AMPKα1 deficiency amplifies proinflammatory myeloid APC activity and CD40 signaling

AMPK attenuates CD40-mediated inflammatory activity in myeloid APC; its activity in both APC and T cells contributes to T cell functional polarization.

Abstract

AMPK is a serine/threonine kinase that regulates energy homeostasis and metabolic stress in eukaryotes. Previous work from our laboratory, as well as by others, has provided evidence that AMPKα1 acts as a negative regulator of TLR-induced inflammatory function. Herein, we demonstrate that AMPKα1-deficient macrophages and DCs exhibit heightened inflammatory function and an enhanced capacity for antigen presentation favoring the promotion of Th1 and Th17 responses. Macrophages and DCs generated from AMPKα1-deficient mice produced higher levels of proinflammatory cytokines and decreased production of the anti-inflammatory cytokine IL-10 in response to TLR and CD40 stimulation as compared with WT cells. In assays of antigen presentation, AMPKα1 deficiency in the myeloid APC and T cell populations contributed to enhanced IL-17 and IFN-γ production. Focusing on the CD154–CD40 interaction, we found that CD40 stimulation resulted in increased phosphorylation of ERK1/2, p38, and NF-κB p65 and decreased activation of the anti-inflammatory Akt -GSK3β-CREB pathway in DCs deficient for AMPKα1. Our data demonstrate that AMPKα1 serves to attenuate LPS and CD40-mediated proinflammatory activity of myeloid APCs and that AMPKα1 activity in both APC and T cells contributes to T cell functional polarization during antigen presentation.

Introduction

AMPK is a highly conserved serine/threonine kinase crucial to the maintenance of energy balance in eukaryotic cells [1]. AMPK becomes activated by stresses causing ATP depletion and functions to turn off ATP-consuming anabolic pathways and turn on ATP-producing catabolic pathways. AMPK is a heterotrimeric complex made up of a catalytic α subunit and regulatory β and γ subunits. Phosphorylation of the α subunit at Thr¹⁷² is essential for AMPK activity [2], and upstream kinases include LKB1 [3–5] and CAMKKβ [6–8]. AMP allosterically activates AMPK [9] and along with ADP, promotes phosphorylation of AMPK by inducing phosphorylation and reducing dephosphorylation by protein phosphatases [10].

Over the past several years, a role of AMPK in the regulation of inflammatory responses has been revealed. Treatment with a pharmacological activator of AMPK, AICAR, has been shown to reduce the clinical severity of experimental autoimmune encephalitis [11], EAU [12], and models of acute and relapsing colitis in mice [13]. However, interpretation of studies using AICAR is complicated by the finding that AICAR has both AMPK-dependent and independent effects on cell function [12, 14, 15]. AICAR is taken into the cell and converted to an AMP analog, AICAR1-β-d-ribofuranotide, making it a nonspecific activator of AMPK and able to activate other AMP-sensitive enzymes. Studies from our lab using macrophage cell lines expressing CA and DN forms of AMPKα1 indicated that AMPKα1 promotes anti-inflammatory activity and suppresses proinflammatory signaling in macrophages [16]. Consistent with these findings, we also demonstrated that treatment of primary macrophages with anti-inflammatory stimuli, such as IL-10 and TGF-β, resulted in rapid phosphorylation of AMPKα1, whereas LPS stimulation resulted in rapid dephosphorylation of AMPKα1 [16]. Subsequent to this work, it was reported that AMPKα1 counter-regulates lipid-induced inflammation in macrophages through Sirtuin1 [17], and AMPKβ1 was shown to reduce adipose tissue macrophage inflammation in obesity [18]. Additionally, evidence has been presented indicating that AMPKα1 activity antagonizes TLR-induced DC maturation [19]. It was also shown recently that AMPK suppresses IFN-γ-induced gene expression in astrocytes and microglia [20].

AMPK has also been shown to play a role in T cell survival [21] and proliferation [22] downstream of LKB1. It has been reported that AMPKα1 is required for CD8 T cell memory [23]. In murine models of inflammatory disease, in vivo AICAR treatment reduced the production of proinflammatory Th cell cytokines [11–13]. Alternatively, studies by McIver et al. showed that AMPKα1 regulates T cell viability and metabolism and promotes CD44 expression and proinflammatory cytokine production through mTORC1 in CD8⁺ T cells but not CD4⁺ T cells [24]. A study of CD4⁺ T cell subset differentiation reported that generation of Tregs was accompanied by elevation of AMPK activity, associated with dependency on lipid oxidation for the Treg functional phenotype [25].

