Candida albicans β-Glucan Differentiates Human Monocytes Into a Specific Subset of Macrophages

Abstract

β-Glucan derived from cell walls of Candida albicans is a potent immune modulator. It has been shown to induce trained immunity in monocytes via epigenetic and metabolic reprogramming and to protect from lethal sepsis if applied prior to infection. Since β-glucan-trained monocytes have not been classified within the system of mononuclear phagocytes we analyzed these cells metabolically, phenotypically and functionally with a focus on monocyte-to-macrophage differentiation and compared them with naïve monocytes and other types of monocyte-derived cells such as classically (M1) or alternatively (M2) activated macrophages and monocyte-derived dendritic cells (moDCs). We show that β-glucan inhibits spontaneous apoptosis of monocytes independent from autocrine or paracrine M-CSF release and stimulates monocyte differentiation into macrophages. β-Glucan-differentiated macrophages exhibit increased cell size and granularity and enhanced metabolic activity when compared to naïve monocytes. Although β-glucan-primed cells expressed markers of alternative activation and secreted higher levels of IL-10 after lipopolysaccharide (LPS), their capability to release pro-inflammatory cytokines and to kill bacteria was unaffected. Our data demonstrate that β-glucan priming induces a population of immune competent long-lived monocyte-derived macrophages that may be involved in immunoregulatory processes.

Keywords: Candida albicans; monocyte survival; monocyte to macrophage differentiation; trained immunity; β-glucan.

Figure 1

β-Glucan inhibits spontaneous human monocyte apoptosis. (A) Human monocytes were left untreated (control) or stimulated with 5 μg/ml β-glucan (+), 50 μg/ml β-glucan (++) or M-CSF (50 ng/ml). After 24 and 48 h, protein extracts were analyzed for cleavage of PARP and caspase 3. One representative of two (24 h) or three (48 h) independent experiments is shown. Densitometry data are given in the text. (B–D) Cell viability was measured after 48 h by flow cytometric analysis of Annexin V (Ann) and propidium iodide (PI). One representative dot plot is shown in (B) and the frequencies of dead (PI+/Ann-), late apoptotic (PI+/Ann+), early apoptotic (PI-/Ann+) and viable cells (PI-/Ann-) from five independent experiments are shown in (C, D). The frequency of viable cells was significantly higher in the groups treated with M-CSF or β-glucan as compared to untreated monocytes (control) (D). Values are means ± SEM, ^*p < 0.05 compared to control.

Figure 2

The anti-apoptotic effect of β-glucan is independent of M-CSF. (A) Human monocytes were left untreated (control) or stimulated for 24 h with β-glucan (5 μg/ml or 50 μg/ml). The release of M-CSF was determined by cytometric bead array in cell supernatants or in medium plus 10% serum only (medium). (B–D) Human monocytes were preincubated with the M-CSF inhibitor GW2580 or vehicle for 60 min and then incubated for 48 h with either M-CSF (50 ng/ml) or β-glucan (5 μg/ml or 50 μg/ml). Controls were left without stimulation. Protein lysates were analyzed in immunoblots for cleavage of PARP and caspase 3. (B) One representative blot is shown. (C,D) Densitometry analyses of protein bands normalized to β-actin of three independent experiments. Values are means ± SEM, ^*p < 0.05 compared to the respective untreated control, §p < 0.05 compared to 5 μg/ml β-glucan, #p < 0.05 compared to M-CSF alone.

Figure 3

Transient treatment of monocytes with β-glucan increases long-term survival. Human monocytes were left untreated (control) or stimulated with β-glucan (5 μg/ml or 50 μg/ml) for 24 h, followed by washing and 6 further resting days. (A–C) May-Grünwald-Giemsa staining of cultured cells after 7 days. The scale bar indicates 100 μm. (D,E) The number of surviving cells was evaluated as cell count per high-power field and DNA content per well. Values are means ± SEM from seven (D) or four (E) independent experiments, ^*p < 0.05 compared to control, #p < 0.05 compared to 5 μg/ml β-glucan.

Figure 4

β-Glucan increases cell size and granularity. Human monocytes were left untreated (control) or stimulated with β-glucan (5 μg/ml) or M-CSF (50 ng/ml) for 48 h. All viable cells (PI-/Ann-) were analyzed by flow cytometry using forward scatter (FSC) and sideward scatter (SSC). Representative dot plots (A) and means ± SEM from five independent experiments are shown (B,C), ^*p < 0.05 compared to control.

