Activation patterns of microglia and their ... - Wiley Online Library

Neuropathology and Applied Neurobiology (2013), 39, 3–18

doi: 10.1111/nan.12011

Review: Activation patterns of microglia and their identification in the human brain D. Boche*, V. H. Perry† and J. A. R. Nicoll*‡ *Clinical Neurosciences, Clinical and Experimental Sciences, Faculty of Medicine, †Centre for Biological Sciences, Faculty of Natural and Environmental Science, University of Southampton, and ‡Neuropathology, Department of Cellular Pathology, University Hospital Southampton NHS Foundation Trust, Southampton, UK

D. Boche, V. H. Perry and J. A. R. Nicoll (2013) Neuropathology and Applied Neurobiology 39, 3–18 Activation patterns of microglia and their identification in the human brain Microglia in the central nervous system are usually maintained in a quiescent state. When activated, they can perform many diverse functions which may be either beneficial or harmful depending on the situation. Although microglial activation may be accompanied by changes in morphology, morphological changes cannot accurately predict the function being undertaken by a microglial cell. Studies of peripheral macrophages and in vitro and animal studies of microglia have resulted in the definition of specific activation states: M1 (classical activation) and M2

(sometimes subdivided into alternative activation and acquired deactivation). Some authors have suggested that these might be an overlapping continuum of functions rather than discrete categories. In this review, we consider translational aspects of our knowledge of microglia: specifically, we discuss the question as to what extent different activation states of microglia exist in the human central nervous system, which tools can be used to identify them and emerging evidence for such changes in ageing and in Alzheimer’s disease.

Keywords: human brain, microglia, neuroinflammation

Introduction Microglia are the representatives in the central nervous system (CNS) of the mononuclear phagocyte series of cells and ultimately have a myeloid origin [1–3]. In this regard, microglia can be compared with the resident tissue macrophages of other organs such as the Kupffer cells of the liver and the dendritic cells of the skin and other epithelial surfaces. During development, there is a major wave of migration of primitive myeloid progenitors into the CNS to become resident microglia (Figure 1A) [4]. There is currently active debate concerning the capacity for proliferation and turnover of microglia subsequently during the lifespan. Particular controversy surrounds the potential for circulating monocytes to enter the brain during adult Correspondence: Delphine Boche, Faculty of Medicine, Clinical and Experimental Sciences, University of Southampton, Mailpoint 806, Southampton General Hospital, Southampon, SO16 6YD, UK. Tel: +44 2380 796107; Fax: +44 2380 796085; E-mail: d.boche@ soton.ac.uk © 2012 British Neuropathological Society

life and supplement or replenish the resident population of microglia. Some evidence from highly manipulated animal models suggests that this can occur in special circumstances [5], although to what extent it is important in the normal responses to ageing or disease is currently unclear. There is better evidence that perivascular macrophages in the CNS (Figure 1B), which are also ultimately of myeloid origin, can be derived from the circulation during life. Other macrophage-related populations in the CNS are present in the leptomeninges and in the choroid plexus. Relatively little is known about the functions of these populations, but presumably they have monitoring and scavenging functions related to their locations. For example, perivascular and leptomeningeal macrophages (Figure 1C) are important in scavenging blood products following subarachnoid haemorrhage [6]. Clearly, if a capacity for circulating cells to enter the brain and function as microglia/macrophages is retained during adult life, or can be induced or enhanced in humans, then this could have important consequences for delivery of 3

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Figure 1. Different morphological states of microglia/macrophages in the CNS. (A) Microglia in the developing brain (germinal matrix, CD68); (B) Perivascular macrophages in the cerebral cortex (CD163); (C) Leptomeningeal macrophages reacting to subarachnoid haemorrhage (CD68); (D) Ramified microglia in the absence of ageing or neurodegenerative disease (HLA-DR); (E) ramified microglia in an activated state in AD (HLA-DR); (F) numerous macrophages phagocytosing necrotic tissue in an infarct (H&E); (G) Macrophages/ phagocytic microglia at the edge of an infarct (CD68); (H) Rod cells in the cerebral cortex in subacute sclerosing panencephalitis (H&E); (I) Multinucleated giant cell in the leptomeninges in a patient with severe cerebral amyloid angiopathy (H&E); (J) Epithelioid macrophages/microglia forming a granuloma (CD68); (K) Dystrophic microglia with beading of cell processes (Iba1); (L) A cluster of microglia associated with an amyloid plaque in AD (macrophage scavenger receptor). Scale bar: A–B and D–L = 5 mm, C = 10 mm. CNS, central nervous system; AD, Alzheimer’s disease; HLA-DR, human major histocompatibility complex class-II; Iba1, ionized calcium-binding adaptor molecule 1.

