL-12 increased sharply in the group treated with5 μg/ml beta-glucan but decreased in the group treated with 10 μg/ml beta-glucan. The mean cytokine levels of the beta-glucan group were found to be significantly different from those of the control group (p=0.001). One-way ANOVA revealed that the highest IL-12 production (77.2 pg/ml) occurred at a dose of 5 μg/ml beta-glucan. This average value was significantly different from the average production of IL-12 in the control group (p=0.001).
Conclusion: The optimum dose of beta-glucan for stimulating PBMCs to produce IFN-γ in vitro was 10 μg/ml, while for the production of IL-12, the dose was 5 μg/ml. Both cytokines can be measured within 6 days of cell culture.
Keywords: beta-glucan, immunomodulator, cytokines
Correspondence: Meira Erawati. Faculty of Medicine, Diponegoro University. Jl. Prof. Soedarto, S.H, Tembalang, Semarang, Indonesia. Email: meiraerawati@gmail.com. Mobile: +62-81915339685
Indonesian Journal of Medicine (2020), 5(2): 170-177
https://doi.org/10.26911/theijmed.2020.05.02.12
References
Budak F, Goral G, Oral HB (2008). Saccharo myces Cerevisiae Beta Glucan Induces Interferon Gamma Production in Human T Cells Via IL-12. Turk J Immunol. 13: 21-6. https://doi.org/10.18585/inabj.v11i2.688
Bustamantea J, Boisson Dupuisa S, Abela L, Casanova J (2014). Mendelian susceptibility to mycobacterial disease: genetic, immunological, and clinical features of inborn errors of IFN-γ immunity. SeminImmunol.26: 454–70. https://doi.org/10.1016/j.smim.2014.09.008
Caragol I, Casanova JL (2003). Inherited disorders of the Interleukin-12/Interferon-gamma axis: Mendelian predisposition to mycobacterial disease in man. Inmunología.22: 263-76. Retrieved from https://www.inmunologia.org/Upload/Articles/6/0/601.pdf
Chan GC, Chan WK, Sze DM (2009). The effects of β-glucan on human immune and cancer cells. J HematolOncol. 2:1-11. https://doi.org/10.1186/1756-8722-2-25
Choromanska A, Kulbacka J, Harasym J, Oledzki R, Szewczyk A, Saczko J (2018). High and low molecular weight oat beta-glucan reveals antitumor activity in human epithelial lung cancer. PatholOncol Res. 24:583–92. https://doi.org/10.1007/s122530170278-3
Goncalves MAD, Dritz SS, DeRouchey JM, Tokach MD (2014). Effects of algaederived β-glucans with zinc onnursery pig growth performance and immuneresponse under commercial conditions. Kansas Agricultural Experiment Station Research Reports.https://doi.org/10.4148/2378-5977.6913
Jabber AJ, Mahdi NR, Yaseen NY (2011). The Effect of Beta Glucan Extract of Saccharomyces cerevieses on Cancer Cell Growth In vitro. Iraqi Journal of Cancer and Medical Genetics. 4:54-8. Retrieved from http://ijcmg.uomustansiriyah.edu.iq/index.php/ijcmg/article/view/79
Javmen A, Nemeikaite Ceniene A, Bratchikov MB, Grigiskis S, Grigas F, Jonauskiene I, et al (2015). β-Glucan from Saccharomyces cerevisiae Induces IFN-γ Production In Vivo in BALB/c Mice. In vivo. 29: 359-64. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/25977381
Kramlich D (2014). Introduction to Complementary, Alternative, and Traditional Therapies. Crit Care Nurse. 34: 50-6. https://doi.org/10.4037/ccn2014807
Lauw FN (2000). IL-12, IL-18 and IFNgamma in the immune response to bacterial infection. UvA-DARE (Digital Academic Repository) University of Amsterdam.: 18-26. Retrieved from https://dare.uva.nl/search?identifier=a3d78cf8-32fa-4e21-b67a-5c8a3d9aa-655
Limberger-Bayer VM, Francisco A, Chan A, Oro T, Ogliari PJ, Barreto PLM (2014). Barley b-glucans extraction and partial characterization. Food Chem. 154:84–9. https://doi.org/10.1016/j.foodchem.2013.12.104
Magnani M, Castro GomEz RH, Aoki MN, Gregório EP, LibosJrF, Watanabe MA B (2010). Effects of carboxymethylglucan from Saccharomyces cerevisiae on the peripheral blood cells of patients with advanced prostate cancer. Exp Ther Med. 1: 859-62. https://dx.doi.org/10.1590%2FS1415-47572010005000103
Marchi LF, Sesti Costa R, Ignacchiti MDC, Chedraoui Silva S, Mantovani B (2014). In vitro activation of mouse neutrophils by recombinant human interferon-gamma: Increased phagocytosis and release of reactive oxygen species and pro-inflammatory cytokines. Int Immunopharmacol.18:228–35. https://doi.org/10.1016/j.intimp.2013.12.010
Newsome CT, Flores E, Ayala A, Gregory S, Reichner JS (2011). Improved Antimicrobial Host Defense in Mice following Poly-(1,6)--D-Glucopyranosyl-(1,3)--D-Glucopyranose Glucan Treatment by a Gender Dependent Immune Mechanism. Clin Vaccine Immunol. 18: 2043–9. https://doi.org/10.1128/CVI.05202-11
Noss I, Doekes G, Thorne PS, Heederik DJJ, Wouters I W (2013). Comparison of the potency of a variety of β-glucans to induce cytokine production in human whole blood. Innate Immun. 19:10–19. https://doi.org/10.1177/1753425912447129
Peters M (1996). Actions of Cytokines on the Immune Response and ViralInteractions: An Overview. Hepatology. 23: 909-16. https://doi.org/10.1053/jhep.1996.v23.ajhep0230909
Ramirez Alejo N, Santos Argumedo L (2014). Innate Defects of the IL-12/IFN-g Axis in Susceptibility to Infections by Mycobacteria and Salmonella. J Interferon Cytokine Res. 34:307-17. https://doi.org/10.1089/jir.2013.0-050
Shah SK, Walker PA, Moore Olufemi SD, Sundaresan A, Kulkarni A, Andrassy RJ (2011). An Evidence Based Review of a Lentinulaedodes Mushroom Extract as Complementary Therapy in the Surgical Oncology Patient. J Parenter Enteral Nutr. 35:44958. https://doi.org/10.1177/0148607110380684
Vetvicka V, Vetvickova J (2015). Glucan supplementation enhances the immune response against an influenza challenge in mice. Ann Transl Med.3:17.https://dx.doi.org/10.3978%2Fj.issn.2305-5839.2015.01.08
Xu H, Zou S, Xu X, Zhang L (2016). Antitumor effect of β-glucan from Lentin use dodes and the underlying mechanism. Nature Scientific Reports. 6:1-13. https://doi.org/10.1038/srep28802
Yun C H, Estrada A, Van Kessel A, Park BC, Laarveld B (2003). L Glucan, extracted from oat, enhances disease resistance against bacterial and parasitic infections. FEMS Immunol Med Microbiol. 35:67-75. https://doi.org/10.-1016/S0928-8244(02)00460-1
