1. Introduction
Phosphatidylcholine (PC) is an essential component found in every cell of the human body. PC is consisted of a choline head group, glycerophosphoric acid, and several fatty acids. Polyunsaturated fatty acids (PUFAs) are common constituents of the diet and have many functionally physiological roles[1,2,3].
Docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3) are major n-3 PUFAs obtained from diet. Calder[2] suggested that an incrmented intake of EPA and DHA resulted in an incremented incorporation of n-3 PUFAs in membrane phospholipids of immune cells. Since dietary n-3 PUFAs have significant immunologically inhibitory and anti-inflammatory properties, they conduct protective effects on inflammatory diseases and immune-mediated diseases[4,5,6]. Studies demonstrated that both EPA and DHA might control the functional responses of immune cells to stimulation by decreaing the production of pro-inflammatory mediators[7]. In autoimmunity[8] and clinical trials in patients with rheumatoid arthritis[9], intake of fish oil including EPA and DHA revealed significant protective effect against inflammatory activity. Intake of PC also possesses a positive effect on anti-inflammation[10]. An animal model study suggested that oral PC supplementation reduced lipopolysaccharide (LPS)-induced plasma tumor necrosis factor- α (TNF- α However, there are less studies on the effect of DHA containing PC on moderation of cytokine production.
There are two distinct patterns of cytokine generation in helper T-cells. Human type 1 helper (Th1) cells generate interleukin-2 (IL-2), interferon- γ (IFN- γ) and tumor necrosis factor- β (TNF- β), whereas human type 2 helper (Th2) cells generate IL-4, IL-5, IL-6 and IL-10. Other cytokines including TNF-α, IL-3 and granulocyte macrophage-colony stimulating factor are generated by both the Th subsets[12]. Different cytokine profiles are associated with strikingly dramatic functions of the two types of Th cells. In general, Th2 cells mediate B cell immunoglobin (Ig) secretion, especially of the IgE class[12]. The helper function of Th1 cells is more uncertain since they have been shown to provide antigen-specific presentation in only few in vitro systems[13]. Under certain circumstances, Th1 cells can also assist to B cells, however, one of their major functions is to lead to delayed type hypersensitivity response[12]. Therefore, Th1 cells may control more effectively with intracellular pathogens, whereas Th2 cells may handle extracellular organisms and their secreted products by inducing strong antibody reactions. In the present study, we synthesized didocosahexaenoyl PC (DHA-PC) and investigated its effects on the expression of Th1 (IL-2 and IFN-γ) and Th2 [IL-4, IL-5, IL-6 and IL-12/IL-23(p40)] cytokines.
2. Materials and methods
2.1. Materials and cell culture
Six-week-old male C57BL6 mice were purchased from Daehan BioLink (Eumsong, Chungcheongbukdo, South Korea). The animal experiment was approved by Institutional Animal Care and Use Committee (GNU-171116-M0051) in Gyungsang National University (Gyungsang, South Korea). LPS, RPMI 1640, fetal bovine serum (FBS), phosphate buffer saline (PBS) and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). DHA-PC were synthesized according to the method of Borsotti et al.[14]. Mouse enzyme-linked immunosorbant assay kits purchased from Biolegent (San Diego, CA, USA) were used to measure the levels of cytokines [IL-2, IL-4, IL-5, IL-6, IL-12/IL-23(p40), IFN- γ)].
2.2. Spleen cell culture supernatants
Spleen is an organ where immune responses against pathogen can be regulated[15] and thus murine splenocytes have been used for immune studies[16,17]. Cell counts within spleen cell suspensions were adjusted to 2 × 106 cells/mL using RPMI 1640 medium with 10% FBS, and the adjusted suspensions were aliquoted into 24-well tissue culture plates (Costar, Cambridge, MA, USA) at 1 mL/well. The cells were treated with sample and set at 37 °C in 5% carbon dioxide condition. Concomitant treatment with sample and LPS also was conducted. As a control, cells were treated with 0.01% DMSO. Cells were incubated for 6, 24, 48, 72 h before collection. The suspensions were centrifuged at 300 × g for 10 min, followed by additional centrifugation at 1 000 × g for 30 min. The supernatants were stored at -70 °C until use in analysis of cytokine expression[18]. We measured the expression of IL-2, IL-4, IL-5, IL-6, IL-12/IL-23(p40) and IFN-γ in samples collected from 6, 24, 48, and 72 hours of treatments.
