Triggering Dectin-1-Pathway Alone Is Not Sufficient to Induce Cytokine Production by Murine Macrophages

Abstract

β-glucans (BG) are abundant polysaccharides of the Saccharomyces cerevisiae cell wall (Sc CW), an industry byproduct. They have immuno-stimulatory properties upon engagement of dectin-1 (Clec7a), their main receptor on particular immune cells, and they actually become of great interest because of their preventive or therapeutic potentials. Zymosan, a crude extract of Sc CW was studied as a prototypic BG, despite its miscellaneous PAMPs content. Here, we examined the response of murine wild type or Clec7a-/- bone marrow-derived macrophages (BMDM) to products with increasing BG content (15, 65 or 75%) and compared their effects with those of other dectin-1 ligands. The enrichment process removed TLR ligands while preserving dectin-1 activity. The most enriched extracts have very low NFκB activity and triggered low amounts of cytokine production in contrast with crude products like zymosan and BG15. Furthermore, MyD88-/- BMDM did not produce TNFα in response to crude Sc CW extracts, whereas their response to BG-enriched extracts was unaffected, suggesting that BG alone are not able to initiate cytokine secretion. Although Sc CW-derived BG stimulated the late and strong expression of Csf2 in a dectin-1-dependent manner, they remain poor inducers of chemokine and cytokine production in murine macrophages.

Fig 1. Saccharomyces cerevisiae (Sc) extracts enriched in β-glucan (BG) are much weaker inducers of NFκB/AP-1 activity in RAW-Blue^™ macrophages than other sources of BG.

NFκB/AP-1 activity was determined by a colorimetric enzyme assay using RAW-Blue^™ macrophages as reporter cell line. Cells were treated for 16 h with 100 or 10 μg/mL of various Sc BG extracts (BG15, BG65 and BG75) or with zymosan, curdlan, dispersible WGPd and soluble WGPs as controls. Data are expressed as the mean OD 650nm ± SD of three independent experiments performed in triplicate. Mean values not sharing the same letter are significantly different according to the Student’s t-test (p < 0.05). Comparisons are shown for BG compounds used at 100 μg/mL.

Fig 2. Enrichment of BG from yeast cell wall extracts strongly abolishes TLR2/4-related NFκB/AP-1 activities conversely to dectin-1.

(A-C) HEK-Blue^™-hTLR2, -hTLR4 and hDectin-1 cells were incubated with serially diluted Sc BG cell wall extracts (BG15, BG65 and BG75) or their BG controls (zymosan, curdlan, dispersible and soluble WGP) for 16 h in culture medium containing the reporter reagent (37°C, 5% CO₂₎. Each cell line was stimulated with a 10-fold serial dilution (from 100 ng/mL to 0.001 ng/mL) of control ligand, Pam3CSK4 for HEK-Blue^™-hTLR2 and ultraPure LPS for HEK-Blue^™-hTLR4, respectively as shown in the panels on the right of A and B. The NFκB/AP-1-related activity of TLR2, TLR4 and dectin-1 was assessed in supernatants by a colorimetric assay. The OD value of a blank control, which corresponds to the OD value of HEK-Blue detection medium, was subtracted from the OD values of samples. The results are presented as OD 650 nm values and are representative of three independent experiments.

Fig 3. Murine macrophages treated with extracts enriched in BG produce less TNFα than those treated with unpurified Sc cell wall extracts.

(A) RAW-Blue^™ macrophages were treated with 100 or 10 μg/mL of Sc BG extracts (BG15, BG65 and BG75) or control (zymosan, curdlan, dispersible WGPd and soluble WGPs) for 16 h and TNFα was then quantified by ELISA. Comparisons are shown for BG compounds used at 100 μg/mL. (B-C) BMDM from C57Bl/6 mice were subcultured in 96-well plates. (B) Cells were stimulated for 1, 4, 8, 16 or 24 h with 100 μg/mL of Sc BG extracts (BG15, BG65 and BG75) or control (curdlan, dispersible WGPd and soluble WGPs). Supernatants were collected immediately after incubation. (C) Cells were incubated for 8 h with a 3-fold serial dilution from 1000 to 10 μg/mL of BG-purified compounds (BG15, BG65 and BG75). Supernatants were harvested immediately after incubation. (A-C) TNFα was measured using ELISA and the data are expressed as the mean ± SD of three independent experiments performed in triplicate. Mean values not sharing the same letter are significantly different according to the Student’s test (p < 0.05). (D) Immortalized BMDM (iBMDM) from WT and MyD88^-/- mice were subcultured in 96-well plates and stimulated for 8 h with 100 μg/mL of Sc BG extracts (BG15, BG65 and BG75) or control (zymosan and dispersible WGPd). LPS (100 ng/mL) was used as a positive control of MyD88 activation. Supernatants were harvested immediately after each incubation time to quantify TNFα by ELISA. Data are expressed as the mean ± SD of two independent experiments performed in triplicate. Mean values not sharing the same letter are significantly different according to the Student’s t-test (p < 0.05).

Fig 4. Murine primary macrophages including peritoneal macrophages and BMDM express similar levels of the BG receptor dectin-1 and exhibit the same ability to respond to Sc BG extracts.

