Substrate Fate in Activated Macrophages: A
Comparison between Innate, Classic, and
Alternative Activation
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of June 15, 2017.
Juan-Carlos Rodríguez-Prados, Paqui G. Través, Jimena
Cuenca, Daniel Rico, Julián Aragonés, Paloma Martín-Sanz,
Marta Cascante and Lisardo Boscá
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J Immunol 2010; 185:605-614; Prepublished online 24 May
2010;
doi: 10.4049/jimmunol.0901698
http://www.jimmunol.org/content/185/1/605
The Journal of Immunology
Substrate Fate in Activated Macrophages: A Comparison
between Innate, Classic, and Alternative Activation
Juan-Carlos Rodrı́guez-Prados,*,1 Paqui G. Través,†,‡,1 Jimena Cuenca,†
Daniel Rico,x Julián Aragonés,{ Paloma Martı́n-Sanz,†,‡ Marta Cascante,* and
Lisardo Boscá†,‡
T
he innate immune system acts as the first line of defense
and functions by recognizing highly conserved sets of
molecular patterns (pathogen-associated molecular patterns [PAMPs]) through a limited number of germline-encoded
*Department of Biochemistry and Molecular Biology, Institute of Biomedicine, University of Barcelona; ‡Centro de Investigación Biomédica en Red de Enfermedades
Hepáticas y Digestivas, Barcelona; †Instituto de Investigaciones Biomédicas ‘Alberto
Sols’, Consejo Superior de Investigaciones Cientı́ficas-Universidad Autónoma de
Madrid; xCentro Nacional de Investigaciones Oncológicas, Melchor Fernández de
Almagro; and {Servicio de Inmunologı́a, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain
1
J.-C.R.-P. and P.G.T., in alphabetical order, contributed equally to this work.
Received for publication May 28, 2009. Accepted for publication April 10, 2010.
This work was supported by European Commission (FP7) Etherpath KBBE-Grant
Agreement no. 222639, Grants SAF2005-01627, SAF2008-00164, and BFU200802161 from Ministerio de Ciencia e Innovación of the Spanish Government,
2005SGR00204 from the Generalitat de Catalunya, S-BIO-0283/2006 from Comunidad de Madrid, Red Temática de Investigación Sobre Cáncer (RETICC RD6/0020/
0046) and Red Temática de Investigación Sobre Enfermedades Cardiovasculares
(RECAVA RD6/0014/006), and FIS-RECAVA RD06/0014/0025. RECAVA and
Ciberehd are funded by Instituto de Salud Carlos III. J.-C.R.-P. is supported by
a fellowship from the University of Barcelona.
Address correspondence and reprint requests to Dr. Lisardo Boscá and Dr. Marta Cascante, Instituto de Investigaciones Biomédicas ‘Alberto Sols’ (Consejo Superior de Investigaciones Cientı́ficas-Universidad Autónoma de Madrid), Arturo Duperier 4, 28029
Madrid, Spain (L.B.), and Department of Biochemistry and Molecular Biology, Faculty
of Biology (edifici Nou) Planta-2.Avinguda Diagonal 645, 08028 Barcelona, Spain
(M.C.). E-mail addresses: [email protected] (L.B.) and [email protected] (M.C.)
The online version of this article contains supplemental material.
Abbreviations used in this paper: COX-2, cyclooxygenase-2; FDR, false discovery rate;
FI, fold induction; Fru-2,6-P2, fructose-2,6-bisphosphate; GCMS, gas chromatography/mass spectrometry; GSEA, gene set enrichment analysis; IP-10, IFN-g–inducible
protein-10; L-PFK2, liver type-PFK2; NOS-2, nitric oxide synthase-2; PAMP, pathogenassociated molecular pattern; PFK2, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase;
PI, propidium iodide; poly(I:C), polyriboinosinic:polyribocytidylic acid; PPP, pentose
phosphate pathway; scRNA, scrambled RNA; siRNA, small interfering RNA; uPFK2,
ubiquitous PFK2.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901698
receptors called pattern-recognition receptors. TLRs, a class of pattern-recognition receptors, have the ability to recognize pathogens
or pathogen-derived products and initiate signaling events leading to
activation of innate host defense (1, 2). Macrophages play an essential role in the immune response and normal tissue development
by producing proinflammatory mediators and by phagocytic clearance of pathogens and apoptotic cells (3, 4). It has been described
that macrophages could undergo different activation processes
depending on the stimuli received (5, 6). The classic activation,
which can be induced by in vitro culture of macrophages with
IFN-g and LPS (inducing TNF-a production), is associated with
high microbicidal activity, proinflammatory cytokine, and reactive
oxygen species production and cellular immunity; the innate
activation, which is mediated in culture by ligation of receptors,
such as TLRs, is associated with microbicidal activity and
proinflammatory cytokine production; the alternative activation,
which can be mimicked in vitro after culture with IL-4, IL-13,
glucocorticoids, immune complexes, or IL-10, is associated with
tissue repair, tumor progression, and humoral immunity (7). Some
authors also distinguish macrophage deactivation, which is induced
by cytokines, such as IL-10 or TGF-b, or by ligation of inhibitory
receptors, such as CD200 receptor or CD172a, and is related to
anti-inflammatory cytokine production and reduced MHC class II
expression (8, 9).
Under normal conditions, macrophages are recruited and phagocyte at sites of infection. In addition to playing a crucial role in immunity, some of the mammalian TLRs have been described to
regulate bodily energy metabolism, mostly through acting on adipose
tissue. This has recently opened new avenues of research on the role
of TLRs in pathologies related to metabolism, such as obesity, insulin resistance, metabolic syndrome, or atherosclerosis (10). The
accumulation of fatty acids, above all cholesterol in low density
lipoprotein form, is the main reason that lipid metabolism has been
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Macrophages play a relevant role in innate and adaptive immunity depending on the balance of the stimuli received. From an analytical and
functional point of view, macrophage stimulation can be segregated into three main modes, as follows: innate, classic, and alternative pathways. These differentialactivations result intheexpression of specific sets ofgenesinvolvedinthe release of pro- or anti-inflammatory stimuli.
