Brain microglia are derived entirely from yolk sac macrophages; therefore, in the steady state, their functions can be directly attributed to this lineage. After birth, a massive expansion of microglia cell numbers is driven exclusively through in situ proliferation via M-CSF and the CX3CR1 ligand IL-34 without monocyte input [17-20]. Resident microglia have critical homeostatic functions in regulating synaptic development. As in the embryo, microglia retain their ability to alter neuronal circuitry during the postnatal period, where they are critical for synaptic pruning through engulfment of neuronal synapses, in addition to phagocytosis of apoptotic neurons [21] - activities that are necessary for healthy brain development. Importantly, microglia dynamically interact with neurons at both pre- and postsynaptic sites through the extension of microglial processes, where prolonged contact leads to neuronal elimination [22] and at neuronal cell bodies through somatic microglial junction [23]. Microglia can also suppress neuronal activity through the catabolism of extracellular ATP [13]. The expression of CX3CR1 is critical in controlling microglia numbers, synaptic pruning, and functional brain connectivity as determined by Cx3cr1 deficient mice studies [24]. Microglia, however, are distinct from other tissue macrophages owing to their unique homeostatic phenotype and tight regulation by the central nervous system (CNS) microenvironment. Microglia are responsible for the elimination of microbes, dead cells, redundant synapses, protein aggregates, and other particulate and soluble antigens that may endanger the CNS. Furthermore, as the primary source of proinflammatory cytokines in the CNS, microglia are pivotal mediators of neuroinflammation and can induce or modulate a broad spectrum of cellular responses. Alterations in microglia functionality are therefore implicated in brain development and aging, as well as in neurodegeneration.

One challenge in defining brain microglial properties and microglia activation states in relation to the neuropathology of neurodegenerative diseases with microglial components has been lack of accessibility to fresh biopsies and the microglia susceptibility to cryopreservation damage. Thus, studies relied heavily on ex vivo phenotyping studies of brain isolate microglia by immunohistochemistry techniques from fixed human tissue sections of postmortem human brain white matter [25]. Alternative approaches to studying microglia involves a combination of technologies such as laser capture microdissection (LCM) from brain tissue sections along with flow cytometry and gene expression profiling or ablation of microglia with, for example, PLX5622, a CSF1R inhibitor [13]. The LCM samples help identify discrete populations of neurons, astrocytes, microglia, and endothelial cells in intact tissue sections [26], flow cytometry allows examination of homogeneous cell populations, and gene expression profiling uniquely permits the study of genes that are not readily responsive to antibody staining (e.g., soluble chemokines / cytokines) [26, 27]. Recent observations about microglia ontogeny combined with extensive gene expression profiling and novel tools to study microglia biology have allowed us to characterize the spectrum of microglial phenotypes during development, homeostasis, and disease [28]. Studies revealed that microglial cells are highly dynamic in their interaction with the microenvironment, response to inflammatory signals [29], and interact with neuronal circuits at the synaptic level [30]. Microglia phagocytic activity can sculpt the brain and affect its physiology [21].

Moreover, microglia population turnover in the brain is a highly dynamic process that is made possible by the fine-tuned temporal and spatial balance of microglial proliferation and apoptosis [31]. It is still unclear whether the microglial function in neurodegenerative diseases is beneficial but insufficient, or whether microglia are only effective at the early disease stages but lose their efficacy or even become detrimental in later stages. Importantly, the pathways and molecular mechanisms of microglia activity at the different stages of Alzheimer’s disease (AD) remain controversial [32].

In studies that aimed to better understand how microglia regulate the synaptic development of neurons it was found that after acute brain injury such as brain ischemia, microglia significantly increase the duration of their interactions with synapses and protect the brain [33]. However, in chronic disorders, such as neurodegenerative diseases, that are often associated with the numerical expansion of activated microglia, a dysfunction in the macrophage degradative pathways develops, which seem to contribute to the disease pathology [30, 33]. In animal models of Alzheimer’s disease, for example, microglia numerically expand, and beta-amyloid plaques progressively accumulate, an outcome that is hypothesized to impair the protective phagocytic function of microglia [34].

