Euglena gracilis paramylon activates human lymphocytes by upregulating pro-inflammatory factors

Abstract

The aim of this study was to verify the activation details and products of human lymphomonocytes, stimulated by different β-glucans, that is Euglena paramylon, MacroGard^®, and lipopolysaccharide. We investigated the gene expression of inflammation-related cytokines and mediators, transactivation of relevant transcription factors, and phagocytosis role in cell-glucan interactions, by means of RT-PCR, immunocytochemistry, and colorimetric assay. Our results show that sonicated and alkalized paramylon upregulates pro-inflammatory factors (NO, TNF-α, IL-6, and COX-2) in lymphomonocytes. A clear demonstration of this upregulation is the increased transactivation of NF-kB visualized by immunofluorescence microscopy. Phagocytosis assay showed that internalization is not a mandatory step for signaling cascade to be triggered, since immune activity is not present in the lymphomonocytes that have internalized paramylon granules and particulate MacroGard^®. Moreover, the response of Euglena β-glucan-activated lymphomonocytes is much greater than that induced by commercially used β-glucans such as MacroGard^®. Our in vitro results indicate that linear fibrous Euglena β-glucan, obtained by sonication and alkaline treatment can act as safe and effective coadjutant of the innate immune system response.

Keywords: Euglena; MacroGard®; glucans; innate immunity; paramylon.

Figure 1

Overall aspect of MacroGard^® and paramylon: (A) optical microscopy image of MacroGard^® aggregates dispersed in distilled water; 20 μm bar; (B) SEM image of a single aggregate; (B’) magnification of the aggregate surface showing its particulate texture; the white contour defines a single particle; (C) homogeneous particulate mixture of sonicated and alkalized MacroGard^®; the white contour defines a single particle; 5 μm bar; (D) SEM image of the particles present in the mixture; the white contour defines a single particle; (E) optical microscopy image of paramylon granules dispersed in distilled water; 5 μm bar; (F) SEM images of the granules; (G) heterogeneous mixture of sonicated and alkalized paramylon; 10 μm bar; (H) SEM image of paramylon sonicated and alkalized showing its linear fibrous structure.

Figure 2

Translocation of nuclear factor nuclear factor kappa light‐chain‐enhancer of activated B cell visualized by fluorescence microscopy: (A) nonstimulated peripheral blood mononuclear cell (PBMC); (B) paramylon granules stimulated PBMC; (C) MacroGard^® stimulated PBMC; (D) sonicated and alkalized paramylon stimulated PBMC; (E) lipopolysaccharide stimulated PBMC. Bar 10 μm.

Figure 3

Relative expression of interleukin‐6, tumor necrosis factor alpha, cyclooxygenase 2, and inducible nitric oxide synthase in peripheral blood mononuclear cell after 24 h stimulation with glucans and lipopolysaccharide. Results were expressed as fold‐increase respect to control and plotted as the mean ± SD. ** = P < 0.01 versus no stimuli.

Figure 4

Nitric oxide production by peripheral blood mononuclear cell stimulated with glucans and lipopolysaccharide after 4 and 24 h, expressed as percent nitrite concentration versus control. ** = P < 0.01 versus no stimuli.

Figure 5

Results of the phagocytic assay: (A) Peripheral blood mononuclear cell (PBMC) surrounded by sonicated and alkalized paramylon and dendritic cells typical of immune activation (arrowheads); (B) paramylon granules visible inside PBMC; (C) MacroGard^® particles visible inside PBMC. Bar 10 μm.