Cellular and molecular mechanisms of fungal β-(1→6)-glucan in macrophages

Abstract

Over the last 40 yr, the majority of research on glucans has focused on β-(1→3)-glucans. Recent studies indicate that β-(1→6)-glucans may be even more potent immune modulators than β-(1→3)-glucans. Mechanisms by which β-(1→6)-glucans are recognized and modulate immunity are unknown. In this study, we examined the interaction of purified water-soluble β-(1→6)-glucans with macrophage cell lines and primary peritoneal macrophages and the cellular and molecular consequences of this interaction. Our results indicate the existence of a specific β-(1→6)-glucan receptor that internalizes the glucan ligand via a clathrin-dependent mechanism. We show that the known β-(1→3)-glucans receptors are not responsible for β-(1→6)-glucan recognition and interaction. The receptor-ligand uptake/interaction has an apparent dissociation constant (KD) of ∼ 4 µM, and was associated with phosphorylation of ERK and JNK but not IκB-α or p38. Our results indicate that macrophage interaction with β-(1→6)-glucans may lead to modulation of genes associated with anti-fungal immunity and recruitment/activation of neutrophils. In summary, we show that macrophages specifically bind and internalize β-(1→6)-glucans followed by activation of intracellular signaling and modulation of anti-fungal immune response-related gene regulation. Thus, we conclude that the interaction between innate immunity and β-(1→6)-glucans may play an important role in shaping the anti-fungal immune response.

Keywords: Fungi; glucan; innate immunity; macrophages; receptor.

Figure 1.

Specific binding and internalization of β-(1→6)-glucan by macrophage cell lines. (A) Human U937 and murine RAW and J774a.1 cell lines were treated with 50 x higher concentration of non-labeled competitors—β-(1→6)-glucan or β-(1—3)-glucan—prior to the treatment with 10 μg/ml (167 nM) labeled β-(1→6)-glucan. (B)J774a.1 cells treated with 12.5×or 50 × higher concentrations of non-labeled competitors—β-(1→6)-glucan or β-(1→3)-glucan—prior to the treatment with 10 μg/ml (167 nM) labeled β-(1→6)-glucan. The data are presented as the percent fluorescence relative to the fluorescence of the corresponding positive controls ± SD of four repeated measurements per group. *Significant difference compared with control (P ≤ 0.05).

Figure 2.

Specific binding and internalization of β-(1→6)-glucan by murine primary macrophages. Macrophages were treated with unlabeled competitors—β-(1→6)-glucan or (β-(1→3)-glucan—prior to the treatment with labeled β-(1→6)-glucan. The data are presented as the percent fluorescence relative to the fluorescence of the corresponding positive controls ± SD of four repeated measurements per group. *Significant difference compared with controls without competitor (P ≤ 0.05).

Figure 3.

Internalization of β-(1→6)-glucan by macrophages is mediated via a clathrin-dependent mechanism. Various inhibitors specific for the different pathways were used. (A) Data as measured by flow cytometry. The data are presented as the percent fluorescence relative to the fluorescence of the corresponding positive controls ± SD of four repeated measurements per group. (B) Data as measured by confocal microscopy. *Significant difference compared with controls without competitor (P ≤ 0.05).

Figure 4.

Saturability and dose dependence of β-(1→6)-glucan in primary macrophages. Data from three replicate experiments were fitted to a single-site hyperbolic binding model using nonlinear least squares regression. Combined saturation and competitive displacement data yielded a K_D of 4 μM and a B_max of 6l.

Figure 5.

Effect of β-(1→6)-glucan on phosphorylation of IκB-α, ERK, JNK and p38 in murine macrophage cell line J774a.l. (A) Representative results of membranes being probed for phosphorylated protein, re-probed for total protein, stripped and re-probed for loading control. (B) Fold regulation (ratio phosphorylated to total protein) of phosphorylation status of the measured proteins at different time points. Data are expressed as the ratio of phospho-protein to total protein (fold regulation) ± SD of three repeated measurements. *Significant difference when compared with non-treated control (P ≤ 0.05).

Figure 6.

Phosphorylation status of IκB-α, ERK and p38 in primary peritoneal macrophages treated with β-(1→6)-glucan. The data are presented as fold regulation (ratio of phosphorylated to total protein) of phosphorylation status of the measured proteins at different time points. Data are expressed as the ratio of phospho-protein to total protein (fold regulation) ± SD of three repeated measurements. *Significant difference when compared with non-treated control (P ≤ 0.05).

Figure 7.

Treatment of the macrophage cell line J774a.l with β-(l→6)-glucan results in (A) up- or (B) down-regulation of mRNAs associated with anti-fungal immune responses. Data are expressed as the ratio of treated samples to non-treated controls (fold regulation) of two repeated measurements. *Significance based on ≥ two-fold changes; **significance based on ≥ four-fold changes as suggested by the manufacturer.