One initial and significant finding that facilitated the definition of microglia inflammation state in AD is the expression of the major histocompatibility complex class II protein HLA-DR, which is used as a marker of activated microglia. This finding promoted the hypothesis that increased expression of HLA-DR by microglia may serve to designate the classically activated, M1 microglia phenotype; however, HLA-DR upregulation could also be a feature of alternatively activated microglia with anti-inflammatory phenotypes, M2 [35, 36]. As shown in Fig 1 below, HLA-DR-stained microglia can be found with various morphologies indicating different levels of activation and ranging from highly ramified in A to highly activated in E, with enlarged cell body and processes. Additional markers that identify microglial polarity were therefore necessary, and the markers that are currently used to describe microglia in human brains include the ionized calcium binding adaptor molecule-1 (IBA-1) [37], CD68 [2], CD14 [38, 39], CD40 [40], the FcγRs CD16, CD32 and CD64 [41] and others (Table 1). A description of each of these is provided below.

As with other tissue resident macrophages, the main goal in microglia research was to define the M1/M2 polarization and any M2 subset markers. However, there is uncertainty about the assignment of the M1 and M2 protein markers. This is because a spectrum of microglial phenotypes and morphologies can be present within a human brain, representing different stages of differentiation, activation, and function in tissue [42]. Instead, markers that identify specific microglia functions, or disease association, have been utilized. For example, markers that are associated with phagocytic microglia; The FcγRs: CD16, CD32 and CD64 [41, 43], and CD36, a phagocytic scavenger receptor for amyloid beta peptide (Aβ) [44]. Markers that are increased in disease; CD45 [45, 46], specifically in AD, CD33 [7], and in plaque-associated microglia, TLR2 [38], cyclooxygenase-1 [47], and CD68 [48].

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Figure 1. Various morphologies in human brain sections from AD case. Immunohistochemistry HLA-DR stain with LN3 antibody, and counterstained with neutral red. Bar=20 uM. Reproduced from Figure 1 of [1].

A breakthrough in this area of research was generated from a range of profiling studies by Gordon and colleagues that identified some valid markers for the classically or activated human microglia [49, 50]. These studies defined the types of microglia that could be mediating inflammatory tissue damage, M1, based on changes in response to the proinflammatory agents LPS, and IFN-gamma. The production of reactive oxygen species further defined the M1 microglia through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, and by increased levels in the pro-inflammatory cytokines TNF-a, and IL-1b - See table 2. CD40 has been consistently shown to be a marker for M1 activation of macrophages/microglia [40].

The alternative activated (M2) microglia was defined as the phenotype that is responding to IL-4 or IL-13. Later this phenotype was further subcategorized into M2a, M2b, and M2c groups. The M2a phenotypes have increased phagocytosis activity and production of growth factors such as insulin-like growth factor-1 and anti-inflammatory cytokines such as IL-10 [36]. These microglia could remove cellular debris and promote tissue repair. The M2b phenotype is induced by ligation of immunoglobulin Fc gamma receptors (FcγRs), CD16, CD32 or CD64, or by immune complexes on LPS or IL-1β primed microglia. This phenotype has been described on microglia in AD brains which are characterized by increased expression of the FcγRs CD32 and CD64, and by phagocytic activity [41]. M2b activation is also characterized by down-regulated expression of IL-12, increased IL-10 secretion, and increased HLA-DR expression. The M2c microglia phenotype represents the acquired deactivation. This phenotype is induced by the anti-inflammatory cytokine IL-10 or glucocorticoids, and is associated with increased expression of TGF-b, sphingosine kinase (SPHK1) and CD163, the membrane-bound scavenger receptor for haptoglobin/ hemoglobin complexes [51].