In the present study, we focus on the role of AMPKα1 in APC function and T cell–APC interactions, including the unexplored role of AMPKα1 in CD40 signal transduction. With the use of an AMPKα1-deficient mouse model, we demonstrate that AMPKα1 polarizes APCs to an anti-inflammatory phenotype and that deficiency of AMPKα1 results in heightened CD40-mediated inflammatory activity. We also demonstrate that deficiency of AMPKα1 in both APCs and T cells contributes to the enhanced capacity for production of proinflammatory T cell responses.

MATERIALS AND METHODS

Mice

Mice deficient for AMPKα1 were generated as described previously [26]. AMPKα1-deficient and WT littermates are bred and maintained in the Research Resources Facility, University of Louisville. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee, University of Louisville.

Reagents

LPS (Escherichia coli serotype O111:B4) was purchased from Sigma-Aldrich (St. Louis, MO, USA). CD40 was activated with FLAG-tagged multimeric CD154 (MegaCD40L; Enzo Life Sciences, Farmingdale, NY, USA). Western blot detection of specific proteins used the following primary antibodies: antiphospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, antiphospho-NF-κB p65 (Ser536), anti-NF-κB p65, antiphospho-p38 MAPK (Thr180/Tyr182), anti-p38 MAPK, antiphospho-GSK3β (Ser9), anti-GSK3β, antiphospho-CREB (Ser133), anti-CREB, antiphospho-Akt (Ser473), and anti-Akt (Cell Signaling Technology, Beverly, MA, USA) and HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

ELISA

Murine bone marrow-derived DCs and macrophages were generated as described previously [27]. Following stimulation in 96-well plates, supernatants were collected and assayed by ELISA for TNF-α, IL-6, IL-10, and IFN-γ (BD Biosciences, San Jose, CA, USA) and IL-17 (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions. Analysis was performed using an EMax Precision Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

Antigen-presentation assays

Murine bone marrow-derived DCs and macrophages were plated (10⁵ cells/well in 100 μl medium) with 100 μg/ml OVA peptide (Sigma-Aldrich) for 4 h. T cells isolated from spleens of OT-II mice (The Jackson Laboratory, Bar Harbor, ME, USA) using CD4 (L3T4) Microbeads (Miltenyi Biotec, Auburn, CA, USA) were then added to culture. Supernatants were collected after 48 h and analyzed via ELISA.

MOG_35–55 peptide, corresponding to the sequence MEVGWYRSPFSRVVHLYRNGK, was purchased from Bio-Synthesis (Lewisville, TX, USA). Mice were injected in the flank with 100 μl of an emulsion containing 150 μg MOG_35–55 in CFA (Sigma-Aldrich), supplemented with 500 ng Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA). Fourteen days later, T cells were isolated from the spleens of immunized mice using Pan T Cell Isolation Kit II (Miltenyi Biotec). T cells (2.5×10⁵) were added to murine bone marrow-derived DCs and macrophages that had been pulsed with 50 μg/ml MOG_35–55 peptide for 1 h. Supernatants were collected and assayed by ELISA at 48 h.

Flow cytometry

Single-cell suspensions of bone marrow-derived DCs and macrophages were stained with fluorescently conjugated antibodies against murine CD11c, CD11b, CD80, CD86, CD40, and MHC II I-Ab (all from BD Biosciences, San Jose, CA, USA) for 30 min at 4°C, washed, and analyzed using a FACSCalibur flow cytometer and FlowJo software (Tree Star, Ashland, OR, USA).

Phagocytosis assay

Murine bone marrow-derived DCs and macrophages were cultured for 24 h with IgG FITC-conjugated latex beads (Cayman Chemical, Ann Arbor, MI, USA). Cells were collected, stained with fluorescently conjugated antibodies against CD11c or CD11b (both from BD Biosciences) for 30 min at 4°C, washed, and analyzed using a FACSCalibur flow cytometer and FlowJo software (Tree Star).

