The document discusses macrophage metabolism and its role in medical diagnosis. It begins by discussing how macrophage polarization involves specific metabolic profiles, with M1 macrophages relying primarily on glycolysis for ATP production while M2 macrophages use both glycolysis and oxidative phosphorylation. Tracer-based metabolomics experiments can help visualize these metabolic signatures and changes that occur during macrophage activation. Specifically, 13C-glucose and 13C-glutamine tracing combined with mass spectrometry can quantify metabolic fluxes and reveal how stimuli like GM-CSF alter glucose and glutamine metabolism in macrophages. This metabolic profiling of macrophages may help study diseases like atherosclerosis and provide methods for visualizing macrophages in lesions using PET tracers like 18F-FDG and 18F-FDM

1. Una nueva visita al metabolismo del macrófago: la
reprogramación de su función y su uso en el diagnóstico médico/
Revisiting macrophage metabolism: Reprogramming macrophage
function and its use in medical diagnosis
Silvia González-Ramos, Marta Paz-García, Marina Mojena, Patricia Prieto-Chinchilla, María
Fernández-Velasco & Lisardo Boscá
IIBm (CSIC-UAM). Madrid, Spain
FUNDACION RAMON ARECES
In the footsteps of Alberto Sols:
a homage on the centennial of his birth
20-21 de febrero, 2017
Alberto Sols
1917-1989

2. Map of age-standardized ischemic heart disease mortality rate per 100 000 persons in 21
world regions, 2010-15, the Global Burden of Disease 2015 Study.
Mozaffarian D et al. Circulation. (2015) doi: 10.1161/CIR0350

3. Inflammation as a pathogenic cause
As initial disorder
•Alzheimer’s disease
•Anaphylaxis
•Asthma
•Atopic dermatitis
•Atherosclerosis
•Crohn’s disease
•Ischaemia-reperfusion injury (occlusive
and embolic stroke and myocardial infarction)
• Cancer
•Multiple sclerosis
•Osteoarthritis
•Psoriasis
•Rheumatoid arthritis
•Systemic lupus
•Type I diabetes mellitus
•Xenograft rejection
As post-inflammatory disorder (fibrosis)
•Pulmonary fibrosis
•Chronic allograft rejection
•Idiopathic pulmonary fibrosis
•Hepatic cirrhosis

4. Macrophages constitute different populations

5. Activation Innate Response
(TLRs, NODs, NLRs LPS, PGN, LTA, dsDNA)
CK, RNI, ROS, MMPs...
Classical Activation: IFNg+TLRs
(CK, RNI, ROS, MMPs...
+MHC class II, APC, CD36, microbicidial
Alternative Activation: IL4/IL-13
Mannose R, dectin-1, arginase, tissue repair, parasite
killing, cell growth, phago/endocytosis
Resolution/deactivation: IL10/TGFb
CD200-CD200R, PPARg, LXR, GR
PGs, reduced MHC-II, increase TGFb
Monocyte
Macrophage
Macrophage stimulation
M1
M2

6. HEATMAP OF Mo/Mφ
Mφ vs. Mo
M1-M2 vs. Mφ
M1: LPS
M2: IL4/IL13
Tresholds: FDR<0.20
Mφ M1 M2
Rodríguez-Prados, J Immunol 185:605-14 (2010)

7. Use of MF metabolic profile in the study
of CVDs (athero-dependent)
oxLDL are main pathogenic molecules in CVDs,
even under statin control of the patient
Bouiller ATVB 26:1169 (2006)

8. The LDL particle
OxLDL are main pathogenic molecules in CVDs,
even under statin control of the patient
ROS
ROS
ROS

10. CumulativerateMACE(%)
•ACS is an inflammatory syndrome
•Systemic Inflammation
•Multifocal vascular inflammation
Stone NEJM 2011
Rogers JIMG 2010
Fused FDG-PET/CT Image
•High residual risk despite statins
•Many events are due to rupture of inflamed, vulnerable plaque
LDL/oxLDL and CVD/ACS: role of macrophages
MACE= major adverse cardiac event
CL/NCL= nonculprit lesion

