Immuno-modulatory effects of yeast cell wall compounds on murine macrophages and their stakes in bacterial infections of mammary gland

Les ß-glucanes (BG) sont les polysaccharides les plus abondants de la paroi de Saccharomyces cerevisiae. Depuis des millénaires, ils sont utilisés pour leurs propriétés immunostimulantes et leurs potentiels thérapeutiques. L'objectif de ce travail était de caractériser la réponse immunitaire induite par les BG et de comprendre leurs modes d'action sur les macrophages murins en contexte infectieux. Nous avons montré que (i) les extraits de paroi enrichis en BG n'induisent qu'une faible production de cytokines par les macrophages contrairement aux extraits bruts, (ii) la réponse inflammatoire médiée par les extraits bruts résulte de la signalisation des TLRs et non de Dectin-1 et (iii) les BG stimulent la synthèse tardive de GM-CSF via Dectin-1. En conditions infectieuses, les BG enrichis confèrent une forte signature inflammatoire aux macrophages prétraités conduisant à l'amplification de la production cytokinique, à la synthèse de ROS et l'optimisation de la clairance bactérienne. En conclusion, cette étude souligne les enjeux de l'utilisation des BG enrichis comme adjuvants dans l'amélioration de la résistance des individus aux infections.ß-glucans (BG) are the most abundant polysaccharides of the Saccharomyces cerevisiae cell wall. For decades, they have been extensively used because of their immuno-modulatory properties and their potential therapeutic effects. The aim of this study was to characterize the immune response induced by BG and to understand their mechanisms of action on murine macrophages occurring upon bacterial infections. We demonstrated that (i) BG-enriched extracts trigger low amounts of cytokine production in contrast with crude products, (ii) the immune response mediated by crude extracts results from TLRs and not from Dectin-1 signaling and (iii) BG-enriched compounds stimulate the late and strong induction of GM-CSF in a Dectin-1-dependent manner. Upon bacteria exposure, BG-enriched extracts confer a strong inflammatory to pretreated macrophages leading to synergistic increase of cytokine release, ROS production and better clearance of pathogens. Altogether, our findings emphasize the relevancy of using BG-enriched extracts for the design of novel adjuvant formulations contributing to individuals' resistance to infections

Etude des propriétés immunostimulantes de composés pariétaux de levure sur les macrophages murins et évaluation dans des modèles infectieux

ß-glucans (BG) are the most abundant polysaccharides of the Saccharomyces cerevisiae cell wall. For decades, they have been extensively used because of their immuno-modulatory properties and their potential therapeutic effects. The aim of this study was to characterize the immune response induced by BG and to understand their mechanisms of action on murine macrophages occurring upon bacterial infections. We demonstrated that (i) BG-enriched extracts trigger low amounts of cytokine production in contrast with crude products, (ii) the immune response mediated by crude extracts results from TLRs and not from Dectin-1 signaling and (iii) BG-enriched compounds stimulate the late and strong induction of GM-CSF in a Dectin-1-dependent manner. Upon bacteria exposure, BG-enriched extracts confer a strong inflammatory to pretreated macrophages leading to synergistic increase of cytokine release, ROS production and better clearance of pathogens. Altogether, our findings emphasize the relevancy of using BG-enriched extracts for the design of novel adjuvant formulations contributing to individuals' resistance to infections.Les ß-glucanes (BG) sont les polysaccharides les plus abondants de la paroi de Saccharomyces cerevisiae. Depuis des millénaires, ils sont utilisés pour leurs propriétés immunostimulantes et leurs potentiels thérapeutiques. L'objectif de ce travail était de caractériser la réponse immunitaire induite par les BG et de comprendre leurs modes d'action sur les macrophages murins en contexte infectieux. Nous avons montré que (i) les extraits de paroi enrichis en BG n'induisent qu'une faible production de cytokines par les macrophages contrairement aux extraits bruts, (ii) la réponse inflammatoire médiée par les extraits bruts résulte de la signalisation des TLRs et non de Dectin-1 et (iii) les BG stimulent la synthèse tardive de GM-CSF via Dectin-1. En conditions infectieuses, les BG enrichis confèrent une forte signature inflammatoire aux macrophages prétraités conduisant à l'amplification de la production cytokinique, à la synthèse de ROS et l'optimisation de la clairance bactérienne. En conclusion, cette étude souligne les enjeux de l'utilisation des BG enrichis comme adjuvants dans l'amélioration de la résistance des individus aux infections

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

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

Molecular Analysis of a Short-term Model of β-Glucans-Trained Immunity Highlights the Accessory Contribution of GM-CSF in Priming Mouse Macrophages Response.