Western blot analysis

Murine bone marrow-derived DCs were lysed in buffer containing 125 mM Tris (pH 6.8), 2% SDS, 20% glycerol, 200 uM PMSF, and protease inhibitor mixture (Promega, Madison, WI, USA) and phosphatase inhibitor mixture (Thermo Fisher Scientific, Rockford, IL, USA). Total protein content of the samples was assessed by BCA protein assay (Thermo Fisher Scientific). Equal amounts of protein were separated on 10% Criterion gels (Bio-Rad, Hercules, CA, USA) by SDS-PAGE. Proteins were transferred to nitrocellulose membranes using the Transblot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). Antibody-bound proteins were detected using an ECL Western blotting analysis system (GE Healthcare, Pittsburgh, PA, USA), and the membranes were exposed to UltraCruz 5 × 7 Autoradiography Film (Santa Cruz Biotechnology, Santa Cruz, CA, USA) Densitometric analysis was performed using UN-SCAN-IT gel analysis software (Silk Scientific, Orem, UT, USA).

Statistical analysis

Statistical significance between groups was evaluated by two-way ANOVA, followed by Bonferroni multiple comparison test using GraphPad Prism software (GraphPad, La Jolla, CA, USA), and a level of P < 0.05 was considered statistically significant.

RESULTS

AMPKα1 activity counter-regulates inflammatory activity of myeloid APCs

The influence of AMPKα1 on APC inflammatory activity was evaluated using bone marrow-derived DCs and macrophages generated from AMPKα1-deficient mice. Cytokine production by AMPKα1-deficient bone marrow-derived DCs and macrophages in response to LPS stimulation was measured by ELISA. AMPKα1-deficient APCs showed increased LPS-induced production of the proinflammatory cytokines IL-6 (Fig. 1A and D) and TNF-α (Fig. 1B and E), whereas production of the anti-inflammatory cytokine IL-10 was decreased with LPS (Fig. 1C and F). The results of these experiments reveal that AMPKα1 counter-regulates LPS-induced inflammatory cytokine production in primary macrophages and DCs and support our previous work that used macrophage cell lines transfected with DN and CA forms of AMPKα1 [16].

Figure 1. AMPKα1 modulates the inflammatory response of DCs and macrophages.

Bone marrow-derived DCs (A–C) and macrophages (D–F) were generated from AMPKα1-deficient (AMPKα1−/−) and AMPKα1+/+ mice and stimulated with LPS at the concentrations shown for 18 h or left unstimulated. IL-6, TNF-α, and IL-10 levels were detected by ELISA. Data are shown as mean ± sem and are the combined results of three independent experiments (*P<0.05; **P<0.01).

AMPKα1-deficient APCs promote proinflammatory T cell responses

To determine the influence of AMPKα1 on T cell–APC interactions, bone marrow-derived DCs and macrophages generated from AMPKα1-deficient mice, were pulsed with OVA and cocultured with OT-II T cells. Cell culture supernatants were assayed for cytokine content. AMPKα1-deficient APCs induced significantly higher levels of T cell production of IFN-γ (Fig. 2A and C) and IL-17 (Fig. 2B and D) than WT APCs, demonstrating that absence of AMPKα1 in APCs promotes Th1 and Th17 responses.

Figure 2. Antigen presentation by AMPKα1-deficient DCs and macrophages promotes proinflammatory T cell responses.

AMPKα1+/+ and AMPKα1-deficient bone marrow-derived DCs (A and B) and macrophages (Mϕ; C and D) were pulsed with OVA and coincubated with OT-II CD4⁺ T cells for 18 h. IFN-γ and IL-17 levels were detected by ELISA. Data are shown as mean ± sem and are the combined results of three independent experiments (**P<0.01).

We also evaluated APC–T cell interactions using T cells isolated from mice immunized with MOG_35–55 peptide. AMPKα1-deficient and AMPKα1+/+ APCs were pulsed with MOG_35–55 peptide and cocultured with splenic CD4⁺ T cells harvested from AMPKα1-deficient and AMPKα1+/+ mice that had been immunized with MOG_35–55 14 d earlier. AMPKα1-deficient APCs induced higher levels of IFN-γ (Fig. 3A, B, E, and F) and IL-17 (Fig. 3C, D, G, and H) secretion than AMPKα1+/+ APCs.

Figure 3. AMPKα1 deficiency in APCs and T cells leads to Th1 and Th17 responses.

Bone marrow-derived DCs and macrophages were generated from AMPKα1-deficient and AMPKα1+/+ mice. DCs (A–D) and macrophages (E–H) were pulsed with MOG_35–55 before coculture with T cells from MOG_35–55-immunized AMPKα1+/+ (A, C, E, and G) or AMPKα1-deficient (B, D, F, and H) mice. Levels of IFN-γ and IL-17 were detected by ELISA. Data shown are mean ± sd and are representative of three independent experiments with similar results (** P<0.01).

