Microglia. Learning objectives. Learning objectives. Wendy Noble

Transcription

1 Microglia Wendy Noble Learning objectives To know the origins, morphologies and main functions of microglia To be able to distinguish different phenotypes of microglia To understand how microglia interact with neurons To appreciate the contribution of microglia to neuroinflammation and neurodegenerative disease Learning objectives To know the origins, morphologies and main functions of microglia To be able to distinguish different phenotypes of microglia To understand how microglia interact with neurons To appreciate the contribution of microglia to neuroinflammation and neurodegenerative disease 1

2 Microglia Resident macrophages of the CNS Constitute 5-15% of total cellular composition of the CNS Dynamic cells - phenotype changes under different conditions: effects on microglia function Macrophages in the CNS Brain has several heterogenous populations of macrophages Have homeostatic and surveillance actions at distinct sites Prinz and Priller, 2014 The origin of microglia Embryonic and postnatal development of microglia in mice Microglia derived from erythroid/myeloid progenitor stem cells that are formed at embryonic day 7.5 (E7.5) E8.0 in the yolk sac in mice. This is quite distinct from the origin of other macrophage populations. As they differentiate (A1-A2), theybegin to express many typical microglial markers, and migrate to the brain These microglia are thought to sustain local microglial populations Prinz and Priller,

3 Microglial morphology Ramified microglia - often found in resting state (M0 phenotype) - cells have many short fine processes that they use to monitor the local environment Microglial morphology Amoeboid microglia (M1) - Usually active microglia - commonly found in developing and damaged brain - spherical in shape, lack processes, and contain numerous phagocytic vacuoles Microglial morphology Rod cells - Commonly found in conditions such as chronic measles infection - Markedly elongated nuclei, scanty cytoplasm and few processes Multinucleated cells - Found in mycobacterial infection and around amyloidcontaining vasculature - Giant cells, multiple nuclei Epithelioid macrophages - Found in chronic infections such as tuberculosis and leprosy - Many microglia cluster to form granulomas Dystrophic microglia - Commonly found in older age/in age-related diseases - Beading of microglial processes 3

4 Main functions of microglia Function Modulation of immune responses and oxidative stress Pathogen recognition (innate immunity) Antigen presentation Recognition of bound antibody (adaptive immune function) CNS development Stem cell regulation Lipid transport Examples Production and secretion of anti- and pro-inflammatory cytokines, reactive oxygen species (ROS) Microglial receptors recognise evolutionarily conserved antigens on pathogen surfaces (pathogen-/damage-associated molecular patterns e.g. LPS, -amyloid) Present pathogens bound to MHC for activation of T lymphocytes. Relevant in neuroinflammation and autoimmune disease. Respond to antibodies bound to pathogens (opsonization). Relevant in autoimmune diseases (e.g. demyelination) Phagocytosis (pruning of redundant synapses/neurons) Influence of microglial secretions (cytokines, neurotrophins, growth factors) Regulation of stem cell proliferation (e.g. granule cells in hippocampus) Secretion of lipoprotein particles that deliver lipids to neurons for membranes/synapse maintenance Main functions of microglia Phagocytosis Ingestion and destruction by digestive enzymes in lysosomes of: Neurons and other damaged cells (e.g. in neurodegenerative diseases, Wallerian degeneration, tract degeneration) Misfolded/aggregated proteins Synapses (during development and in disease) Myelin (e.g. multiple sclerosis) Micro-organisms (e.g. abscess) Virally infected cells (e.g. herpes encephalitis) Erythrocytes and haemoglobin breakdown products (e.g. haemosiderin) following haemorrhage Part 1 Summary Microglia are resident macrophages of the CNS They are dynamic cells, with alterations in morphology reflecting changes in their activation state/function They play many important roles in the CNS, including: - During development and disease - presentation of antigens, - induction and mediation of inflammatory states - phagocytosis of damaged cells, excess and damaged synapses, abnormal proteins and other pathogens 4

