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Mitochondrial dysfunction prevents repolarization of inflammatory macrophages
Jan Van den Bossche, PhD
j.vandenbossche@amc.nl Departement of Medical Biochemistry Experimental Vascular Biology group
Postdoc Grant 2013T003
Mitochondrial dysfunction prevents repolarization of inflammatory - - PowerPoint PPT Presentation
Mitochondrial dysfunction prevents repolarization of inflammatory - - PowerPoint PPT Presentation
Mitochondrial dysfunction prevents repolarization of inflammatory macrophages Jan Van den Bossche, PhD j.vandenbossche@amc.nl Departement of Medical Biochemistry Experimental Vascular Biology group Postdoc Grant 2013T003 Macrophage functions
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Macrophage plasticity and repolarization are key features of macrophages
Sica and Mantovani, JCI, 2012
M1 M2
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Macrophages are plastic
1/ Respond to distinct environmental stimuli 2/ Reverse their phenotype = repolarization
M1 M(LPS+IFNg) M2 M(IL-4)
Kill tumor cells Promote tumor growth Promote inflammation Anti-atherogenic
Colombo, 1992 IFNg
Atherosclerosis
epigenetics metabolism
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Reshaping macrophage (re)polarization for atherosclerosis therapy
iNOS+ CD206+
A
Stöger et al., 2012
M(LPS+IFNg) M(IL-4)
M1 M2
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Reshaping macrophage (re)polarization for atherosclerosis therapy
iNOS+ CD206+
A
Stöger et al., 2012
M(LPS+IFNg) M(IL-4)
M1 M2
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M1s fail to repolarize to M2s
M1 M2
LPS+IFNg M2 M1 M2 N
BMM
IL-4 untreated IL-4 untreated untreated
compare
M2 surface markers and function
LPS+IFNg→IL-4 isotype naive IL-4
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M1s fail to repolarize to M2s in vivo
M1 M2
CD71 CD301 CD206
i: CD45.1+ F4/80 + macrophages
Ex vivo stimulation
- Naive (N)
- LPS+IFNg
transfer
CD45.1 CD45.2
IL-4c/PBS BMM F4/80 CD45.1
i
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Human M1s fail to repolarize to M2s
M1 M2
LPS+IFNg M2 M1 M2 N IL-4 untreated IL-4 untreated untreated
compare
MoDM
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Why are M1 macrophages not plastic?
Because they don’t have a IL-4Ra? Because they have dysfunctional STAT6 activation?
IL-4Ra
STAT6
JAK
IL-4
IL-4Ra isotype IL-4Ra DMFI (x10E3) STAT6-P b-actin
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Why are M1 macrophages not plastic?
Because they are dead?
SDH
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Why are M1 macrophages not plastic?
Because their metabolism doesn’t allow it?
LPS+IFNg IL-4 Naive
Glycolysis stress test Mito stress test
OM FCCP ROT/AA GLUC OM 2-DG
M1 = high glycolysis, low OXPHOS M2 = high OXPHOS
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Seahorse intermezzo
Because their metabolism doesn’t allow it?
Glycolysis & mito stress test combined
Van den Bossche et al., JoVE, 2015
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Seahorse intermezzo
Because their metabolism doesn’t allow it?
Glycolysis & mito stress test combined :
Van den Bossche et al., JoVE, 2015
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Seahorse intermezzo
Because their metabolism doesn’t allow it?
Glycolysis & mito stress test combined
Van den Bossche et al., JoVE, 2015
Add pyruvate together with FCCP to allow max respiration Use 2-DG to verify that ECAR is indeed ‘true’ glycolysis
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Why are M1 macrophages not plastic?
Because their metabolism doesn’t allow it?
M1 = high glycolysis, low OXPHOS M2 = high OPXHOS
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Why are M1 macrophages not plastic?
IL-4 does not induce OXPHOS upon M1→M2 repolarization
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Why are M1 macrophages not plastic?
IFNg+LPS induces mitochondrial dysfunction Which complexes are affected?
SDH = complex II assay
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Measure complex I-IV activity of the ETC with the Seahorse
Nature protocols, 2014
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Measure complex I-IV activity of the ETC with the Seahorse
Nature protocols, 2014 complex II activity
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Measure complex I-IV activity of the ETC with the Seahorse
IFNg+LPS mainly hit complex I and II activity
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M2 polarization needs mitochondrial function
FA ETO
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30 min 24h M1 (or naive)
iNOS inhibition prevents the drop in OXPHOS in M1
N 2DG NAC (ROS scavenger) 1400W (iNOS inhibitor)
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24h 24h Stimulation 1 Stimulation 2 (after wash) M2 M1 1400W + M1
iNOS inhibition improves metabolic reprogramming by IL-4
isotype naive 14000+LPS+IFNg → IL-4 IL-4 LPS+IFNg → IL-4
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24h 24h Stimulation 1 Stimulation 2 (after wash) M2 M1 1400W + M1
iNOS inhibition improves M1 to M2 repolarization
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a
isotype naive LPS+IFNg WT iNOS-/- IL-4 DMFI (%) CD206 CD71 CD301 WT iNOS-/- WT iNOS-/- WT iNOS-/-
b c d e
OM FCCP AA/Rot
f
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Macrophage Immunometabolism : Where Are We?
Van den Bossche et al., Cell Reports, 2016
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Van den Bossche et al., Trends in Immunology, 2017
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Junior Postdoc Grant 2013T003