Glycolysis upregulation upon <i>Candida</i> stimulation.

<p>(A) Schematic pathway map of the gene expression in the main metabolic pathways in PBMCs stimulated with heat-killed <i>C</i>. <i>albicans</i> yeast 24 h after stimulation. The dots represent metabolites, and the arrows indicate reactions converting these metabolites. For each reaction it is known which enzymes (and thus which genes) are involved in catalyzing the reaction. The arrows marked in red indicate an overall upregulation of genes involved in those reactions in <i>C</i>. <i>albicans</i> versus RPMI, whereas the blue indicates a downregulation. A darker color indicates a larger change in transcript levels. The complete map stimulation as created by Escher for 4 h and 24 h stimulation is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006632#ppat.1006632.s001" target="_blank">S1 Fig</a>. (B) Fold increase of mRNA expression for the indicated enzymes analyzed by RT-PCR in monocytes 24 h after stimulation with heat-killed <i>C</i>. <i>albicans</i> conidia or heat-killed <i>C</i>. <i>albicans</i> hyphae (mean ± SEM, n = 6–9; pooled from 2–3 experiments). *p<0.05, **p<0.01 Wilcoxon signed-rank test. HK2: Hexokinase 2; PFKP: Phosphofructokinase, platelet; α-KGDH: alpha-ketoglutarate dehydrogenase; LDH: Lactate dehydrogenase; mTOR: Mammalian target of rapamycin; GLS: Glutaminase; GLUD: Glutamine dehydrogenase.</p

Inhibition of glucose metabolism impaired <i>in vivo</i> responses to systemic <i>C</i>. <i>albicans</i> infection.

<p>(A) Fungal burden measured in the kidneys of C57BL/6 mice treated with PBS, 2-DG or BPTES during 5 days after i.v. <i>C</i>. <i>albicans</i> challenge (mean ± SEM, n = 6; similar results were obtained in 2 independent experiments). *p<0.05, Student’s t test. Each dot represents one mouse. (B) Candidacidal activity of neutrophils isolated from blood of C57BL/6 mice treated with PBS, 2-DG or BPTES during 5 days after i.v. <i>C</i>. <i>albicans</i> challenge (mean ± SEM, n = 6; similar results were obtained in 2 independent experiments) *p < 0.05, Student’s t test. Each dot represents one mouse. (C) IL-1β, IL-6, IL-10, IFNγ and TNFα production by mouse splenocytes obtained from PBS, 2-DG or BPTES-treated mice 5 days after <i>C</i>. <i>albicans</i> i.v. infection were measured by ELISA 48 h after <i>in vitro</i> restimulation with medium, LPS, heat-killed <i>C</i>. <i>albicans</i> yeast or heat-killed <i>C</i>. <i>albicans</i> hyphae (mean ± SEM, n = 6; similar results were obtained in 2 independent experiments) *p<0.05, Student’s t test.</p

<i>Candida</i> stimulation induced glycolysis in human monocytes.

<p>(A) Lactate production and glucose consumption by monocytes after overnight stimulation with heat-killed <i>C</i>. <i>albicans</i> yeast or heat-killed <i>C</i>. <i>albicans</i> hyphae (mean ± SEM, n = 12 for lactate, n = 6 for glucose; pooled from 2–4 independent experiments). *p<0.05, ***p<0.001 Wilcoxon signed-rank test. (B-D) Basal and maximum extracellular acidification rates (ECAR; B), basal and maximum oxygen consumption rates (OCR; C) or spare respiratory capacity (SRC; D) of monocytes were determined by Seahorse analysis at 4 and 24 h after stimulation with medium or heat-killed <i>C</i>. <i>albicans</i> yeast (mean ± SEM, n = 6–8; pooled from 2 independent experiments). *p<0.05, **p<0.01 Wilcoxon signed-rank test. (E) Intracellular metabolite levels of monocytes 4 and 24 h after heat-killed <i>C</i>. <i>albicans</i> yeast or heat-killed <i>C</i>. <i>albicans</i> hyphae stimulation (mean ± SEM, n = 6–8; pooled from 2 independent experiments). *p<0.05, **p<0.01 Wilcoxon signed-rank test. (F) Lactate production by monocytes after overnight stimulation with live <i>hgc1</i> or <i>Δhgc1 C</i>. <i>albicans</i>. (mean ± SEM, n = 6; pooled from 2 independent experiments). *p<0.05, Wilcoxon signed-rank test. (G) Intracellular metabolite levels of monocytes 4 and 24 h after <i>hgc1</i> or <i>Δhgc1</i> live <i>C</i>. <i>albicans</i> stimulation (mean ± SEM, n = 6; pooled from 2 independent experiments). *p<0.05, **p<0.01 Wilcoxon signed-rank test.</p

Overview of the immune and metabolic processes taking place in monocytes after systemic <i>C</i>. <i>albicans</i> recognition.

<p><i>C</i>. <i>albicans</i> recognition by monocytes triggers a complex network of metabolic pathways that lead to the production of proinflammatory cytokines and ROS, and also the release of lactate to the extracellular space. Heat killing of <i>C</i>. <i>albicans</i> resulted in higher β-glucan exposure and subsequent dectin-1 recognition while shielding of β-glucans by mannans in hyphae prevents activation of the CTL signaling pathways. While heat-killed hyphae-derived responses mainly rely on the participation of glycolysis and the pentose phosphate pathway, the response to heat-killed yeast is a much more demanding process that requires the participation of glycolysis, oxidative phosphorylation, glutaminolysis and the pentose phosphate pathway through a process driven by an enhanced C-type lectin-derived signaling.</p

ROS production by monocytes involved glycolysis and the pentose phosphate pathway.