5 Learning objectives To know the origins, morphologies and main functions of microglia To be able to distinguish different phenotypes of microglia To understand how microglia interact with neurons To appreciate the contribution of microglia to neuroinflammation and neurodegenerative disease Microglial phenotypes Originally described for peripheral macrophages broadly apply to microglia (M0 phenotype) ramified amoeboid M1: Proinflammatory M2: Antiinflammatory amoeboid/ ramified Promote inflammation, cause cytotoxicity, demyelination and axonal damage Scavenging of debris, tissue modelling and repair Phenotyping of microglia - morphology 5

6 Immunophenotyping of microglia Microglial marker Iba1 CD163 HLA-DR CD68 MSR-A CD64 (Fc RI) CD32 (Fc RII) CD16 (Fc RIII) CD206 (Mannose R) Fizz1 CD14 (TLR-4) Function Ionized calcium-binding adaptor molecule 1: resting and activated microglia Perivascular macrophage and macrophage-like microglia in areas of blood brain barrier breakdown/scavenger receptor for the haemoglobin haptoglobin complex Cell surface homologue of MHC II antigen presenting function Microglial lysosomes phagocytosis Macrophage scavenger receptor-a cell surface protein lipoprotein receptor involved in direct ligand recognition High affinity receptor for IgG role in mounting immune response Low affinity receptor for IgG phagocytosis of immune complexes and regulation of inflammation Antibody-dependent binding, uptake and killing pathogens Phagocytosis and endocytosis of endogenous and exogenous proteins Inhibitor of inflammation Receptors for bacterial lipopolysaccharide, Gram-/associated with classical (M1) activation Immunophenotyping of microglia Resting Activated Phenotyping of microglia cytokine arrays E.g. Antibody arrays: controls Membranes pre-spotted with antibodies against a panel of cytokines Cytokines in sample bind to their respective antibodies Detection of relative signal intensities allows comparison of changes in protein amounts Increase in proinflamm mediators Might suggest a change to which phenotype? 6

7 Techniques for assessing microglial phenotype Technique Advantages Disadvantages Morphological analyses Immunohistochemistry Cytokine detection (WB, ELISA, array) Easy, quick, cheap. Can use stored/fixed tissues. Provides info on distribution/localisation. Can be combined with morphological assessment. Multiplexing possible. Relatively quick. Good quality data. Limited information on microglial function (except phagocytosis) Antibodies provide limited information on phenotype. Needs to be sensitive to detect small amounts. Fresh tissue needed. Gene expression analyses Proteomics In vivo imaging Definitive detection of microglial activation status (M1, M2 etc) mrna is labile. Needs fresh tissue. Can identify large numbers Not very sensitive (most existing of proteins simultaneously in techniques detect only few small sample amounts. proteins of interest) Real time information about changes in microglial activation Requires live animals/patients. Expensive PET ligands (freshly synthesised). Part 2 Summary Several techniques are used to distinguish between microglial phenotype, each with their own advantages and disadvantages The most commonly used methods are immunohistochemistry (IHC) combined with morphological assessment. Most accurate method is analysis of changes in gene expression pathways In vivo analysis allows determination of changes in mocroglial activity and localisation in real time, but resolution is poor Learning objectives To know the origins, morphologies and main functions of microglia To be able to distinguish different phenotypes of microglia To understand how microglia interact with neurons To appreciate the contribution of microglia to neuroinflammation and neurodegenerative disease 7

8 Neuronal on/off switches for microglia Neuron on/off switches for microglia 1. Resting signals keep microglia in non-activated surveillance state CD200 CD200R Neuronal glycoprotein CD200 activates microglial CD200R to keep microglia in resting state CX3CL1 CX3CL1R (Fractalkine R) CX3CL1R activation keeps microglia in quiescent state Also contributes to basal microglial surveillance (motility and dynamics of microglial processes) Neuron on/off switches for microglia 2. Don t eat me signals cell does not need to be phagocytosed/cleared CD47 Sirp alpha CD47 (membrane protein) interacts with the myeloid inhibitory immunoreceptor SIRPα to drive a downregulatory signal that inhibits host cell phagocytosis. 8

