Rewiring monocyte glucose metabolism via C-type lectin signaling protects against disseminated candidiasis

Abstract

Monocytes are innate immune cells that play a pivotal role in antifungal immunity, but little is known regarding the cellular metabolic events that regulate their function during infection. Using complementary transcriptomic and immunological studies in human primary monocytes, we show that activation of monocytes by Candida albicans yeast and hyphae was accompanied by metabolic rewiring induced through C-type lectin-signaling pathways. We describe that the innate immune responses against Candida yeast are energy-demanding processes that lead to the mobilization of intracellular metabolite pools and require induction of glucose metabolism, oxidative phosphorylation and glutaminolysis, while responses to hyphae primarily rely on glycolysis. Experimental models of systemic candidiasis models validated a central role for glucose metabolism in anti-Candida immunity, as the impairment of glycolysis led to increased susceptibility in mice. Collectively, these data highlight the importance of understanding the complex network of metabolic responses triggered during infections, and unveil new potential targets for therapeutic approaches against fungal diseases.

Fig 1. Glycolysis upregulation upon Candida stimulation.

(A) Schematic pathway map of the gene expression in the main metabolic pathways in PBMCs stimulated with heat-killed C. albicans 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 C. albicans 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 S1 Fig. (B) Fold increase of mRNA expression for the indicated enzymes analyzed by RT-PCR in monocytes 24 h after stimulation with heat-killed C. albicans conidia or heat-killed C. albicans 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.

Fig 2. Candida stimulation induced glycolysis in human monocytes.

(A) Lactate production and glucose consumption by monocytes after overnight stimulation with heat-killed C. albicans yeast or heat-killed C. albicans 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 C. albicans 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 C. albicans yeast or heat-killed C. albicans 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 hgc1 or Δhgc1 C. albicans. (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 hgc1 or Δhgc1 live C. albicans stimulation (mean ± SEM, n = 6; pooled from 2 independent experiments). *p<0.05, **p<0.01 Wilcoxon signed-rank test.

Fig 3. Glycolysis, glutaminolysis and oxidative phosphorylation are involved in cytokine production after Candida stimulation.

(A) Scheme of the chemical inhibitors used, their target enzymes, and the main intracellular metabolic pathways studied. (B) IL-1β, IL-6 and TNFα production by human monocytes treated with different metabolic inhibitors and stimulated with heat-killed C. albicans yeast or heat-killed C. albicans hyphae for 24 h (mean ± SEM, n = 6; pooled from 2 independent experiments). *p<0.05, **p<0.01 Wilcoxon signed-rank test. (C-D) Basal OCR (C) and ECAR (D) of monocytes were determined 24 h after stimulation with medium or heat-killed C. albicans yeast by extracellular flux measurements in basal conditions of after injection of 2-DG (mean ± SEM, n = 6; pooled from 2 independent experiments). *p<0.05, **p<0.01 Wilcoxon signed-rank test.

Fig 4. C-type lectins triggered glycolysis after stimulation with Candida yeasts.

(A-B) Lactate production by human monocytes was measured after blockade of dectin-1, CR3, TLR2, TLR4 and MR and subsequent 24 h stimulation with medium, heat-killed C. albicans yeast or heat-killed C. albicans hyphae (A) or hgc1 or Δhgc1 live C. albicans (B) (mean ± SEM, n = 6; pooled from 2 independent experiments). *p< 0.05, **p<0.01 Wilcoxon signed-rank test. Results of stimulation with isotype controls are displayed in S3 Fig.

Fig 5. ROS production by monocytes involved glycolysis and the pentose phosphate pathway.

(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 C. albicans yeast or heat-killed C. albicans 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.

Fig 6. Inhibition of glucose metabolism impaired in vivo responses to systemic C. albicans infection.

(A) Fungal burden measured in the kidneys of C57BL/6 mice treated with PBS, 2-DG or BPTES during 5 days after i.v. C. albicans 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. C. albicans 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 C. albicans i.v. infection were measured by ELISA 48 h after in vitro restimulation with medium, LPS, heat-killed C. albicans yeast or heat-killed C. albicans hyphae (mean ± SEM, n = 6; similar results were obtained in 2 independent experiments) *p<0.05, Student’s t test.

Fig 7. Overview of the immune and metabolic processes taking place in monocytes after systemic C. albicans recognition.

C. albicans 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 C. albicans 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.