Assays using AMPKα1-deficient T cells provided evidence for the role of AMPKα1 in the regulation of effector T cell responses. In assays with AMPKα1+/+ APCs, deficiency of AMPKα1 in T cells led to increased production of IFN-γ and IL-17 (Fig. 3). It is clear that AMPKα1 deficiency in both cell types promotes Th1 and Th17 responses. When both APCs and T cells were deficient in AMPKα1 (Fig. 3B, D, F, and H), the most profound influence on production of IFN-γ and IL-17 was observed.

APC deficiency of AMPKα1 leads to increased expression of CD80 and CD86

To explore further the role of AMPKα1 in antigen presentation, the impact of AMPKα1 deficiency on expression of antigen presentation machinery was evaluated. LPS-stimulated AMPKα1-deficient DCs had significantly higher expression of costimulatory molecules CD80 (Fig. 4C–E) and CD86 (Fig. 4H–J) compared with DCs generated from control mice. AMPKα1 deficiency in LPS-stimulated macrophages resulted in similar increases of CD80 (Fig. 4C–E) and CD86 (Fig. 4H–J) but did not reach statistical significance. We did not see a significant difference in CD40 (Supplemental Fig. 1) or MHC II (Supplemental Fig. 2) expression on AMPKα1-deficient bone marrow-derived DCs or macrophages compared with those generated from control mice nor did we see differences in phagocytosis of latex beads (Supplemental Fig. 3).

Figure 4. AMPKα1-deficient APCs have increased expression of CD80 and CD86.

AMPKα1+/+ and AMPKα1-deficient bone marrow-derived DCs and macrophages were left unstimulated (A, B, F, and G) or stimulated with LPS (100 ng/ml; C, D, H, and I) for 18 h. Cells were stained and surface expression of CD80 and CD86 was analyzed via flow cytometry. Histograms show fluorescence of costimulatory molecule expression (black line) of CD11c+ (for DCs) or CD11b+ (for macrophages) cells and unstained cells (solid gray peak). Mean fluorescence intensity (MFI) of populations in brackets is stated numerically. Data shown in A–D and F–I are representative of three independent experiments with similar results. Bar graphs describe fold change in MFI of CD80 (E) and CD86 (J) compared with unstimulated WT cells, and values are mean ± sem and are the combined results of three independent experiments (*P<0.05; **P<0.01).

AMPKα1 regulates APC responses to CD40 stimulation

The CD40:CD154 receptor:ligand interaction is well-established as critical to productive APC–T cell interactions [28, 29]. With the demonstration that AMPKα1 influences antigen presentation and the development of effector T cell responses, we investigated the role of AMPKα1 in the response of myeloid APCs to CD154 stimulation. AMPKα1-deficient bone marrow-derived DCs and macrophages were generated and evaluated for their cytokine production in response to stimulation with a soluble, multimeric CD154. AMPKα1-deficient APCs displayed increased production of the proinflammatory cytokine IL-6 in response to CD154 stimulation (Fig. 5A and C) and alternatively, decreased production of the anti-inflammatory cytokine IL-10 (Fig. 5B and D). In contrast to previous observations by our laboratory and others that proinflammatory stimuli result in decreased phosphorylation of AMPKα1 [16, 17, 19, 20], stimulation of macrophages and DCs with CD154 at a concentration that effectively induced a proinflammatory response (1.0 μg/ml) had no effect on phosphorylation of AMPKα1 (data not shown).

Figure 5. AMPKα1 regulates the inflammatory cytokine response of APCs to CD154 stimulation.

Bone marrow-derived DCs (A and B) and macrophages (C and D) were generated from AMPKα1-deficient and AMPKα1+/+ mice and stimulated with CD154 at the concentrations shown for 18 h or left unstimulated. IL-6 and IL-10 cytokine levels were detected by ELISA. Data shown are mean ± sem and are the combined results of three independent experiments (*P<0.05; **P<0.01).

We considered a number of downstream targets of AMPKα1 as possible mediators of the anti-inflammatory activity that we observed. CD154-mediated TRAF signaling activates the NF-κB and MAPK pathways, including p38 and ERK1/2 kinases [30–35]. We evaluated CD154-induced NF-κB activation through phosphorylation of p65. As shown in Fig. 6A, CD154 induced p65 phosphorylation in AMPKα1-deficient DCs to a much greater degree than in DCs generated from WT mice. Phosphorylation of the MAPKs ERK1/2 and p38, which have been implicated in the production of proinflammatory cytokines in response to CD40 activation [31, 32, 34], was also evaluated. AMPKα1-deficient DCs showed much higher phosphorylation of ERK1/2 and p38 in response to CD154 stimulation (Fig. 6B and C).

