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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.


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LPS stimulated IL-10 production in macrophages is controlled via ATP abundance, which acts through AMPK activation and mTOR inhibition. ATP content was measured in BMDM±pretreatment of 2-DG (5 mM) for 30 min followed by stimulation of LPS (100 ng/mL) for 4 h (a). BMDM were pretreated with 2-DG ±LPS and immunoblotted for AMPK, pAMPK, pMTOR, and β-actin (b). IL-10 and TNFα mRNA (c, d) and protein content (e, f) was measured in BMDM ±pretreatment of Rapamycin (100 nM) followed by stimulation of LPS (100 ng/mL) for 4–5 h. Data are a combination of at least 3 (a, c-d) independent experiments or a representative figure of at least 3 independent experiments (b, e-f) *p<0.05. Statistical significance was assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.
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f0020: LPS stimulated IL-10 production in macrophages is controlled via ATP abundance, which acts through AMPK activation and mTOR inhibition. ATP content was measured in BMDM±pretreatment of 2-DG (5 mM) for 30 min followed by stimulation of LPS (100 ng/mL) for 4 h (a). BMDM were pretreated with 2-DG ±LPS and immunoblotted for AMPK, pAMPK, pMTOR, and β-actin (b). IL-10 and TNFα mRNA (c, d) and protein content (e, f) was measured in BMDM ±pretreatment of Rapamycin (100 nM) followed by stimulation of LPS (100 ng/mL) for 4–5 h. Data are a combination of at least 3 (a, c-d) independent experiments or a representative figure of at least 3 independent experiments (b, e-f) *p<0.05. Statistical significance was assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.

Mentions: Mammalian target of rapamycin (mTOR) is a mediator of IL-10 production in macrophages, and is in turn regulated by AMP kinase (AMPK). AMPK is a metabolic sensor that detects elevations in ATP/AMP ratios within the cell and initiates programs to increase ATP production [16]. Therefore, we hypothesized that glucose deprivation of glycolytically committed macrophages might result in reductions in cellular ATP levels, activation of AMPK, repression of mTOR and reduced IL-10 production. To test this possibility, we stimulated macrophages with LPS in the presence or absence of 2-DG and assayed cellular ATP (Fig. 4A). In addition, we stimulated cells with LPS and 2-DG before western blotting to detect the levels of phosphorylated AMPK (pAMPK) and phosphorylated mTOR (pMTOR) (Fig. 4B). BMDM stimulated with LPS had lower levels of ATP but these levels were not sufficient to elicit the activation of AMPK. Rather, in LPS stimulated macrophages levels of pAMPK fell and mTORC activation proceeded unabated (Fig. 4B). In contrast, incubation of macrophages with 2-DG or 2-DG followed by LPS administration reduced ATP levels to lower than LPS treatment alone (Fig. 4A). Accordingly, BMDM treated with 2-DG or 2-DG and LPS now showed enhanced pAMPK and concomitant repression of mTOR activation consistent with reductions in IL-10 production (Fig. 4B). Consistent with this mechanism we found that similar to 2-DG, rapamycin potently suppressed LPS-induced IL-10 at mRNA and protein levels without affecting TNFα, confirming mTOR signaling as a key component in LPS-induced IL-10 production (Fig. 4C-F). Taken together, these results suggest a model where LPS stimulation leads to transient reductions in ATP that are not sufficient to activate AMPK allowing mTOR activation to proceed and IL-10 production to occur. However, in the presence of 2-DG, glycolysis is suppressed and ATP levels fall sufficiency low to activate AMPK, suppressing mTOR and blocking IL-10 production.


Autocrine IL-10 functions as a rheostat for M1 macrophage glycolytic commitment by tuning nitric oxide production ☆ ☆ ☆
LPS stimulated IL-10 production in macrophages is controlled via ATP abundance, which acts through AMPK activation and mTOR inhibition. ATP content was measured in BMDM±pretreatment of 2-DG (5 mM) for 30 min followed by stimulation of LPS (100 ng/mL) for 4 h (a). BMDM were pretreated with 2-DG ±LPS and immunoblotted for AMPK, pAMPK, pMTOR, and β-actin (b). IL-10 and TNFα mRNA (c, d) and protein content (e, f) was measured in BMDM ±pretreatment of Rapamycin (100 nM) followed by stimulation of LPS (100 ng/mL) for 4–5 h. Data are a combination of at least 3 (a, c-d) independent experiments or a representative figure of at least 3 independent experiments (b, e-f) *p<0.05. Statistical significance was assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.
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Related In: Results  -  Collection

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f0020: LPS stimulated IL-10 production in macrophages is controlled via ATP abundance, which acts through AMPK activation and mTOR inhibition. ATP content was measured in BMDM±pretreatment of 2-DG (5 mM) for 30 min followed by stimulation of LPS (100 ng/mL) for 4 h (a). BMDM were pretreated with 2-DG ±LPS and immunoblotted for AMPK, pAMPK, pMTOR, and β-actin (b). IL-10 and TNFα mRNA (c, d) and protein content (e, f) was measured in BMDM ±pretreatment of Rapamycin (100 nM) followed by stimulation of LPS (100 ng/mL) for 4–5 h. Data are a combination of at least 3 (a, c-d) independent experiments or a representative figure of at least 3 independent experiments (b, e-f) *p<0.05. Statistical significance was assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.
Mentions: Mammalian target of rapamycin (mTOR) is a mediator of IL-10 production in macrophages, and is in turn regulated by AMP kinase (AMPK). AMPK is a metabolic sensor that detects elevations in ATP/AMP ratios within the cell and initiates programs to increase ATP production [16]. Therefore, we hypothesized that glucose deprivation of glycolytically committed macrophages might result in reductions in cellular ATP levels, activation of AMPK, repression of mTOR and reduced IL-10 production. To test this possibility, we stimulated macrophages with LPS in the presence or absence of 2-DG and assayed cellular ATP (Fig. 4A). In addition, we stimulated cells with LPS and 2-DG before western blotting to detect the levels of phosphorylated AMPK (pAMPK) and phosphorylated mTOR (pMTOR) (Fig. 4B). BMDM stimulated with LPS had lower levels of ATP but these levels were not sufficient to elicit the activation of AMPK. Rather, in LPS stimulated macrophages levels of pAMPK fell and mTORC activation proceeded unabated (Fig. 4B). In contrast, incubation of macrophages with 2-DG or 2-DG followed by LPS administration reduced ATP levels to lower than LPS treatment alone (Fig. 4A). Accordingly, BMDM treated with 2-DG or 2-DG and LPS now showed enhanced pAMPK and concomitant repression of mTOR activation consistent with reductions in IL-10 production (Fig. 4B). Consistent with this mechanism we found that similar to 2-DG, rapamycin potently suppressed LPS-induced IL-10 at mRNA and protein levels without affecting TNFα, confirming mTOR signaling as a key component in LPS-induced IL-10 production (Fig. 4C-F). Taken together, these results suggest a model where LPS stimulation leads to transient reductions in ATP that are not sufficient to activate AMPK allowing mTOR activation to proceed and IL-10 production to occur. However, in the presence of 2-DG, glycolysis is suppressed and ATP levels fall sufficiency low to activate AMPK, suppressing mTOR and blocking IL-10 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, &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