One potential limitation to the M1 or M2 immune classification is that it excludes microglia undergoing cell division in response to the macrophage CSF-1 or IL-34. These cytokines signal through the same receptor (CSF-1R) to induce microglia cell division and affect their development, maturation, and survival [52]. Recent findings suggest that CSF-1 treated microglia have neither an M1 or M2a polarization state [53], although other works have classified CSF-1R signaling as similar to M2a activation [36]. Moreover, cell division by microglia can be considered an ongoing feature of microglia in pathology-rich areas and is required to replace these short-lived cells. Thus, an M3 phenotype classification has been suggested for proliferating microglia [1], also included in Table 2.

In conclusion, to better understand the role of microglia in neurodegenerative diseases, a panel of antigenic markers and reagents that can detect microglial activities in human brain tissues that are associated with immune phenotypes and functions are needed. For example, markers that can be used to identify the production of reactive oxygen species or the secretion of cytokines would be the optimal choice. This would also require alternative access to human brain tissues that can preserve this information rather than be limited by the availability of antibodies that react well with fixed human brain tissues. In addition, reliable fluid biomarkers that can be measured in cerebrospinal fluid (CSF) or blood may enable more effective drug development and the establishment of a more personalized medicine approach for AD diagnosis and treatment. Below is a description of some of the primary markers that are used in the study of brain microglia.

P2Y12 is a G-protein coupled receptor for adenosine diphosphate (ADP). It is higly enriched in brains. Its expression in brain, limited to microglia, decreases upon the activation of microglia [54]. Zrzavy T et al identified resting / homeostatic microglia in cortices and white matters of septic patients with P2RY12 labeling [9]. Cserép C et al demonstrated the crucial role of P2Y12 in establishing somatic microglia–neuron junctions [23].

Also known as allograft inflammatory factor (AIF), ionized calcium-binding adapter molecule 1 (IBA1) is a 17kDa calcium-binding helix-loop-helix protein that is consistently expressed on all microglial subtypes (pan marker). Encoded by the AIF gene located within the major histocompatibility complex region on human chromosome 6, IBA1 is involved in microglia motility and phagocytosis and is associated with microglial cell activation. Microglial localization of IBA-1 was first identified by Ito et al by immunohistochemistry on formalin-fixed rat brain sections [37]. In humans, IBA1 is an IFN-γ-inducible protein predominantly expressed on the membrane and cytoplasm of ramified and amoeboid microglia (Figure 2) [2]. Ising C et al labelled microglia in mouse hippocampi with IBA1 antibodies [55]. Dominy SS et al used IBA1 as a marker of microglia in IHC staining of hippocampi from control subjects and Alzheimer's disease patients [56]. C Adaikkan et al identified Iba1 immunoreactive cells was microglia in mice [57].

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Figure 2. Expression of microglia markers in ramified/inactive microglia (A), rounded amoeboid microglia (B), and foamy/phagocytic macrophages (C). Immunohistochemistry staining, Bar=20 uM, reproduced from Figure 1 of [2].

MHC class II molecule (HLA-DR) is a heterodimer consisting of an alpha (DRA) and a beta chain (DRB), both anchored in the membrane. The alpha chain is approximately 33-35 kDa, and the beta chain is around 26-28 kDa. This heterodimer plays a central role in the immune system by presenting peptides derived from extracellular proteins. Class II MHC molecules are expressed in antigen presenting cells (APC: B lymphocytes, dendritic cells, macrophages) and are constitutively expressed at a steady state in resting microglial cells [2] with consistent up-regulation in neuroinflammatory conditions such as experimental autoimmune encephalitis, Alzheimer’s disease, Parkinson’s disease (PD) and multiple sclerosis (Figure 2) [58-60].

Classified as a myeloid cell marker, CD68 is a 110kDa transmembrane glycoprotein encoded by CD68 gene located on human chromosome 17. CD68 is one of the most useful and descriptive markers for microglial function (macrosialin in mice). This protein is localized to the lysosomal membrane in microglia and monocytes and is upregulated in actively phagocytic cells [48]. Both M1 polarized and M2 polarized microglia/macrophages can express CD68 [49]. CD68 is a prominent member of LAMP (lysosome-associated membrane protein) family, and functionally it regulates phagocytosis (Figure 2).