9 Neuron on/off switches for microglia 3. Eat me signals this cell should be cleared by phagocytosis 1. Phosphatidylserine (phospholipid membrane component) exposed on neurons: When bound by opsonins milk fat globule EGF factor 8 (MFG-E8) or growth-arrest specific protein 6 (GAS6) bind and activates vitronectin receptor (VNR) When bound by GAS6 or Protein S bind and activates MER receptor tyrosine kinase (MERTK) Binds directly to brain-specific angiogenesis inhibitor 1 (BAI1). 2. Neuron-exposed calreticulin or C1q activate low-density lipoprotein receptor related-protein (LRP). 3. C1q deposition on de-sialylated glycoproteins converts C3 to the opsonin C3b which activates CR3 and its signalling partner, DAP12. Neuron on/off switches for microglia 4: Help signals this cell is injured. Often results in a toxic pro-inflammatory microglial response (P2X7) Activation of purinoceptors (P2X, P2Y) by ATP, ADP etc leads to: Microglial branch extension towards the injury site. Synthesis and release of proinflammatory cytokines (e.g. IL-1beta) Neuron on/off switches for microglia 5: Survival signals provides neuroprotection, support or regeneration Activation of colony stimulating factor 1 receptor (Csf1R) by Csf1 and IL-34 induces cell survival or proliferation 9

10 Part 3 Summary Signalling from neurons to microglia is complex. There are five main types of signal: Resting Don t eat me Eat me Help Survival Generally involves signal released from neurons activating a microglial receptor Results in different phenotypic states in microglia inducing specific functions relevant to neighbouring cells High dynamism and flexibility of microglial responses essential to allow them to maintain CNS environment. Microglial interactions with astrocytes and neurons adds another level of complexity... Will be discussed in cytokine lecture Learning objectives To know the origins, morphologies and main functions of microglia To be able to distinguish different phenotypes of microglia To be aware of the physiological and pathological interactions of microglia with astrocytes and neurons To appreciate the contribution of microglia to neuroinflammation and Alzheimer s disease Alzheimer s disease AD brain is characterised by deposits of -amyloid (A ) in plaques and intracellular neurofibrillary tangles of hyperphosphorylated aggregated tau The amyloid cascade hypothesis positions A upstream of tau changes and neurodegeneration Inflammation is also a prominent feature of AD brain that accompanies A and tau accumulations and neurodegeneration 10

11 Microglia in Alzheimer s disease Microglia (blue) within A plaques (brown) in AD brain Microglia in AD brain produce the proinflammatory cytokine IL-1 Activated microglia (green) associate with plaques (red) in a transgenic mouse model of AD that forms plaques Heneka et al., 2015 Microglia in healthy brain Heneka et al., 2014 Microglia in Alzheimer s brain A and other damage-associated molecular pathogens activate microglia via pattern recognition receptors (PRRs) Heneka et al.,

12 Microglia in Alzheimer s brain A and other damage-associated molecular pathogens activate microglia via pattern recognition receptors (PRRs) Heneka et al., 2014 The NLRP3 inflammasome Three main components: 1. NLRP3: NLR family, pyrin (PYD) domaincontaining ASC: Apoptosisassociated speck-like protein containing a caspase-recruitment domain (CARD) 3. Pro-caspase-1 The NLRP3 inflammasome in microglia in AD Neurodegeneration A activates microglial receptors to induce the inflammasome and NF B driven increased transcription of inflammatory mediators Altered tau phosphorylation and aggregation A NMDAR IL-1R microglia neuron 12

13 The NLRP3 inflammasome in microglia in AD Goldmann et al., 2013 Part 4 Summary Activated microglia associate with A plaques in Alzheimer s disease (AD) Microglial phenotype is altered by the local environment in AD Microglia contain all components of the NLRP3 inflammasome The NLRP3 inflammasome is activated by A to drive neuroinflammation, including the increased synthesis and release of proinflammatory mediators. Knocking out components of the NLRP3 inflammasome in microglia is protective in transgenic mouse models of AD. Recommended Reading 1. Boche D, Perry VH, Nicoll JAR (2013). Activation patterns of microglia and their identification in the human brain. Neuropath Applied Neurobiol. 39(1), Prinz M, Priller J (2014). Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease. Nat Rev Neurosci. 15, Heneka MT, Kummer MP, Latz E. (2014). Innate immune activation in neurodegenerative disease. Nat Rev Immunol. 14(7): Heneka MT et al. (2015). Neuroinflammation in Alzheimer's disease. Lancet Neurol. 14(4):