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 Anti-inflammatory Promote inflammation Mediate Type 2 inflammation in allergy Bacterial killing Tissue repair Kill tumor cells Wound healing Promote tumor growth Host defense
Macrophage plasticity and repolarization are key features of macrophages M2 M1 Sica and Mantovani, JCI, 2012
Macrophages are plastic 1/ Respond to distinct environmental stimuli 2/ Reverse their phenotype = repolarization Atherosclerosis M1 Promote inflammation Kill tumor cells M (LPS+IFN g ) epigenetics metabolism Colombo, 1992 IFN g M2 Anti-atherogenic Promote tumor growth M (IL-4)
Reshaping macrophage (re)polarization for atherosclerosis therapy M1 iNOS+ M (LPS+IFN g ) M2 A CD206+ Stöger et al., 2012 M (IL-4)
Reshaping macrophage (re)polarization for atherosclerosis therapy M1 iNOS+ M (LPS+IFN g ) M2 A CD206+ Stöger et al., 2012 M (IL-4)
M1s fail to repolarize to M2s untreated untreated N untreated IL-4 M2 BMM LPS+IFN g IL-4 compare M1 M2 M2 surface markers and function isotype M1 naive IL-4 LPS+IFN g → IL-4 M2
M1s fail to repolarize to M2s in vivo i Ex vivo stimulation CD45.1 - Naive (N) BMM transfer IL-4c/PBS CD45.1 - LPS+IFN g CD45.2 F4/80 i: CD45.1 + F4/80 + macrophages M1 CD71 CD206 CD301 M2
Human M1s fail to repolarize to M2s untreated untreated N untreated IL-4 M2 MoDM LPS+IFN g IL-4 compare M1 M2 M1 M2
Why are M1 macrophages not plastic? Because they don’t have a IL-4R a ? IL-4 IL-4R a JAK isotype IL-4R a IL-4R a D MFI (x10 E 3) STAT6 Because they have dysfunctional STAT6 activation? STAT6-P b -actin
Why are M1 macrophages not plastic? Because they are dead? SDH
Why are M1 macrophages not plastic? Because their metabolism doesn’t allow it? Glycolysis stress test Mito stress test GLUC OM 2-DG OM FCCP ROT/AA Naive LPS+IFN g IL-4 M1 = high glycolysis, low OXPHOS M2 = high OXPHOS
Seahorse intermezzo Because their metabolism doesn’t allow it? Glycolysis & mito stress test combined Van den Bossche et al., JoVE, 2015
Seahorse intermezzo Because their metabolism doesn’t allow it? Glycolysis & mito stress test combined : Van den Bossche et al., JoVE, 2015
Seahorse intermezzo Because their metabolism doesn’t allow it? Glycolysis & mito stress test combined Use 2-DG to verify that ECAR is indeed ‘true’ glycolysis Add pyruvate together with FCCP to allow max respiration Van den Bossche et al., JoVE, 2015
Why are M1 macrophages not plastic? Because their metabolism doesn’t allow it? M1 = high glycolysis, low OXPHOS M2 = high OPXHOS
Why are M1 macrophages not plastic? IL-4 does not induce OXPHOS upon M1→M2 repolarization
Why are M1 macrophages not plastic? IFN g +LPS induces mitochondrial dysfunction Which complexes are affected? = complex II assay SDH
Measure complex I-IV activity of the ETC with the Seahorse Nature protocols, 2014
Measure complex I-IV activity of the ETC with the Seahorse Nature protocols, 2014 complex II activity
Measure complex I-IV activity of the ETC with the Seahorse IFN g +LPS mainly hit complex I and II activity
M2 polarization needs mitochondrial function FA ETO
iNOS inhibition prevents the drop in OXPHOS in M1 30 min 24h N M1 (or naive) 2DG NAC (ROS scavenger) 1400W (iNOS inhibitor)
iNOS inhibition improves metabolic reprogramming by IL-4 24h 24h Stimulation 1 Stimulation 2 (after wash) M2 M1 1400W + M1 naive isotype IL-4 LPS+IFN g → IL-4 14000+LPS+IFN g → IL-4
iNOS inhibition improves M1 to M2 repolarization 24h 24h Stimulation 1 Stimulation 2 (after wash) M2 M1 1400W + M1
OM FCCP AA/Rot b a c CD71 WT iNOS -/- CD206 WT iNOS -/- CD301 WT iNOS -/- LPS+IFN g naive IL-4 D MFI (%) WT d iNOS -/- e isotype f
Macrophage Immunometabolism : Where Are We? Van den Bossche et al., Cell Reports, 2016
Van den Bossche et al., Trends in Immunology, 2017
Junior Postdoc Grant 2013T003 Medical Biochemistry Genetic Metabolic diseases Experimental Vascular Biology group Michel van Weeghel Riekelt Houtkooper Jeroen Baardman Vincent de Boer Menno de Winther & Esther Lutgens
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