2.3. Measurement of cytokines
Capture antibodies for IL-2, IL-4, IL-5, IL-6, IL-12/IL-23(p40) and IFN-γ were diluted with coating buffer, loaded into 96-well plates at 100 μg/well, and incubated at 4 °C overnight. The wells were washed with washing buffer 4 times, and 200 μL of assay diluents was supplemented to each well. The plates were then set at room temperature for 1 h. The wells then were washed with washing buffer 4 times again, and 100 μL of the sample was supplemented to each well. The plates were then set at room temperature for 2 h. After washing 4 times, 100 μL of the detection antibody for the appropriate cytokine was supplemented to each well, and the plates were set at room temperature for 1 h. The wells were washed with washing buffer 4 times, 100 μL of avidin-horseradish peroxides was supplemented to each well, and plates were set at room temperature for 30 min. After washing 5 times with washing buffer, 100 μL of substrate fluid containing tetramethylbenzidine was supplemented, and the plates were set at room temperature for 20 min. One hundred μL of stop solution was supplemented to stop reaction, and the optical density was assessed at 450 nm by ELISA reader (Model 550 microplate reader, Bio-Rad, Richmond, VA, USA)[16].
2.4. Statistical analysis
Data were presented as mean ± standard deviation. To determine normal distribution, Kolmogorove-Smirnov test was done. Significance of differences observed between the control and experiment groups using Student's t test was set at P<0.05. Analyses were conducted using STATISTICA package.
3. Results
3.1. Effect of DHA-PC on Th1 cytokine expression
Changes in levels of Th1 cytokines (IL-2 and IFN-γ) were investigated upon treatment of murine splenocyte suspensions with only LPS- or by co-administration of LPS and different concentrations of DHA-PC. Co-administration of DHA-PC (0.3 μg/mL) with LPS significantly reduced the IL-2 expression after 6 and 72 hours of treatment, respectively, as compared to treatment with only LPS (P<0.05) (Table 1). Table 2 showed the modulation of IFN- γ expression induced by DHA-PC. The expression of IFN-γ in spleen cells increased after 48 hours of LPS treatment. However, no such significant difference was observed with DHA-PC treatment.
;)
Table 1: Effect of DHA-PC on production of LPS induced IL-2 at different times in mouse spleen cells.
Table 2: Effect of DHA-PC on production of LPS induced IFN- γ at different times in mouse spleen cells.
3.2. Effect of DHA-PC on Th2 cytokine expression
We investigated changes in the levels of Th2 cytokines [IL-4, IL-5, IL-6, and IL-12/IL-23(p40)] upon treatment of murine splenocyte suspensions with LPS, or with co-administration of different concentrations of DHA-PC. Table 3 showed the expression of IL-4 after treatment with DHA-PC. The treatment of spleen cells with only LPS increased the expression of IL-4 after 48 h. However, there were no significant differences in IL-4 expression induced by DHA-PC. Co-administration of DHA-PC (1 μg/mL) with LPS significantly increased the IL-5 expression after 24 hours of treatment, as compared to treatment with only LPS (P<0.05) (Table 4). Table 5 showed the expression of IL-6 induced by DHA-PC. Co-administration of DHA-PC (0.3 μg/mL) with LPS significantly decreased IL-6 expression after 48 and 72 hours of treatment, respectively in comparision with the treatment with LPS alone (P<0.05). As shown in Table 6, co-administration of DHA-PC at concentration of 0.3 jig/mL with LPS significantly decreased IL-12/ IL-23(p40) expression after 48 and 72 hours of treatment compared with the treatment with only LPS (P<0.05).
Table 3: Effect of DHA-PC on production of LPS induced IL-4 at different times in mouse spleen cells.
Table 4: Effect of DHA-PC on production of LPS induced IL-5 at different times in mouse spleen cells.
Table 5: Effect of DHA-PC on production of LPS induced IL-6 at different times in mouse spleen cells.
Table 6: Effect of DHA-PC on production of LPS induced IL-12/IL-23(p40) at different times in mouse spleen cells.
4. Discussion
N-3 highly unsaturates, such as DHA and EPA, have been known to protect against immune disease and inflammatory side effects in rodents[19] and humans[20]. Meydani et al.[21] indicated that feeding with n-3 fatty acid deficiency for 3 mon inhibits production of IL-1 β, TNF, IL-6 and IL-2 induced by LPS. Liu et al.[22] found that fish oil and its functional components including EPA and DHA conducted an anti-inflammatory effect by decreasing cytokine production (IL-1 β and IL-2) in a weaning pigs, which might be associated with the modulation of intracellular signaling, especially that of protein kinase C. However, there have been few studies on the effects of DHA-PC on cytokine production. Huang et al.[23] suggested that the anti-inflammatory action of lysophosphatidylcholine containing DHA was partially linked to the decreased formation of leukotriene C4, TNF-α and IL-6. IL-2 is a soluble, locally released inflammatory mediator with a multitude of regulatory roles, which therefore is a possible marker that can be applied to measure the functional effects of anti-inflammatory intervention[24]. We found that DHA-PC decreased the expression of pro-inflammatory cytokine, IL-2.
