Syk-dependent glycolytic reprogramming in dendritic cells regulates IL-1β production to β-glucan ligands in a TLR-independent manner

Abstract

Dendritic cells (DCs) activated via TLR ligation experience metabolic reprogramming, in which the cells are heavily dependent on glucose and glycolysis for the synthesis of molecular building blocks essential for maturation, cytokine production, and the ability to stimulate T cells. Although the TLR-driven metabolic reprogramming events are well documented, fungal-mediated metabolic regulation via C-type lectin receptors such as Dectin-1 and Dectin-2 is not clearly understood. Here, we show that activation of DCs with fungal-associated β-glucan ligands induces acute glycolytic reprogramming that supports the production of IL-1β and its secretion subsequent to NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. This acute glycolytic induction in response to β-glucan ligands requires spleen tyrosine kinase signaling in a TLR-independent manner, suggesting now that different classes of innate immune receptors functionally induce conserved metabolic responses to support immune cell activation. These studies provide new insight into the complexities of metabolic regulation of DCs immune effector function regarding cellular activation associated with protection against fungal microbes.

Keywords: NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3); dectin-1/2; dendritic cells (DCs); glycolysis; inflammasome; oxidative phosphorylation (OXPHOS); pattern recognition receptor (PRR); spleen tyrosine kinase (Syk); toll-like receptor (TLR).

Figure 1

Dectin‐mediated activation drives glycolytic reprogramming. (A) Bone marrow‐derived dendritic cells (BMDCs) were stimulated with LPS (10 ng/mL), zymosan (Zy; 100 µg/mL), depleted zymosan (ZD; 100 µg/mL), and extracellular acidification rate (ECAR) is measured in real time using a Seahorse Extracellular Flux Analyzer (Agilent, Seahorse Biosciences). (B) Relative ECAR of BMDCs stimulated with 5 mM 2DG followed by media or corresponding ligands (LPS, Zy, or ZD). (C) Surface expression of CD40 and CD86 on BMDCs stimulated for 24 h with LPS, Zy, ZD, Curdlan, or whole glucan particles (WGP). (D) Intracellular cytokine staining of TNF and IL‐12 of BMDCs stimulated for 6 h with LPS, Zy, ZD in 5 mM glucose in the presence or absence of 2DG was performed. Percentage of cytokine‐positive cells is indicated. Bar graphs shows mean ± sd for 3 biological replicates; asterisk indicates significance of P < 0.05; and dotted line represents the mean background percentage for unstimulated cells. Data are representative of more than 3 independent experiments

Figure 2

Dectin‐dependent glycolytic burst and maturation requires Syk signaling. (A) Real‐time Relative extracellular acidification rate (ECAR) of bone marrow‐derived dendritic cells (BMDCs) treated with media control (M) or Syk inhibitor (PRT) followed by stimulation with LPS, Zy (zymosan), or ZD (depleted zymosan). The legend indicates the treatment order at the 2 injection points, where “M:M” indicates media injection at both time points. (B) CD40 and CD86 surface expression of BMDCs stimulated for 24 h with LPS, Zy, ZD, Curdlan, or whole glucan particles (WGP) in the presence or absence of Syk inhibitor (PRT). Data are representative of more than 3 independent experiments

Figure 3

Dectin‐mediated glycolytic reprogramming is independent of TLR2/MyD88 signals. (A) Relative extracellular acidification rate (ECAR) of bone marrow‐derived dendritic cells (BMDCs) from wild‐type (WT), TLR2^−/−, and MyD88^−/− mice stimulated in real time response to ZD. (B) Relative ECAR of BMDCs from WT, TLR2^−/−, and MyD88^−/− mice stimulated in real time response to Syk inhibitor (PRT) followed by ZD. (C) CD40 and CD86 surface expression of BMDCs WT, TLR2^−/−, and MyD88^−/− stimulated for 24 h with Pam2CSK4 (TLR2 ligand), CpG (TLR9 ligand), LPS (TLR4 ligand), Zy (TLR2 and Dectin‐1 ligand), ZD, or Curdlan (Dectin‐1 ligands). Data are representative of more than 3 independent experiments

Figure 4

Dectin‐mediated glycolytic reprogramming requires a PI3k/TBK1/Akt signaling axis. (A) Protein expression of pAkt (T308), pan Akt, and β‐actin in BMDCs stimulated with Zy and ZD for 1, 5, 15, and 30 min. (B) Protein expression of pAkt (T308) and total Akt in BMDCs stimulated for 5, 15, and 30 min with ZD in the presence or absence of PI3K inhibitor (LY). (C) Protein expression of pAkt (T308) and total Akt in BMDCs stimulated with ZD in the presence or absence of TBK1 inhibitor (BX) for 5, 15, and 30 min. (D) Relative extracellular acidification rate (ECAR) of BMDCs stimulated in real time response to Zy and ZD in the presence or absence of PI3K or Akt inhibitors. Data represents n = 4; ^* P < 0.05 Student's t test

Figure 5

Dectin‐mediated acute glycolytic reprogramming is required for NLRP3‐dependent IL‐1β secretion. (A) Relative mRNA expression of IL‐1β in BMDCs stimulated for 6 h with LPS, Zy, or ZD in the presence or absence of 2DG, normalized to β‐actin expression. Data represents n = 4; ^* P < 0.05 ANOVA test. (B) ELISA determination of total (cleaved and uncleaved) secreted IL‐1β in dendritic cells stimulated for 4 h with indicated agonists and 1 h of nigericin. Data represents n = 4; ^* P < 0.05 ANOVA test. (C) Cleaved caspase‐1 and cleaved IL‐1β protein expression in cell lysates and concentrated culture supernatants from BMDCs stimulated with LPS, Zy, and ZD for 5 h with an addition of nigericin in the last hour. (D) Cleaved caspase‐1 and cleaved IL‐1β protein expression in BMDC concentrated culture supernatants stimulated with LPS, Zy, and ZD in the presence or absence of 2DG or PRT for 5 h with an addition of nigericin in the last hour. (E) Cleaved caspase‐1 and cleaved IL‐1β protein expression in BMDC concentrated culture supernatants stimulated with LPS, Zy, and ZD for 5 h with an addition of nigericin in the last hour in the presence or absence of 2DG throughout (2DG “0”) or 2DG at the time of nigericin addition (2DG “4”). Western blot data are representative of at least 3 independent experiments