Figure 5

β-Glucan increases metabolic activity. Human monocytes were left untreated (control) or incubated with β-glucan (5 μg/ml or 50 μg/ml) for 24 h, followed by washing and a resting period for 6 further days. (A) Medium was exchanged on day 6 and cells were stimulated with 10 ng/ml LPS (black bars) or left untreated (gray bars). Lactate levels of cell supernatants were determined 24 h later (day 7). (B–E) extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were determined at baseline, after oligomycin treatment (inhibition of mitochondrial ATP synthase), after 2,4-dinitrophenol (DNP, mitochondrial uncoupling agent) and after antimycin A [inhibition of electron transport chain (ETC) complex III]. All ECAR and OCR results were normalized to the DNA content of cells. Values are means ± SEM from five (A) or four (B–E) independent experiments, ^*p < 0.05 compared to control, #p < 0.05 compared to 5 μg/ml β-glucan.

Figure 6

β-Glucan-induced metabolic phenotype resembles differentiated monocyte-derived cells. (A–F) Human monocytes prepared from one donor were either directly analyzed for metabolic data (monocytes) or were differentiated to monocyte-derived cells. To obtain β-glucan-differentiated cells, monocytes were incubated for 24 h with β-glucan (5 μg/ml or 50 μg/ml), followed by washing and resting for 6 further days. M1-like macrophages (M1) were differentiated with GM-CSF and polarized with LPS + IFNγ. M2-like macrophages (M2) were differentiated with M-CSF and polarized with IL-4. Dendritic cells (DC) were differentiated with GM-CSF + IL-4 (immature DC) and were matured with LPS (mature DC). Monocytes left untreated for 7 days (control) served as additional control. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were determined at baseline and after treatment with oligomycin [inhibition of complex V of the electron transport chain (ETC)] plus 2,4-dinitrophenol (DNP, mitochondrial uncoupling agent). (A–C) OCR and ECAR are shown under baseline conditions. (D–F) Mitochondrial ATP production, non-mitochondrial respiration and spare capacity were calculated from OCR. All ECAR and OCR results were normalized to the DNA content of cells. Values are means ± SEM from four independent experiments. *p < 0.05 compared to monocytes.

Figure 7

β-Glucan differentiates monocytes into macrophage-like cells. (A-B) Human monocytes (Mo) from one donor were either analyzed for expression of cell surface markers by FACS or differentiated for 7 days to monocyte-derived cells. To obtain β-glucan-differentiated cells (β5, β50), monocytes were incubated for 24 h with 5 μg/ml (β5) or 50 μg/ml β-glucan (β50), followed by washing and resting for 5 further days. On day 6, cells were stimulated with 10 ng/ml LPS or left untreated. M1-like macrophages (M1) were differentiated with GM-CSF and polarized with LPS + IFNγ. M2-like macrophages (M2) were differentiated with M-CSF and polarized with IL-4. Dendritic cells were differentiated with GM-CSF + IL-4 (immature dendritic cells, iDC) and were matured with LPS (mature dendritic cells, mDC). Heat map displaying the z-normalized median fluorescence intensity (MFI) of surface expression markers (A) and principal component analysis (B) of isolated monocytes and monocyte-derived cells are shown. One representative experiment out of 2.

Figure 8

β-Glucan-differentiated cells release pro- and anti-inflammatory cytokines and kill live bacteria. Human monocytes were left untreated (control) or stimulated with 5 μg/ml β-glucan for 24 h, followed by washing and resting for 5 further days. (A–C) Medium was exchanged on day 6. Cells were stimulated with 10 ng/ml LPS for 24 h or left untreated. Cytokine release in supernatants was determined by cytometric bead array and normalized to cell proteins. Values are means ± SEM from six independent experiments. (D,E) Cells were detached and reseeded to 6-well plates on day 6. After 24 h, cell numbers were determined and infection with S. aureus was performed at a MOI of 5 for 90 min. Extra- and intracellular bacteria were quantified by CFU measurements. The killing activity was calculated as difference between the initial number of bacteria and the number of surviving extra- and intracellular bacteria in % of the initial inoculum. Values are means ± SEM from six independent experiments, n.s. not significant, ^*p < 0.05.