In the present work, we have analyzed whether specific metabolic patterns depend on the signaling pathway activated. A [1,2-13C2]glucose
tracer-based metabolomics approach has been used to characterize the metabolic flux distributions in macrophages stimulated through the
classic, innate, and alternative pathways. Using this methodology combined with mass isotopomer distribution analysis of the new formed
metabolites, the data show that activated macrophages are essentially glycolytic cells, and a clear cutoff between the classic/innate activation
and the alternative pathway exists. Interestingly, macrophage activation through LPS/IFN-g or TLR-2, -3, -4, and -9 results in similar flux
distribution patterns regardless of the pathway activated. However, stimulation through the alternative pathway has minor metabolic
effects. The molecular basis of the differences between these two types of behavior involves a switch in the expression of 6-phosphofructo-2kinase/fructose-2,6-bisphosphatase (PFK2) from the liver type-PFK2 to the more active ubiquitous PFK2 isoenzyme, which responds to
Hif-1a activation and increases fructose-2,6-bisphosphate concentration and the glycolytic flux. However, using macrophages targeted for
Hif-1a, the switch of PFK2 isoenzymes still occurs in LPS/IFN-g–activated macrophages, suggesting that this pathway regulates
ubiquitous PFK2 expression through Hif-1a-independent mechanisms. The Journal of Immunology, 2010, 185: 605–614.
606
METABOLIC FLUXES IN MACROPHAGE ACTIVATION
studied in macrophages in the atherosclerosis framework. Recent
works have built a fatty acid bridge between diet and immune system due to the induction of TLR expression by some fatty acids (11).
However, few are known on central metabolism patterns in macrophages since the work of Newsholme and collaborators (12–14)
in the 1990s. In this study, we will apply a system biology approach
to shed some light in the cross-talk between signal transduction
and central metabolism in macrophages. We studied whether the
distinct stimulation pathways required different energy demands or
have different metabolic patterns. To achieve these goals, tracerbased metabolomics experiments have been used and combined with
analysis of the expression of markers of activation. Following
the normal activation process of the macrophages, cells were fed
with [1,2-13C2]glucose, a nonradioactive isotope of glucose that
will incorporate [13C] carbons into metabolite end products (i.e.,
lactate, glutamate) and the ribose of RNA. This tracer has been
broadly used before (15). Our data show that activated macrophages are essentially glycolytic cells, and a clear cutoff between
the classic/innate activation and the alternative pathway exists. Interestingly, activation through TLR-2, -3, -4, and -9 results in similar
patterns of metabolic activation, despite the use of at least in part
distinct signaling pathways and expression of different sets of genes.
In the classic and innate activation we observe a shift in the expression of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
(PFK2) isoforms, from the liver type-PFK2 (L-PFK2), which has
a low net activity, to the more active ubiquitous PFK2 (uPFK2)
that maintains higher fructose-2,6-bisphosphate (Fru-2,6-P2)
concentrations due to a minor bisphosphatase activity and, therefore, potentiates the glycolytic flux (16–18). This isoenzyme is
expressed in response to hypoxia through Hif-1a activation (16).
Interestingly, LPS challenge results in an increase in Hif-1a levels
that might contribute to this uPFK2 expression (19–21). Using
macrophages targeted for this transcription factor, we have investigated the contribution of Hif-1a to the enhancement of the
glycolytic flux and to the expression of inflammatory molecules in
response to classic and alternative activation (22, 23).
phages to the plastic. An aliquot of the cell suspension was used to determine the cell density in the peritoneal fluid. The cells were centrifuged at
200 3 g for 10 min at 4˚C, and the pellet was washed twice with 25 ml icecold PBS. Cells were seeded at 1 3 106/cm2 in RPMI 1640 medium supplemented with 10% of heat-inactivated FCS and antibiotics. After incubation for 3 h at 37˚C in a 5% CO2 atmosphere, nonadherent cells were
removed by extensive washing with PBS. Experiments were carried out
in phenol-red free RPMI 1640 medium and 1% of heat-inactivated FCS
plus antibiotics (26). When glucose concentration was changed, this was
added to glucose-free RPMI 1640 medium. Prior to stimulation, the medium was aspirated and replaced by warm medium containing the indicated
TLR ligand or cytokine. When the murine macrophage RAW 264.7 cells
were used, they were processed as indicated for the peritoneal macrophages.
Materials and Methods
Cytokine assay
Reagents were from Sigma-Aldrich (St. Louis, MO), Roche (Basel, Switzerland), Invitrogen (Carlsbad, CA), or Merck (Darmstadt, Germany). TLR
ligands were from InvivoGen (San Diego, CA). Cytokines were from PeproTech (Rocky Hill, NJ). Commercial Abs were from Santa Cruz Biotechnology (Santa Cruz, CA), Cell Signaling Technology (Danvers, MA), Abcam
(Cambridge, U.K.), R&D Systems (Minneapolis, MN), Sigma-Aldrich, or
PeproTech. Serum and media were from BioWhittaker (Walkersville,
MD). Commercial Pfkfb3 small interfering RNAs (siRNAs) were from
Ambion (Austin, TX), and Pfkfb3 TaqMan gene expression assays were
from Applied Biosystems (Carlsbad, CA). [1,2-13C2]glucose (.99%
enriched) was purchased from Isotec (Miamisburg, OH).
