Contribution of microglia to tissue injury and repair in MS

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1 Contribution of microglia to tissue injury and repair in MS MS disease course histologic features Courtesy of Samuel Ludwin I ACUTE CHRONIC s ACTIVE CHRONIC Clinical Course Intra CNS Extra CNS Imaging Course - Is inflammation needed for new lesions - What drives the inflammatory process? - role of CNS micro-environment - Is their reversible sub-lethal injury early and late? - Is their potential for CNS directed therapies? 1

2 Model of initial outside in lesion formation in MS 1. Activation Th 3. Attraction 4. Invasion 2. Adhesion 5. Reactivation 1 4 targets of current therapies Periphery BBB CNS Events behind the BBB what is contribution of CNS microenvironment? MTR decreases in pre-lesions Time 1 Time 2 Time 3 Lesion MTR (%) Normal WM MS NAWM Time Since Lesion Appearance (years) Bruce Pike 2

3 Myeloid cells in the CNS microglia/macrophages Courtesy of 3

4 Identification of a microglia signature by gene expression and quantitative mass spectrometry (AffyExon1 & MG400 chip) Nat. Neurosci Jan;17(1): qpcr validation of microglial genes in human microglia Nat. Neurosci Jan;17(1):

5 Figure 2 Micro-RNA (Mir 155 ) expression in microglia vs macrophages A Control Tissue Low magnification High magnification Post-LCM of single C cells Parenchyma Control vs. MS B Perivascular Inflamed MS Tissue D Control Low magnification High magnification Post-LCM of single cells E MS Parenchyma Moore/Rao Functions of CNS Myeloid Cells M1/M2 Model Adapted from Shechter et al., J. Pathol.,

6 Antigen presentation Cytotoxicity Tissue remodeling Neurodegenerative role Tumor destruction Astrogliosis TNF-α IL-6 IL-1β il-23 * GM-CSF LPS IFNγ CD80 HLA-DR CCR 7 TLR4 Activation M1 Inhibition/Q uiescence M2 CD163 CX3CR1 SIRP1α CD206 CD209 M-CSF IL-4 IL-13 TREM-2 M-CSFR M-CSF * CD200R CX3CL1 CD47 CD200 TGF-β IL-10 IL-13 Immunoregulation Phagocytosis Resting signals Survival signals Tumor promotion Neuron: Soluble/surface quiescence receptors ligands Pro-Inflammation Quiescence / Anti-Inflammation Members of the innate immune system that sense and respond to events occurring in the CNS environment Stranger Signals products of infectious agents Pathogen Associated Molecules (PAMPs) - Microglia - Blood-derived myeloid cells Toll-like Receptor (TLR) Signals from immune system Danger Signals CNS tissue injury Danger Associated Molecules (DAMPs) Effect on neural cells: - phagocytosis - toxic products Effect on Immune cells: recruitment antigen presentation 6

7 Functional Properties of Microglia Migration Clearance of injured Tissue Phagocytosis Tissue Injury soluble factor and cell contact dependent Tissue repair support remyelination Figure 3 - Lambert et al. ATP effects on human myeloid cells Comparison of migration responses of adult human microglia and macrophages to chemokines and ATP macrophages microglia B % of max CCR2 number of cells microglia 0.5% BSA M-CSF (25 ng/ml) ATP (um) Number of cells macrophages M-CSF (ng/ml) MCP-1 ATP (um) 0.5% BSA (ng/ml) 7

8 In vitro P2Y12 expression in human myeloid cells Human Adult Microglia CD68 P2Y12 MERGE ADP is the natural agonist and leads to intracellular increases in calcium, and receptor desensitization Human Monocyte-derived Macrophages Clopidogrel (Plavix), Prasugrel, and Ticlopidine (Ticlid) are P2Y12 antagonists and clinically used as antiplatelet agents P2Y12 Expression is Increased in M2-polarized Human Myeloid Cells Human Adult Microglia Human Fetal Microglia Fold Change (P2Y12 expression) * Unpol. M1 M2 Fold Change (P2Y12 expression) *** Unpol. M1 M2 Fold Change (P2Y12 expression) Human Macrophages * Unpol. M1 M2 8

9 Using single cell calcium imaging, a P2RY12 agonist (ADP) induces an intracellular calcium response in human microglia that is blocked by a selective P2RY12 antagonist P2Y12 agonist (ADP) B P2Y12 agonist (ADP) 340/380 P2Y12 antagonist Human Microglia Rat Microglia Using single cell calcium imaging, a P2Y12 agonist (ADP) failed to induce an intracellular calcium response in human macrophages 340/380 P2Y12 agonist (ADP) ATP ATP Time (sec) Time (sec) Human Macrophages 9

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11 In SPMS white matter, P2Y12 is expressed by phagocytic microglia. lesion decreased P2Y12 receptor immunoreactivity in proximity to the lesions is directly correlated with the extent of demyelination found in all types of gray matter cortical plaques (I-III) and subcortical white matter. Amadio S et al. Cereb. Cortex 2010;20: The Author Published by Oxford University Press. All rights reserved. For permissions, please journals.permissions@oxfordjournals.org M2c M2a M1 M TGF M0 Flow cytometry analysis of polarized microglia s ability to phagocytose human myelin, indicating an increase in phagocytosis by M2c polarized microglia and a decrease in M1 associated phagocytosis. Confocal image of human adult microglia showing a cell containing phagocytosed, fluorescing myelin adjacent to a nonphagocytizing cell. 11

12 Non antigen determined selective injury of neural cells by innate immune mechanisms lymphocyte Microglia/macrophage glutamate TCR TNFα TRAIL FasL MHC TNFR DR4/5 Fas Glutamate receptor JNK activation Caspase cascade Ca 2+ Apoptotic cell death Oligodendrocyte or progenitor cell Human M1 supernatants significantly decrease percentage of O4+ cells in vitro Human Macrophages Human Microglia Media alone M1 media M2 media 12

13 Oxidized lipids in multiple sclerosis lesions. Haider L et al. Brain 2011;134: free radical iron has the same distribution The Author(s) Published by Oxford University Press on behalf of Brain. Microglia directed therapy DMF/MMF effects on myeloid cells Michel Robinson AAN 2015 Anti Oxidant Anti Inflammatory Neuroprotection Induces anti oxidant response element genes Binds KEAP 1, activates Nrf2 others? Increase the cell s ability to mitigate oxidative stress Inhibits production of inflammatory mediators Effects via NFkB, HO 1, others? Inhibits migration of neutrophils into CNS during EAE HCAR

14 50uM DMF is not cytotoxic to microglia AQUA Stain Live/Dead 5000 total events Utx UTx + 0.1% Triton X 100 Vehicle + LPS LPS DMF 10 um + LPS DMF 50 um + LPS MMF 10 um + LPS MMF 50 um + LPS Calcein AM (Green) + Propidium Iodide (Red) DMF induces HMOX1 in microglia in vitro adult fetal 14

15 DMF has anti inflammatory activity on human microglia in vitro Expression of MMF receptor HCAR2 in Human Microglia greater than Astrocytes 15

16 In vivo imaging of myeloid cells TSPO directed CNS directed therapies Will they work? 16