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

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

<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

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

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