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Autocrine IL-10 functions as a rheostat for M1 macrophage glycolytic commitment by tuning nitric oxide production ☆ ☆ ☆

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ABSTRACT

Inflammatory maturation of M1 macrophages by proinflammatory stimuli such as toll like receptor ligands results in profound metabolic reprogramming resulting in commitment to aerobic glycolysis as evidenced by repression of mitochondrial oxidative phosphorylation (OXPHOS) and enhanced glucose utilization. In contrast, “alternatively activated” macrophages adopt a metabolic program dominated by fatty acid-fueled OXPHOS. Despite the known importance of these developmental stages on the qualitative aspects of an inflammatory response, relatively little is know regarding the regulation of these metabolic adjustments. Here we provide evidence that the immunosuppressive cytokine IL-10 defines a metabolic regulatory loop. Our data show for the first time that lipopolysaccharide (LPS)-induced glycolytic flux controls IL-10-production via regulation of mammalian target of rapamycin (mTOR) and that autocrine IL-10 in turn regulates macrophage nitric oxide (NO) production. Genetic and pharmacological manipulation of IL-10 and nitric oxide (NO) establish that metabolically regulated autocrine IL-10 controls glycolytic commitment by limiting NO-mediated suppression of OXPHOS. Together these data support a model where autocine IL-10 production is controlled by glycolytic flux in turn regulating glycolytic commitment by preserving OXPHOS via suppression of NO. We propose that this IL-10-driven metabolic rheostat maintains metabolic equilibrium during M1 macrophage differentiation and that perturbation of this regulatory loop, either directly by exogenous cellular sources of IL-10 or indirectly via limitations in glucose availability, skews the cellular metabolic program altering the balance between inflammatory and immunosuppressive phenotypes.

No MeSH data available.


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Prolonged stimulation of macrophages with LPS leads to glycolytic commitment. OCR, ECAR and OCR/ECAR ratio of mouse BMDM 24 h after LPS administration (100 ng/mL) (a–c). mRNA levels of glycolytic enzymes HK2, LDHA, and PFK1 were evaluated 24 h after LPS administration to BMDM in which the dotted line indicates control levels (d). Glucose uptake in LPS treated macrophages (24 h) was assessed by fluorescent 2-NBDG (100 μM) uptake via flow cytometry (e). OCR/ECAR ratio was evaluated in human MDM 24 h after exposure to 100 ng/mL LPS (f). Metabolic data (a–c, f) are from one experiment representative of at least 3 independent experiments (mean±SEM n=5). mRNA analysis (d) is a combination of 2 independent experiments (N=5). 2-NBDG analysis (e) is a combination of 3 independent experiments *p <0.05. Statistical significance was assessed by Student's t test.
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f0010: Prolonged stimulation of macrophages with LPS leads to glycolytic commitment. OCR, ECAR and OCR/ECAR ratio of mouse BMDM 24 h after LPS administration (100 ng/mL) (a–c). mRNA levels of glycolytic enzymes HK2, LDHA, and PFK1 were evaluated 24 h after LPS administration to BMDM in which the dotted line indicates control levels (d). Glucose uptake in LPS treated macrophages (24 h) was assessed by fluorescent 2-NBDG (100 μM) uptake via flow cytometry (e). OCR/ECAR ratio was evaluated in human MDM 24 h after exposure to 100 ng/mL LPS (f). Metabolic data (a–c, f) are from one experiment representative of at least 3 independent experiments (mean±SEM n=5). mRNA analysis (d) is a combination of 2 independent experiments (N=5). 2-NBDG analysis (e) is a combination of 3 independent experiments *p <0.05. Statistical significance was assessed by Student's t test.

Mentions: In contrast, prolonged stimulation (24 h) of BMDM with LPS leads to a glycolytic phenotype characterized by enhancement of ECAR in combination with significant reductions to OCR (Fig. 2A and B). Best quantitated by a decline in OCR/ECAR ratios; this state has been termed “glycolytic commitment” in inflammatory DC [11] (Fig. 2C). Notably, the reduction in OXPHOS due to LPS affects both basal OCR levels as well as spare respiratory capacity indicating a global suppression of OXPHOS. As expected, glycolytic commitment was associated with transcriptional metabolic reprogramming. BMDM stimulated with LPS for 24 h display significant increases to multiple glycolytic genes including hexokinase-2 (Hk2), lactate dehydrogenase A (Ldha) and phosphofructokinase 1 (Pfk1) (Fig. 2D). LPS-treated BMDM import ~50% more glucose than unstimulated controls as measured with the fluorescent glucose analog 2-NBDG (Fig. 2E). Notably, as shown in Fig. 2F, human MDM also undergo a strong glycolytic commitment as evidenced by a reduction in OCR/ECAR ratios after overnight culture with LPS. Taken together, these results show that endotoxin stimulated BMDM undergo an immediate increase in aerobic glycolysis followed by a sustained glycolytic commitment associated with transcriptional modification of metabolic genes.


