JOURNAL OF NEUROCHEMISTRY
| 2014 | 130 | 165–171
doi: 10.1111/jnc.12705
*MRC Centre for Reproductive Health, The Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK †Wellcome Trust and MRC Cambridge Stem Cell Institute and Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
Abstract Microglia are the resident macrophages of the central nervous system that survey the microenvironment for signals of injury or infection. The response to such signals induces an inflammatory response involving macrophages derived from both resident microglia and recruited circulating monocytes. Although implicated as contributors to autoimmune-mediated injury, microglia/ macrophages have recently been shown to be critical for the important central nervous system regenerative process of remyelination. This functional dichotomy may reflect their ability to be polarized
along a continuum of activation states including the wellcharacterized cytotoxic M1 and regenerative M2 phenotypes. Here, we review the roles of microglia, monocytes and the macrophages which they give rise to in creating lesion environments favourable to remyelination, highlighting the specific roles of M1 and M2 phenotypes and how the pro-regenerative role of the innate immune system is altered by ageing. Keywords: central nervous system, inflammation, macrophage, microglia, myelin, remyelination. J. Neurochem. (2014) 130, 165–171.
Macrophages are myeloid cells involved in surveying tissue environments for damage or infection. Signals indicative of injury induce macrophage responses such as phagocytosis of debris/apoptotic cells, host defence against infections (e.g. antigen presentation), secretion of growth factors, toxic molecules and cytokines/chemokines. Macrophage functions extending beyond a response to injury include regulation of angiogenesis and proliferation, differentiation, migration, and survival of progenitors in healthy tissue. In the central nervous system (CNS), the resident macrophage is the microglia, which represents 5–12% of brain cells (Lawson et al. 1990). Microglia regulate fundamental developmental processes such as neurogenesis, neural precursor migration, survival and apoptosis, axonal pruning and growth and angiogenesis (Aarum et al. 2003; Marin-Teva et al. 2004; Miller et al. 2009; Antony et al. 2011; Lu et al. 2011). Loss of microglia causes severe perturbations in brain development (Erblich et al. 2011). In the adult brain, regional variability in microglial densities exists, being highest in the grey matter of the hippocampus, olfactory telencephalon, basal ganglia and substantia nigra (Lawson et al. 1990; Yang et al. 2013). Even within a given brain region, microglia can show variability in function and gene expression (Kremlev et al. 2004; Olah et al. 2011; Lai and McLaurin 2012). Following injury in the developing or adult CNS, microglia
activation is the first cellular response, as shown in models of traumatic brain injury, demyelination, inflammation, excitotoxic injury and ischaemia–hypoxia. Following injury, microglia proliferate and extend processes to the site of damage, responding to chemoattractant signals released from injured cells (e.g adenosine-50 -triphosphate (ATP), CXCL10) (Davalos et al. 2005; Haynes et al. 2006). Whereas in the healthy CNS circulation-derived macrophages are not present, monocytes from the circulation are recruited into the
Received January 24, 2014; accepted February 24, 2014. Address correspondence and reprint requests to Veronique E. Miron, MRC Centre for Reproductive Health, The Queen’s Medical Research Institute, Edinburgh, EH16 4TJ, UK. E-mail: [email protected] Abbreviations used: ATP, adenosine triphosphate; CCL, chemokine C-C motif ligand; CCR, C-C chemokine receptor; CD, cluster designation; CNS, central nervous system; CSF1, colony stimulating factor 1; CXCL, chemokine C-X-C motif ligand; DAMPS, damage-associated molecular patterns; EAE, experimental autoimmune encephalomyelitis; GFP, green fluorescent protein; Gpr, Probable G protein-coupled receptor; IGF1, insulin-like growth factor 1; IL1Ra, interleukin 1 receptor antagonist; IL, interleukin; iNOS, inducible nitric oxide synthase; NLR, nod-like receptor; OPC, oligodendrocyte progenitor cell; PAMPS, pathogen-associated molecular patterns; RLR, RIG-1-like receptor; TGF-b, transforming growth factor receptor beta; TLR, tolllike receptor; Tmem, transmembrane protein; TNF-a, tumour necrosis factor alpha; TREM2, triggering receptor expressed on myeloid cells 2.
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CNS following injury or infection where they then typically undergo differentiation into macrophages. Microglia/ macrophages subsequently phagocytose debris/dead cells and secrete a multitude of factors (e.g. cytokines, growth factors, reactive species) that influence regenerative processes. This article reviews the diverse functions and activation states of microglia/ macrophages and how these cells contribute to the CNS regenerative process of remyelination.