Treatment of animals and preparation of peritoneal
macrophages
Mice were housed and bred in our pathogen-free facility. C57BL/6 mice were
used for macrophage isolation. Mice with myeloid lineage-specific HIF1a
floxed-UBC-cre/ERT2 mice for conditional HIF1a inactivation were used
(24, 25) and were a generous gift of M. Landázuri and J. Aragonés (Hospital
La Princesa, Madrid, Spain). Experimental procedures were approved by
the Committee for Research Ethics of the Universidad Autonoma de Madrid
in accordance with Spanish and European guidelines. Animals were used
aged 8–12 wk, as follows: 4 d prior to the assay, mice were i.p. injected
2.5 ml 3% (w/v) of thioglycolate broth (26). Elicited peritoneal macrophages were prepared from light-ether anesthetized mice (four to six animals per condition), killed by cervical dislocation, and injected i.p. 10 ml
sterile RPMI 1640 medium. The peritoneal fluid was carefully aspirated,
avoiding hemorrhage, and kept at 4˚C to prevent the adhesion of the macro-
Cells were harvested and washed in cold PBS. After centrifugation at 4˚C
for 5 min and 1000 3 g, cells were resuspended in annexin V-binding
buffer (10 mM HEPES [pH 7.4], 140 mM NaCl, 2.5 mM CaCl2). Cells
were labeled with annexin V FITC solution (BD Pharmingen, San Jose,
CA) and/or propidium iodide (PI; 100 mg/ml) for 15 min at room temperature in the dark. PI is impermeable to living and apoptotic cells, but stains
necrotic and apoptotic dying cells with impaired membrane integrity, in
contrast to annexin V, which stains early apoptotic cells.
Assay of PFK2 activity
Cultured macrophages (6-cm dishes) were homogenized in 1 ml medium
containing 20 mM potassium phosphate (pH 7.4, 4˚C), 1 mM DTT,
50 mM NaF, 0.5 mM PMSF, 10 mM leupeptin, and 5% poly(ethylene)
glycol. After centrifugation (7000 3 g for 15 min), poly(ethylene)glycol
was added to the supernatant up to 15% (mass/v) to fully precipitate the
PFK2. After resuspension of the pellet in the extraction medium, PFK2
activity was assayed at pH 8.5 with 5 mM MgATP, 5 mM Fru-6-P, and
15 mM Glc-6-P. One unit of PFK2 activity is the amount of enzyme that
catalyzes the formation of 1 pmol Fru-2,6-P2 per minute (17).
Metabolite assays
Fru-2,6-P2 was extracted from cells (24-well dishes) after homogenization
in 100 ml 50 mM NaOH, followed by heating at 80˚C for 10 min. The
metabolite was measured by the activation of the pyrophosphate-dependent
phosphofructo-1-kinase (17). Lactate and glucose were measured enzymically in the culture medium. NO release was determined spectrophotometrically by the accumulation of nitrite and nitrate in the medium (phenol red
free). Nitrate was reduced to nitrite, and this was determined after reduction of nitrate to nitrite and the latter with Griess reagent (26) by adding
1 mM sulfanilic acid and 100 mM HCl (final concentration).
The accumulation of TNF-a, IL-6, and IL-10 in the culture medium was
measured per triplicate using commercial kits (Biotrak, GE Healthcare,
Barcelona, Spain, [TNF-a and IL-6] and eBioscience, San Diego, CA
[IL-10]), following the indications of the supplier.
Preparation of cell extracts
The macrophage cultures (six-well dishes) were washed twice with ice-cold
PBS, and the cells were homogenized in 0.2 ml buffer containing 10 mM
Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.5%
CHAPS, 1 mM 2-ME, and 0.1 mM PMSF and a protease inhibitor mixture
(Sigma-Aldrich). The extracts were vortexed for 30 min at 4˚C and centrifuged for 15 min at 13,000 3 g. The supernatants were stored at 220˚C.
Proteins levels were determined using the Bio-Rad (Richmond, CA)
detergent-compatible protein reagent. All steps were carried out at 4˚C.
Western blot analysis
Samples of cell extracts containing equal amounts of protein (30 mg/lane)
were boiled in 250 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 2%
2-ME, and size separated in 10–15% SDS-PAGE. The gels were blotted
onto a polyvinylidene difluoride membrane (GE Healthcare) and processed
as recommended by the supplier of the Abs against the murine Ags: NO
synthase-2 (NOS-2; sc-7271), cyclooxygenase-2 (COX-2; sc-1999),
MHC-II (sc-59322), hemeoxygenase-1 (AB-1284), Arg-1 (sc-20150), suppressor of cytokine signaling-3 (2923s), KC/CXCL1 (AF-453-NA), IFNg–inducible protein-10 (IP-10)/CXCL10 (500-p129), L-PFK2 (sc-10096),
Hif-1a (MAB1536), and b-actin (A-5441). For uPFK2, specific peptides of
the isoenzyme were used to generate polyclonal Abs by immunization of
rabbits (New Zealand White) with multiple intradermal injections with
300 mg Ag in 1 ml CFA, followed by boosters with 100 mg Ag in IFA
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Chemicals
Flow cytometry
The Journal of Immunology
(a gift of R. Barrtrons, University of Barcelona, Barcelona, Spain). The
blots were developed by ECL protocol (GE Healthcare), and different
exposition times were performed for each blot with a charged coupling
device camera in a luminescent image analyzer (Molecular Imager, BioRad) to ensure the linearity of the band intensities.
RNA isolation and RT-PCR analysis
A total of 1 mg total RNA, extracted with TRIzol reagent (Invitrogen)
according to the manufacturer’s instructions, was reverse transcribed using
Transcriptor First Strand cDNA Synthesis Kit for RT-PCR following the
indications of the manufacturer (Roche). Real-time PCR was conducted
with SYBR Green on a MyiQ Real-Time PCR System (Bio-Rad) using the
SYBR Green method. PCR thermocycling parameters were 95˚C for 10
min, 40 cycles of 95˚C for 15 s, and 60˚C for 1 min. All samples were
analyzed for 36B4 expression in parallel. Each sample was run in duplicate
and was normalized to 36B4. The replicates were then averaged, and fold
induction was determined in DDCt-based fold-change calculations. Primer
sequences are available on request.