TMEM119 is an evolutionarily conserved protein that is encoded on the human chromosome 12. It was identified in a comparative analysis of microglial transcriptome datasets and was shown to be expressed exclusively on a subset of Iba1(+) CD68(+) microglia with ramified and amoeboid morphologies in the brains of neurodegenerative diseases, such as AD. Because TMEM119 was excluded from Iba1(+) CD68(+) infiltrating macrophages, the authors of the study postulated that this protein may serve as a reliable microglia marker that discriminates resident microglia from blood-derived macrophages in human brain [61]. To further study TMEM119 during microglia development and after an immune challenge, Bennett ML and colleagues developed monoclonal antibodies to TMEM119 intracellular and extracellular domains to enable the immunostaining of microglia in histological sections in healthy and diseased mouse brains, and in FACS isolated nonactivated microglia. The authors demonstrated that Tmem119 is developmentally regulated in the mouse, and it matures in vivo by postnatal day 14 (P14). In addition, it is a stable microglia marker in that Tmem119 expression is maintained even in the presence of CNS injury as assessed in three mouse models of injury and disease including sciatic nerve injury-induced microglial activation, lipopolysaccharide (LPS)-induced systemic inflammation, and optic nerve crush injury [3]. The study tools established in this study, monoclonal anti-mouse Tmem119 antibodies, are available to the research community via Abcam ( ab209064 and ab210405). Fig 3 confocal images illustrate the expression of TMEM119 in normal human brain. Pluvinage JV et al labeled microglia from young and old mice to study the role of CD22 in homeostatic microglial phagocytosis during aging [62]. Zrzavy T et al used TMEM119 labeling to distinguish microglia from blood-derived macrophages in the cortices and white matter of septic patients [9].

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Figure 3. TMEM119 is enriched in human microglia. Representative confocal images of normal cortical tissue showing localization of TMEM119 (green) to highly ramified, Iba1+ cells (red) at low (upper) and high (lower) magnification. Bars=30 um. Reproduced from Figure 6B of [3].

CD14 is encoded by the CD14 gene located on human chromosome 5. The protein encoded by this gene is a surface antigen that is preferentially expressed on monocytes / macrophages. Greenwood DJ et al, for example, isolated human monocyte-derived primary macrophages with anti-CD14 magnetic beads from Miltenyi (130-050-201) from preparations of white blood cells [63]. It cooperates with other proteins to mediate the innate immune response to bacterial lipopolysaccharide. CD14 has been used to determine the degrees of activation in macrophages and microglia by flow cytometric analyses. Some view CD14 as a constitutive macrophage/microglia marker, however, in populations of monocytes/ microglia, high or low expression of CD14 has been useful in defining the levels of microglial activation [27]. Few studies characterized the cellular localization of CD14 expression in microglia in human AD-affected brains [38, 39]. CD14 antibody stains blood monocytes in brain vessels abundantly, and can also detect plaque-associated microglia.

As members of immunoglobulin (Ig) superfamily of proteins, Fcγ receptors (FcγRs) are involved in immune cell activation and are considered phagocytic. Macrophages and microglia express multiple types of the FcγR family. These include FcγRI-CD64 [64], FcγRII-CD32, and FcγRIII-CD16, all located within human chromosome 1. CD64 and CD16 activate proinflammatory signaling, and CD32 activate inhibitory signaling. Increased expression of these receptors has been associated with the acquisition of the M2b phenotype [65]. Microglia expressing CD16, CD32 and CD64 have been described in AD brains with increased levels of expression in pathology-associated microglia [41, 43].