Microglia/ macrophage function and activation Microglia/ macrophages have long been implicated in inducing neural pathology by secretion of toxic molecules (e.g. glutamate and reactive oxygen/nitrogen species), antigen presentation to cytotoxic T lymphocytes and degradation of synapses (Blinzinger and Kreutzberg 1968; Banati et al. 1993; Cash et al. 1993). In support of this concept, dampening macrophage activation in spinal cord injury, traumatic brain injury and hypoxic-ischaemic injury using minocycline improves recovery in adult rodents (Sanchez Mejia et al. 2001; Arvin et al. 2002; Wells et al. 2003). In addition, infiltration of the CNS by monocytes has been identified as a trigger for disease progression in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) (Ajami et al. 2011). However, recent studies have elucidated the regenerative role of macrophages in the CNS, and in particular in the regeneration of myelin (remyelination), the focus of the current review. Remyelination following a demyelinating insult is inhibited when macrophage activation or number is reduced by minocycline or toxin-encapsulated liposome administration respectively (Li et al. 2005, Kotter et al. 2001; Miron et al. 2013). In the CNS, the regenerative capacity of macrophages involves 1) phagocytosis of myelin debris (which normally inhibits remyelination) and 2) secretion of factors that stimulate axonal re-growth and oligodendrocyte differentiation (Setzu et al. 2006; Zhao et al. 2006; Kigerl et al. 2009; Ruckh et al. 2012; Miron et al. 2013). Indeed, decreased remyelination efficiency with ageing is associated with reduced phagocytosis of myelin debris and growth factor expression (Hinks and Franklin 2000; Ruckh et al. 2012). Macrophages are highly dynamic cells that rapidly respond to cues in their microenvironment by changing their activation state (Fig. 1). These cues come in the form of small protein sequences from pathogens ‘pathogen associated molecular patterns’ or injured cells ‘damage associated molecular patterns’. Pathogen associated molecular patterns and damage associated molecular patterns are recognized by pattern recognition receptors on the surface of macrophages, including toll-like receptors (TLRs), nod-like receptors and RIG1-like receptors. Although these can be expressed by other cells in the CNS, the engagement of these receptors on macrophages has been well characterized to initiate major inflammatory responses to infection or injury.
Fig. 1 Activation and Function of Microglia/ Macrophages. Microglia/ Macrophages survey their microenvironment for signals associated with pathogens (PAMPS) or damage (damage associated molecular patterns, DAMPS), which activates them along a continuum of activation states including pro-inflammatory M1 phenotypes and antiinflammatory/ immune-regulatory M2 phenotypes. M1 microglia/ macrophages are associated with oligodendrocyte progenitor cell (OPC) recruitment whereas M2 macrophages are associated with myelin phagocytosis and promoting OPC differentiation and remyelination.
The diversity of macrophage functions can be related to their capacity to assume a diversity of distinct activation states, which often manifests as polarization into proinflammatory M1 macrophages and anti-inflammatory/ immuno-regulatory M2 macrophages, representing either end of a spectrum (Fig. 1). Although clearly an oversimplification of macrophage activation, these dynamic M1/M2 activation states are a starting point for investigation into the role of macrophages in injury and regeneration. ‘Classically activated’ M1 macrophages typically peak immediately following injury and are associated with host defence, cytotoxicity and secretion of pro-inflammatory cytokines, proteases, and reactive oxygen and nitrogen species (Edwards et al. 2006). M1 activation is modelled by exposure to inflammatory stimuli such as interferon-c and lipopolysaccharide. M1 markers include inducible nitric oxide synthase, CD86, CD16/32, tumour necrosis factor alpha, chemokine (C-C) motif ligand 2, interleukin(IL)-6 and IL-12, and chemokine (C-X-C) motif ligand 11 (CXCL11) (Edwards et al. 2006; Miron et al. 2013). Conversely, ‘alternatively activated’ (M2a) or ‘deactivated’ (M2c) phenotypes have increased phagocytic and angiogenic capacity and secrete anti-inflammatory cytokines, growth factors and neurotrophic factors (Edwards et al. 2006; Durafourt et al. 2011; Miron et al. 2013). They are activated by IL-4/IL-13 or IL-10, respectively, and express markers such as mannose receptor (CD206), CD163, arginase-1, interleukin 1 receptor antagonist, insulin-like growth factor 1, transforming growth factor beta, and IL-10. Interestingly, following spinal cord
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injury M1 macrophages (Ly6Chi CX3CR1lo) and M2 macrophages (Ly6Clo CX3CR1hi) derived from monocytes enter the CNS via distinct routes: the leptomeninges and choroid plexus/cerebrospinal fluid, respectively (Shechter et al. 2013).
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is driven by a temporally limited inflammatory response (i.e. controlled by a switch to M2 activation), whereas this regeneration is inhibited by persistent inflammation (i.e. M1 activation) (Fig. 1).