(A-B) Freshly isolated 4-days thioglycollate-elicited peritoneal macrophages (TEPM), resident peritoneal macrophages (RPM) and BMDM from WT and Clec7a^-/- C57Bl/6, and DBA/2 mice, and the macrophages cell line RAW-Blue^™, used as control, were assessed for surface expression of dectin-1 by flow cytometry (MACSQuant^®, Miltenyi Biotech, Germany). Doublets of cells were excluded at the beginning of the acquisition and a 7-AAD staining was used to discriminate death cells. The surface expression of dectin-1 was determined for each type of primary macrophages on CD115⁺ cells. (A) Data are expressed as the frequency of dectin-1⁺ among CD115⁺ cells. (B) The mean of fluorescence intensity (MFI) of dectin-1 was established on CD115⁺ cells. Results are presented as the mean ± SD MFI of dectin-1 corrected by either the MFI of isotype control for RAW macrophages, or the MFI of Clec7a^-/- C57Bl/6 for WT C57Bl/6 or DBA/2 primary macrophages. (C) Remaining TEPM and BMDM from WT and Clec7a^-/- C57Bl/6 mice isolated from these experiments were subcultured and stimulated with 100 μg/mL of crude or enriched Sc BG CW extracts (BG15, BG65 or BG75) or zymosan for 8 h (37°C, 5% CO₂). Supernatants were collected immediately after incubation and were assessed for TNFα production by ELISA. Results are shown as the mean ± SD. All data are representative of three mice per group from three independent experiments performed in triplicate. *p < 0.05 (two-tailed unpaired Student’s t-test); mean values not sharing the same letter are significantly different.

Fig 5. The timing of Clec7a-dependent Csf2 expression depends on Sc BG content.

WT and Clec7a (dectin-1)^-/- BMDM were stimulated with Sc BG extracts for 4, 8 or 16 h. After incubation, supernatants were removed and cells were lysed in RLT lysis buffer. Total RNA was extracted using the RNeasy Mini Kit and retro-transcribed with the SuperScript III First-Strand Synthesis Super Mix Kit. The expression of cytokine and chemokine genes was measured by quantitative PCR and normalized with three housekeeping genes (Sdha, Rpl9 and Hprt1) selected with GeNorm Software. Individual quantitative PCR was performed on the LightCycler^® 480 system (Roche, Switzerland) using Power SYBR Green PCR Master Mix (Applied Biosystems) to confirm the results obtained with the Biomark method. Data are expressed as fold change related to the mock value, representative of two experiments performed in triplicate.

Fig 6. Sc cell wall crude products- and zymosan-induced cytokine productions are mainly dectin-1-dependent.

WT and Clec7a^-/- BMDM were stimulated with Sc extracts enriched in BG for 8 h. After incubation, cell culture supernatants were harvested and stored at -20°C. Chemokines and cytokines (TNFα, IL-6, CCL2, IL-10 and IL-1β) were quantified using a customized multiplex assay and GM-CSF was measured with a cytokine detection kit. Data are expressed as the mean ± SD of three independent experiments performed in triplicate. Mean values not sharing the same letter are significantly different according to the Student’s t-test (p < 0.05).

Fig 7. Sc BG-enriched extracts poorly induce neutrophil recruitment via dectin-1 but strongly stimulate ROS production via CR3 and not dectin-1.

(A) Transwell chemotaxis assay to assess the chemotactic activity of supernatants from WT and Clec7a^-/- BMDM stimulated with Sc BG extracts (BG15, BG65 and BG75) for 8 h. BM neutrophils of WT C57Bl/6 mice (5x10⁵cells/well) were incubated for 30 min at 37°C with the supernatants and the neutrophils migrating into the lower chamber of the 96-well transwell plate were quantified by flow cytometry with an absolute counting system. Control wells containing medium (RPMI supplemented with 10% heat-inactivated FBS) were included in the chemotaxis assay. Data are expressed as the mean ± SD of three independent experiments performed in triplicate. (B) C57Bl/6 mice (n = 4/group) were challenged ip with 100 μg of BG15, BG75 or zymosan under 500 μL of PBS. After 6 h of incubation, peritoneal cells were collected and the number of recruited neutrophils and inflammatory monocytes were assessed by flow cytometry as described in Material and Methods section. Data are presented as the mean ± SD of four individual mice measurements and are representative of two independent experiments. (C) WT and Clec7a^-/- BMDM were stimulated with Sc BG extracts for 8 h. After incubation, cell culture supernatants were collected and stored at -20°C. CXCL1 and CXCL2 were quantified using a customized multiplex assay. Data are expressed as the mean ± SD of three independent experiments performed in triplicate. (A-C) Mean values not sharing the same letter are significantly different according to the Student’s test (p < 0.05). (C-D) WT (D-E) and Clec7a^-/- BMDM (E) were subcultured in 96-well plates and incubated simultaneously with Luminol and 100 μg/mL of Sc BG extracts (BG15, BG65 and BG75) or control (zymosan, curdlan, dispersible WGPd and soluble WGPs). ROS production was assessed immediately from the intensity of luminescence in each well, which was measured every 5 min for 2 h with a Tecan plate reader. (D) Results are expressed as the mean of luminescence intensity ± SD of two independent experiments performed in triplicate. (E) WT BMDM were incubated for 1 h with LEAF^™ anti-mouse CD11b (αCD11b) or with its rat control isotype IgG2a mAb and were then treated with BG products. Data were recorded into Prism 6 (GraphPad Software, San Diego, CA) and analyzed by the area under curve (AUC) method. Levels of ROS produced by αIgG2a-WT BMDM were comparable to those of untreated cells. Results are expressed as the mean of three independent experiments performed in triplicate. Mean values not sharing the same letter are significantly different according to the Student’s t-test (p < 0.05).