Figure 6. AMPKα1 expression modulates CD40 signaling in DCs.

Bone marrow-derived DCs were generated from AMPKα1-deficient and AMPKα1+/+ mice and stimulated with 1.0 μg/ml CD154 for the time-points indicated. After cell lysis, Western blot was performed using antibodies against phosphorylated (p)-NF-κB p65 (Ser536) and total NF-κB p65 (A), phosphorylated ERK1/2 (Thr202/Tyr204) and total ERK1/2 (B), phosphorylated p38 MAPK (Thr180/Tyr182) and p38 MAPK (C), phosphorylated Akt (Ser473) and total Akt (D), phosphorylated CREB (Ser133) and total CREB (E), and phosphorylated GSK3β (Ser9) and total GSK3β (F). Bands were analyzed by densitometry and displayed as bar histograms. Data shown are representative of three (B–D and F) and four (A and E) independent experiments with similar results.

AMPK is known as a modulator of signaling events downstream of the PI3K-Akt pathway [36], and in previous studies of macrophage function, we found that deficiency of AMPKα1 resulted in decreased Akt activity in response to LPS stimulation [16]. In the unphosphorylated state, GSK3β is active and prevents IL-10 expression via its inactivation of the transcription factor CREB. Akt can phosphorylate and inactivate GSK3β (Ser9), such that it can no longer prevent CREB-dependent IL-10 production [37, 38]. We evaluated phosphorylation of Akt, CREB, and GSK3β in AMPKα1-deficient DCs. As shown in Fig. 6D–F, DCs lacking AMPKα1 expression displayed decreased phosphorylation of Akt, CREB, and GSK3β when stimulated with CD154 compared with DCs generated from WT mice.

DISCUSSION

AMPK has been recognized recently as a counter-regulator of inflammatory pathways, including those induced by TLR and IFN-γ stimulation [16, 19, 20] and fatty acids [17, 18]. Our previous work using macrophage cell lines transfected with CA and DN forms of AMPKα1, as well as AMPKα1 small interfering RNA knockdown, revealed the role of AMPKα1 in the negative control of TLR-mediated inflammatory activity. In this report, use of primary myeloid APCs generated from AMPKα1-deficient mice confirms the function of AMPK in the suppression of TLR-mediated induction of proinflammatory cytokine production and the enhancement of anti-inflammatory IL-10 production (Fig. 1) and establishes AMPK as a regulator of APC activity and CD40 signal transduction (Figs. 4–6). These data suggest that the influence of AMPK activators on development of autoimmune disease in mice [11, 12] could be largely a result of the regulatory role of AMPKα1 in the outcome of CD154:CD40 interactions during initial APC events, as well as during T cell activation of myeloid cells at the sites of inflammation.

We investigated a number of downstream effectors of AMPKα1 to determine the mechanism by which AMPKα1 exerts its anti-inflammatory influence on CD40 signaling, including the Akt-GSK3β pathway known to be suppressive of TLR-mediated inflammatory signaling [37, 38]. Previous reports on the association of AMPK and Akt activation have shown mixed results, with some suggesting that AMPK activation is positively correlated with Akt activation and others reporting an association of AMPK activity with decreased Akt activity (reviewed in ref. [36]). We have shown previously a positive association of AMPKα1 and Akt in macrophages stimulated with LPS [16]. Likewise, in the present study, AMPKα1-deficient DCs responded to CD154 stimulation with decreased Akt activity, enhanced GSK3β activity, and decreased CREB activation (Fig. 6D–F).

Akt must be phosphorylated at two sites to become active: Thr308 by phosphoinositide-dependent kinase 1 [39, 40] and Ser473 by mTORC2 [41]. When cellular energy becomes low, AMPK phosphorylates and activates TSC2, an inhibitor of mTOR [42], and alternatively, Akt can phosphorylate and inactivate TSC2, thereby activating mTORC1 [43, 44]. mTORC1 regulates mRNA translation, in part, through phosphorylation and activation of S6K1 [45], which like Akt, has been shown to phosphorylate and inactivate GSK3β [46]. Our data fit with this latter scenario in that decreased AMPKα1 activity is associated with decreased Akt phosphorylation (Ser473) and decreased phosphorylation (Ser9) of GSK3β in CD154-stimulated DCs (Fig. 6D and F).