Microglia, macrophages and remyelination
Endogenous versus circulation-derived macrophages
Several studies have suggested that in the CNS, M1 macrophages are deleterious whereas M2 macrophages support regeneration. High M1 : M2 ratios are associated with inhibition of axonal re-growth following spinal cord injury and the peak of clinical severity in EAE (Kigerl et al. 2009; Mikita et al. 2011). Skewing this balance towards M2 activation by transplant of exogenous M2 cells improves clinical presentation and oligodendrocyte differentiation in EAE (Butovsky et al. 2006; Mikita et al. 2011) and induces functional recovery following spinal cord injury (Rapalino et al. 1998). In addition, a switch from M1 to M2 activation in injured spinal cord, induced by transplantation of stem cells (neural and other), prevents axonal damage and improves locomotor function (Busch et al. 2011; Cusimano et al. 2012). In our recent study, we showed that both M1 and M2 macrophages play a role in remyelination and that there is a critical switch from M1 to M2 macrophage activation at a key stage in the regenerative process (Miron et al. 2013). Remyelination is mediated by a widespread population of multipotent adult CNS progenitor cells commonly referred to as oligodendrocyte progenitor cells (OPCs), but giving rise to both oligodendrocytes and Schwann cells following demyelination (Zawadzka et al. 2010). In response to demyelination, these progenitor cells assume an activated state in response to signals from microglia (that are activated by the primary injury), which enables them to proliferate and divide (recruitment phase) and then differentiate into new myelin-forming cells (differentiation phase) (Franklin and ffrench-Constant 2008). While the M1 macrophages, which dominate early on after demyelination, contribute to the recruitment phase, a switch to an M2-dominant macrophage profile is required for the differentiation phase to ensue in a timely manner (Miron et al. 2013). Indeed, only M2-conditioned media is sufficient to drive oligodendrocyte differentiation in vitro, a critical step in remyelination (Miron et al. 2013). Consistent with this, in post-mortem tissue of multiple sclerosis lesions, active lesions with ongoing remyelination have relatively higher numbers of M2 macrophages than chronic lesions which do not remyelinate (Boven et al. 2006; Zhang et al. 2011; Miron et al. 2013). We identified that activin-A, an M2-derived factor and member of the transforming growth factor-b superfamily, contributes to this regenerative function of M2 macrophages (Miron et al. 2013). Together, these findings demonstrate that successful CNS regeneration
Until recently, microglia-derived and circulation-derived macrophages were considered to be functionally similar. This was largely a consequence of the significant overlap between the two macrophage sources as well as difficulty in distinguishing between them by immune-histochemical means. Methods to differentiate between the two sources include exploiting differential expression levels of CD45 or C-C chemokine receptor 2 (CCR2), bone marrow chimeras with reporter-expressing monocytes/myeloid cells and parabiosis. Recent studies using novel transgenic, gene profiling and methodological approaches have elucidated key differences between macrophages derived from microglia and circulating monocytes. Although they share many functions and gene expression profiles, considerable differences between them include their origin, expression of genes involved in sensing endogenous danger signals, and their requirement for CNS pathology induction and progression. Microglia are derived from primitive myeloid progenitors that invade the brain during rodent embryonic development and early post-natal life, and during the first two trimesters of human gestation (Esiri et al. 1991; Monier et al. 2006, 2007). Recent studies tracing the origin of microglial precursors (CX3CR1+CCR2) during development demonstrated that these arise from the yolk sac at embryonic day 9 (E9) in rodents prior to vascularization and blood–brain barrier formation (Cuadros et al. 1993; Ginhoux et al. 2010; Mizutani et al. 2012). These cells penetrate the neuroepithelium at E10.5, adopt an ameboid morphology and form clusters, then undergo a significant expansion in the cortex, optic vesicles and spinal cord (Rezaie et al. 2004; Mizutani et al. 2012). This process is dependent on colony-stimulating factor 1 receptor (Erblich et al. 2011) and IL34 (Wang et al. 2012) and transcription of genes regulated PU.1, but not Myb (Gomez et al. 2013). Conversely, peripherally derived macrophages are derived from circulating Ly6Chi monocytes originating from bone marrow haematopoietic stem cells in a Myb-dependent manner (Schulz et al. 2012). The distinct origin of microglia and monocyte-derived macrophages is confirmed by Myb-deficient mice having no haematopoietic stem cell-derived macrophages yet normal microglia development (Schulz et al. 2012). Circulating monocytes are short-lived, represent < 2% of peripheral blood mononuclear cells, and are continuously replaced by haematopoietic stem cell-derived precursors, in comparison to long-lived and selfrenewing microglia (Ajami et al. 2007). During pathology,
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these lineages remain distinct as monocyte-derived macrophages do not contribute to the microglia pool in EAE (Ajami et al. 2011). A significant overlap in gene expression profiles between microglia and circulation-derived macrophages renders it difficult to distinguish between these populations during injury and repair. Indeed, a recent study comparing transcriptome profiles between microglia (CD11b+ CD45lo) and macrophages (CD11b+ CD45hi) in adult mice by direct RNA sequencing found a considerable number of shared transcripts (Hickman et al. 2013). However, each cell type expresses over 600 transcripts not expressed by the other, identifying novel markers enriched in microglia (e.g. P2ry12/13, Tmem119, Gpr34, Siglech, Trem2) or peripherally derived macrophages (e.g. fibronectin, CXCL13, Endothelin B receptor, toll-like receptors 8, CCR1) (Hickman et al. 2013). The transcripts enriched in microglia were largely related to sensing endogenous ligands (Hickman et al. 2013). Both microglia and circulation-derived macrophages have been implicated in inducing pathology and promoting regeneration/repair. For example, both are involved in promoting remyelination. In our recent study using CCR2/ mice (in which endogenous monocytes cannot extravasate to enter CNS lesions) injected with green fluorescent protein (GFP)-expressing monocytes, we showed that both microglia (GFP) and circulation-derived macrophages (GFP+) adopt regenerative M2 phenotypes during remyelination (Miron et al. 2013). In our earlier studies, we showed that remyelination is impaired following specific depletion of circulation-derived macrophages by peripheral administration of blood–brain barrier impermeable toxinencapsulating liposomes (Kotter et al. 2001); the level of lesion macrophages is, however, quickly restored to normal, presumably by a compensatory increase in microglia-derived macrophages (Kotter et al. 2005). Nevertheless, gene expression profiling of microglia during active demyelination show that these phagocytose debris and release regenerative/growth factors (Olah et al. 2012; Voss et al. 2012). Other studies suggest some differences between microglia and circulation-derived macrophages with regard to response to injury. For instance, peripherally derived macrophages are limited to the lesion area, whereas activated microglia are more widely distributed throughout the core of the lesion and surrounding area (Rolls et al. 2008; Shechter et al. 2009; Saederup et al. 2010). In addition, following resolution from injury, microglia can return to quiescence but circulation-derived macrophages vanish (Ajami et al. 2011). Finally, inhibition of monocyte infiltration to the CNS by complete red fluorescent protein (RFP) knock-in at the CCR2 locus demonstrated the requirement for circulation-derived macrophages (CCR2RFP+ CX3CR1GFP+) but not microglia (CCR2RFP CX3CR1GFP+) for EAE induction and progression (Saederup et al. 2010).
