In vitro and in vivo Effects of Lactate on Metabolism and Cytokine Production of Human Primary PBMCs and Monocytes

Abstract

Lactate, the end product of anaerobic glycolysis, is produced in high amounts by innate immune cells during inflammatory activation. Although immunomodulating effects of lactate have been reported, evidence from human studies is scarce. Here we show that expression of genes involved in lactate metabolism and transport is modulated in human immune cells during infection and upon inflammatory activation with TLR ligands in vitro, indicating an important role for lactate metabolism in inflammation. Extracellular lactate induces metabolic reprogramming in innate immune cells, as evidenced by reduced glycolytic and increased oxidative rates of monocytes immediately after exposure to lactate. A short-term infusion of lactate in humans in vivo increased ex vivo glucose consumption of PBMCs, but effects on metabolic rates and cytokine production were limited. Interestingly, long-term treatment with lactate ex vivo, reflecting pathophysiological conditions in local microenvironments such as tumor or adipose tissue, significantly modulated cytokine production with predominantly anti-inflammatory effects. We found time- and stimuli-dependent effects of extracellular lactate on cytokine production, further emphasizing the complex interplay between metabolism and immune cell function. Together, our findings reveal lactate as a modulator of immune cell metabolism which translates to reduced inflammation and may ultimately function as a negative feedback signal to prevent excessive inflammatory responses.

Keywords: cytokines; glycolysis; immunometabolism; innate immune cells; lactate; monocytes.

Figure 1

Lactate dehydrogenase and lactate transporters are important for inflammatory responses of human immune cells. (A) Gene expression analysis from microarray data of PBMCs isolated from patients with acute infections. Fold change of gene expression from PBMCs isolated from patients with infections compared with healthy controls is shown. (B) Gene expression analysis from microarray data of human monocytes stimulated for 24 h with LPS or Pam3Cys. Fold change of gene expression from monocytes stimulated with LPS or Pam3Cys compared with unstimulated cells is shown. (C) Lactate levels measured in supernatants of CD14⁺ monocytes stimulated for 24 h with LPS or Pam3Cys. (D,E) Relative quantity of intracellular lactate (D) and pyruvate (E) in CD14⁺ monocytes stimulated for 24 h with LPS or Pam3Cys. (F–H) Correlation of the change in LDH expression upon LPS- or Pam3Cys-stimulation with IL-1β production. *p < 0.05, **p < 0.01. Friedman test with post-hoc Dunn's test (C–E). Spearman's rank test (F–H).

Figure 2

In vitro, lactate acutely shifts metabolism of human monocytes to oxidative phosphorylation. (A–D) Change of ECAR (A,B) and OCR (C,D) in human CD14⁺ monocytes after exposure to lactate, presented as raw data or % baseline (before lactate injection). (E) OCR/ECAR after lactate injection. (F) Spare respiratory capacity (SRC) after lactate injection. Each dot represents one healthy individual. n = 7–9, *p < 0.05, **p < 0.01, Wilcoxon signed rank test (A,C), Friedman test with post-hoc Dunn's test (B,D–F).

Figure 3

LDH-inhibition abolishes acute effects of lactate on metabolism. CD14⁺ monocytes were pretreated for 1 h with 40 mM sodium oxamate and medium as a control (A–D) or with 0.5 mM α-cyano-4-hydroxycinnamic acid (α-CHCA) and DMSO as a control (E–H) before cells were exposed to lactate and metabolism was assessed. Change of ECAR (A,E) OCR (B,F) OCR/ECAR (C,G) and SRC (D,H) in human CD14⁺ monocytes after exposure to lactate. Each dot represents one healthy individual. n = 2.

Figure 4

Lactate acutely shifts metabolism of inflammatory human monocytes to oxidative phosphorylation. CD14⁺ monocytes were stimulated with LPS for 4 h before metabolism was assessed. (A–D) Change of ECAR (A,B) and OCR (C,D) in human CD14⁺ monocytes stimulated for 4 h with LPS before exposure to lactate, presented as raw data or % baseline (before lactate injection). (E) OCR/ECAR after lactate injection. (F) Spare respiratory capacity (SRC) after lactate injection. Each dot represents one healthy individual. n = 4, Wilcoxon signed rank test.