GSK3β is a negative regulator of the transcription factor CREB, and phosphorylation and inactivation of GSK3β enhances CREB activity [37, 38]. It is thought that inhibition of GSK3β allows CREB to compete for the nuclear coactivator CREB-binding protein, which is required for NF-κB activity. This results in reduced NF-κB activation and enhanced expression of CREB-activated IL-10 synthesis [38]. Accordingly, we observe decreased CREB phosphorylation (Fig. 6E) and increased phosphorylation of p-65 NF-κB (Fig. 6A) in AMPKα1-deficient DCs stimulated with CD154. AMPKα1-deficient DCs and macrophages also display increased NF-κB production of proinflammatory cytokines and decreased synthesis of IL-10 in response to TLR and CD154 stimulation (Figs. 1 and 5).

Additionally, we found that absence of AMPKα1 in APCs led to increased costimulatory molecule expression (Fig. 4). It has been reported previously that treatment of DCs with AICAR and LPS results in an AMPKα1-independent decrease in expression of costimulatory molecules [12]. Although AICAR has been shown to have AMPKα1-independent effects, our results confirm that AMPKα1 activity is involved in the regulation of CD80 and CD86 expression (Fig. 4). The elevated expression of costimulatory molecules by AMPKα1-deficient APCs is associated with robust induction of Th1 and Th17 responses (Figs. 2 and 3).

In addition to AMPKα1 expression by APCs, we confirm a role for T cell-expressed AMPKα1 in the development of Th immune responses (Fig. 3). Previous reports on AMPK function in CD4⁺ T cells have generated mixed results. A recent study implicated AMPKα1 in the regulation of inflammatory cytokine production by CD8⁺ T cells but not CD4⁺ T cells [24], and a requirement for AMPK activity for Treg function has been reported [25]. It has also been shown that in vivo AICAR treatment in a murine model of EAU resulted in decreased CD4⁺ Th1 and Th17 T cell responses [12], which is in agreement with our findings that absence of AMPKα1 in CD4⁺ T cells leads to development of proinflammatory T cell responses (Fig. 3).

Excessive Th1 and Th17 responses have been associated with a variety of autoimmune diseases, and therapeutic strategies include targeting factors that can dampen these T cell responses. Our results indicate that AMPKα1 is one such potential target, as AMPKα1 expression in APCs and T cells leads to reduced IFN-γ and IL-17 T cell production in antigen presentation assays (Figs. 2 and 3). The anti-inflammatory effect of AMPK on leukocyte behavior has been associated with the involvement of AMPK in cellular metabolism. As described in a recent review by O'Neill and Hardie [47], inflammatory cells, such as activated macrophages [48] and Th17 cells [49, 50], display high rates of glycolysis and lower rates of oxidative metabolism. Alternatively, anti-inflammatory macrophages [48] and Tregs [49, 50] have lower rates of glycolysis. Much about this association is still unclear, but higher glycolytic activity is thought to provide a quicker, albeit less efficient, source of energy for the cell to rapidly alter its functional phenotype from resting to inflammatory. The transition to glycolysis in activated cells mirrors the “Warburg effect” observed in cancer cells. It has been shown that resting DCs generate ATP through oxidative phosphorylation and upon LPS stimulation, switch to glycolysis [19]. Recent findings suggest that AMPKα1 may repress the switch from oxidative phosphorylation to glycolysis upon activation. AMPKα1 activation through AICAR, along with IL-10, antagonizes the LPS-induced switch to glycolysis in DCs [19]. Additionally, T cells isolated from AMPKα1-deficient mice display elevated levels of glycolysis [24]. Our finding that AMPKα1 antagonizes CD154-induced proinflammatory signaling and cytokine production raises questions regarding the role of AMPKα1 in regulation of the glycolytic switch in response to CD154. It is unknown whether CD154 stimulation is associated with a transition from oxidative to glycolytic metabolism. It is also important to consider that aside from its regulatory role in cell metabolism, AMPKα1 activity modulates numerous signaling pathways that influence cell behavior and gene transcription.

AUTHORSHIP

K.C.C. contributed to experimental design, performed all experimental work shown, analyzed data, prepared figures, and cowrote the manuscript. B.V. generated mice deficient for AMPKα1 and provided valuable consultation on the project. J.S. designed the project and experimental protocols, contributed to data analysis, and cowrote the manuscript.

DISCLOSURES

The authors declare no conflict of interest.