Ageing, macrophages and remyelination Like all regenerative processes, the efficiency of remyelination declines progressively throughout adulthood. This is likely to have a significant bearing on chronic demyelinating diseases such as multiple sclerosis (MS), which can be of decades’ duration. With age, there is a decrease in OPC activation, recruitment and especially differentiation (Sim et al. 2002; Fancy et al. 2004; Woodruff et al. 2004) owing to effects on intrinsic mechanisms of differentiation (Shen et al. 2008) and changes in the extrinsic/ environmental factors governing the behaviour of OPCs during remyelination (Hinks and Franklin 2000; Zhao et al. 2006). We have previously discussed how macrophages contribute to remyelination by secreting signalling molecules that regulate OPC function, either directly or via their effects on other cells types (e.g. astrocytes) and by phagocytosis of myelin debris which contains potent inhibitors of OPC differentiation (Kotter et al. 2006; Baer et al. 2009). Both these contributions are affected by ageing – the increase in macrophage numbers associated with the early stages of remyelination becomes slower with ageing resulting in changes in the kinetics of the cytokine/chemokine profiles (Hinks and Franklin 2000; Zhao et al. 2006), whereas there is a delay in the clearing of myelin debris (Ruckh et al. 2012). The question therefore arises whether the effects of ageing on remyelination might be reversed by creating an innate immune response to demyelination more similar to that which occurs in younger animals. To do this, we exploited the experimental technique of heterochronic parabiosis in which an old adult and a young adult mice are surgically joined such that they share a common circulation. When focal demyelinating lesions were made in the old adult mouse, they underwent remyelination at the same rate as a young adult mouse. In other words, the failing remyelination of ageing had been rejuvenated. We were able to show using young CCR2/ mice, in which the recruitment of young monocytes into lesions in old mouse partners is blocked, that this was in large part due to the improved myelin debris clearance that occurs when young monocytes contribute to the macrophage population in lesions in old mice (Ruckh et al. 2012). In a further study, we showed that the switch from M1 to M2 is delayed with ageing, consistent with the role of the M2 macrophages in OPC differentiation and the delay in differentiation that occurs with ageing (Miron et al. 2013). However, in the heterochronic parabiosis model, the timing of the M1 to M2 shift in demyelinating lesions in old adult animals was brought forward such that it more closely resembled what occurs in young adult animals, consistent with the earlier onset of OPC differentiation and the more rapid rate of remyelination. Intriguingly, and somewhat unexpectedly, the young monocytes not only contributed to the M2 population but also pre-maturely
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affected the M1 to M2 switch in old microglia/macrophage cells, revealing a capacity for the young monocytes to influence cell behaviour within the lesion beyond their own direct contribution.
Conclusions Given the dominant position of the autoimmune condition multiple sclerosis amongst demyelinating diseases, it is hardly surprising that immunological research on myelin disease has focused on the role of the adaptive immune response (and by implication, the innate immune response as its accomplice) in causing tissue damage. It is only relatively recently that the role of the innate immune system in remyelination, the regenerative response to demyelination, has been recognized and explored. With hindsight, it is perhaps puzzling why this central role for the innate immune system in remyelination has remained hidden given that one of the textbook functions of inflammation is to create an environment that allows regeneration, and the pro-regenerative role of macrophages is well recognized in the healing of many other tissues such as skin, bone and skeletal muscle. Much remains to be learned about how macrophages and microglia contribute to remyelination. For example, what are the signals and mechanisms that bring about the critical M1 to M2 switch, and are there pro-regenerative elements within the adaptive immune system such as regulatory T-cells? Above all, it is to be hoped that a better appreciation of the role of inflammation in remyelination will open up new opportunities for the development of medicines by which this important regenerative process can be enhanced in clinical disease.
Acknowledgements and conflict of interest disclosure V.E.M. is supported by the Wellcome Trust Institute Strategic Support Fund at the University of Edinburgh. R.J.M.F. is supported the UK Multiple Sclerosis Society. V.E.M. and R.J.M.F. are co-inventors on a file patent for the use of activin-A to promote oligodendrocyte differentiation and remyelination. All experiments were conducted in compliance with the ARRIVE guidelines.