Transient transfections
Microarray analysis
Normalized expression data were obtained from National Center for Biotechnology Information GEO dataset GDS2429 (27) using GEOquery
package from Bioconductor (Berkeley, CA) (28). Differential expression for
the following comparisons was tested using limma Bioconductor package
(29): 1) naive mature macrophage versus classic activated macrophage, and
2) alternatively activated macrophage. Two gene lists were generated after
each comparison, and they were ranked according to the test statistic for
subsequent gene set enrichment analysis (GSEA). Enrichment of gene sets
of interest in each list was accomplished using the GSEA method, as described by Mootha et al. (30). We used “Which genes?” (www.whichgenes.
org/) to retrieve the REACTOME (31) pathways as gene sets. The genes
from the two lists were also mapped into canonical pathways using Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood, CA; see
Supplemental Material).
Gas chromatography/mass spectrometry sample preparation
and procedure
The macrophage cultures (10-cm dishes) were washed twice with ice-cold
PBS, and culture media were replaced by 50% enriched [1,2-13C2]glucose
containing the indicated TLR ligand or cytokine. After incubation, cells
were centrifuged (200 3 g for 5 min), and incubation medium and cell
pellets were obtained and stored at 280˚C until processing. Glucose,
lactate, glutamine, and glutamate incubation medium concentrations were
determined, as previously described (32, 33), using a Cobas Mira Plus
chemistry analyzer (HORIBA ABX, Montpelier, France). Lactate from
the cell culture media was extracted by ethyl acetate after acidification
with HCl. Lactate was derivatized to its propylamide-heptafluorobutyric
form, and the m/z 328 (carbons 1–3 of lactate, chemical ionization) was
monitored for the detection of m1 (lactate with a [13C] in one position) and
m2 (double-labeled lactate) for the estimation of pentose cycle activity
versus anaerobic glycolysis (34). RNA/Ribose was isolated by acid hydrolysis of cellular RNA after TRIzol purification of cell extracts. Ribose
isolated from RNA was derivatized to its aldonitrile acetate form using
hydroxylamine in pyridine and acetic anhydride. We monitored the ion
cluster around the m/z 256 (carbons 1–5 of ribose, chemical ionization) to
find the molar enrichment of [13C] labels in ribose (34). Glutamate was
separated from the medium using ion-exchange chromatography (35). Glutamate was converted to its n-trifluoroacetyl-n-butyl derivative, and the ion
clusters m/z 198 (carbons 2–5 of glutamate, electron impact ionization) and
m/z 152 (carbons 2–4 of glutamate, electron impact ionization) were monitored. Isotopomeric analysis of C2–C5 and C2–C4 fragments of glutamate
in the medium was done to estimate the relative contributions of pyruvate
carboxylase and pyruvate dehydrogenase to the Krebs cycle (36, 37). Mass
spectral data were obtained on the QP2010 Shimadzu mass selective detector connected to a GC-2010 gas chromatograph. The settings were as
follows: gas chromatography inlet for glucose and ribose 250˚C and 200˚C
for lactate and glutamate, transfer line 250˚C, mass spectrometry source
200˚C. A Varian VF-5 capillary column (30-m length, 250-mm diameter,
0.25-mm film thickness) was used to analyze all of the compounds studied.
Statistical analysis
The data shown are the means 6 SD of three or five experiments. Statistical
significance was estimated with Student t test for unpaired observations. A
p value of ,0.05 was considered significant.
Results
Distribution of substrate fluxes in activated macrophages
Elicited peritoneal macrophages were isolated, maintained in
culture overnight, and activated for the indicated periods of time
through TLRs involved in innate immunity (TLR4, LPS; TLR2,
lipoteichoic acid; TLR3, polyriboinosinic:polyribocytidylic acid;
and TLR9, CpG), classic activation (LPS/IFN-g), and alternative
activation (IL-4/IL-13; IL-10) (5, 6). Fig. 1A shows the expression
of a panel of representative markers of macrophage activation.
NOS-2 and COX-2 were expressed in cells activated by the innate and classic response, except after CpG stimulation. MHC-II
upregulation was very sensitive to LPS signaling and to the classic
activation pathway, and Arg-1 was notably upregulated in response to IL-4/IL-13. Hemeoxygenase-1 was moderately induced
by innate and alternative pathways of activation. Suppressor
of cytokine signaling-3 was upregulated in response to LPS or
LPS/IFN-g. The chemokine CXCL1/KC was expressed in cells
activated through classic activation pathway, and innate immunity
mediated by TLR2 and TLR4 and CXCL10/IP-10 was upregulated through the classic and the innate response. In addition to
these parameters related to macrophage activation, cell viability
was determined; apoptosis was evaluated by annexin V exposure
of the cells and by positivity for PI labeling, the later characteristic
of dying cells. As Fig. 1B shows, an increase of annexin V was
observed in cells treated with LPS/IFN-g, and to a lesser extent
with LPS; however, the percentage of PI-positive cells at this time
(12 h) was modest, suggesting that the integrity of the plasma
membrane remained preserved. These data were reminiscent of
the expression of NOS-2, indicating that NO plays an important
role in the induction of apoptosis in these cells (38). Staurosporine
was used as a reference for apoptosis induction in macrophages
(39). Moreover, the accumulation in the culture medium of nitrite/
nitrate, IL-6, TNF-a, and IL-10 contributed to establish the profile
of activation elicited by the different stimuli used; for example,
CpG failed to promote NOS-2 expression, but notably increased
IL-6 and TNF-a synthesis (Fig. 1C).