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Microglia in human brain

extraneous cells, genes and other substances for therapeutic purposes.

Functions of microglia Microglia can exhibit widely differing functions at different stages in life both physiological and in response to, and in contribution to, various pathological situations as summarized in Table 1. Although in reality it seems probable that at any given point in time, an individual microglial cell is likely to be undertaking several different

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functions, a major thrust of the microglial literature in recent years has been to define a number of ‘activation states’ in which microglia are performing a restricted set of functions. This development has taken place largely as a consequence of microglia being regarded as resident CNS macrophages; the results of studies performed on peripheral macrophages are extrapolated and anticipated to apply also to the response of microglia to an insult, injury or disease occurring in the CNS. Here we consider the extent, in the human CNS, to which the function or functions of a microglial cell can be deduced from

Table 1. Functions of microglia Function

Examples

CNS development

• Phagocytic activity during neuronal/synaptic development likely represents ‘pruning’ of redundant neurons and connections • Development influenced by secretion of cytokines, neurotrophins and growth factors

Recognition of pathogens (innate immune function)

• Receptors (e.g. Toll-like receptors, TLRs) recognize evolutionarily conserved antigens on surface of pathogens known as pathogen-associated molecular patterns (PAMPs) such as the endotoxin lipopolysaccharide (LPS) • Similar mechanisms possibly also involved in response to extracellular protein accumulations (e.g. amyloid plaques)

Phagocytosis

Ingestion and destruction by digestive enzymes in lysosomes of: • Multiple types of damaged cells (e.g. infarct) • Neurons (e.g. neuronophagia, Wallerian degeneration, tract degeneration) • Micro-organisms (e.g. abscess) • Virally infected cells (e.g. herpes encephalitis) • Erythrocytes and haemoglobin breakdown products (e.g. haemosiderin) following haemorrhage

Antigen presentation

• Presentation of pathogens (e.g. in bacterial, fungal, viral infections) bound to MHC for activation of T lymphocytes • Possibly relevant also in autoimmune disease

Recognition of bound antibody (adaptive immune function)

• Respond to antibodies bound to pathogens (opsonization) • Possibly also relevant to autoimmune disease (e.g. demyelination, paraneoplastic syndromes

Cytotoxicity

• Reactive oxygen species/respiratory burst (H2O2, NO) • Cytokines (e.g. IL, TNF, interferons, TGF, CSF) • Secretion of glutamate, aspartate

Extracellular matrix remodelling

• Proteases (MMPs degrade extracellular matrix)

Modulation of inflammation/ immune responses

• Chemokines (attract other inflammatory cells) • CD200 receptor (CD200 secreted by neurons has anti-inflammatory role) • Interferon-g (promotes further microglial activation)

Repair

• Removal of cell debris facilitates plasticity and synaptogenesis

Stem cells

• Regulation of stem cell proliferation (e.g. granule cell neurons of hippocampus)

Tumours

• Response to neoplastic cells, possible regulation of tumour cell proliferation

Lipid transport

• Secretion of lipoprotein particles which deliver lipids to neurons for maintenance of cell membranes and synapses, facilitating synaptic plasticity

Viral entry into CNS

• CCR5 and CD4 are receptors for entry of HIV into macrophages and hence into CNS

Support mycobacteria

• Permits intracytoplasmic survival of mycobacteria (e.g. tuberculosis)

Demyelination

• Myelin destruction/phagocytosis (e.g. multiple sclerosis)

CNS, central nervous system; MHC, major histocompatibility complex; IL, interleukin; TGF, transforming growth factor; CSF, colony-stimulating factor; MMP, matrix metalloproteinase; HIV, human immunodeficiency virus; CCR5, C–C chemokine receptor 5. © 2012 British Neuropathological Society

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observations of its morphology, gene expression and protein composition.