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doi: 10.1111/jnc.12705
*MRC Centre for Reproductive Health, The Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK †Wellcome Trust and MRC Cambridge Stem Cell Institute and Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
Abstract Microglia are the resident macrophages of the central nervous system that survey the microenvironment for signals of injury or infection. The response to such signals induces an inflammatory response involving macrophages derived from both resident microglia and recruited circulating monocytes. Although implicated as contributors to autoimmune-mediated injury, microglia/ macrophages have recently been shown to be critical for the important central nervous system regenerative process of remyelination. This functional dichotomy may reflect their ability to be polarized
along a continuum of activation states including the wellcharacterized cytotoxic M1 and regenerative M2 phenotypes. Here, we review the roles of microglia, monocytes and the macrophages which they give rise to in creating lesion environments favourable to remyelination, highlighting the specific roles of M1 and M2 phenotypes and how the pro-regenerative role of the innate immune system is altered by ageing. Keywords: central nervous system, inflammation, macrophage, microglia, myelin, remyelination. J. Neurochem. (2014) 130, 165–171.
Macrophages are myeloid cells involved in surveying tissue environments for damage or infection. Signals indicative of injury induce macrophage responses such as phagocytosis of debris/apoptotic cells, host defence against infections (e.g. antigen presentation), secretion of growth factors, toxic molecules and cytokines/chemokines. Macrophage functions extending beyond a response to injury include regulation of angiogenesis and proliferation, differentiation, migration, and survival of progenitors in healthy tissue. In the central nervous system (CNS), the resident macrophage is the microglia, which represents 5–12% of brain cells (Lawson et al. 1990). Microglia regulate fundamental developmental processes such as neurogenesis, neural precursor migration, survival and apoptosis, axonal pruning and growth and angiogenesis (Aarum et al. 2003; Marin-Teva et al. 2004; Miller et al. 2009; Antony et al. 2011; Lu et al. 2011). Loss of microglia causes severe perturbations in brain development (Erblich et al. 2011). In the adult brain, regional variability in microglial densities exists, being highest in the grey matter of the hippocampus, olfactory telencephalon, basal ganglia and substantia nigra (Lawson et al. 1990; Yang et al. 2013). Even within a given brain region, microglia can show variability in function and gene expression (Kremlev et al. 2004; Olah et al. 2011; Lai and McLaurin 2012). Following injury in the developing or adult CNS, microglia
activation is the first cellular response, as shown in models of traumatic brain injury, demyelination, inflammation, excitotoxic injury and ischaemia–hypoxia. Following injury, microglia proliferate and extend processes to the site of damage, responding to chemoattractant signals released from injured cells (e.g adenosine-50 -triphosphate (ATP), CXCL10) (Davalos et al. 2005; Haynes et al. 2006). Whereas in the healthy CNS circulation-derived macrophages are not present, monocytes from the circulation are recruited into the
Received January 24, 2014; accepted February 24, 2014. Address correspondence and reprint requests to Veronique E. Miron, MRC Centre for Reproductive Health, The Queen’s Medical Research Institute, Edinburgh, EH16 4TJ, UK. E-mail: [email protected] Abbreviations used: ATP, adenosine triphosphate; CCL, chemokine C-C motif ligand; CCR, C-C chemokine receptor; CD, cluster designation; CNS, central nervous system; CSF1, colony stimulating factor 1; CXCL, chemokine C-X-C motif ligand; DAMPS, damage-associated molecular patterns; EAE, experimental autoimmune encephalomyelitis; GFP, green fluorescent protein; Gpr, Probable G protein-coupled receptor; IGF1, insulin-like growth factor 1; IL1Ra, interleukin 1 receptor antagonist; IL, interleukin; iNOS, inducible nitric oxide synthase; NLR, nod-like receptor; OPC, oligodendrocyte progenitor cell; PAMPS, pathogen-associated molecular patterns; RLR, RIG-1-like receptor; TGF-b, transforming growth factor receptor beta; TLR, tolllike receptor; Tmem, transmembrane protein; TNF-a, tumour necrosis factor alpha; TREM2, triggering receptor expressed on myeloid cells 2.
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CNS following injury or infection where they then typically undergo differentiation into macrophages. Microglia/ macrophages subsequently phagocytose debris/dead cells and secrete a multitude of factors (e.g. cytokines, growth factors, reactive species) that influence regenerative processes. This article reviews the diverse functions and activation states of microglia/ macrophages and how these cells contribute to the CNS regenerative process of remyelination.
