Effect of glycerolipid preparations from fern and horsetail on human peripheral blood mononuclear cells under ex vivo conditions

Introduction. Human peripheral blood mononuclear cells (PBMCs) are a pool of immune cells and they are also a convenient model system for studying immune pathologies.

Aim. Testing for bioactivity of glycerolipid prepa­rations from fern and horsetail species containing long chain polyunsaturated fatty acids (LCPUFAs) towards PBMCs without exogenous stimulation and after phorbol-12-myristate-13-acetate (PMA) plus ionomycin stimulation.

Materials and methods. Glycerolipid preparations were produced by fractionation of total lipids, isolated from young fronds of the fern Matteuccia struthiopteris and shoots of the horsetail Equisetum arvense, on silica. Egg phosphatidylcholine was used for comparison. Fatty acids were analyzed by gas chromatography. Mononuclear cells were isolated from blood of patients with asthma. Parameters of cell viability and activation were estimated by flow cytometry.

Results. The glycerolipid prep­arations from the fern and horsetail were found to have a cytotoxic effect while egg phosphatidylcholine was not. The most active was the fraction of fern lipids eluted with methanol which reduced cell viability by 64.6 (51.1-79.0)% in the concentration 2 pg/ml and caused complete cell death in 20 pg/ml. After cell stimulation with PMA/ionomycin, the cyto­toxic effect of the preparation increased although the level of PBMCs expressing the marker CD69 did not change. The cytotoxic effect of other glycerolipid preparations was observed in the higher concentrations (20 and/or 80 pg/ml) and it was less pronounced: the cell viability reduced by 7.1 (6.7-9.4)% for the fraction of fern lipids eluted by the mixture chlo­roform - methanol - water (3:5:2), by 39.8 (26.4-41.6)% and 12.0 (10.0-15.5)% for the fractions of the horsetail lipids eluted with methanol and the chloroform-methanol-water mixture, respectively, in the concentration 80 pg/ml.

Conclu­sion. Comparison of fatty acid composition of the glycerolipid preparations did not confirm a contribution of LCPUFAs to the observed effects. Identification of an active component may allow development of a drug for the local application in a hyperimmune response or for model experiments.

1. Sen P., Kemppainen E., Oresic M. Perspectives on systems modeling of human peripheral blood mononuclear cells. Front. Mol. Biosci. 2018; 4:96. https://doi.org/10.3389/fmolb.2017.00096

2. Falcai A., Soeiro-Pereira P.V., Kubo C.A., Aranda C.S., Sole D., Condino-Neto A. Peripheral blood mononuclear cells from severe asthmatic children release lower amounts of IL-12 and IL-4 after LPS stimulation. Allergol. Immunopathol. (Madr.) 2015; 43(5):482-486. https://doi.org/10.1016/j.aller.2014.10.005

3. Zambalde E.P., Teixeira M.M., Favarin D.C., de Oliveira J.R., Magalhaes M.L., Cunha M.M., Silva W.C., Okuma C.H., Rodrigues V., Levy B.D., de Paula Rogerio A. The anti-inflammatory and pro-resolution effects of aspirin-triggered RvD1 (AT-RvD1) on peripheral blood mononuclear cells from patients with severe asthma. Int. Immunopharmacol. 2016; 35:142-148. https://doi.org/10.1016/j.intimp.2016.03.014

4. Kytikova O., Novgorodtseva T., Denisenko Y., Antonyuk M., Gvozdenko T. Pro-resolving lipid mediators in the pathophysiology of asthma. Medicina (Kaunas) 2019; 55(6):284. https://doi.org/10.3390/medicina55060284

5. Sokolowska M., Rovati G.E., Diamant Z., Untersmayr E., Schwarze J., Lukasik Z., Sava F., Angelina A., Palomares O., Akdis C.A., O'Mahony L., Sanak M., Dahlen S.-E., Woszczek G. Current perspective on eicosanoids in asthma and allergic diseases: EAACI Task Force consensus report, part I. Allergy 2021;76(1):114-130. https://doi.org/10.1111/all.14295

6. Capra V., Rovati G.E., Mangano P., Buccellati C., Murphy R.C., Sala A. Transcellular biosynthesis of eicosanoid lipid mediators. Biochim. Biophys. Acta 2015; 1851(4):377-382. http://dx.doi.org/10.1016/j.bbalip.2014.09.002

7. Powell W.S. Eicosanoid receptors as therapeutic targets for asthma. Clin. Sci. (Lond.) 2021; 135(16):1945-1980. http://dx.doi.org/10.1042/CS20190657

8. Nekrasov E.V., Svetashev V.I., Khrapko O.V., Vyssotski M.V. Variability of fatty acid profiles in ferns: Relation to fern taxonomy and seasonal development. Phytochemistry 2019; 162:47-55. http://dx.doi.org/10.1016/j.phytochem.2019.02.015

9. Nekrasov E.V., Shelikhan L.A., Svetashev V.I. Fatty acid composition of gametophytes of Matteuccia struthiopteris (L.) Tod. (Onocleaceae, Polypodiophyta). Botanica Pacifica 2019; 8(1):63-66. http://dx.doi.org/10.17581/bp.2019.08104