β-Glucans (BGs) are glucose polymers present in the fungal cell wall (CW) and, as such, are recognized by innate immune cells as microbial-associated pattern through Dectin-1 receptor. Recent studies have highlighted the ability of the pathogenic yeast Candida albicans or its CW-derived β(1,3) (1,6)-glucans to increase human monocytes cytokine secretion upon secondary stimulation, a phenomenon now referred as immune training. This ability of monocytes programming confers BGs an undeniable immunotherapeutic potential. Our objective was to determine whether BGs from Saccharomyces cerevisiae, a non-pathogenic yeast, are endowed with such a property. For this purpose, we have developed a short-term training model based on lipopolysaccharide re-stimulation of mouse bone marrow-derived macrophages primed with S. cerevisiae BGs. Through a transcriptome analysis, we demonstrated that BGs induced a specific gene expression signature involving the PI3K/AKT signaling pathway as in human monocytes. Moreover, we showed that over-expression of Csf2 (that encodes for GM-CSF) was a Dectin-1-dependent feature of BG-induced priming of macrophages. Further experiments confirmed that GM-CSF up-regulated Dectin-1 cell surface expression and amplified macrophages response along BG-mediated training. However, the blockade of GM-CSFR demonstrated that GM-CSF was not primarily required for BG-induced training of macrophages although it can substantially improve it. In addition, we found that mouse macrophages trained with BGs upregulated their expression of the four and a half LIM-only protein 2 (Fhl2) in a Dectin-1-dependent manner. Consistently, we observed that intracellular levels of FHL2 increased after stimulation of macrophages with BGs. In conclusion, our experiments provide new insights on GM-CSF contribution to the training of cells from the monocytic lineage and highlights FHL2 as a possible regulator of BG-associated signaling

Molecular Analysis of a Short-term Model of β-Glucans-Trained Immunity Highlights the Accessory Contribution of GM-CSF in Priming Mouse Macrophages Response.

β-Glucans (BGs) are glucose polymers present in the fungal cell wall (CW) and, as such, are recognized by innate immune cells as microbial-associated pattern through Dectin-1 receptor. Recent studies have highlighted the ability of the pathogenic yeast Candida albicans or its CW-derived β(1,3) (1,6)-glucans to increase human monocytes cytokine secretion upon secondary stimulation, a phenomenon now referred as immune training. This ability of monocytes programming confers BGs an undeniable immunotherapeutic potential. Our objective was to determine whether BGs from Saccharomyces cerevisiae, a non-pathogenic yeast, are endowed with such a property. For this purpose, we have developed a short-term training model based on lipopolysaccharide re-stimulation of mouse bone marrow-derived macrophages primed with S. cerevisiae BGs. Through a transcriptome analysis, we demonstrated that BGs induced a specific gene expression signature involving the PI3K/AKT signaling pathway as in human monocytes. Moreover, we showed that over-expression of Csf2 (that encodes for GM-CSF) was a Dectin-1-dependent feature of BG-induced priming of macrophages. Further experiments confirmed that GM-CSF up-regulated Dectin-1 cell surface expression and amplified macrophages response along BG-mediated training. However, the blockade of GM-CSFR demonstrated that GM-CSF was not primarily required for BG-induced training of macrophages although it can substantially improve it. In addition, we found that mouse macrophages trained with BGs upregulated their expression of the four and a half LIM-only protein 2 (Fhl2) in a Dectin-1-dependent manner. Consistently, we observed that intracellular levels of FHL2 increased after stimulation of macrophages with BGs. In conclusion, our experiments provide new insights on GM-CSF contribution to the training of cells from the monocytic lineage and highlights FHL2 as a possible regulator of BG-associated signaling

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

<p>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 <i>Sc</i> 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 <i>Student’s t-test</i> (<i>p</i> < 0.05). Comparisons are shown for BG compounds used at 100 μg/mL.</p

Murine macrophages treated with extracts enriched in BG produce less TNFα than those treated with unpurified <i>Sc</i> cell wall extracts.

<p>(A) RAW-Blue^™ macrophages were treated with 100 or 10 μg/mL of <i>Sc</i> 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 <i>Sc</i> 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 <i>Student’s test</i> (<i>p</i> < 0.05). (D) Immortalized BMDM (iBMDM) from WT and <i>MyD88</i>^-/- mice were subcultured in 96-well plates and stimulated for 8 h with 100 μg/mL of <i>Sc</i> 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 <i>Student’s t-test</i> (<i>p</i> < 0.05).</p

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

<p>(A-C) HEK-Blue^™-hTLR2, -hTLR4 and hDectin-1 cells were incubated with serially diluted <i>Sc</i> 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.</p

The timing of <i>Clec7a</i>-dependent <i>Csf2</i> expression depends on <i>Sc</i> BG content.

<p>WT and <i>Clec7a</i> (dectin-1)^-/- BMDM were stimulated with <i>Sc</i> 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 (<i>Sdha</i>, <i>Rpl9</i> and <i>Hprt1</i>) 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.</p

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 <i>Sc</i> BG extracts.

<p>(A-B) Freshly isolated 4-days thioglycollate-elicited peritoneal macrophages (TEPM), resident peritoneal macrophages (RPM) and BMDM from WT and <i>Clec7a</i>^<i>-/-</i> 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 <i>Clec7a</i>^<i>-/-</i> C57Bl/6 for WT C57Bl/6 or DBA/2 primary macrophages. (C) Remaining TEPM and BMDM from WT and <i>Clec7a</i>^<i>-/-</i> C57Bl/6 mice isolated from these experiments were subcultured and stimulated with 100 μg/mL of crude or enriched <i>Sc</i> 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. *<i>p</i> < 0.05 (two-tailed unpaired <i>Student’s t-test)</i>; mean values not sharing the same letter are significantly different.</p