Microglia/ macrophage function and activation Microglia/ macrophages have long been implicated in inducing neural pathology by secretion of toxic molecules (e.g. glutamate and reactive oxygen/nitrogen species), antigen presentation to cytotoxic T lymphocytes and degradation of synapses (Blinzinger and Kreutzberg 1968; Banati et al. 1993; Cash et al. 1993). In support of this concept, dampening macrophage activation in spinal cord injury, traumatic brain injury and hypoxic-ischaemic injury using minocycline improves recovery in adult rodents (Sanchez Mejia et al. 2001; Arvin et al. 2002; Wells et al. 2003). In addition, infiltration of the CNS by monocytes has been identified as a trigger for disease progression in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) (Ajami et al. 2011). However, recent studies have elucidated the regenerative role of macrophages in the CNS, and in particular in the regeneration of myelin (remyelination), the focus of the current review. Remyelination following a demyelinating insult is inhibited when macrophage activation or number is reduced by minocycline or toxin-encapsulated liposome administration respectively (Li et al. 2005, Kotter et al. 2001; Miron et al. 2013). In the CNS, the regenerative capacity of macrophages involves 1) phagocytosis of myelin debris (which normally inhibits remyelination) and 2) secretion of factors that stimulate axonal re-growth and oligodendrocyte differentiation (Setzu et al. 2006; Zhao et al. 2006; Kigerl et al. 2009; Ruckh et al. 2012; Miron et al. 2013). Indeed, decreased remyelination efficiency with ageing is associated with reduced phagocytosis of myelin debris and growth factor expression (Hinks and Franklin 2000; Ruckh et al. 2012). Macrophages are highly dynamic cells that rapidly respond to cues in their microenvironment by changing their activation state (Fig. 1). These cues come in the form of small protein sequences from pathogens ‘pathogen associated molecular patterns’ or injured cells ‘damage associated molecular patterns’. Pathogen associated molecular patterns and damage associated molecular patterns are recognized by pattern recognition receptors on the surface of macrophages, including toll-like receptors (TLRs), nod-like receptors and RIG1-like receptors. Although these can be expressed by other cells in the CNS, the engagement of these receptors on macrophages has been well characterized to initiate major inflammatory responses to infection or injury.
Fig. 1 Activation and Function of Microglia/ Macrophages. Microglia/ Macrophages survey their microenvironment for signals associated with pathogens (PAMPS) or damage (damage associated molecular patterns, DAMPS), which activates them along a continuum of activation states including pro-inflammatory M1 phenotypes and antiinflammatory/ immune-regulatory M2 phenotypes. M1 microglia/ macrophages are associated with oligodendrocyte progenitor cell (OPC) recruitment whereas M2 macrophages are associated with myelin phagocytosis and promoting OPC differentiation and remyelination.
The diversity of macrophage functions can be related to their capacity to assume a diversity of distinct activation states, which often manifests as polarization into proinflammatory M1 macrophages and anti-inflammatory/ immuno-regulatory M2 macrophages, representing either end of a spectrum (Fig. 1). Although clearly an oversimplification of macrophage activation, these dynamic M1/M2 activation states are a starting point for investigation into the role of macrophages in injury and regeneration. ‘Classically activated’ M1 macrophages typically peak immediately following injury and are associated with host defence, cytotoxicity and secretion of pro-inflammatory cytokines, proteases, and reactive oxygen and nitrogen species (Edwards et al. 2006). M1 activation is modelled by exposure to inflammatory stimuli such as interferon-c and lipopolysaccharide. M1 markers include inducible nitric oxide synthase, CD86, CD16/32, tumour necrosis factor alpha, chemokine (C-C) motif ligand 2, interleukin(IL)-6 and IL-12, and chemokine (C-X-C) motif ligand 11 (CXCL11) (Edwards et al. 2006; Miron et al. 2013). Conversely, ‘alternatively activated’ (M2a) or ‘deactivated’ (M2c) phenotypes have increased phagocytic and angiogenic capacity and secrete anti-inflammatory cytokines, growth factors and neurotrophic factors (Edwards et al. 2006; Durafourt et al. 2011; Miron et al. 2013). They are activated by IL-4/IL-13 or IL-10, respectively, and express markers such as mannose receptor (CD206), CD163, arginase-1, interleukin 1 receptor antagonist, insulin-like growth factor 1, transforming growth factor beta, and IL-10. Interestingly, following spinal cord
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injury M1 macrophages (Ly6Chi CX3CR1lo) and M2 macrophages (Ly6Clo CX3CR1hi) derived from monocytes enter the CNS via distinct routes: the leptomeninges and choroid plexus/cerebrospinal fluid, respectively (Shechter et al. 2013).
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is driven by a temporally limited inflammatory response (i.e. controlled by a switch to M2 activation), whereas this regeneration is inhibited by persistent inflammation (i.e. M1 activation) (Fig. 1).
