Adenosine 5'-monophosphate-activated protein kinase regulates IL-10-mediated anti-inflammatory signaling pathways in macrophages

Abstract

AMP-activated protein kinase (AMPK) is a conserved serine/threonine kinase with a critical function in the regulation of metabolic pathways in eukaryotic cells. Recently, AMPK has been shown to play an additional role as a regulator of inflammatory activity in leukocytes. Treatment of macrophages with chemical AMPK activators, or forced expression of a constitutively active form of AMPK, results in polarization to an anti-inflammatory phenotype. In addition, we reported previously that stimulation of macrophages with anti-inflammatory cytokines such as IL-10, IL-4, and TGF-β results in rapid activation of AMPK, suggesting that AMPK contributes to the suppressive function of these cytokines. In this study, we investigated the role of AMPK in IL-10-induced gene expression and anti-inflammatory function. IL-10-stimulated wild-type macrophages displayed rapid activation of PI3K and its downstream targets Akt and mammalian target of rapamycin complex (mTORC1), an effect that was not seen in macrophages generated from AMPKα1-deficient mice. AMPK activation was not impacted by treatment with either the PI3K inhibitor LY294002 or the JAK inhibitor CP-690550, suggesting that IL-10-mediated activation of AMPK is independent of PI3K and JAK activity. IL-10 induced phosphorylation of both Tyr(705) and Ser(727) residues of STAT3 in an AMPKα1-dependent manner, and these phosphorylation events were blocked by inhibition of Ca(2+)/calmodulin-dependent protein kinase kinase β, an upstream activator of AMPK, and by the mTORC1 inhibitor rapamycin, respectively. The impaired STAT3 phosphorylation in response to IL-10 observed in AMPKα1-deficient macrophages was accompanied by reduced suppressor of cytokine signaling 3 expression and an inadequacy of IL-10 to suppress LPS-induced proinflammatory cytokine production. Overall, our data demonstrate that AMPKα1 is required for IL-10 activation of the PI3K/Akt/mTORC1 and STAT3-mediated anti-inflammatory pathways regulating macrophage functional polarization.

Conflict of interest statement

Conflict-of-Interest Disclosure:

The authors declare no competing financial interests

Copyright © 2015 by The American Association of Immunologists, Inc.

Figures

Figure 1

AMPK mediates IL-10-induced gene expression in macrophages. BMDM generated from AMPKα1+/+ and AMPKα1−/− mice were treated with recombinant mouse (rm)-IL-10 (20 ng/ml) for the duration of 30 min, 1 h, 3 h, 6 h, and 18 h. Total cellular lysates were collected for real-time PCR analysis. The time point representing the peak level of expression for each gene is shown (representative result of two or more independent experiments). The mRNA expression of each gene was normalized to β-actin and compared to the AMPKα1+/+ untreated group. Data shown are the mean ± SD of triplicate determinations. Statistical significance between groups was calculated with an unpaired Student’s t test, with a value of p < 0.050 considered statistically significant. (**, p < 0.001. *, p < 0.050. n.s., p > 0.050).

Figure 2

IL-10 activation of the PI3K/Akt pathway requires AMPK. (A and B), IL-10 activates PI3K and Akt in AMPKα1+/+ BMDM. BMDM generated from AMPKα1+/+ and AMPKα1−/− mice were treated with rm-IL-10 (20 ng/ml) for the time points indicated. Total cellular lysates were collected for Western blot assessment of (A) AMPK-Thr172, ACC-Ser79, PI3K-Tyr199, and (B) Akt-Thr308 and Akt-Ser473 phosphorylation. (C), PI3K inhibition does not block IL-10 activation of AMPK. BMDM generated from C57BL/6 mice were incubated with either media alone, or with LY294002 (20 µM) for 1 h, then exposed to rm-IL-10 (20 ng/ml) for the time points indicated. Total cellular lysates were collected for Western blot assessment of Akt-Thr308 and Akt-Ser473 and AMPK-Thr172 phosphorylation. Protein phosphorylation levels were analyzed by densitometry and are displayed as a bar histogram. Results shown are representative of three independent experiments.

Figure 3

IL-10-induced AMPK activity promotes mTORC1 activation. BMDM generated from AMPKα1+/+ and AMPKα1−/− mice were treated with rm-IL-10 (20 ng/ml) for the time points indicated. Total cellular lysates were collected for Western blot assessment of (A) mTOR-Ser2448, (B) p70 S6K-Ser371, and (C) TSC2-Ser939 and TSC2-Ser1387 phosphorylation. Protein phosphorylation levels were analyzed by densitometry and are displayed as a bar histogram. Results shown are representative of two to four independent experiments.