Previous data suggested the relevance of carbohydrate metabolism in the commitment for activation of macrophages (40–43). In
this regard, we investigated the glucose consumption and fate in
macrophages activated through the innate, classic, and alternative
pathways. As Fig. 2A shows, two clear profiles were observed in
terms of glucose consumption and lactate release. TLR activation
promoted an enhancement in the glycolytic flux to lactate that can
be divided into two time-dependent steps: one from 0 to 4 h, and
a second flux rate from 4 to 12 h. Indeed, when the same experiment was performed with [1,2-13C2]-glucose, the incorporation of
[13C] atoms from glucose in different metabolites, according to the
scheme depicted in Fig. 2B, allowed the measurement of the precise contribution of these time-dependent fluxes in activated macrophages. From 0 to 4 h, the glycolytic activity was much lower
than from 4 to 12 h (Fig. 2C). Interestingly, alternative stimulation
exhibited a metabolic profile that essentially matched that of control cells, despite the influence of the expression of a specific set of
genes (vide infra). Also, macrophages displayed a metabolic flux
that involved the conversion of glucose into lactate by more than
95%, regardless of the activation pathway considered (Fig. 3A).
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For transient transfections, macrophages were seeded at 80% confluence and
transfected using the Cell Line Nucleofector Kit V, following the manufacturer’s instructions (Amaxa, Cologne, Germany). Forty-eight hours after
nucleofection with pfkfb3 Silencer Predesigned siRNA or a scrambled RNA
(scRNA), macrophages were stimulated for 12 h and mRNA isolated. Transfections were performed in triplicate, and expression of a GFP vector was
used as a control for transfection efficiency.
607
608
METABOLIC FLUXES IN MACROPHAGE ACTIVATION
FIGURE 1. Characterization of macrophages
stimulated through the classic, innate, and alternative
pathways. Peritoneal macrophages were maintained in
culture and stimulated for 12 h with the indicated
stimuli: LPS/IFN-g (250 and 20 ng/ml, respectively);
LPS (100 ng/ml); IL-13, IL-4, and IL-10 (20 ng/ml
each); lipoteichoic acid (5 mg/ml); polyriboinosinic:
polyribocytidylic acid (25 mg/ml); CpG (3 mg/ml);
and staurosporine (100 ng/ml). The levels of the indicated proteins were determined by Western blot (A).
The extent of apoptosis/necrosis was determined by
the staining with annexin V and PI, respectively (B).
The release of nitrite plus nitrate, TNF-a, IL-6, and
IL-10 to the culture medium was determined, as described in Materials and Methods (C). Results show
the mean 6 SD of five experiments. pp , 0.01 versus
the none condition.
Gene profiling in stimulated macrophages
The previous experiments showed that the carbohydrate metabolism
in macrophages is fundamentally glycolytic, and that the rate of
glucose consumption is lower in alternatively activated macrophages
than in those activated through the classic pathway. Therefore, the
expression levels of genes involved in energetic metabolism
may differ between the two types of macrophage activation. To test
this hypothesis, we compared the functional enrichment (in up- or
downregulated genes) of the following REACTOME (31) pathways:
glycolysis, pyruvate metabolism and TCA cycle, and electron transport chain. Glycolysis was significantly enriched in upregulated genes
after classic activation (false discovery rate [FDR] 0.032; Fig. 4A);
however, the enrichment trend of glycolysis upon alternative activation was not significant (FDR 0.547; Fig. 4A, Supplemental Fig. 1).
Moreover, pyruvate/TCA cycle pathways were most clearly enriched
in upregulated genes after IL-4/IL-13 activation (FDR 0.097),
whereas the opposite trend was observed for LPS/IFN-g activation,
although statistically not significant (FDR 0.455) (Fig. 4A, Supplemental Fig. 2). Noteworthily, the most significant enrichment (FDR
0.000) was found in electron-transport chain genes that were downregulated during classic activation, an effect not observed for the alternative activation (Fig. 4A, Supplemental Fig. 3). These data suggest
that classic activation has a stronger effect on the expression of genes
related with the energetic metabolism, favoring the upregulation of
genes from the glycolytic pathway and the repression of genes encoding for proteins that participate in oxidative phosphorylation.
PFK2 isoenzyme changes in activated macrophages
The aforementioned differences in transcription of the glycolysis
gene set between classic and alternative activation of macrophages
allowed us to hypothesize that PFK2, one of the key enzymes of
the pathway, could be regulated at the protein and/or enzymatic
level. Accordingly, the expression of the isoforms of PFK2 and the
levels of Fru-2,6-P2 were determined under these conditions as an
indication of the capacity of these cells to metabolize glucose (17,
44). As Fig. 4B shows, resting macrophages expressed the L-type
isoenzyme of PFK2, but not the uPFK2 isoenzyme, resulting in
low steady state levels of Fru-2,6-P2. However, as result of the
activation through the classic and innate pathway, but not the
alternative pathway, a robust expression of the uPFK2 isoenzyme occurred, concomitant with a 9-fold rise in the PFK2
activity and a 5-fold increase in the levels of Fru-2,6-P2. Interestingly, the levels of expression of uPFK2, and more importantly, the enzyme activity and intracellular concentration of Fru2,6-P2 exhibited parallel profiles for each activation condition.
Moreover, neither IL-4/IL-13 nor IL-10 was able to change the
expression pattern of PFK2 or to increase Fru-2,6-P2 levels. To
evaluate the capacity of these ILs to influence the response to LPS
costimulation, studies were done. As Fig. 4C shows, when combining IL-10 and IL-4/IL-13 with LPS, the intracellular levels of
Fru-2,6-P2 were only minimally influenced with respect to the
LPS condition, and an important expression of uPFK2 occurred,
despite maintaining a certain level of L-PFK2 expression. The
time course of the changes in Fru-2,6-P2 (Fig. 5A) was compared with that of uPFK2 (Fig. 5B) and exhibited a parallel pattern. Because uPFK2 expression has been described to be regulated by Hif-1a, the levels of this protein were determined and
only the classic pathway was able to promote its expression (Fig.
