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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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Inhibition of glycolysis in LPS treated macrophages leads to a reduction in IL-10 but not TNFα. IL-10 and TNFα mRNA (a, b) and protein content (c, d) was measured in BMDM ±pretreatment of 2-DG (2 mM, 5 mM) followed by stimulation of LPS (100 ng/mL) for 4–5 h. IL-10 and TNFα (e, f) protein content was measured in LPS (100 ng/mL) stimulated BMDM incubated in titrating amount of glucose for 5 h. Human MDM IL-10 and TNFα (g, h) protein content was assessed 5 h post LPS and/or 2-DG pre-treatment. Data are a combination of 3 (a, b) independent experiments or a representative of at least 2 (e, f) or 3 (c, d, g, h) independent experiments *p<0.05. Statistical significance was assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.
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f0015: Inhibition of glycolysis in LPS treated macrophages leads to a reduction in IL-10 but not TNFα. IL-10 and TNFα mRNA (a, b) and protein content (c, d) was measured in BMDM ±pretreatment of 2-DG (2 mM, 5 mM) followed by stimulation of LPS (100 ng/mL) for 4–5 h. IL-10 and TNFα (e, f) protein content was measured in LPS (100 ng/mL) stimulated BMDM incubated in titrating amount of glucose for 5 h. Human MDM IL-10 and TNFα (g, h) protein content was assessed 5 h post LPS and/or 2-DG pre-treatment. Data are a combination of 3 (a, b) independent experiments or a representative of at least 2 (e, f) or 3 (c, d, g, h) independent experiments *p<0.05. Statistical significance was assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.

Mentions: Given that macrophages preferentially utilize glycolysis upon endotoxin stimulation, we sought to determine the ramifications of interference of LPS-induced glycolysis via blockade of glucose utilization with 2-DG might have on inflammatory outcome. We assayed levels of the LPS-inducible cytokines TNFα and IL-10. Interestingly, quantitative RT-PCR analyses revealed a substantial reduction in Il10 mRNA and a modest increase in Tnfa when BMDM were pretreated with 2-DG 30 min prior to a 4 h stimulation with LPS (Fig. 3A and B). 2-DG suppression of IL-10 production was confirmed by assaying secreted IL-10, with no significant change in secreted TNFα (Fig. 3C and D). To ensure these results were not off-target effects of 2-DG, BMDM were stimulated with LPS in decreasing amounts of glucose. BMDM stimulated in media with less than 0.5 mM glucose produced less IL-10 than those cultured in higher levels with no significant change in TNFα (Fig. 3E and F). Additionally, human MDM cultured under identical conditions mirrored the preferential reduction of IL-10 with 2-DG pre-stimulation (Fig. 3G and H). Together these data show that LPS-induced IL-10 production is sensitive to the availability of glucose in macrophages.


Autocrine IL-10 functions as a rheostat for M1 macrophage glycolytic commitment by tuning nitric oxide production ☆ ☆ ☆
Inhibition of glycolysis in LPS treated macrophages leads to a reduction in IL-10 but not TNFα. IL-10 and TNFα mRNA (a, b) and protein content (c, d) was measured in BMDM ±pretreatment of 2-DG (2 mM, 5 mM) followed by stimulation of LPS (100 ng/mL) for 4–5 h. IL-10 and TNFα (e, f) protein content was measured in LPS (100 ng/mL) stimulated BMDM incubated in titrating amount of glucose for 5 h. Human MDM IL-10 and TNFα (g, h) protein content was assessed 5 h post LPS and/or 2-DG pre-treatment. Data are a combination of 3 (a, b) independent experiments or a representative of at least 2 (e, f) or 3 (c, d, g, h) independent experiments *p<0.05. Statistical significance was assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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f0015: Inhibition of glycolysis in LPS treated macrophages leads to a reduction in IL-10 but not TNFα. IL-10 and TNFα mRNA (a, b) and protein content (c, d) was measured in BMDM ±pretreatment of 2-DG (2 mM, 5 mM) followed by stimulation of LPS (100 ng/mL) for 4–5 h. IL-10 and TNFα (e, f) protein content was measured in LPS (100 ng/mL) stimulated BMDM incubated in titrating amount of glucose for 5 h. Human MDM IL-10 and TNFα (g, h) protein content was assessed 5 h post LPS and/or 2-DG pre-treatment. Data are a combination of 3 (a, b) independent experiments or a representative of at least 2 (e, f) or 3 (c, d, g, h) independent experiments *p<0.05. Statistical significance was assessed by ANOVA with a Bonferroni post-test. Error bars represent ±SEM.
Mentions: Given that macrophages preferentially utilize glycolysis upon endotoxin stimulation, we sought to determine the ramifications of interference of LPS-induced glycolysis via blockade of glucose utilization with 2-DG might have on inflammatory outcome. We assayed levels of the LPS-inducible cytokines TNFα and IL-10. Interestingly, quantitative RT-PCR analyses revealed a substantial reduction in Il10 mRNA and a modest increase in Tnfa when BMDM were pretreated with 2-DG 30 min prior to a 4 h stimulation with LPS (Fig. 3A and B). 2-DG suppression of IL-10 production was confirmed by assaying secreted IL-10, with no significant change in secreted TNFα (Fig. 3C and D). To ensure these results were not off-target effects of 2-DG, BMDM were stimulated with LPS in decreasing amounts of glucose. BMDM stimulated in media with less than 0.5 mM glucose produced less IL-10 than those cultured in higher levels with no significant change in TNFα (Fig. 3E and F). Additionally, human MDM cultured under identical conditions mirrored the preferential reduction of IL-10 with 2-DG pre-stimulation (Fig. 3G and H). Together these data show that LPS-induced IL-10 production is sensitive to the availability of glucose in macrophages.

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