10. Nekrasov E.V., Svetashev V.I. Edible Far Eastern ferns as a dietary source of long-chain polyunsaturated fatty acids. Foods 2021; 10(6):1220. http://dx.doi.org/10.3390/foods10061220

11. Schlenk H., Gellerman J.L. Arachidonic, 5, 11, 14, 17-eicosatetraenoic and related acids in plants - identification of unsaturated fatty acids. J. Am. Oil Chem. Soc. 1965; 42:504-511. https://doi.org/10.1007/BF02540092

12. Cifre M., Diaz-Rua R., Varela-Calvino R., Reynes B., Pericas-Beltran J., Palou A., Oliver P., Human peripheral blood mononuclear cell in vitro system to test the efficacy of food bioactive compounds: Effects of polyunsaturated fatty acids and their relation with BMI. Mol. Nutr. Food Res. 2017; 61(4):1600353. https://doi.org/10.1002/mnfr.201600353

13. Ameer F., Munir R., Usman H., Rashid R., Shahjahan M., Hasnain S., Zaidi N. Lipid-load in peripheral blood mononuclear cells: Impact of food-consumption, dietary-macronutrients, extracellular lipid availability and demographic factors. Biochimie 2017; 135:104-110. https://doi.org/10.1016/j.biochi.2017.01.015

14. Rundblad A., Holven K., Bruheim I., Myhrstad M., Ulven S. Effects of fish and krill oil on gene expression in peripheral blood mononuclear cells and circulating markers of inflammation: A randomised controlled trial. J. Nutr. Sci. 2018; 7:e10. https://doi.org/10.1017/jns.2018.2

15. Sureda A., Martorell M., Bibiloni M.d.M., Bouzas C., Gallardo-Alfaro L., Mateos D., Capo X., Tur J.A., Pons A. Effect of free fatty acids on inflammatory gene expression and hydrogen peroxide production by ex vivo blood mononuclear cells. Nutrients 2020; 12(1):146. https://doi.org/10.3390/nu12010146

16. Nekrasov E.V., Tallon S.J., Vyssotski M.V., Catchpole O.J. Extraction of lipids from New Zealand fern fronds using near-critical dimethyl ether and dimethyl ether-water-ethanol mixtures. J. Supercrit. Fluids 2021; 170:105137. https://doi.org/10.1016/j.supflu.2020.105137

17. Boyum A. Separation of leukocytes from blood and bone marrow. Introduction. Scand. J. Clin. Lab. Invest. Suppl. 1968; 97:7. PMID: 5707208

18. Hartnell A., Robinson D.S., Kay A.B., Wardlaw A.J. CD69 is expressed by human eosinophils activated in vivo in asthma and in vitro by cytokines. Immunology 1993; 80(2):281-286. PMID: 8262555; PMCID: PMC1422202.

19. Kew S., Banerjee T., Minihane A.M., Finnegan Y.E., Williams C.M., Calder P.C. Relation between the fatty acid composition of peripheral blood mononuclear cells and measures of immune cell function in healthy, free-living subjects aged 25-72 y. Am. J. Clin. Nutr. 2003; 77(5):1278-1286. https://doi.Org/10.1093/ajcn/77.5.1278

20. Quasney M.E., Carter L.C., Oxford C., Watkins S.M., Gershwin M.E., German J.B. Inhibition of proliferation and induction of apoptosis in SNU-1 human gastric cancer cells by the plant sulfolipid, sulfoquinovosyldiacylglycerol. J. Nutr. Biochem. 2001; 12(5):310-315. https://doi.org/10.1016/S0955-2863(01)00146-2

21. Mizushina Y., Watanabe I., Ohta K., Takemura M., Sahara H., Takahashi N., Gasa S., Sugawara F., Matsukage A., Yoshida S., Sakaguchi K. Studies on inhibitors of mammalian DNA polymerase a and в: Sulfolipids from a pteridophyte, Athyrium niponicum. Biochem. Pharm. 1998; 55(4):537-541. https://doi.org/10.1016/S0006-2952(97)00536-4

22. Murakami C., Kumagai T., Hada T., Kanekazu U., Nakazawa S., Kamisuki S., Maeda N., Xu X., Yoshida H., Sugawara F., Sakaguchi K., Mizushina Y. Effects of glycolipids from spinach on mammalian DNA polymerases. Biochem. Pharm. 2003; 65(2):259-267. https://doi.org/10.1016/s0006-2952(02)01483-1

23. Stemmer U., Dunai Z.A., Koller D., Purstinger G., Zenzmaier E., Deigner H.P., Aflaki E., Kratky D., Hermetter A. Toxicity of oxidized phospholipids in cultured macrophages. Lipids Health Dis. 2012; 11:110. https://doi.org/10.1186/1476-511X-11-110

24. Mayhew E., Ito M., Lazo R. Toxicity of non-drug-containing liposomes for cultured human cells. Exp. Cell Res. 1987; 171(1):195-202. https://doi.org/10.1016/0014-4827(87)90262-X