Figure 5

A short-term lactate infusion only has minor effects on human immune cell metabolism. (A) Plasma lactate concentrations measured during lactate infusion. (B–E) Change of ECAR (B,C) and OCR (D,E) in human CD14⁺ monocytes after exposure to lactate, presented as raw data and % baseline (before lactate injection). (F) OCR/ECAR after lactate injection. (G–I) Glucose concentration assessed in the medium of cultured PBMCs from patients with diabetes. PBMCs were isolated and stimulated either before (T0) or after (T1) lactate infusion. PBMCs were stimulated with LPS or Pam3Cys for 24 h. Each dot represents one patient. (A,G–I) n = 12, (B–F) n = 3. *p < 0.05, Friedman test with post-hoc Dunn's test.

Figure 6

A short-term lactate infusion only has minor effects on cytokine production of human PBMCs. PBMCs were isolated and stimulated either before (T0) or after (T1) lactate infusion. (A,B) Change in LPS- (A) and Pam3Cys- (B) induced cytokine production after lactate infusion. (C) Correlation heatmap indicating correlations between changes in cytokine production after lactate infusion. Colors indicate Spearman correlation coefficients. FC, Fold change T1/T0. (D) Correlation heatmap indicating correlations between changes in LPS-induced IL-1β production after lactate infusion and various clinical and experimental factors. (E,F) Correlation of changes in LPS-induced IL-1β production after lactate infusion with percentage of monocytes in the PBMC fraction (E) and gene expression levels of LDHB (F). (G–N) Cytokine production of human PBMCs pretreated with lactate for 1 h before LPS (G–J) or Pam3Cys (K–N) was added for 24 h. n = 12. *p < 0.05, **p < 0.01, ***p < 0.001. Wilcoxon signed rank test (A,B) Spearman's rank test (C–F) Friedman test with post-hoc Dunn's test (G–N).

Figure 7

In vitro, lactate has anti-inflammatory effects on PBMCs. Cytokine production of PBMCs from healthy individuals (4 men, 5 women). PBMCs were pretreated with lactate for 1 h before LPS (A–D) or Pam3Cys (E–H) was added for 24 h. (A–H) n = 9. *p < 0.05, **p < 0.01, ***p < 0.001, Friedman test with post-hoc Dunn's test.

Figure 8

In vitro, lactate reduces IL-1β production of monocytes. Cytokine production of CD14⁺ monocytes from healthy individuals. Monocytes were pretreated with lactate for 1 h before LPS (A,C,E) or Pam3Cys (B,D,F) was added for 24 h. (A–F) n = 6–8. *p < 0.05, **p < 0.01, Friedman test with post-hoc Dunn's test.

Figure 9

Anti-inflammatory effects of lactate are time-dependent. Cytokine production of monocytes from healthy individuals. (A,B) Monocytes were pretreated with lactate for 1 h before LPS (A) or Pam3Cys (B) was added for 24 h. (C,D) Monocytes were pretreated with lactate for 1 h, lactate was removed and cells were washed once before LPS (C) or Pam3Cys (D) was added for 24 h. (E) Monocytes were treated as described for (A) or (C) before metabolism was measured. (F) Monocytes were stimulated with LPS. 4 h after stimulation, lactate was added. (A,B) n = 7, (C,D) n = 5, (E) n = 3, (F) n = 4. *p < 0.05, **p < 0.01, Friedman test with post-hoc Dunn's test.

Figure 10

Inhibition of lactate metabolism modulates cytokine production. (A–D) Cytokine production of PBMCs from healthy individuals. PBMCs were pretreated with 40 mM sodium oxamate and medium as a control (A,B) or 0.5 mM α-cyano-4-hydroxycinnamic acid (α-CHCA) and DMSO as a control (C,D) for 1 h, before first lactate was added for 1 h and then LPS (A,C) or Pam3Cys (B,D) was added for 24 h. (E–H) Cytokine production of CD14⁺ monocytes from healthy individuals. Monocytes were pretreated with 40 mM sodium oxamate and medium as a control (E–F) or 0.5 mM α-CHCA and DMSO as a control (G,H) for 1 h, before first lactate was added for 1 h and then LPS (E,G) or Pam3Cys (F,H) was added for 24 h. (A–D) n = 6, (E,F) n = 4, (G,H) n = 3. *p < 0.05, Wilcoxon signed rank test.

Figure 11

Lactate metabolism in human immune cells. Overview of the role of lactate in modulating metabolism and function in human immune cells. Expression of genes marked in red was upregulated and expression of genes marked in blue was downregulated in human immune cells upon stimulation with LPS.