11. ROS

12. Cryosection from human
coronary atheroma; LDL
immunolocalized (green
fluorescence) in macrophage-
derived foam cells (M) and in
smooth muscle cells (SMC) from
an area next to the internal
elastic lamina (iel). The red
fluorescence represents the Oil
Red-O staining of the lipid
deposits. Nikon Microphot- FXA
microscope (x 500).
Atherosclerosis
 High resolution x-Ray (calcification)
 Echo-doppler (fluxes, share-stress)
 PET/CT with FDG

13. • Innate immune inflammation is a major cause
of residual risk in statin-treated patients
• oxLDL is a key mediator of plaque inflammation
• Anti-oxLDL has profound effects on systemic
inflammation and insulin resistance in non-
hypertense patients, and to reduce residual risk
after ACS
• Use of molecular inflammation imaging with
FDG-PET/CT to test biological hypothesis early
in development
• Opportunity to be best in a new class of
therapies for ACS: macrophage viability, PCSK9
targeting, etc.
LDL/oxLDL and CVD: role of macrophages

14. Macrophage self-renewal in atherogenesis
M H Sieweke, and J E Allen Science 2013;342:1242974

15. How to visualize/interfere macrophages in the
atheroma?
Using PET/SPEC imaging
1: metabolic tracers
18F-2D-glucose
18F-2D-mannose
Other 18F-TRACERS? (FMISO)
2: improving metabolic fitness
increasing the use of glucose  GM-CSF
3: interfering macrophages in the atheroma

16. Profiling of macrophage glucose metabolism
Sols A J Biol Chem 210:581-95 (1954)

17. 2-FDM2-FDGF
none LDL LDLox
1
0
[U-14C]2DGlc,
nmol/mgprotein
2
**
**
*
FDG
FDM
0 5 10 15
Time (min)
hypoxia
2
1
0
**
**
Time (min)
2
1
0
[U-14C]2DGlc,
nmol/mgprotein
0 5 10 15
normoxia
Visualization of macrophages with 18F-2D-Hx
Glc
Tahara N Nat Med 20:215-19 (2014)

18. Visualization of macrophages with 18F-2D-Hx
PET 120 min
FDM in athero
FDG in athero
FDG FDM FDG FDM
ApoE -/-
HFD
Tahara N Nat Med 20:215-19 (2014)

19. none
FDG
FDM
0
5
10
15
20
25
30 MR
GLUT
C ath C ath
18F-2D-Glc vs. 18F-2D-Man
TBR
Time (h)
Lactaterelease,
µmol/mgprotein
1 5
1.5
0.5
0
1 **
Incubation (min)
HKactivity(a.u)
20
40
60
80
100
0 5 10 15
**
***
**
Control
FDG6P
FDM6P
FDG FDM

20. Questions:
• Does MF polarization involve specific fates in glucose/energy
metabolism?
• Are metabolic changes primary events (essential?) for the
polarization? Is oxidative stress involved in commitment?
• Is it possible to ‘visualize’ metabolic inputs in MF polarization?
M1
Mo
M2
metabolic signatures in the development of atherogenesis and CVDs: A dominant
role for ROS and macrophages
Option 2: Metabolic fitness of MF
Rodríguez-Prados, J Immunol 185:605-14 (2010)

21. Adapted from A Garedew and S Moncada, J Cell Science 2010
The main source of ATP in M1 MF is glycolysis

22. Cell Death and Differentiation (2010)
•Glucose used for glycolytic
and mitochondrial ATP synthesis
•Galactose used for mitochondrial
but not glycolytic ATP synthesis
and this ‘glycolytic’ ATP is relevant to maintain MF viability

23. Argus, Science 2013;341:37-38
Dual source of ATP in T cells

24. Tracer-based metabolomics experiments
EXPERIMENTAL DESIGN
Murine or human macrophages
Media from the lower compartment was
collected after 0, 4, 8 and 18 h incubation
in the presence or absence of stimuli
Spectrophotometric analysis
using COBAS Mira for
lactate, glutamine and
glutamate concentrations
GC/MS mass isotopomer
analysis for label distribution:
m0, m1, m2… species
(glucose, lactate, glutamine +
glutamate)
EXPECTED
MASS ISOTOPOMER
INCORPORATIONS