Morphological states of microglia

Ramified microglia Microglia have a distinct morphology when compared with macrophages in other tissues. The typical morphology of human microglia is in the form of cells with many short, fine processes (Figure 1D) which provide a large surface area and which extend into their surroundings, putting them in a good position to sense and monitor changes in their local environment. This morphology, often referred to as ‘ramified’, has in the past been thought to reflect a ‘resting’ or relatively inactive state. However, time-lapse video microscopy in vivo in the mouse shows the processes of microglia to be in constant motion as if they are constantly actively sampling their environment [7]. Microglial processes extend towards those of their neighbours with the consequence that together they form a matrix extending throughout the CNS. The morphology of microglia bears a striking resemblance to epithelial dendritic cells (Langerhans cells). The function of epithelial dendritic cells is to sample the external environment for foreign antigens, for example those of bacteria and viruses, by interaction with the presence of cell surface pattern recognition receptors and Toll-like receptors. The activated dendritic cells then travel to local lymph nodes and present the antigens in conjunction with major histocompatibility complex (MHC) class II to lymphocytes in order to stimulate an adaptive immune response. In contrast, despite a similar morphology, microglia appear not to have the capacity to form this ‘afferent’ arm of the immune system, one of the reasons the CNS is a relatively ‘immune privileged’ site [8]. Nevertheless, in human post mortem brain tissue MHC II-positive microglia are often abundant and, consequently, MHC II immunohistochemistry is commonly employed in neuropathological practice to identify microglia (Figure 1E).

Macrophages/amoeboid microglia Cells with macrophage morphology develop in the CNS in situations in which phagocytosis takes place. Such cells are spherical in shape, lack processes, and contain numerous phagocytic vacuoles (Figure 1F,G). Abundant cells with macrophage morphology appear in response to acute © 2012 British Neuropathological Society

destruction of CNS tissue such as by trauma, infarction and infection. Macrophage-like cells are also common in the developing brain, potentially functioning in a ‘pruning’ capacity, removing aberrant neurons and synapses. The label ‘amoeboid’ implies that such cells are capable of motility. These cells are essentially morphologically indistinguishable from macrophages elsewhere in the body – an observation which is a source of potential confusion. First, in terms of terminology, should such cells in the CNS simply be called ‘macrophages’ to reflect the similarity in appearance and function to macrophages elsewhere in the body or, alternatively, should they be called ‘amoeboid microglia’ to recognize that potentially they may have a different origin? Second, there is genuine uncertainty as to the origin of these ‘macrophage-like’ cells in the CNS. Are they derived by alterations in local resident microglia or are they derived from circulating monocytes entering the brain in response to tissue damage, as happens elsewhere in the body? Animal studies indicate that, at least in some circumstances, circulating monocytes can enter the CNS and develop into microglia during adult life [5,9]. However, observations from clinical neuropathology practice demonstrate that at the margin of a lesion, for example a tumour or infarct, there is a gradation from ramified microglia with multiple fine processes relatively distant from the lesion, through to microglia with relatively few stubby processes to macrophage-like cells in the epicentre of the lesion. Such observations seem to support an origin of the macrophage-like cells from pre-existing resident microglia rather than from circulating monocytes. These two potential origins of macrophages in the brain are, of course, not mutually exclusive and it is possible that both mechanisms can occur.

Other morphological states Rod cells Rod cells are microglia with markedly elongated nuclei, scanty cytoplasm and few processes. They are most notable in chronic disorders in which a reaction lasts for several years, the classic example being subacute sclerosing panencephalitis due to chronic measles infection (Figure 1H). Multinucleated cells Multinucleated cells form as a reaction to indigestible material. They are commonly seen in mycobacterial infection, in response to foreign material such as sutures or haemostatic gel and rarely around amyloid-laden blood vessels in cerebral amyloid NAN 2013; 39: 3–18


angiopathy (Figure 1I). Multinucleated giant cells, which contains human immunodeficiency virus (HIV), also occur in a characteristically perivascular location in the white matter in HIV encephalopathy. Epithelioid macrophages Epithelioid macrophages cluster to form granulomas in chronic infections such as tuberculosis and leprosy and non-infective conditions including sarcoid (Figure 1J). ‘Dystrophic’ microglia This term has been applied to a particular morphological appearance with ‘beading’ of microglial processes (Figure 1K). It has been suggested that this is due to microglial dysfunction due to ageing [10,11]. From the discussion above, it can be seen that to a limited extent microglial function can be deduced from the morphology, the best example being the association of macrophage-like morphology with phagocytosis. In the normal brain, microglia do not express many of the cell surface or cytoplasmic proteins that are typical of other tissue macrophages, so they appear to be relatively downregulated or switched off by the CNS micro-environment [3,12–14]. However, as discussed below, microglia can be readily activated, even though retaining a ramified morphology, for example during ageing [15–20], and it is postulated that such activation is important in the pathogenesis of chronic age-related neurodegenerative diseases such as Alzheimer’s disease (AD) and prion disease [14,21–24].