Microglia, macrophages and remyelination
Endogenous versus circulation-derived macrophages
Several studies have suggested that in the CNS, M1 macrophages are deleterious whereas M2 macrophages support regeneration. High M1 : M2 ratios are associated with inhibition of axonal re-growth following spinal cord injury and the peak of clinical severity in EAE (Kigerl et al. 2009; Mikita et al. 2011). Skewing this balance towards M2 activation by transplant of exogenous M2 cells improves clinical presentation and oligodendrocyte differentiation in EAE (Butovsky et al. 2006; Mikita et al. 2011) and induces functional recovery following spinal cord injury (Rapalino et al. 1998). In addition, a switch from M1 to M2 activation in injured spinal cord, induced by transplantation of stem cells (neural and other), prevents axonal damage and improves locomotor function (Busch et al. 2011; Cusimano et al. 2012). In our recent study, we showed that both M1 and M2 macrophages play a role in remyelination and that there is a critical switch from M1 to M2 macrophage activation at a key stage in the regenerative process (Miron et al. 2013). Remyelination is mediated by a widespread population of multipotent adult CNS progenitor cells commonly referred to as oligodendrocyte progenitor cells (OPCs), but giving rise to both oligodendrocytes and Schwann cells following demyelination (Zawadzka et al. 2010). In response to demyelination, these progenitor cells assume an activated state in response to signals from microglia (that are activated by the primary injury), which enables them to proliferate and divide (recruitment phase) and then differentiate into new myelin-forming cells (differentiation phase) (Franklin and ffrench-Constant 2008). While the M1 macrophages, which dominate early on after demyelination, contribute to the recruitment phase, a switch to an M2-dominant macrophage profile is required for the differentiation phase to ensue in a timely manner (Miron et al. 2013). Indeed, only M2-conditioned media is sufficient to drive oligodendrocyte differentiation in vitro, a critical step in remyelination (Miron et al. 2013). Consistent with this, in post-mortem tissue of multiple sclerosis lesions, active lesions with ongoing remyelination have relatively higher numbers of M2 macrophages than chronic lesions which do not remyelinate (Boven et al. 2006; Zhang et al. 2011; Miron et al. 2013). We identified that activin-A, an M2-derived factor and member of the transforming growth factor-b superfamily, contributes to this regenerative function of M2 macrophages (Miron et al. 2013). Together, these findings demonstrate that successful CNS regeneration
Until recently, microglia-derived and circulation-derived macrophages were considered to be functionally similar. This was largely a consequence of the significant overlap between the two macrophage sources as well as difficulty in distinguishing between them by immune-histochemical means. Methods to differentiate between the two sources include exploiting differential expression levels of CD45 or C-C chemokine receptor 2 (CCR2), bone marrow chimeras with reporter-expressing monocytes/myeloid cells and parabiosis. Recent studies using novel transgenic, gene profiling and methodological approaches have elucidated key differences between macrophages derived from microglia and circulating monocytes. Although they share many functions and gene expression profiles, considerable differences between them include their origin, expression of genes involved in sensing endogenous danger signals, and their requirement for CNS pathology induction and progression. Microglia are derived from primitive myeloid progenitors that invade the brain during rodent embryonic development and early post-natal life, and during the first two trimesters of human gestation (Esiri et al. 1991; Monier et al. 2006, 2007). Recent studies tracing the origin of microglial precursors (CX3CR1+CCR2) during development demonstrated that these arise from the yolk sac at embryonic day 9 (E9) in rodents prior to vascularization and blood–brain barrier formation (Cuadros et al. 1993; Ginhoux et al. 2010; Mizutani et al. 2012). These cells penetrate the neuroepithelium at E10.5, adopt an ameboid morphology and form clusters, then undergo a significant expansion in the cortex, optic vesicles and spinal cord (Rezaie et al. 2004; Mizutani et al. 2012). This process is dependent on colony-stimulating factor 1 receptor (Erblich et al. 2011) and IL34 (Wang et al. 2012) and transcription of genes regulated PU.1, but not Myb (Gomez et al. 2013). Conversely, peripherally derived macrophages are derived from circulating Ly6Chi monocytes originating from bone marrow haematopoietic stem cells in a Myb-dependent manner (Schulz et al. 2012). The distinct origin of microglia and monocyte-derived macrophages is confirmed by Myb-deficient mice having no haematopoietic stem cell-derived macrophages yet normal microglia development (Schulz et al. 2012). Circulating monocytes are short-lived, represent < 2% of peripheral blood mononuclear cells, and are continuously replaced by haematopoietic stem cell-derived precursors, in comparison to long-lived and selfrenewing microglia (Ajami et al. 2007). During pathology,
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these lineages remain distinct as monocyte-derived macrophages do not contribute to the microglia pool in EAE (Ajami et al. 2011). A significant overlap in gene expression profiles between microglia and circulation-derived macrophages renders it difficult to distinguish between these populations during injury and repair. Indeed, a recent study comparing transcriptome profiles between microglia (CD11b+ CD45lo) and macrophages (CD11b+ CD45hi) in adult mice by direct RNA sequencing found a considerable number of shared transcripts (Hickman et al. 2013). However, each cell type expresses over 600 transcripts not expressed by the other, identifying novel markers enriched in microglia (e.g. P2ry12/13, Tmem119, Gpr34, Siglech, Trem2) or peripherally derived macrophages (e.g. fibronectin, CXCL13, Endothelin B receptor, toll-like receptors 8, CCR1) (Hickman et al. 2013). The transcripts enriched in microglia were largely related to sensing endogenous ligands (Hickman et al. 2013). Both microglia and circulation-derived macrophages have been implicated in inducing pathology and promoting regeneration/repair. For example, both are involved in promoting remyelination. In our recent study using CCR2/ mice (in which endogenous monocytes cannot extravasate to enter CNS lesions) injected with green fluorescent protein (GFP)-expressing monocytes, we showed that both microglia (GFP) and circulation-derived macrophages (GFP+) adopt regenerative M2 phenotypes during remyelination (Miron et al. 2013). In our earlier studies, we showed that remyelination is impaired following specific depletion of circulation-derived macrophages by peripheral administration of blood–brain barrier impermeable toxinencapsulating liposomes (Kotter et al. 2001); the level of lesion macrophages is, however, quickly restored to normal, presumably by a compensatory increase in microglia-derived macrophages (Kotter et al. 2005). Nevertheless, gene expression profiling of microglia during active demyelination show that these phagocytose debris and release regenerative/growth factors (Olah et al. 2012; Voss et al. 2012). Other studies suggest some differences between microglia and circulation-derived macrophages with regard to response to injury. For instance, peripherally derived macrophages are limited to the lesion area, whereas activated microglia are more widely distributed throughout the core of the lesion and surrounding area (Rolls et al. 2008; Shechter et al. 2009; Saederup et al. 2010). In addition, following resolution from injury, microglia can return to quiescence but circulation-derived macrophages vanish (Ajami et al. 2011). Finally, inhibition of monocyte infiltration to the CNS by complete red fluorescent protein (RFP) knock-in at the CCR2 locus demonstrated the requirement for circulation-derived macrophages (CCR2RFP+ CX3CR1GFP+) but not microglia (CCR2RFP CX3CR1GFP+) for EAE induction and progression (Saederup et al. 2010).