Figure 4

AMPK is required for IL-10-induced activation of the JAK/STAT3/SOCS3 pathway. BMDM generated from AMPKα1+/+ and AMPKα1−/− mice were treated with rm-IL-10 (20 ng/ml) for the time points indicated. Total cellular lysates were collected for Western blot assessment of (A) STAT3-Tyr705 and STAT3-Ser727 phosphorylation, and (B) JAK1-Tyr1022/1023 phosphorylation. (C), BMDM generated from C57BL/6 mice were incubated with either media alone, or with JAK inhibitor CP-690550 (10 µM) for 1 h, then exposed to rm-IL-10 (20 ng/ml) for the time points indicated. Cell lysates were analyzed by Western blot for JAK1-Tyr1022/1023 and AMPK-Thr172 phosphorylation. (D), Cells were generated and treated as in (A). SOCS3 and β-actin expression were assessed by Western blot. Protein phosphorylation and expression levels were analyzed by densitometry and are displayed as a bar histogram. The results shown are representative of two to four independent experiments.

Figure 5

IL-10 activation of STAT3 requires AMPK, JAK and mTORC1 activity. (A), BMDM generated from C57BL/6 mice were incubated with either media alone, or with the CaMKKβ inhibitor STO-609 (5 µM) for 1 h and then exposed to rm-IL-10 (20 ng/ml) for the time points indicated. Total cellular lysates were collected for Western blot assessment of AMPK-Thr172, STAT3-Tyr705 and STAT3-Ser727 phosphorylation. (B), BMDM generated from C57BL/6 mice were incubated with either media alone, or with JAK inhibitor CP-690550 (10 µM) for 1 h, then exposed to rm-IL-10 (20 ng/ml) for the time points indicated. Cell lysates were analyzed by Western blot for STAT3-Ser727 and STAT3-Tyr705 phosphorylation. (C), BMDM generated from C57BL/6 mice were incubated with either media alone, or with the mTORC1 inhibitor rapamycin (100 ng/ml) for 1 h and then exposed to rm-IL-10 (20 ng/ml) for the time points indicated. Total cellular lysates were collected for Western blot assessment of p70 S6K-Ser371 and AMPK-Thr172 phosphorylation. (D), BMDM generated from C57BL/6 mice were incubated with either media alone, with LY294002 (20 µM) for 1 h (top panels), or with the mTORC1 inhibitor rapamycin (100 ng/ml) for 1 h (bottom panels), then exposed to rm-IL-10 (20 ng/ml) for the time points indicated. Total cellular lysates were collected for Western blot assessment of STAT3-Ser727 and STAT3-Tyr705 phosphorylation. Protein phosphorylation levels were analyzed by densitometry and are displayed as a bar histogram. Results shown are representative of two to three independent experiments.

Figure 6

AMPK contributes to IL-10-mediated suppression of LPS-induced NF-κB activation and proinflammatory cytokine production. (A), BMDM generated from AMPKα1+/+ and AMPKα1−/− mice were treated with rm-IL-10 (20 ng/ml) for 6h, then exposed to LPS (10 ng/ml) for 3 h. Total cellular lysates were collected for Western blot assessment of IκB degradation and NF-κB p65 phosphorylation. Protein expression or phosphorylation levels were analyzed by densitometry and are displayed as a bar histogram. (B), BMDM generated from AMPKα1+/+ and AMPKα1−/− mice were treated with rm-IL-10 (20 ng/ml) for 6h, then exposed to LPS (10 ng/ml) for 3 h. Total cellular lysates were collected for real-time PCR analysis of TNFα, IL-6 and IL-12p40 mRNA expression (top panels). The mRNA expression of each gene was normalized to β-actin and compared to the AMPKα1+/+ untreated group. Supernatants were collected for analysis by ELISA (bottom panels). The production level of each cytokine was compared to the AMPKα1+/+ untreated group. RT-PCR data shown are mean ± SD of triplicate determinations. ELISA data shown are mean ± SEM of triplicate determinations. Statistical significance between groups was calculated with an unpaired Student’s t test, with a value of p < 0.050 considered statistically significant. (**, p < 0.001. *, p < 0.050. n.s., p > 0.050). The data shown are representative of two or three independent experiments.

Figure 7

AMPK activation promotes the anti-inflammatory properties of IL-10 through bifurcated activation of the Akt/mTORC1 and JAK/STAT signaling pathways. IL-10 signaling promotes the rapid phosphorylation of JAK1 in an AMPK-dependent manner. AMPK’s influence on JAK1 phosphorylation is indirect, as indicated by the dotted line. Activation of JAK1 subsequently leads to the phosphorylation and activation of STAT3 (Tyr 705), which positively regulates STAT3 (Ser727) phosphorylation and is critical for SOCS3 production. In addition to its role in JAK/STAT signaling, AMPK also simultaneously promotes the activation of PI3K by enhancing the phosphorylation of the p55 subunit (indirectly, as indicated by the dotted line). Phosphorylation/ activation of Akt follows leading to an increase in mTORC1 activity, reflected by an increase in S6K phosphorylation (Ser371). Activation of mTORC1 leads to an increase in phosphorylation of STAT3 (Ser727), which further enhances STAT3 transcriptional activity leading to SOCS3 gene expression. STAT3-regulated genes, possibly including SOCS3, in turn, suppresses TLR-activated inflammatory cytokine production.