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The concept of different activation states of peripheral macrophages In non-neural tissues, the prior state of activation and the nature of the activating stimulus play a significant role in determining the spectrum of molecules that are secreted by a macrophage (Table 2) [25]. Activation of macrophages, although potentially helpful to the organisms for example in killing pathogens, can also be harmful leading to a destructive profile associated with tissue damage (known as ‘classical activation’ or M1). Furthermore, according to the type of injury or insult, activated macrophages can expressed a tolerant profile, also known as ‘alternative activation’ or M2 [26]. The definition of the types of activation was based initially on peripheral monocytes/macrophages characterized in in vitro experiments, and has been confirmed in rodents. Classical activation (M1) is defined by the response of macrophages to challenge by micro-organisms resulting in the expression of high levels of pro-inflammatory cytokines and an enhanced microbicidal capacity. Interferon-g causes M1 activation, a response usually associated with host defence to intracellular pathogens [27–29]. Classical activation has been the activation state most widely explored in animal models. Alternative activation (M2) was originally defined following exposure to the Th2 cytokine, interleukin (IL)-4, and upregulation by macrophages of the mannose receptor [30]. The mannose receptor can bind structures on

Table 2. Different activation states of macrophages which by extrapolation may apply to microglia

M1 (Classic activation)

M2 (Alternative activation: wound healing) Tissue repair

Anti-inflammatory

Stimulus

Interferon-g, TNF-a

IL-4, IL-13, TREM2?

IL-10, glucocorticoids

Source

Natural killer, T helper 1 lymphocytes.

Granulocytes responding to tissue injury, fungi and parasites (chitin), T helper 2 lymphocytes

Macrophage

Macrophage products

Pro-inflammatory cytokines: IL-1b, TNF-a, IL-6, IL-23 Oxygen free radicals

Extracellular matrix components Arginase 1 Chitinase

TGFb1, IL-10

Cell surface proteins

MHC II?

Mannose receptor (CD206)

Functions

Kill micro-organisms and other cellular targets. Phagocytosis Present antigen to lymphocytes. May cause collateral damage to host cells.

Tissue repair/wound healing Phagocytosis Increases production/remodelling of extracellular matrix

Alternative terms

M2 (Alternative activation: regulatory)

Inhibits inflammation Phagocytosis

Adapted from Mosser and Edwards (2008) [22]. TNF, tumour necrosis factor; IL, interleukin; TREM2, Triggering Receptor Expressed on Myeloid cells; MHC, major histocompatibility complex. © 2012 British Neuropathological Society

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the surface of viruses, bacteria and fungi enabling their phagocytosis. Macrophages activated by the Th2 cytokines IL-4 and IL-13 [31] are implicated in a range of physiological and pathological processes including homeostasis, inflammation, allergy, malignancy and repair [26,31]. The M2 category has been further divided into functions relating, first, to tissue repair and wound healing and, second, a state of acquired deactivation (Table 2). Recent evidence suggests that the same macrophage cell has the potential to adopt M1 or M2 profiles based on either the type of the stimulus (e.g. ageing, injury or chronic disease) or on the initial cell status, as already activated or not, before the stimulus [26]. Some investigators have proposed that viewing the categories of activation as colours on a wheel permits overlap and emphasises that activation states are part of a spectrum of plastic functional states rather than discrete categories [26].

Do microglia have a similar range of functional profiles to peripheral macrophages?

Evidence from animal studies In contrast to peripheral macrophages, the mechanisms by which the microglial phenotype is regulated in the CNS are poorly understood at present. Microglia can develop a range of functional phenotypes that broadly correspond to M1/M2 activation of macrophages as described above [32–35]. This classification, derived mainly from animal studies, is based on the expression of either pro- or antiinflammatory cytokines in association with pro- or antiinflammatory receptors (Table 2). These observations highlight the point that, rather than simply demonstrating that microglia have been activated, it is the specific manner in which microglia are activated and the phenotype that they adopt that is important in determining the influence of microglia, for example, in the context of neurodegeneration [36].