5B); COX-2 and NOS-2 levels exhibited the expected profile. In
addition to these protein levels, the time course of the expression
of Hif-1a, uPFK2, IL-10, and Glut-1 and Glut-4 was determined,
showing a kinetics compatible with the changes in Fru-2,6-P2
concentration (Fig. 5C). Interestingly, when the mRNA levels
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The metabolic features revealed by glucose consumption and lactate release are followed by a basal consumption of glutamine and
a release of glutamate (Figs. 2A, 3A). Besides, low enrichment in
glutamate and RNA (Fig. 3A) evidenced the high glycolytic rate in
peritoneal macrophages. Peritoneal macrophages are quiescent
cells; however, when the macrophage cell line RAW 264.7 was
used and kept overnight with 1% FCS, stimulation for 12 h under
these conditions revealed that the basal proliferation rate of these
cells did not influence the metabolic profile associated with the
macrophage activation process. As Fig. 3B shows, RAW 264.7 cells
followed a similar m0 (nonlabeled lactate) and m2 (double-labeled
lactate produced from [1,2-13C2]glucose through glycolysis) mass
isotope distribution than peritoneal macrophages, without
differences in the distribution among the different pathways of
stimulation, and with ~80% of the glucose label converted into
lactate. However, proliferation induces higher metabolic fluxes
and [13C] enrichment in ribose and glutamate (enrichment is the
weighted sum of all the labeled species; Fig. 3C).
The Journal of Immunology
609
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FIGURE 2. Metabolic fluxes in macrophages stimulated through the classic, innate, and alternative pathways. The time course of glucose and glutamine
consumption and lactate and glutamate release was determined enzymically by sampling the culture medium at periods of 2 h (A). Macrophages were
loaded with [1,2-13C2]-glucose, and the [13C] trace was followed by GCMS to establish unambiguously the fate of glucose in cells stimulated by the classic,
innate, and alternative pathways. [1,2-13C2]-glucose could follow the oxidative branch of PPP to provide [1-13C1]-ribose (═ line) or the upper part of
glycolysis driving to the [1,2-13C2]-glyceraldehyde-3-phosphate formation (▬ line). Recycling through the nonoxidative branch of PPP allows the formation
of [1-13C1]-glyceraldehyde-3-phosphate and [1,2-13C2]-ribose, respectively. Label pattern does not change in the lower part of glycolysis, and the lactate
formed could drive into the four depicted isotopomers of glutamate, depending on whether pyruvate dehydrogenase or pyruvate carboxykinase is used to
enter in the Krebs cycle (B). The fluxes of glucose consumption and lactate release using [1,2-13C2]-glucose were quantified from 0 to 4 h and from 4 to 12 h
(C). Results show the mean of five experiments (A), and the mean 6 SD of four experiments (C). pp , 0.01 versus the none condition. GCMS, gas
chromatography/mass spectrometry.
of the four PFK2 isoenzymes were determined at 12 h in LPS/
IFN-g–treated macrophages, minimal differences in L-PFK2 expression were observed, whereas uPFK2 and testis-PFK2 were
upregulated by classic activation and downregulated in alternatively activated cells (Fig. 5D).
Macrophage activation in the absence of Hif-1a
uPFK2 has been described to be a gene regulated by Hif-1a (18, 45)
and, therefore, the differences in transcription of the glycolysis gene
set between classic and alternative activation of macrophages might be
explained through the changes in Hif-1a promoted after LPS
activation (23). However, as Fig. 6A shows, targeting of Hif-1a in
macrophages failed to induce changes in the levels of Fru-2,6-P2
(cells treated with IL-10 exhibited a similar behavior as with IL-4/
IL-13), or in the glycolytic flux measured as lactate release (Fig. 6B),
or in the expression of uPFK2 in these cells (Fig. 6C). Moreover,
the mRNA levels of the Hif-1a– and the Hif-1a–targets PHD3,
Glut-1, and, to a lesser extent, Glut-4 exhibited the expected drop in
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METABOLIC FLUXES IN MACROPHAGE ACTIVATION
Hif-1a2/2 macrophages (Fig. 6D); however, those of the proinflammatory cytokines IL-6 and IP-10, and the inflammatory markers NOS-2 and Arg-1, showed a similar expression in Hif-1a+/+ and
Hif-1a2/2 macrophages activated with LPS/IFN-g or IL-4/IL-13,
respectively (Fig. 6E). In agreement with these data, the protein
levels of COX-2, NOS-2, KC, IP-10, uPFK2, and L-PFK2 did not
FIGURE 4. GSEA of energy metabolism pathways
and PFK2 isoenzyme switch in activated macrophages.
The normalized enriched score calculated by GSEA
was shown in the bars, and numbers indicated the FDR
for the enrichment. Positive NES indicates enrichment
in upregulated genes, whereas negative normalized
enriched score corresponds to enrichment in downregulated genes; see Supplemental Material for more
details (A). Peritoneal macrophages were maintained
in culture and stimulated for 12 h, as indicated in Fig.
1. The levels of the L-PFK2 and uPFK2 isoenzymes
were determined by Western blot using specific Abs.
Cell extracts were prepared, and the activity of PFK2
was determined in vitro. The levels of Fru-2,6-P2 were
determined in cell extracts after collection of the cell
pellets in 50 mM NaOH at 80˚C (B). Cotreatment of
cells with LPS and IL-10 or IL-4 results in a decrease
in Fru-2,6-P2 content (C). Results show the mean 6
SD of five experiments. pp , 0.05; ppp , 0.01;
pppp , 0.001 versus resting macrophages (A). pp ,
0.01 versus untreated cells; #p , 0.05 versus the LPS
condition (C).
