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

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, “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.


Related in: MedlinePlus

IL-10 control of macrophage metabolism is via regulation of NO. Metabolic analyses were performed on WT and Il10-/- BMDM 24 h after LPS (100 ng/mL) stimulation including ECAR fold change of LPS treated BMDM compared to non-LPS treated BMDM (a), percent mitochondrial OCR (b), and OCR/ECAR ratio (c). BMDM were treated with for 24 h ±IFNγ (50 ng/mL), ±LPS (100 ng/mL), ±Pam3Csk4 (100 ng/mL) in which nitrite concentrations (d) and OCR (e) were measured. Percent mitochondrial OCR was measured after the addition of NO donor DETA/NO (1 mM) (f). WT and Nos2-/- BMDM mitochondrial OCR were measured after 24 h treatment of IFNγ±LPS (g). Nitrite concentrations were evaluated in WT and IL-10−/− BMDM 24 h post treatment ±IL-10 (50 ng/mL), ±LPS (h). Metabolic data (a–c, e–g) are representative of 2 independent experiments (mean±SEM, n=5) *p<0.05. Figs. d and h are representative figures from 3 independent experiments *p<0.05. Statistical significance for a–d, f–h were assessed by Student's t test. Statistical significance for e and g were assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.
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f0025: IL-10 control of macrophage metabolism is via regulation of NO. Metabolic analyses were performed on WT and Il10-/- BMDM 24 h after LPS (100 ng/mL) stimulation including ECAR fold change of LPS treated BMDM compared to non-LPS treated BMDM (a), percent mitochondrial OCR (b), and OCR/ECAR ratio (c). BMDM were treated with for 24 h ±IFNγ (50 ng/mL), ±LPS (100 ng/mL), ±Pam3Csk4 (100 ng/mL) in which nitrite concentrations (d) and OCR (e) were measured. Percent mitochondrial OCR was measured after the addition of NO donor DETA/NO (1 mM) (f). WT and Nos2-/- BMDM mitochondrial OCR were measured after 24 h treatment of IFNγ±LPS (g). Nitrite concentrations were evaluated in WT and IL-10−/− BMDM 24 h post treatment ±IL-10 (50 ng/mL), ±LPS (h). Metabolic data (a–c, e–g) are representative of 2 independent experiments (mean±SEM, n=5) *p<0.05. Figs. d and h are representative figures from 3 independent experiments *p<0.05. Statistical significance for a–d, f–h were assessed by Student's t test. Statistical significance for e and g were assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.

Mentions: Previous literature has suggested that exogenous IL-10 is capable of repressing LPS-driven glycolysis and inflammatory function in DC [12]. Thus, we hypothesized that because LPS-stimulated macrophages are a substantial source of IL-10, and that IL-10 is regulated by the rate of glycolysis, this cytokine may act in an autocrine fashion as a rheostat tempering glycolytic commitment. If endogenous LPS-induced IL-10 production is a regulator of metabolic programing, BMDM lacking IL-10 should exhibit a greater degree of metabolic commitment. To test this possibility, we generated BMDM from mice lacking Il10. ECAR rates of Il10−/− BMDM stimulated with LPS overnight were significantly higher when compared to LPS stimulated wild type (WT) BMDM confirming that autocrine IL-10 production participates in glycolytic regulation (Fig. 5A). Furthermore, OCRs of Il10−/− BMDM stimulated overnight with LPS were significantly repressed when compared to LPS stimulated WT BMDM suggesting that autocrine IL-10 might regulate both arms of glycolytic commitment (Fig. 5B). Accordingly, the OCR/ECAR ratio of Il10−/− BMDM stimulated overnight with LPS was significantly reduced compared to LPS stimulated WT BMDM indicating an enhanced glycolytic commitment in Il10−/− BMDM (Fig. 5C).


Autocrine IL-10 functions as a rheostat for M1 macrophage glycolytic commitment by tuning nitric oxide production ☆ ☆ ☆
IL-10 control of macrophage metabolism is via regulation of NO. Metabolic analyses were performed on WT and Il10-/- BMDM 24 h after LPS (100 ng/mL) stimulation including ECAR fold change of LPS treated BMDM compared to non-LPS treated BMDM (a), percent mitochondrial OCR (b), and OCR/ECAR ratio (c). BMDM were treated with for 24 h ±IFNγ (50 ng/mL), ±LPS (100 ng/mL), ±Pam3Csk4 (100 ng/mL) in which nitrite concentrations (d) and OCR (e) were measured. Percent mitochondrial OCR was measured after the addition of NO donor DETA/NO (1 mM) (f). WT and Nos2-/- BMDM mitochondrial OCR were measured after 24 h treatment of IFNγ±LPS (g). Nitrite concentrations were evaluated in WT and IL-10−/− BMDM 24 h post treatment ±IL-10 (50 ng/mL), ±LPS (h). Metabolic data (a–c, e–g) are representative of 2 independent experiments (mean±SEM, n=5) *p<0.05. Figs. d and h are representative figures from 3 independent experiments *p<0.05. Statistical significance for a–d, f–h were assessed by Student's t test. Statistical significance for e and g were assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.
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f0025: IL-10 control of macrophage metabolism is via regulation of NO. Metabolic analyses were performed on WT and Il10-/- BMDM 24 h after LPS (100 ng/mL) stimulation including ECAR fold change of LPS treated BMDM compared to non-LPS treated BMDM (a), percent mitochondrial OCR (b), and OCR/ECAR ratio (c). BMDM were treated with for 24 h ±IFNγ (50 ng/mL), ±LPS (100 ng/mL), ±Pam3Csk4 (100 ng/mL) in which nitrite concentrations (d) and OCR (e) were measured. Percent mitochondrial OCR was measured after the addition of NO donor DETA/NO (1 mM) (f). WT and Nos2-/- BMDM mitochondrial OCR were measured after 24 h treatment of IFNγ±LPS (g). Nitrite concentrations were evaluated in WT and IL-10−/− BMDM 24 h post treatment ±IL-10 (50 ng/mL), ±LPS (h). Metabolic data (a–c, e–g) are representative of 2 independent experiments (mean±SEM, n=5) *p<0.05. Figs. d and h are representative figures from 3 independent experiments *p<0.05. Statistical significance for a–d, f–h were assessed by Student's t test. Statistical significance for e and g were assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.
Mentions: Previous literature has suggested that exogenous IL-10 is capable of repressing LPS-driven glycolysis and inflammatory function in DC [12]. Thus, we hypothesized that because LPS-stimulated macrophages are a substantial source of IL-10, and that IL-10 is regulated by the rate of glycolysis, this cytokine may act in an autocrine fashion as a rheostat tempering glycolytic commitment. If endogenous LPS-induced IL-10 production is a regulator of metabolic programing, BMDM lacking IL-10 should exhibit a greater degree of metabolic commitment. To test this possibility, we generated BMDM from mice lacking Il10. ECAR rates of Il10−/− BMDM stimulated with LPS overnight were significantly higher when compared to LPS stimulated wild type (WT) BMDM confirming that autocrine IL-10 production participates in glycolytic regulation (Fig. 5A). Furthermore, OCRs of Il10−/− BMDM stimulated overnight with LPS were significantly repressed when compared to LPS stimulated WT BMDM suggesting that autocrine IL-10 might regulate both arms of glycolytic commitment (Fig. 5B). Accordingly, the OCR/ECAR ratio of Il10−/− BMDM stimulated overnight with LPS was significantly reduced compared to LPS stimulated WT BMDM indicating an enhanced glycolytic commitment in Il10−/− BMDM (Fig. 5C).

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