Evidence from human studies The concept of different activation states of macrophages and microglia has been derived mainly from animal studies; to what extent has their relevance to the human brain been confirmed? In vitro studies Human macrophages, macrophage-like cell lines and cultured microglia derived from human foe© 2012 British Neuropathological Society

tuses or adults have been used to decipher the mechanisms underlying human diseases, but this approach has been criticized as it strongly relies on the cellular model used. Foetal microglia have been derived from therapeutic abortions [37,38] and adult microglia from surgical biopsies [39]. One of the first challenges of primary cell culture is the maintenance of the cells in in vitro conditions [40], with the additional difficulty that the highly sensitive microglia become stimulated by the in vitro conditions acquiring an activation state characterized by the amoeboid morphology, but which is unlikely to be representative of their in vivo status [41]. In vitro tissue slice cultures have the advantage that the microglia retain their brain tissue environment; this approach has been used in rodent studies [42] and in principle could be used for studies of the human brain. However, even in these models, there is evidence that the process of creating the slice culture modifies microglia [42]. To circumvent these effects, which are suggested to be initiated in part by the neonatal origin of the cells and their co-culture with astrocytes [43], isolation of microglia from human adult brain autopsy tissue was developed [44]. The specificity of microglia was then tested based on their ramified morphology and phagocytic properties [44,45], and subsequently, activation of microglia isolated from the adult brain demonstrated that they were able to express complement and cytokine genes as expected from animal studies [45]. Further studies have shown that isolated human microglia in the presence of amyloid-b protein (Ab) express the pro-inflammatory cytokines IL-1b, IL-6, IL-8 and tumour necrosis factor (TNF)-a and the chemokines monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory peptide (MIP)-a [46,47]. However, these studies used an acute challenge which does not necessarily mimic the situation in chronic disease in the aged human brain. Microarray studies have demonstrated changes in gene expression of these pro-inflammatory cytokines and chemokines and also of the matrix metalloproteinase family [48]. Investigation of the mechanisms underlying the inflammatory reaction in AD using this experimental approach has revealed that: (i) expression of the microglial receptor for advanced glycation endproducts (RAGE) which can bind Ab, was increased in its presence and was associated with an increase of macrophage colony-stimulating factor (M-CSF) [49]; (ii) CD87 receptor expression, a marker of macrophage activation known as the urokinase plasminogen-activator receptor, was strongly increased by Ab possibly as the result of NAN 2013; 39: 3–18


oxidative-stress-related mechanisms [50]; and that (iii) calcium-mediated signal transduction was impaired in microglia from AD patients [51]. Recently, a protocol involving microglial isolation from human post mortem brain has been optimized to obtain a high purity microglial population from a range of diseased brains and different cerebral regions [52] with the aim that the rapidity of the technique (less than 90 min) avoids the influence that cell culture might have on microglial phenotype. The in vitro conditions still do not reflect the brain environment but may be relevant to study the microglial inflammatory response rather than their resting state. Human brain tissue studies Study of human brain tissue, particularly if obtained post mortem, presents a number of practical difficulties resulting from variables such as the age, gender, genetic heterogeneity, agonal status, preterminal medication, cause of death, concomitant disease and post mortem interval (Table 3) [53]. In addition, the potential role of the history of systemic inflammatory diseases and infections may also influence microglial activation status [54–58]. Identification of the microglial cytokine expression profile as pro-inflammatory (M1 activation) or anti-inflammatory (M2 activation) ideally requires the availability of unfixed tissue. With the rise of genomic and proteomic technologies, the quality of preservation of the human tissue samples has become an issue and standardized protocols have been developed and optimized by brain banks to minimize the impacts of the clinical pre- and post mortem conditions. Brain tissue pH and RIN (RNA integrity number) values are proxy indices of agonal status and are useful indicators of the quality of the frozen tissue [59–61]. However, a study of 32 AD cases compared with 36 non-demented controls matched for age and gender found that mRNA integrity was not affected by the post mortem interval [62]. It is clear that the debate about the effects of confounding factors on mRNA integrity in human post mortem tissue remains open and it is crucial that the experimental group should be matched as well as possible with the controls and their selection based on the question to be investigated [63,64]. M1 activation of microglia, characterized by an upregulation of IL-1, IL-6 and TNF-a cytokines, has been identified in the context of traumatic brain injury and stroke [65–68], whereas exploration of the microglial profile in chronic neurodegenerative diseases appears more challenging. This is a reflection of microglia acting as sensors of changes in brain homeostasis by taking on the classical © 2012 British Neuropathological Society