<p>(A-D) Human monocytes were treated with medium (A), 2-DG (B), DCA (C) or 6-AN (D) and subsequently stimulated with medium, heat-killed <i>C</i>. <i>albicans</i> yeast or heat-killed <i>C</i>. <i>albicans</i> hyphae. Luminescence generated from ROS production was measured every 145 seconds during 60 minutes (n = 4; pooled from 2 independent experiments). Dashed lines show maximum ROS levels reached without inhibitor treatment to be used as a visual reference.</p

GAG induces interleukin 1 receptor antagonist.

<p>(A) IL-1 bioactivity measured as IL-2 production by NOB-1 cells stimulated with 50 ng/ml IL-1β in the presence of culture supernatant of unstimulated PBMCs (medium) or culture supernatants of PBMCs that were exposed to 10 µg/ml GAG for 24 hours (GAG conditioned medium) (n = 6 donors). (B) IL-1Ra, IL-1β and IL-1α concentrations in culture supernatants of human PBMCs stimulated with for 24 hours with 10 µg/ml GAG. Data are represented as mean +/− SEM.</p

Suppression of IL-17 and IL-22 by GAG is dependent on IL-1Ra.

<p>(A) IL-17, IL-22 and IFN-γ concentrations in culture supernatants of PBMCs stimulated for 7 days with heat inactivated <i>A. fumigatus</i> conidia 1×10⁷/ml, IL-1β/IL-23 (50/100 ng/ml) or IL-12/IL-18 (50/100 ng/ml) in the presence or absence of recombinant human IL-1Ra (10 ng/ml). Data are represented as mean +/− SEM. (B) Inhibition of IL-1β/IL-23 (50/100 ng/ml) induced IL-17 and IL-22 by GAG (10 µg/ml) in human PBMCs in the presence of isotype control (10 µg/ml) or anti-IL-1Ra (10 µg/ml). The IL-17 and IL-22 production by IL-1β/IL-23 in absence of GAG was set at 100% and mean percentage changes relative to the control are represented +/− SEM.</p

GAG protects mice from experimental DSS-induced colitis.

<p>(A) Body weight losses, (B) stool and histological score, (C) histology of colonic sections and (D) cytokine concentrations present in total colonic cells a day after the 7-day of DSS rest in C57BL/6 and <i>p47^phox−/−</i> (CGD) mice with or without GAG treatment. *P<0.05, GAG treated vs. untreated mice.</p

GAG induces IL-1Ra <i>in vivo</i> and IL-1Ra increases susceptibility to aspergillosis.

<p>BALB/c and <i>Il1ra^−/−</i> mice were intranasally infected with <i>Aspergillus</i> conidia and treated with GAG (250 μg/kg intranasally) the day of infection, and 1, 2 and 3 days post-infection. (A) <i>Il1ra</i> mRNA expression in lung homogenates of mice with invasive aspergillosis, (B) <i>Il1ra</i> mRNA expression in purified cells from lungs of naive mice pre-stimulated with <i>Aspergillus</i> conidia or LPS for 1 hour, and exposed to different GAG concentrations for an additional 18 hours. (C) Survival, (D) fungal growth (CFU/lung, mean +/−SEM), (E) protein levels of IL-1Ra, (F) <i>Mpo</i> expression in lung homogenates, and (G) BAL morphometry [% polymorphonuclear (PMNs) cells and lung histology (PAS stained sections, bars indicate 20× magnification) of <i>Aspergillus</i>-infected mice with or without GAG treatment. Assays were done a day after the last GAG treatment. (H) BAL morphometry [% PMNs or eosinophils (Eo)] and lung histology (PAS stained sections, bars indicate 20× magnification), and (I) expression of Th transcription factors and cytokines in total cells from the draining lymph nodes in mice with ABPA and treated with or without GAG. Naïve means uninfected mice, and none means untreated mice and/or unstimulated cells. (J) IL-5, IL-13 and IL-17 concentrations in culture supernatants of PBMCs pre-incubated 1 h either with medium or GAG (10 μg/ml). After washing, the cells were stimulated for 7 days with heat inactivated 1×10⁷/ml <i>A. fumigatus</i> conidia (n = 4 donors). Data are represented as mean +/− SEM. *, p<0.05; **, p<0.01.</p

GAG inhibits <i>Aspergillus</i>-induced human T-helper cell cytokine production.

<p>(A) TNFα, IL-6, IL-8 and IL-10 concentrations in culture supernatants of human PBMCs stimulated for 24 hours with 10 µg/ml GAG and IFN-γ, IL-17, IL-5 and IL-9 concentrations after 7 days of stimulation. (B) TNFα, IL-6 and IL-10 concentrations in culture supernatants of human PBMCs (n = 6 donors) stimulated for 24 hours with heat inactivated <i>A. fumigatus</i> conidia (1×10⁷/ml) in the presence or absence of 10 µg/ml GAG. (C,D) IL-17, IL-22 and IFN-γ concentrations in culture supernatants of human PBMCs stimulated for 7 days with heat inactivated <i>A. fumigatus</i> conidia (1×10⁷/ml) (n = 10 donors for IL-17 and IL-22, n = 6 donors for IFN-γ) (c), IL-1β/IL-23 (50/100 ng/ml) (n = 14 donors) or IL-12/IL-18 (50/100 ng/ml) (n = 10 donors) in the presence or absence of GAG (10 µg/ml). Data are represented as mean +/− SEM.</p