There is big significant evidence that these n-3 fatty acids are capable to inhibit development of inflammatory cells and generation of eicosanoids from n-6 fatty acids[25,26]. According both in vitro and clinical studies, administration with EPA, DHA and fish oil could reduce production of pro-inflammatory cytokines[27]. Wang et al.[28] demonstrated that macrophages supplemented with EPA and DHA resulted in decreased levels of TNF-α and IL-6 compared to controls, suggesting the ability of EPA, DHA and EPA+DHA to decrease pro-inflammatory markers. For maintaining health, it is important to balance pro-inflammatory and anti-inflammatory cytokines. Excessive chronic production of pro-inflammatory cytokines contributes to inflammatory diseases[29,30,31]. Here we evaluated the effects of synthesized DHA-PC on the production of cytokines in murine splenocytes induced by LPS. There was few studies on relationship between DHA-PC and cytokine expression. Our results suggest that DHA-PC inhibits expression of pro-inflammatory cytokines [IL-2, IL-6 and IL-12/IL-23(p40)] on induction of inflammation. Further studies are warranted including dose response trials and gene expression studies.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2017R1A2B4005915).
REFERENCES
1. Wysoczanski T, Sokola-Wysoczanska E, Pekala J, Lochynski S, Czyz K, Bodkowski R, et al Omega-3 fatty acids and their role in central nervous system-A review
Curr Med Chem. 2016;23(8):816–831
2. Calder PC, Newsholme EA. Unsaturated fatty acids suppress interleukin-2 production and transferring receptor expression by concanavalin A-stimulated rat lymphocytes
Mediat Inflamm. 1992;1(2):107–115
3. Saidane O, Semerano L, Sellam J. Could omega-3 fatty acids prevent rheumatoid arthritis?
Joint Bone Spine 2018 Doi:10.1016/j.jbspin.2018.05.007
4. Lin N, Shi JJ, Li YM, Zhang XY, Chen Y, Calder PC, et al What is the impact of n-3 PUFAs on inflammation markers in Type 2 diabetic mellitus populations?: A systematic review and meta-analysis of randomized controlled trials
Lipids Health Dis. 2016;15:133 Doi: 10.1186/s12944-016-0303-7
5. Tian T, Zhao Y, Huang Q, Li J. n-3 Polyunsaturated fatty acids improve inflammation via inhibiting sphingosine kinase 1 in a rat model of parenteral nutrition and CLP-induced sepsis
Lipids. 2016;51:271–278
6. Marion-Letellier R, Savoye G, Ghosh S, Dinarello CA. Polyunsaturated fatty acids and inflammation
Inter Union Biochem Mol Biol Life. 2017;67:659–667
7. Allam-Nudoul B, Guenard F, Barbier O, Vohl MC. Effect of n-3 fatty acids on the expression of inflammatory genes in THP-1 macrophages
Lipids Health Dis. 2016;15:69 Doi:10.1186/s12944-016-0241-4
8. Leslie CA, Gonnermann WA, Ullman MD, Hayes KC, Rfanz-Blau C, Cathcart ES. Dietary fish oil modulates macrophage fatty acids and decreases arthritis susceptibility in mice
J Exp Med. 1985;162(4):1336–1349
9. Kremer JM, Bigaucette J, Michalek AV, Timchalk MA, Lininger L, Rynes RI, et al Effects of manipulation of dietary fatty acids on clinical manifestations of rheumatoid arthritis
Lancet. 1985;1(8422):184–187
10. Li Y, Ren X, Lio C, Sun W, Lai K, Liu Y, et al A Chlorogenic acid-phospholipid complex ameliorates post-myocardial infarction inflammatory response mediated by mitochondrial reactive oxygen species in SAMP8 mice
Pharmacol Res. 2018;130:110–122
11. Tokes T, Eros G, Bebes A, Hartmann P, Varszegi S, Varga G, et al Protective effects of a phosphatidylcholine-enriched diet in lipopolysaccharide-induced experimental neuroinflammation in the rat
Shock. 2011;36(5):458–465
12. Cherwinski HM, Shumacher JH, Brown KD, Mosmann TR. Two types of mouse helper T cell clone III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays and monoclonal antibodies
