Particulate and solubilized β-glucan and non-β-glucan fractions of Euglena gracilis induce pro-and anti-inflammatory innate immune cell responses and exhibit antioxidant properties

Abstract

Purpose: The purpose of this work was to determine the pro-and anti-inflammatory properties of the single-cell organism Euglena gracilis (EG) and various fractions of its whole biomass.

Methods: Heterotrophically grown EG was tested, along with its aqueous fraction (E-AQ), the intact linear β-glucan paramylon granules (PAR), and alkaline-solubilized paramylon. Peripheral blood mononuclear cell cultures were treated with the test products and analyzed for a variety of cellular responses. Immune cell activation was evaluated by flow cytometry detection of CD69 levels on CD3^-CD56⁺ NK cells, CD3⁺CD56⁺ NKT cells, and monocytes, and cytokines were analyzed from the cell culture supernatants. Antioxidant capacity was measured by Folin-Ciocalteu assay and cellular antioxidant protection and MTT assays.

Results: EG and E-AQ were the most effective in driving immune cell responses as measured by CD69 upregulation on NK and NKT cells and proinflammatory (tumor necrosis factor, IL-6, IL-1β) cytokine production. None of the test products effectively stimulated monocyte. EG and PAR inhibited reactive oxygen species under conditions of oxidative stress. E-AQ contained antioxidants capable of providing cellular antioxidant protection from oxidative damage and protection of mitochondrial function under inflammatory conditions.

Conclusion: The effects of EG on immune function are only partially attributable to the content of the β-glucan, paramylon. The regulation of additional cellular responses, such a reactive oxygen species production and resistance to oxidative stress, is likely mediated by currently unknown molecules found in the EG cell.

Keywords: CAP-e; Dectin-1; PAMP; mannitol; oxidative stress; paramylon.

Figure 1

CD69 expression on human PBMC subsets is modulated by EG, but not E-AQ, PAR, or PAR-S. Notes: Human PBMCS from three healthy donors were cultured untreated or stimulated in vitro for 24 hours with LPS (10 ng/ml), poly I:C (2.5 μg/ml), IL-2 (100 IU/ ml), or each of the four test products (EG, E-AQ, PAR, AND PAR-S), stained and analyzed by flow cytometry. (A) Representative flow plots of gating strategy for identifying innate cell subsets. These subsets were analyzed for the expression of CD69, which is represented as a fold increase in MFI over the untreated control (dotted line) in (B) Monocytes, (C) NK cells, and (D) NKT cells. In the table, the average fold increase in CD69 MFI for each donor by the positive controls is shown. All samples were analyzed in triplicate. Symbols represent mean ± SD. Abbreviations: E-AQ, aqueous fraction of EG; EG, Euglena gracilis whole algae; LPS, lipopolysaccharide; MFI, mean fluorescence intensity; PAR, granular paramylon; PaR-s, alkaline-solubilized paramylon; PBMCS, peripheral blood mononuclear cells; poly I:C, polyinosinic–polycytidylic acid.

Figure 2

cytokine and growth factor production by human PBMCs is modulated by EG, but not E-AQ, PAR, or PAR-S. Notes: Human PBMCs from three healthy donors were cultured untreated or stimulated in vitro for 24 hours with LPS (10 ng/ml) or each of the four test products (EG, E-AQ, PAR, and PAR-S). Cell culture supernatants were analyzed by luminex multiplex for (A) TNF, (B) IL-6, (C) IL-1B, (D) IL-10, (E) IL-RA, and (F) G-CSF. All samples were analyzed in triplicate. Symbols represent the mean ± sD. Abbreviations: E-AQ, aqueous fraction of EG; EG, Euglena gracilis whole algae; G-CSF, granulocyte colony stimulating factor; IL-1RA, IL-1 receptor antagonist; LPS, lipopolysaccharide; PAR, granular paramylon; PAR-S, alkaline-solubilized paramylon; PBMCs, peripheral blood mononuclear cells; TNF, tumor necrosis factor.

Figure 3

H₂O₂-induced ROS production in human PMN cells is inhibited by β-glucan treatment. Notes: Human PMN cells from three healthy donors were pretreated with the four test products and H₂DCFDA and then water to induce ROs production. Oxidized, fluorescent DCF is represented as MFI as a percentage of DCF MFI average from the H₂O₂-treated cells without pretreatment (dotted line). All samples were analyzed in triplicate. Symbols represent mean ± SD. Abbreviations: DCF, dichlorofluorescein; E-AQ, aqueous fraction of EG; EG, Euglena gracilis whole algae; H₂DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; MFI, mean fluorescence intensity; PAR, granular paramylon; PAR-S, alkaline-solubilized paramylon; PMN, polymorphonuclear.

Figure 4

E-AQ has antioxidant properties and improves mitochondrial metabolism in stressed PBMCs. Notes: The four test products were tested for (A) total antioxidant capacity, (B) antioxidant protection from oxidative stress in erythrocytes, and (C) protection of mitochondrial function under LPS-induced inflammatory culture conditions. Samples in (A–C) were tested in duplicate, quadruplicate, and triplicated, respectively. Symbols represent mean ± SD of replicates, n=1. Abbreviations: CAP-e, cellular antioxidant protection of erythrocytes; E-AQ, aqueous fraction of EG; EG, Euglena gracilis whole algae; LPS, lipopolysaccharide; PAR, granular paramylon; PAR-S, alkaline-solubilized paramylon; PBMCs, peripheral blood mononuclear cells.