Autocrine IL-10 functions as a rheostat for M1 macrophage glycolytic commitment by tuning nitric oxide production ☆ ☆ ☆
Prolonged stimulation of macrophages with LPS leads to glycolytic commitment. OCR, ECAR and OCR/ECAR ratio of mouse BMDM 24 h after LPS administration (100 ng/mL) (a–c). mRNA levels of glycolytic enzymes HK2, LDHA, and PFK1 were evaluated 24 h after LPS administration to BMDM in which the dotted line indicates control levels (d). Glucose uptake in LPS treated macrophages (24 h) was assessed by fluorescent 2-NBDG (100 μM) uptake via flow cytometry (e). OCR/ECAR ratio was evaluated in human MDM 24 h after exposure to 100 ng/mL LPS (f). Metabolic data (a–c, f) are from one experiment representative of at least 3 independent experiments (mean±SEM n=5). mRNA analysis (d) is a combination of 2 independent experiments (N=5). 2-NBDG analysis (e) is a combination of 3 independent experiments *p <0.05. Statistical significance was assessed by Student's t test.
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f0010: Prolonged stimulation of macrophages with LPS leads to glycolytic commitment. OCR, ECAR and OCR/ECAR ratio of mouse BMDM 24 h after LPS administration (100 ng/mL) (a–c). mRNA levels of glycolytic enzymes HK2, LDHA, and PFK1 were evaluated 24 h after LPS administration to BMDM in which the dotted line indicates control levels (d). Glucose uptake in LPS treated macrophages (24 h) was assessed by fluorescent 2-NBDG (100 μM) uptake via flow cytometry (e). OCR/ECAR ratio was evaluated in human MDM 24 h after exposure to 100 ng/mL LPS (f). Metabolic data (a–c, f) are from one experiment representative of at least 3 independent experiments (mean±SEM n=5). mRNA analysis (d) is a combination of 2 independent experiments (N=5). 2-NBDG analysis (e) is a combination of 3 independent experiments *p <0.05. Statistical significance was assessed by Student's t test.
Mentions: In contrast, prolonged stimulation (24 h) of BMDM with LPS leads to a glycolytic phenotype characterized by enhancement of ECAR in combination with significant reductions to OCR (Fig. 2A and B). Best quantitated by a decline in OCR/ECAR ratios; this state has been termed “glycolytic commitment” in inflammatory DC [11] (Fig. 2C). Notably, the reduction in OXPHOS due to LPS affects both basal OCR levels as well as spare respiratory capacity indicating a global suppression of OXPHOS. As expected, glycolytic commitment was associated with transcriptional metabolic reprogramming. BMDM stimulated with LPS for 24 h display significant increases to multiple glycolytic genes including hexokinase-2 (Hk2), lactate dehydrogenase A (Ldha) and phosphofructokinase 1 (Pfk1) (Fig. 2D). LPS-treated BMDM import ~50% more glucose than unstimulated controls as measured with the fluorescent glucose analog 2-NBDG (Fig. 2E). Notably, as shown in Fig. 2F, human MDM also undergo a strong glycolytic commitment as evidenced by a reduction in OCR/ECAR ratios after overnight culture with LPS. Taken together, these results show that endotoxin stimulated BMDM undergo an immediate increase in aerobic glycolysis followed by a sustained glycolytic commitment associated with transcriptional modification of metabolic genes.

View Article: PubMed Central - PubMed

ABSTRACT

Inflammatory maturation of M1 macrophages by proinflammatory stimuli such as toll like receptor ligands results in profound metabolic reprogramming resulting in commitment to aerobic glycolysis as evidenced by repression of mitochondrial oxidative phosphorylation (OXPHOS) and enhanced glucose utilization. In contrast, &ldquo;alternatively activated&rdquo; macrophages adopt a metabolic program dominated by fatty acid-fueled OXPHOS. Despite the known importance of these developmental stages on the qualitative aspects of an inflammatory response, relatively little is know regarding the regulation of these metabolic adjustments. Here we provide evidence that the immunosuppressive cytokine IL-10 defines a metabolic regulatory loop. Our data show for the first time that lipopolysaccharide (LPS)-induced glycolytic flux controls IL-10-production via regulation of mammalian target of rapamycin (mTOR) and that autocrine IL-10 in turn regulates macrophage nitric oxide (NO) production. Genetic and pharmacological manipulation of IL-10 and nitric oxide (NO) establish that metabolically regulated autocrine IL-10 controls glycolytic commitment by limiting NO-mediated suppression of OXPHOS. Together these data support a model where autocine IL-10 production is controlled by glycolytic flux in turn regulating glycolytic commitment by preserving OXPHOS via suppression of NO. We propose that this IL-10-driven metabolic rheostat maintains metabolic equilibrium during M1 macrophage differentiation and that perturbation of this regulatory loop, either directly by exogenous cellular sources of IL-10 or indirectly via limitations in glucose availability, skews the cellular metabolic program altering the balance between inflammatory and immunosuppressive phenotypes.

No MeSH data available.


Related in: MedlinePlus