J Exp Med. 1987;166(5):1229–1244
13. Mosmann TR, Coffman RL. Th1 and Th2 cells: Different patients of lymphokine secretion lead to different functional properties
Annu Rev Immunol. 1989;7(7):145–173
14. Borsotti G, Guglielmetti G, Spera S, Battistel E. Synthesis of phosphatidylcholines containing ricinoleic acid
Tetrahedron. 2001;57(51):10219–10227
15. Bronte V, Pittet M. The spleen in local and systemic regulation of immunity
Immunity. 2013;39(5):806–818
16. Kim KH, Kim SH, Park KY. Effects of kimchi extracts on production of nitric oxide by activated macrophages, transforming growth factor-beta 1 of tumor cells and interleukin-6 in splenocytes
J Food Sci. 2001;6(5):126–132
17. Taghavi M, Mortaz E, Khosravi A, Vahedi G, Folkerts G, Varahram M, et al Zymosan attenuated melanoma growth progression, increases splenocyte proliferation and induces TLR-2/4 and TNF- α expression in mice
J Inflamm. 2018;15:5 Doi:10.1186/s12950-018-0182-y
18. Hwang SA, Dasgupta A, Actor JK. Cytokine production by non-adherent mouse splenocyte cultures to
Echinacea extracts
J Clin Chim Acta. 2004;343(1-2):161–166
19. Ferramosca A, Conte A, Burri L, Berge K, De Nuccio F, Gludetti AM, et al A krill oil supplemented diet suppresses hepatic steatosis in high-fat fed rats
PLoS One. 2012;7(6):e38797 doi:10.1371/journal. pone.0038797
20. Itariu BK, Zeyda M, Hochbrugger EE, Neuhofer A, Prager G, Schindler K, et al Long-chain n-3 PUFAs reduce adipose tissue and systemic inflammation in severely obese nondiabetic patients: A randomized controlled trial
Am J Clin Nutr. 2012;96:1137–1149
21. Meydani SN. Dietary modulation of cytokine production and biological functions
Nutr Rev. 1990;48(10):361–369
22. Liu Y, Gong L, Li D, Feng Z, Zhao L, Dong T. Effects of fish oil on lymphocyte proliferation, cytokine production and intracellular signaling in weaning pigs
Arch Anim Nutr. 2003;57(3):151–165
23. Huang LS, Hung ND, Sok DE, Kim MS. Lysophosphatidylcholine containing docosahexaenoic acid at the sn-1 position is anti-inflammatory
Lipids. 2010;45(3):225–236
24. Mizel SB. The interleukins
FASEB J. 1989;3(12):2379–2389
25. Yaqcob O, Calder P. Effect of dietary lipid manipulation upon inflammatory mediator production by murine macrophages
Cell Immunol. 1995;163(1):1201–1208
26. Simopoulos AP. Omega-3 fatty acids in inflammation and autoimmune disease
J Am Coll Nutr. 2002;21(6):495–505
27. Baumann KH, Hessel F, Larass I, Muller T, Angerer P, Kiefl R, et al Dietary omega-3, omega-6, and omega-9 unsaturated fatty acids and growth factor and cytokine gene expression in unstimulated and stimulated monocytes. A randomized volunteer study
Arterioscler Thromb Vasc Biol. 1999;19(1):59–66
28. Wang S, Wu D, Lamon-Fava S, Mattan NR, Honda KL, Lichtenstein AH.
In vitro fatty acids enrichment of macrophages alters inflammatory response and net cholesterol accumulation
Br J Nutr. 2009;102(4):497–501
29. Kristensen S, Schmidt EB, Schlemmer A, Rasmussen C, Johansen MB, Christensen JH. Beneficial effect of n-3 polyunsaturated fatty acids on inflammation and analgesic use in psoriatic arthritis: A randomized, double blind, placebo-controlled trial
Scad J Rheumatol. 2018;47(1):27–36
30. Tortosa-Caparrós E, Navas-Carrillo D, Marín F, Orenes-Piñero E. Anti-inflammatory effects of omega 3 and omega 6 polyunsturated fatty acids in cardiovascular disease and metabolic syndrome
Crit Rev Food Sci Nutr. 2017;57(16):3421–3429
31. Brinkhoff A, Sieberichs A, Engler H, Dolff S, Benson S, Korth J, et al Pro-inflmmatory Th1 and Th17 cells are suppressed during human experimental endotoxemiz whereas anti-inflammatory IL-10 producing T-cells are unaffected
Front Immunol. 2018;9:1133 Doi:10.3389/fimmu.2018.01133
