Adenosine 5'-monophosphate-activated protein kinase promotes macrophage polarization to an anti-inflammatory functional phenotype

Abstract

Herein, we demonstrate a role of AMP-activated protein kinase (AMPK) as a potent counterregulator of inflammatory signaling pathways in macrophages. Stimulation of macrophages with anti-inflammatory cytokines (i.e., IL-10 and TGFbeta) resulted in the rapid phosphorylation/activation of AMPK, whereas stimulation of macrophages with a proinflammatory stimulus (LPS) resulted in AMPK dephosphorylation/inactivation. Inhibition of AMPKalpha expression by RNA interference dramatically increased the mRNA levels of LPS-induced TNF-alpha, IL-6, and cyclooxygenase-2. Likewise, expression of a dominant negative AMPKalpha1 in macrophages enhanced TNF-alpha and IL-6 protein synthesis in response to LPS stimulation, while diminishing the production of IL-10. In contrast, transfection of macrophages with a constitutively active form of AMPKalpha1 resulted in decreased LPS-induced TNF-alpha and IL-6 production, and heightened production of IL-10. In addition, we found that AMPK negatively regulated LPS-induced IkappaB-alpha degradation and positively regulated Akt activation, accompanied by inhibition of glycogen synthase kinase beta and activation of CREB. Thus, AMPK directs signaling pathways in macrophages in a manner that suppresses proinflammatory responses and promotes macrophage polarization to an anti-inflammatory functional phenotype.

Figure 1. Mouse and human macrophages express predominantly the AMPKα1 isoform

A, Lysates of mouse bone marrow derived macrophages (m-BMDM), the B6J2 macrophage cell line and human monocyte-derived macrophages (hu-MDM) were analyzed by Western blot using antibodies against the AMPKα1 and AMPKα2 proteins. Tissue lysate prepared from mouse liver was used as a positive control for expression of AMPKα2 protein. Data shown are representative of 3 independent experiments with similar results. B–D, Real-time RT-PCR analysis was performed for analysis of AMPKα1 and AMPKα2 mRNA expression. Data shown are mean ± S.D. of triplicate determinations (* p< 0.05, ** p< 0.01) and representative of 3 independent experiments with similar results.

Figure 2. AMPK activity is rapidly modulated by anti-inflammatory and proinflammatory stimuli

A and B, Anti-inflammatory cytokines IL-10 and TGFβ enhance the levels of phosphorylated AMPK in mouse macrophages. Bone marrow derived macrophages were stimulated with 20 ng/ml IL-10 or 5 ng/ml TGFβ for the time points indicated. C, LPS stimulation diminishes AMPK phosphorylation in mouse macrophages. Bone marrow derived macrophages were stimulated with 100 ng/ml LPS for the time points indicated. D, LPS stimulation decreases AMPK phosphorylation in human macrophages. Human macrophages were stimulated with 100 ng/ml LPS for the time points indicated. Western blot was performed using antibodies against phospho-AMPKα and total AMPKα. The p-AMPKα/AMPKα ratio for each was analyzed by densitometry and shown as bar graphs. Data shown are representative of 4 independent experiments with similar results.

Figure 3. Suppression of AMPK expression by RNA interference increases LPS induced pro-inflammatory activity of macrophages

A, Bone marrow derived macrophages were transfected with AMPKα1/α2 siRNA or control siRNA or left untransfected. 72 h post-transfection, the cells were lysed and Western blot was performed with antibodies against AMPKα and β-actin. Data shown are representative of 3 independent experiments with similar results. B–D, 72 h post-siRNA transfection, macrophages were stimulated with 100 ng/ml LPS for 4hrs (TNFα, IL-6) or 2 hrs (COX-2) or left unstimulated. Real-time RT-PCR was performed for analysis of TNFα, IL-6 and COX-2 mRNA expression. Data shown are mean ± S.D. of triplicate determinations (* p< 0.05, ** p< 0.01) and representative of 3 independent experiments with similar results.

Figure 4. Expression of dominant negative or constitutively active AMPKα1 modulates the inflammatory response of macrophages

The B6J2 macrophage cell line was stably transfected with DN-AMPKα1, (A–D) or CA-AMPKα1, (A,E–G). Empty vector (pcDNA-Zeo) B6J2 transfectants served as a control (A–G). Lysates of transfected macrophages were analyzed by Western blot using antibodies against p-ACC and ACC. The p-ACC/total ACC ratio was analyzed by densitometry and shown as a bar histogram. Data shown are representative of 2 independent experiments with similar results (A). Transfected macrophages were stimulated with LPS at the concentrations shown for 18 hrs or left unstimulated. TNFα, IL-6 and IL-10 levels were detected by ELISA. Data shown are mean ± S.D. of triplicate determinations (* p< 0.05, ** p< 0.01) and representative of 3 independent experiments with similar results.

Figure 5. Expression of dominant negative or constitutively active AMPKα1 modulates LPS induced IκB-α, Akt, GSK3β and CREB activity

A, B6J2 macrophages stably transfected with DN-AMPKα1 or empty vector were stimulated with 100 ng/ml LPS for the time points indicated. After cell lysis, Western blot was performed using an anti-IκB-α antibody. Bands were analyzed by densitometry, displayed as a bar histogram. B, B6J2 macrophages stably transfected with CA-AMPKα1 or empty vector were treated and analyzed as in A. C, Western blot was performed with antibodies against p-Akt (Ser473) and total Akt. The p-Akt/total Akt ratio was analyzed by densitometry and shown as a bar histogram. D, B6J2 macrophages stably transfected with CA-AMPKα1 were stimulated and assayed as in C. E, DN-AMPKα1 transfectants were stimulated as in A and Western blot was performed with antibodies against p-GSK3-β (Ser9) and total GSK3-β. The p-GSK3-β/total GSK3-β ratio was analyzed by densitometry and shown as a bar histogram. F, CA-AMPKα1 transfectants were stimulated and assayed as in E. G, DN-AMPKα1 transfectants were stimulated as in A and Western blot was performed with antibodies against p-CREB (Ser133) and total CREB. The p-CREB/total CREB ratio was analyzed by densitometry and shown as a bar histogram. H, CA-AMPKα1 transfectants were stimulated and assayed as in G. Data shown are representative of 2 (B,D,E,G) and 3 (A,C,F,H) independent experiments with similar results.