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FIGURE 3. Metabolic fluxes using tracer-based
distribution of [13C] in stimulated macrophages. Peritoneal macrophages were maintained in culture and
stimulated for 12 h in the presence of [1,2-13C2]
glucose, as indicated in Fig. 1. Samples of culture
medium were collected at 4, 8, and 12 h, and the
[13C] label distribution in lactate (m0, nonlabeled
[13C0]lactate; m1, one label [13C1]lactate; and m2,
two labels [13C2]lactate) was determined by GCMS
to establish the metabolic fluxes (A). The macrophage
cell line RAW 264.7 was stimulated in the presence of
[1,2-13C2]glucose as indicated for peritoneal macrophages, samples of culture medium were collected at
6 and 12 h, and the distribution of the [13C] label was
determined by GCMS to establish the metabolic
fluxes (B). The comparison between peritoneal macrophages and the RAW 264.7 cell line of the main metabolic fluxes and [13C] enrichment in ribose and
glutamate is shown in C. Results show the mean 6
SD of four experiments. pp , 0.001 versus peritoneal
macrophages.
The Journal of Immunology
exhibit differences between Hif-1a+/+ and Hif-1a2/2 macrophages;
Arg-1 protein levels were slightly higher in Hif-1a2/2 cells
activated with IL-4/IL-13 (Fig. 6F).
uPFK2 interference attenuates macrophage activation
In view of the minimal effects observed on metabolic fluxes after
Hif-1a targeting, the interference of uPFK2 expression was investigated. Using a GFP expression vector as a tracer for the estimation of the percentage of transfected cells, the maximal efficiency
was ∼30%, and higher levels of uPFK2 siRNA or scRNA resulted in
a loss of macrophage viability (Fig. 7A). Fig. 7B shows the analysis
of PI-positive cells upon stimulation with LPS/IFN-g or IL-4/IL-13.
Interference of uPFK2 with 100 nM siRNA increased significantly
the apoptosis of LPS/IFN-g–activated cells at 18 h. For this reason,
further analyses were carried out in cells after 10 h of stimulation. As
Fig. 7C and 7D shows, treatment with uPFK2 siRNA decreased the
protein levels of uPFK2, NOS-2, and COX-2, as well as those of the
corresponding mRNAs in macrophages stimulated with LPS/IFN-g.
These data suggest that impairment of the expression of uPFK2
results in attenuation of the classic activation of macrophages.
However, the loss of viability observed after transfection with
siRNA and the moderate efficiency of the process required further
approaches to validate the effects due to a reduction in the expression (or activity) of uPFK2. An additional way to circumvent this
FIGURE 6. Macrophages lacking Hif-1a fail to show changes in the
expression of uPFK2 and in the glycolytic flux upon activation. Peritoneal
macrophages from Hif-1a+/+ and myeloid Hif-1a2/2 mice were activated
through the innate, classic, and alternative pathways, and the
concentrations of Fru-2,6-P2 (A), the lactate flux (B), and the FI of
uPFK2 mRNA (C) were determined. The response of a series of genes
involved in hypoxia (Hif-1a, PHD3, Glut-1, and Glut-4) and inflammation
(IL-6, IL-10, NOS-2, and Arg-1) was determined at different times upon
challenge with LPS/IFN-g or IL-4/IL-13 (D, E). The levels of a panel of
proteins involved in the activation through the innate (polyriboinosinic:
polyribocytidylic acid [poly(I:C)]), classic, and alternative pathways were
determined at 18 h (F). Results show the mean 6 SD of five experiments
(B, C), the means of three independent experiments (A, D, E), or a representative experiment of three (F).
metabolic aspect is to modulate glucose availability, from 5 to 100
mM, and therefore to modify the glycolytic flux. As Fig. 7E shows,
the intracellular levels of Fru-2,6-P2 and the synthesis of lactate and
nitrite in LPS/IFN-g–activated macrophages were modulated by
glucose concentration. However, the protein levels of COX-2,
NOS-2, uPFK2, and Arg-1 apparently did not show significant differences among the range of glucose concentrations assayed (Fig.
7F). At the mRNA level, small, but reproducible differences in
NOS-2 and Arg-1 at 5 mM glucose versus 25–100 mM glucose were
observed in macrophages activated for 8 h with LPS/IFN-g or IL-4/
IL-13, respectively (Fig. 7G). Taken together, these data suggest
a role for uPFK2 expression and activity in the metabolic activation
elicited after LPS/IFN-g treatment of macrophages.
Discussion
Previous work established that peritoneal macrophages are essentially glycolytic cells (13, 14, 41, 42, 46, 47). Compared with
other cell types, macrophages use mainly anaerobic glycolysis in
the metabolization of glucose, and the flux in isolated cells is
∼10% of the actual capacity through 6-phosphofructo-1-kinase,
one of the enzymes that controls the glycolytic pathway (42).
In addition to this, glutamine is converted into glutamate and
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FIGURE 5. Time course of glycolytic changes in activated macrophages. Peritoneal macrophages were activated through the innate, classic,
and alternative pathways, and the time course of the levels of Fru-2,6-P2
was determined (A). The time-dependent changes in uFPK-2/L-PFK2, Hif1a, COX-2, NOS-2, and b-actin (for normalization of lane charge) were
determined by Western blot (B). The changes in mRNA levels as fold
induction (FI) of Hif-1a, uPFK2, IL-10, Glut-1, and Glut-4 were determined during the initial 12 h after activation and using 36B4 mRNA for
normalization (C). The changes in the expression of the mRNA isoforms of
PFK2 (pfkfb) in macrophages activated through the classic and alternative
pathways were compared at 12 h (D). Results show the mean 6 SD of four
experiments.
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METABOLIC FLUXES IN MACROPHAGE ACTIVATION
aspartate, and less than 10% is being oxidized, at the time that
fatty acids appear to be the main substrates for oxidative metabolism (41). In the present work, we have assessed the metabolic
profiles associated with stimulation of macrophages through three
well-defined activation pathways, using a metabolomic approach.