Ageing, macrophages and remyelination Like all regenerative processes, the efficiency of remyelination declines progressively throughout adulthood. This is likely to have a significant bearing on chronic demyelinating diseases such as multiple sclerosis (MS), which can be of decades’ duration. With age, there is a decrease in OPC activation, recruitment and especially differentiation (Sim et al. 2002; Fancy et al. 2004; Woodruff et al. 2004) owing to effects on intrinsic mechanisms of differentiation (Shen et al. 2008) and changes in the extrinsic/ environmental factors governing the behaviour of OPCs during remyelination (Hinks and Franklin 2000; Zhao et al. 2006). We have previously discussed how macrophages contribute to remyelination by secreting signalling molecules that regulate OPC function, either directly or via their effects on other cells types (e.g. astrocytes) and by phagocytosis of myelin debris which contains potent inhibitors of OPC differentiation (Kotter et al. 2006; Baer et al. 2009). Both these contributions are affected by ageing – the increase in macrophage numbers associated with the early stages of remyelination becomes slower with ageing resulting in changes in the kinetics of the cytokine/chemokine profiles (Hinks and Franklin 2000; Zhao et al. 2006), whereas there is a delay in the clearing of myelin debris (Ruckh et al. 2012). The question therefore arises whether the effects of ageing on remyelination might be reversed by creating an innate immune response to demyelination more similar to that which occurs in younger animals. To do this, we exploited the experimental technique of heterochronic parabiosis in which an old adult and a young adult mice are surgically joined such that they share a common circulation. When focal demyelinating lesions were made in the old adult mouse, they underwent remyelination at the same rate as a young adult mouse. In other words, the failing remyelination of ageing had been rejuvenated. We were able to show using young CCR2/ mice, in which the recruitment of young monocytes into lesions in old mouse partners is blocked, that this was in large part due to the improved myelin debris clearance that occurs when young monocytes contribute to the macrophage population in lesions in old mice (Ruckh et al. 2012). In a further study, we showed that the switch from M1 to M2 is delayed with ageing, consistent with the role of the M2 macrophages in OPC differentiation and the delay in differentiation that occurs with ageing (Miron et al. 2013). However, in the heterochronic parabiosis model, the timing of the M1 to M2 shift in demyelinating lesions in old adult animals was brought forward such that it more closely resembled what occurs in young adult animals, consistent with the earlier onset of OPC differentiation and the more rapid rate of remyelination. Intriguingly, and somewhat unexpectedly, the young monocytes not only contributed to the M2 population but also pre-maturely
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affected the M1 to M2 switch in old microglia/macrophage cells, revealing a capacity for the young monocytes to influence cell behaviour within the lesion beyond their own direct contribution.
Conclusions Given the dominant position of the autoimmune condition multiple sclerosis amongst demyelinating diseases, it is hardly surprising that immunological research on myelin disease has focused on the role of the adaptive immune response (and by implication, the innate immune response as its accomplice) in causing tissue damage. It is only relatively recently that the role of the innate immune system in remyelination, the regenerative response to demyelination, has been recognized and explored. With hindsight, it is perhaps puzzling why this central role for the innate immune system in remyelination has remained hidden given that one of the textbook functions of inflammation is to create an environment that allows regeneration, and the pro-regenerative role of macrophages is well recognized in the healing of many other tissues such as skin, bone and skeletal muscle. Much remains to be learned about how macrophages and microglia contribute to remyelination. For example, what are the signals and mechanisms that bring about the critical M1 to M2 switch, and are there pro-regenerative elements within the adaptive immune system such as regulatory T-cells? Above all, it is to be hoped that a better appreciation of the role of inflammation in remyelination will open up new opportunities for the development of medicines by which this important regenerative process can be enhanced in clinical disease.
Acknowledgements and conflict of interest disclosure V.E.M. is supported by the Wellcome Trust Institute Strategic Support Fund at the University of Edinburgh. R.J.M.F. is supported the UK Multiple Sclerosis Society. V.E.M. and R.J.M.F. are co-inventors on a file patent for the use of activin-A to promote oligodendrocyte differentiation and remyelination. All experiments were conducted in compliance with the ARRIVE guidelines.
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