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phenotype [26], and also the rapid nature of the acute insult, which excludes some of the confounding factors previously described and thus facilitating its study. Literature on chronic neurodegenerative disease modelled in animals has highlighted an M2 profile of microglia characterized by expression of transforming growth factor (TGF)-b1 and the lack of the typical pro-inflammatory cytokines IL-1b and TNF-a [69,70]. By adopting the M2 profile, it is suggested that microglia avoid bystander neuronal damage [71]. Inflammation in human AD brain has been associated with M1 activation in the vicinity of the amyloid deposits [72–74] mainly observed by immunoreactivity for IL-1b and complement proteins. However, this contrasts with a study showing a significant increase in AD of the genes associated with M2 activation, AG1 (arginase) and CHI3L1/CH3L2 (chitinase 3-like 1/2) with no difference in IL-1b mRNA level [75]. This is consistent with previous observation of the presence of TGF-b1 in AD using what at the time was pioneering microarray technology [76]; however, neither study indicates whether the proteins have an active role in disease progression. Recently, isolation of specific glial populations by immunolaser capture microdissection has been performed from post mortem human brain tissue using rapid immunohistochemistry [77]. In combination with microarray technology, this technique will allow identification of the changes in the gene expression of microglia specifically. Despite the practical difficulties, a recent study of brain gene expression in ageing and in AD using microarray technology identified changes in very many immune/ inflammation-related genes with marked upregulation of genes reflecting activation of microglia and perivascular macrophages, particularly those associated with innate immune responses [14]. Of note, the transcriptional changes were related more to cognitively normal ageing than to AD, with upregulation of the complement pathway, TLR signalling, inflammasome activation and immunoglobulin receptors all of which can promote release of pro-inflammatory molecules from microglia. Expression profiles of some genes below the microarray sensitivity, analysed by qualitative polymerase chain reaction (qPCR), showed increased expression of the proinflammatory cytokines IL-1b, IL-6, TNF-a and also the anti-inflammatory cytokine IL-10. As the changes were more strongly associated with cognitively normal ageing than the transition to AD, the authors suggested that they may be associated with priming of microglia as a prelude to the subsequent development of AD. NAN 2013; 39: 3–18

Advantages Definitive elaboration of microglial activation status (e.g. M1, M2) Can be detected by Western blot. Multiplex methods developed which can be used for fluid samples could possibly be applied to tissue. Applicable to brain tissue obtained by biopsy or post mortem. Tolerant of state of tissue preservation. Easy, quick, cheap. Provides spatial and anatomical information. Can be quantified (% area stained of total area examined). Can correlate findings with microglial morphology. Multi-label studies examined with confocal microscopy can relate microglia to specific features of disease (e.g. amyloid plaques) and other cell types at high resolution. Can be performed on formalin fixed paraffin embedded post mortem brain tissue. Can identify and quantify huge numbers of proteins simultaneously using very small tissue samples. Applicable to human post mortem tissue. Helps identify genetic variation that may influence disease susceptibility and corresponding proteins and pathways relevant to disease Can relate in real time to disease status.

Technique

Cytokine/chemokine gene expression (mRNA)

Cytokines/chemokines

Microglial morphology

Immunohistochemistry

Proteomics

Studies of genetic variants and genome wide association studies

In vivo imaging

Table 3. Advantages and disadvantages of different techniques for assessing microglial activity in the human brain


Currently expensive and requires positron emission tomography scanning. Available ligands currently very limited (e.g. PK11195)

Comparisons made between large groups of individuals.

Limited ability to detect small peptides and proteins of low abundance (e.g. cytokines).

Currently available antibodies cannot reliably indicate functional state of microglia – with the exception of phagocytosis (e.g. CD68). ‘Immunophenotyping’ may be useful.

Provides very limited information about what microglial are doing – with the exception of phagocytosis.

Difficult to detect. Present in small amounts. Very labile/short half-life. Needs fresh tissue.

mRNA is labile. Needs fresh tissue.