25. Constraint-based method for flux distribution analysis from 13C-label
STEP2: STEADY-STATE CONSTRAINTS
The flux distribution is set to satisfy the
mass balance at steady state:
{Glucose_uptake} x 2 –
{Lactate_excretion} + {Other_sources} –
{PC_utilization} – {PDH_utilization} = 0
{Glutamine_uptake} –
{Glutamate_excretion} +
{α-ketoglutarate_to_Glutamate} –
{other_metabolites_prod} = 0
STEP1: CONSIDERED ASSUMPTIONS
 Primary Mo/MF do not consume lactate
 Lactate produced from glucose
corresponds to 95% of total lower
glycolytic flux
 Only anaplerotic {PC_utilization} and
cataplerotic reaction {α-ketoglutarate 
Glutamate} have been considered
 In the control condition, the flux
{Glutamate_to_other metabolites} is = 0
 Flux {α-ketoglutarate_to_Glutamate} is
proportional to % of 13C incorporation into
glutamate
Rodríguez-Prados, J Immunol 185:605-14 (2010)

26. GC/MS of 13C-glucose in MF

27. Rodríguez-Prados, J Immunol 185:605-14 (2010)
Constraint-based method for flux
distribution from 13C-label
propagation

28. Constraint-based method for flux
distribution from 13C-label propagation
after GM-CSF challenge:
 1-13C-Glc
 U-13C-Gln
Singh P J Nucl Med 57:1428-35 (2016)

29. GM-CSF and CVDs: role of macrophages
Singh P J Nucl Med 57:1428-35 (2016)

30. GM-CSF and CVD: role of macrophages
Singh P J Nucl Med 57:1428-35 (2016)

31. GM-CSF and CVD: human macrophages

32. Key steps in glucose metabolism in Mφ:
GM-CSF-dependent increase
Glc
===========================
Fru-2,6-P2
HK-I
L-PFK2
HK-II
PFKFB3
PKM2 ↓

33. Option 2: GM-CSF infusion allows a direct and functional
view of macrophages in athero lesions
Singh P J Nucl Med 57:1428-35 (2016)

34. Option 3: … and inhibition of glycolysis decreases the
labeling of macrophages in athero lesions after 18F-2D-Glc
uptake
Tawakol A ATVB 35:1463-71 (2015)

35. … exhibiting good fitness btw glycolysis, inflammation and
18F-2D-Glc uptake
Tawakol A ATVB 35:1463-71 (2015)

36. FDM plus GM-CSF infusion to view macrophages
in athero lesions
0
2
4
6
8
10
12
14
16
18
20
GM-CSF
TBR
Basal
HFD
FDG FDM
̶ + ̶ + + (+αCD206)
Singh P J Nucl Med 57:1428-35 (2016) Tawakol A ATVB 35:1463-71 (2015)

37. FDG/FDM/GM-CSF and CVD: role of Mφ
Conclusions
• 2FDM > 2FDG uptake track this response closely (30-40% FDM vs FDG)
• GM-CSF enhances Glut-1/Glut-3 HKII and PFKFBP3 expression in rodent and human Mφ
• Relationship between glycolytic flux and macrophage activation state remains
linear:
• across hypoxic and normoxic conditions
• across several different stimuli
• Hypoxia potentiates the response to GM-CSF in Mφ in glc uptake, which can be
used to trace active plaque formation (ca. 70% vs non-GMCSF)
• Viability of Mφ in the plaque is strictly dependent on anaerobic glycolysis.
 Short infusion of GM-CSF allows the labeling of alive Mφ in the plaque.

38. Participants
Madrid:
M. Mojena
S. González
M. Paz-García
C.E. Rosales
P. Prieto
M. Fernandez
Barcelona:
J. C Rodríguez-Prados
P. de Atauri
S. Marín
M. Cascante
Boston (MGH):
A. Tawakol
P. Signh
NY (MSMS)
J. Narula

40. Key steps in metabolism in MF