The data obtained show that regardless of the stimulation pathway
involved, macrophages remain glycolytic cells and accelerate notably the conversion of glucose into lactate when challenged through
the classic/innate activation pathways; however, the activation
through the alternative pathway exerted minimal effects on the basal
consumption of glucose. One of the relevant regulators of glucose
metabolism is the rise in the levels of Fru-2,6-P2, which in turn
activates the flux through 6-phosphofructo-1-kinase (43, 48, 49).
Fru-2,6-P2 is synthesized/degraded by the bifunctional enzyme.
Four genes encode the PFK2 in higher mammals. The L type is
encoded by the PFKFB1 gene and is mainly expressed in liver,
and a splicing variant is expressed in muscle. This enzyme has
a balanced kinase/bisphosphatase ratio and, unless the bisphosphatase activity is inhibited, it maintains low levels of Fru-2,6-P2
through a futile cycle of synthesis and degradation of the metabolite
(16, 48–51). The uPFK2 encoded by the PFKFB3 gene has a higher
kinase activity (∼10:1 kinase/bisphosphatase), is induced by
hypoxia, and can be regulated by phosphorylation, playing a role
in the high glycolytic rate of various cell types, such as cancer cells
(18, 43, 45, 49, 52). A relevant finding of this work is the observation
that concomitant to the classic/innate activation, there is a significant change in the expression of the PFK2 from the L type to
the uPFK2 isoenzyme. Interestingly, uPFK2 exhibits a higher capacity to accumulate Fru-2,6-P2 in macrophages, due to its lower
bisphosphatase activity compared with L-PFK2, which results in an
increase in the enzymatic activity determined in vitro, and in the
glycolytic flux. Moreover, the substitution between the two isoenzymes is remarkable, and, in fact, the protein levels of L-PFK2
almost disappear and are substituted by the uPFK2 form in the
course of TLR-2, -3, -4, or -9 activation, despite the use of nonredundant signaling pathways (i.e., MyD88-dependent and independent pathways). One likely mechanism to explain this switch
between PFK2 isoenzymes after classic activation is the rise in the
levels of Hif-1a because a functional relationship has been
described between the hypoxia-responsive element motifs present
in the uPFK2 promoter and the expression of this isoenzyme (16, 18,
19, 22, 23, 53). Accordingly, experiments in macrophages lacking
Hif-1a were carried out with the idea to impair uPFK2 expression.
However, our data clearly show that classic activation retains almost
intact the ability to promote uPFK2 expression, and, indeed, the
glycolytic flux was identical to that of parental macrophages
expressing Hif-1a after LPS/IFN-g challenge. These results were
unexpected in view of the decreased glycolysis observed in B cells
lacking Hif-1a (54). Moreover, a cross-talk between Hif-1a and
LPS signaling has been reported in macrophages, and it appears to
be involved in the development of immune tolerance to LPS and in
sepsis (21, 55, 56). More recently, a specific polarization of classic
and alternative activation of macrophages has been attributed to
Hif-1a and Hif-2a, with a predominant role of Hif-1a in classic
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FIGURE 7. Attenuation of uPFK2 expression
impairs macrophage viability upon activation. Peritoneal macrophages were incubated with different concentrations of a GFP expression vector and siRNA
specific for uPFK2 or the corresponding scrambled sequence scRNA. The levels of expression of GFP are
shown in A. Cells treated with 100 nM scRNA, siRNA
of uPFK2 or the transfection reagent (vehicle) were
stimulated with LPS/IFN-g or IL-4/IL-13, and viability
was determined after 18 h by PI staining (B). The protein
levels of uPFK2, COX-2, and NOS-2 (C) and the FI of
the mRNA of these genes (D) were determined at 10 h.
Alternatively, macrophages were maintained in culture
with 5, 25, or 100 mM glucose in RPMI 1640 medium,
and the levels of Fru-2,6-P2 and the accumulation
of lactate and nitrite in the culture medium were
determined (E). The protein levels at 18 h of genes
related to inflammation and uPFK2 were measured by
immunoblot (F). The mRNA fold inductions of these
genes were determined at 8 h (G). Results show the
mean 6 SD of four experiments. pp , 0.01 versus the
vehicle condition in nonstimulated cells; #p , 0.05
versus the corresponding vehicle condition (B, D);
pp , 0.01 versus the corresponding 5 mM glucose
condition (E).
The Journal of Immunology
due to the recycling through the nonoxidative branch of PPP. Finally, the lower glycolytic flux in RAW 264.7 indicates that proliferation promotes the recruitment of other carbon source like
glutamine in an anaplerotic flux, corroborating the role of glutamine metabolism in macrophage activation already described (12).
In conclusion, the data show that the stimulation of macrophages
through the classic, innate, and alternative pathways exhibits, as
expected, a distinct expression of activation markers due to different signaling events involved in each pathway. However, the
innate and classic activation show a higher glycolytic rate versus
alternative activation, due to a switch in the expression of PFK2
isoenzymes that appears to be a common event after LPS/IFN-g or
TLR-2, -3, -4, and -9 stimulation. Despite the differences in glucose consumption and lactate release, label distribution analysis
shows that there is a common anaerobic pattern followed regardless of the activation pathway. Further studies on the molecular
mechanisms that govern the switch between the PFK2 isoenzymes
might contribute to unravel metabolic aspects related to accommodation of macrophages to biological microenvironments, such
as sepsis, endotoxin tolerance, or tumor growth.
Acknowledgments
We thank Dr. Simon Bartlett for discussion, advice, and critical reading of the
manuscript. We thank Dr. Ramón Bartrons (University of Barcelona) for the
generous gift of the uPFK2 Ab and Dr. M. de Landázuri (Hospital La
Princesa, Madrid) and Dr. Luis del Peso (Instituto de Investigaciones Biomédicas, Madrid) for help on Hif-1.
Disclosures
The authors have no financial conflicts of interest.
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