AMP-activated protein kinase restricts IFN-γ signaling

Abstract

Inflammation in the CNS contributes to neurologic disorders. Neuroinflammation involves the release of inflammatory molecules from glial cells, such as astrocytes and microglia, and can lead to neuronal damage if unabated. In multiple sclerosis, peripheral immune cells, including IFN-γ-producing Th1 cells, infiltrate the CNS and are important in shaping the inflammatory microenvironment, in part through cytokine-mediated interactions with glial cells. Recent evidence suggests that AMP-activated protein kinase (AMPK), a central regulator of energetic metabolism, can regulate inflammatory gene expression. In this study, we identified that IFN-γ induces biphasic AMPK signaling, suggestive of negative-feedback mechanisms. Activation of AMPK suppresses several IFN-γ-induced cytokines and chemokines in primary astrocytes and microglia. IFN-γ regulates gene expression through activation of STAT1, and deletion of AMPK results in a marked increase in basal expression of STAT1. Conversely, activation of AMPK blocks IFN-γ-induced STAT1 expression. Deletion of AMPK leads to increased basal and IFN-γ-induced expression of inflammatory molecules, including TNF-α, CXCL10, and CCL2. AMPK does not affect the phosphorylation of STAT1, but instead attenuates nuclear translocation of STAT1, DNA binding, and subsequent gene expression. In vivo, AMPK signaling during experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, is downregulated in the brain at onset and peak of disease. Diminution of AMPK signaling in vivo correlates with increased expression of IFN-γ and CCL2 in the CNS. Overall, these findings provide the first link between AMPK and STAT1 and may provide important clues about how bioenergetics and inflammation are linked.

Figure 1. AMPK Activation is Altered by IFN-γ and Energy Restriction Dampens IFN-γinduced Gene Expression

A. Primary astrocytes were stimulated with IFN-γ (10 ng/ml) under high energy (25 mM glucose/1 mM pyruvate) and low energy conditions (24 h no glucose^#/no pyruvate) followed by immunoblotting for P-AMPK, P-ACC, AMPKα, P-STAT1 and STAT1. B. Astrocytes were stimulated with IFN-γ (10 ng/ml) for 30 min followed by isolation of cytosolic and nuclear fractions and immunoblotting for P-AMPK, AMPK and GAPDH. C. Astroyctes were cultured as in A, were left untreated (UT) or stimulated with IFN-γ (10 ng/ml, 4 h), and gene expression was analyzed by qRT-PCR. ^#Media contains an additional ~0.6 mM glucose from serum. N=3, *p < 0.05.

Figure 2. AMPK Activation Attenuates IFN-γ-induced Inflammatory Gene Expression

A and B. Astrocytes were stimulated with IFN-γ (10 ng/ml) in the absence or presence of the AMPK agonist AICAR (1 mM) for the indicated times, and STAT1 expression examined by immunoblotting and qRT-PCR. C. Astrocytes were stimulated with IFN-γ (10 ng/ml) for 4 h in the absence or presence of AICAR and gene expression analyzed by qRT-PCR. D. Astroyctes were transfected with an IFN-γ inducible reporter (GAS-Luciferase) followed by stimulation with IFN-γ (10 ng/ml, 16 h) with or without 1 mM AICAR. E. Astrocytes were transfected with control or STAT1 siRNA, then stimulated with IFN-γ (10 ng/ml, 4 h), and STAT1 and CCL2 levels measured by qRT-PCR. F. Astrocytes were stimulated with IFN-γ (10 ng/ml, 30 min) in the absence or presence of AICAR (1 mM), followed by chromatin immunoprecipitation with antibodies to STAT1 or control IgG. The precipitated DNA was then analyzed by PCR using primers directed to the STAT1 binding region of murine CCL2. G. and H. Astrocytes were stimulated with OSM (1 ng/ml) or GM-CSF (10 ng/ml) for 4 h in the absence or presence of AICAR (1 mM) or P6 (0.5 μM) and gene expression analyzed by qRT-PCR. N= 3, *p < 0.05.