Disadvantages

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Very recent studies implicating variants in the TREM2 (Triggering Receptor Expressed on Myeloid cells) gene with risk of AD are of particular relevance to this discussion. TREM2 is considered to be a ‘gateway’ influencing the balance between phagocytic and pro-inflammatory microglial activity [78,79] with high levels of TREM2 promoting alternative activation and phagocytosis, whereas low levels of TREM2 induce a pro-inflammatory state. These studies suggest that control of microglial activation status is pivotal in AD pathogenesis. TREM2 was already known to be expressed by microglia surrounding amyloid plaques [80]. The implication for AD is that low TREM2 activity induces microglia to produce pro-inflammatory cytokines which in turn promote neurodegeneration, and that increasing TREM2 activity, thereby inducing alternative activation, may be a potential therapeutic strategy [78–80]. Proteomic technology has the ability to generate quantitative data about very many different proteins in very small samples of post mortem brain tissue [81], but the results seem somewhat biased in that small peptides (e.g. Ab) and molecules of low abundance (e.g. cytokines) may not be reliably detected (J.A.R. Nicoll, pers. obs.). However, some proteomic studies of cerebrospinal fluid and peripheral blood raise the possibility of the use of microgliarelated proteins as biomarkers for AD [82]. Histological immunophenotyping One of the challenges in the identification of microglia is the absence of a specific reliable maker to detect all microglia independently of their activation state. Lectins, which are carbohydratebinding proteins that bind specific sugar groups on cell

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membranes have been used but are not specific for microglia and stain endothelial cells in addition. Immunohistochemistry performed on formalin-fixed tissue is widely used to detect microglial proteins in situ and is highly informative [2] (Table 4). Antibody to CD45 (Leucocyte Common Antigen) has been suggested to label all microglia but as its name indicates it also detects other leucocytes present in the brain, such as lymphocytes, and in our hands does not stain human microglia well. In 1996, a group at Keio University in Tokyo published their discovery of a new protein they called ionized calciumbinding adaptor molecule 1 (Iba1), a 17 kDa protein consisting of 147 amino acids and coded for by the allograft inflammatory factor-1 gene (aif-1), residing in the major histocompatibility class-III region [83]. Iba1 expression is specific for microglia [84], is said to be constitutive and independent of the microglial state, whether resting or activated, and increases during inflammatory states [85]. Antibody to Iba1 is becoming widely used in animal, and increasingly in human studies although whether it genuinely stains all microglia, in our view, remains to be proven. Direct detection of cytokines in the human brain by immunohistochemistry, which could be used for determination of microglial activation status, has been achieved by only a few investigators, presumably reflecting the challenges associated with the detection of labile, soluble proteins which are present at a low concentration [86]. Currently, there is no simple link between the M1/M2 microglial phenotype as defined above and currently available immunohistochemical microglial markers, examples of which are listed in Table 4. Immunophenotyping of

Table 4. Immunophenotyping of microglia Microglial marker

Detection/function

Iba1 CD163

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 activation

HLA-DR CD68 MSR-A CD64 (FcgRI) CD32 (FcgRII) CD16 (FcgRIII) CD206 (Mannose Receptor) Fizz1 CD14/Toll-like receptor (TLR)-4


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Figure 2. Multi-label confocal microscopy of an amyloid plaque in a patient with AD actively immunized with Ab42. There is evidence of abundant phagocytic activity demonstrated by CD68 immunostaining of lysosomes (red) and a moth-eaten appearance to the plaque (Ab42, cyan) indicating that some Ab removal has already occurred. Activated microglia also showed by immunostaining for HLA-DR (green). AD, Alzheimer’s disease; Ab, amyloid-b protein; HLA-DR, human major histocompatibility complex class-II.


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microglia has limitations due to: (i) the detection of only one or a few proteins for colocalization in one experiment, in contrast to the large numbers interrogated by the recent genomic and proteomic technologies; (ii) the time-consuming nature of qualitative and quantitative histological analysis; and (iii) constraints imposed by currently available antibodies suitable for formalin-fixed paraffin-embedded tissue. Commonly used antibodies are CD68, a marker of microglial lysosomes indicative of phagocytic microglia, and HLA-DR (human major histocompatibility complex class-II), the antigen presenting protein complex extensively used to identify activated microglia [87] and although explore to some extent in vitro [88]. The degree to which increased immunoreactivity for microglial cell surface receptors that are associated with specific microglial functions (Table 4) correlates with upregulation of those functions is still unclear in the human brain. A further phenotype of microglia has been proposed in which microglia become dysfunctional with ageing, or senescent, characterized by structural deterioration and increased apoptosis [10,89]. It is suggested that microglia may lose their neuroprotective properties with advancing age [11] leading to chronic neurodegeneration. This observation is currently based purely on morphology changes following HLA-DR and Iba1 immunostaining rather than functional changes. The concept of macrophage priming has been well studied in vitro where it has been shown that priming of macrophages by a first stimulus, such as the cytokine g-interferon, leads to the synthesis of molecules such as receptors and signalling pathways so that a second

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triggering stimulus, for example LPS, leads to a more robust and exaggerated response when compared with the response of a naïve cell exposed to either stimulus alone [90]. The microglia present in the diseased brain in a relatively benign or anti-inflammatory but primed state may be subsequently activated or triggered by signalling molecules, for example from acute or chronic systemic inflammation. This may lead in turn to the generation of cytokines and other molecules with the capacity to cause either dysfunction or degeneration of the neurons, and consequently acute exacerbation of symptoms. This phenomenon of microglial switching from a benign state to a tissue-damaging phenotype by systemic infection and inflammation was first described in murine prion disease [91–94] but has now been described in diverse animal models [95–99] and may underpin the clinical decline associated with systemic inflammation [57,100]. Whether human microglia, as observed in the murine prion model, can switch from M2 to M1 activation with a detrimental effect on the brain needs to be confirmed [93]. However, there is now evidence that ageing in humans is associated with substantial pro-inflammatory microglial activation and the transition to AD is associated with relatively modest further changes. One interpretation of these observations is that ageing primes microglia which then respond to AD pathology [14]. With increasing interest in the concept of modifying microglial activation for therapeutic purposes it is interesting to note that treatment-induced changes in microglia have already been observed. In patients with AD who were actively immunized with Ab, plaque-associated

Table 5. Differences between rodent and human microglia Rodent

Human

Environment

Pathogen-free laboratory environment

Life expectancy Genetics Other variables Anatomical distribution

2 years Identical Defined by experimental design More numerous in grey vs. white matter [104,105] 11–14% [104,105] Extensive Yes, in certain circumstances Relatively well defined

Lifetime exposure to pathogens and common occurrence of terminal systemic infection (e.g. bronchopneumonia) 70–80+ years Genetic variation in microglial activation (e.g. TREM2) Requires careful selection of controls More numerous in white vs. grey matter [87,106]

White matter volume Knowledge of distribution, turnover, dynamics Recruitment of peripheral monocytes to CNS Inflammatory profile

49% [105] Limited Limited evidence Relatively limited knowledge

CNS, central nervous system. © 2012 British Neuropathological Society

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microglia (Figure 1L) acquire the ability to avidly phagocytose Ab plaques, demonstrated by confocal microscopy observations of Ab granules within lysosomes [87,101] (Figure 2), which they were previously unable to do. This provides evidence of a functional switch induced by the treatment, presumably mediated by opsonization of plaques by IgG stimulating their phagocytosis by microglia [87,101–103]. Such manipulation of microglial functional state may in future be of therapeutic benefit in a wide range of neurological disorders.

Conclusion Despite the number of publications in the microglial field, our knowledge of human microglia remains inadequate, partly as a result of the limitation in obtaining high quality mRNA from human post mortem material, the lability of cytokines and the potentially confounding effects of terminal events such as bronchopneumonia. Important gaps in our knowledge of human microglia in addition to identification of activation state, include factors such as their anatomical distribution, dynamic turnover capacity and differences between individuals resulting from different genetic background lifetime events (Table 5). Despite these obstacles, the data retrieved from human autopsy-derived material may be more relevant than results obtained from cell or animal disease models, which do not necessarily correspond with human biology and pathological processes. Communication between the animal and human spheres is essential to increase our understanding. Indeed, animal models by mimicking some aspects of the human disease are essential to study disease mechanisms and test therapeutic strategies involving manipulation of microglial activity with their findings then translated to humans.

Acknowledgements Author contribution: Delphine Boche wrote the first draft and all co-authors revised the manuscript.

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Received 5 December 2012 Accepted after revision 7 December 2012 Published online Article Accepted on 18 December 2012

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