Figure 3. Deletion of AMPKα1 and AMPKα2 in Primary Astrocytes Enhances STAT1 Expression

A. RNA was isolated from brain (cerebrum), spinal cord and primary cultures of astrocytes or microglia. The expression of both catalytic AMPKα isoforms and the astrocyte marker GFAP was analyzed by RT-PCR. B. Isolated AMPKα^fl/fl astrocytes were transduced with GFP or GFP-Cre adenovirus and stimulated with AICAR (1 mM, 1 h) 5 days post transduction followed by immunoblotting. C. Isolated AMPKα^fl/fl astrocytes were transduced with GFP or GFP-Cre adenovirus, and STAT1 expression quantified by immunoblotting 5 days post transduction. D. Isolated AMPKα^fl/fl astrocytes were transduced with GFP or GFP-Cre adenovirus, stimulated with IFN-γ (10 ng/ml, 4 h) 5 days post transduction, and inflammatory gene expression examined by qRT-PCR. E. The effect of AICAR on STAT1 expression is attenuated in cells lacking AMPKα. Isolated AMPKα^fl/fl astrocytes were transduced with GFP or GFP-Cre adenovirus followed by treatment with IFN-γ (10 ng/ml, 16 h) in the absence or presence of AICAR (0.5 mM) and STAT1 expression measured by immunoblotting. N=3, *p < 0.05.

Figure 4. AMPK Regulates STAT1 in Microglia

A. Primary microglia were stimulated with IFN-γ (10 ng/ml, 16 h) in the absence or presence of AICAR (1 mM), and STAT1 expression examined by immunoblotting. B. Primary microglia were stimulated with IFN-γ (10 ng/ml, 4 h) in the absence or presence of AICAR (1 mM), and CXCL10, TNF-α and iNOS gene expression measured by qRT-PCR, and MHC class II expression (IFN-γ 10 ng/ml, 24 h) measured by flow cytometry. N=3, *p < 0.05.

Figure 5. AMPK Signaling Does Not Influence STAT1 Phosphorylation but Attenuates Nuclear Translocation

A. Astrocytes were stimulated with IFN-γ (10 ng/ml, 30 min) in the absence or presence of AICAR (1 mM) followed by quantification of immunoblots for P-STAT1 and STAT1. Values are expressed as the ratio of P-STAT1 to total STAT1. B. Astrocytes were stimulated with IFN-γ (10 ng/ml, 30 min), OSM (1 ng/ml, 30 min) or GM-CSF (10 ng/ml, 30 min) in the absence or presence of AICAR (1 mM) and P-STAT1, P-STAT3 and P-STAT5 measured by flow cytometry. C. Astrocytes were stimulated with IFN-γ (10 ng/ml) for 30 min in the absence or presence of AICAR (1 mM) followed by fractionation into cytosolic and nuclear fractions. STAT1, P-STAT1, AMPKα and P-AMPK subcellular distribution was examined by immunoblotting. The results for total STAT1 were quantified. The separation of GAPDH and RNA pol II demonstrates efficient separation of cytosolic and nuclear fractions, respectively. D and E. STAT1 localization was examined by immunofluorescent microscopy and quantified. The percentage of cells with nuclear/cytosolic STAT1 > 2 is reported in the table. F. AMPKα^fl/fl astrocytes were transduced with GFP or GFP-Cre adenovirus followed by stimulation with IFN-γ (10 ng/ml, 30 min) and subcellular fractionation. N=3, *p < 0.05.

Figure 6. Reduced AMPK Signaling Correlates With EAE Disease Severity and Inflammatory Gene Expression

A. C57Bl/6 mice were immunized with MOG35-55 peptide to induce EAE and mice were scored daily starting at day 5. B. AMPKα phosphorylation in the brain during EAE was measured by immunoblotting. C. Brains from EAE mice were analyzed over time for IFN-γ and CCL2 expression by qRT-PCR.