CD163 is a member of scavenger receptor cysteine-rich (SRCR) superfamily class B of proteins that is mapped to human chromosome 12p13.31. CD163 is a 130kDa cysteine-rich transmembrane glycoprotein that binds to hemoglobin-haptoglobin (Hb-Hp) complexes with high affinity and mediates their Ca2+-dependent endocytosis, thus facilitating the Hb clearance from plasma [66]. In microglia, CD163 immunoreactivity is selectively increased both under acute and chronic inflammatory conditions such as AD, and PD [67]. As indicated in Table 6, CD163 is a marker for anti-inflammatory M2-microglia (M2c), and it is involved in the suppression of inflammatory responses by inducing the production of IL-4 and IL-10 [10].

CD33 (Siglec-3) is a sialic acid-activated receptor encoded on the human chromosome 19. CD33 expression is generally restricted to myeloid cells and is expressed on microglia in human brains [7, 34]. Interest in this marker started when a protective single nucleotide polymorphism (SNP rs3865444] that modulate the gene splicing was identified adjacent to the coding region. Possession of the SNP allele was associated with reduced levels of CD33 in human brains [7, 34], enhanced the microglial activity, less accumulated brain β-amyloid [68, 69], and late-onset Alzheimer's disease [68]. The mechanism of the SNP allele is hypothesized to be the result of localization and accumulation in peroxisomes instead of the cell surface, which interferes with engagement in cell-surface signaling, leading to enhanced β-amyloid clearance [69]. The wild type CD33 allele is constitutively expressed on microglia with increased levels on hypertrophic microglia and is suggested to polarize microglia towards the M2 phenotype [7].

The CD40 gene is a member of the TNF-receptor superfamily. The encoded protein is a receptor on antigen-presenting cells of the immune system and is essential for mediating a broad variety of immune and inflammatory responses. The adaptor protein TNFR2 interacts with this receptor and serves as a mediator of the signal transduction. CD40 is necessary for amyloid-beta-induced microglial activation, and in Alzheimer disease, CD40 is reactive to microglia. CD40 expression by microglia is also up-regulated in a variety of brain insults and is not limited to lesions with amyloid beta-protein deposits, and is thought to polarize microglia towards the M1 phenotype [40, 57].

CD45 is a member of protein tyrosine phosphatase (PTP) family and is officially known as protein tyrosine phosphatase, receptor type C (PTPRC). PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitosis, and oncogenic transformation. CD45 has multiple isoforms and is expressed by all hematopoietic cells [70]. The usefulness of phenotyping human brain microglia for CD45 (leukocyte common antigen) is unclear because this marker appears to identify all microglia in human brains, regardless of the activation state and increased expression in AD pathology-associated microglia [45]. For example, phenotyping microglia for CD45 from fresh human brain tissues reveal lower expression levels in microglia compared to macrophages, and higher CD45 expression in white matter microglia compared to gray matter cells [45, 46]. Additional studies are necessary to evaluate the usefulness of this marker in brain microglia.

This gene encodes a membrane protein that forms a receptor signaling complex with the TYRO protein tyrosine kinase binding protein. The encoded protein functions in immune response and displays a broad expression in the brain. A study that compared its expression with IBA1 and CD68 showed that it did not stain microglia, but specifically labeled monocytes within vascular lumens [71] suggesting that TREM2 may serve as a systemic immune response marker. TREM2 expression is indeed upregulated in AD brains, where it may have a protective effect in the early stages, through the phagocytic clearance of amyloid beta (Aβ) plaques, but a pathogenic effect in the later stages through activation of the inflammatory response [72]. Thus TREM-2 marker appears similar to the M2a anti-inflammatory receptors. Rare TREM2 variants have been associated with an increased risk of developing AD [73], and a soluble variant, sTREM2, can be detected in CSF and has the potential to be used as a fluid biomarker for AD [15, 16].

This antimicrobial gene is one of several Cys-Cys (CC) cytokine genes clustered on the q arm of chromosome 16 (16q21). Cytokines are a family of secreted proteins involved in immunoregulatory and inflammatory processes. The product of this gene binds to chemokine receptor CCR4 and may play a role in the trafficking of activated T lymphocytes to inflammatory sites and other aspects of activated T lymphocyte physiology. One recent study defined CCL22 as an M2a marker by showing increased secretion when culturing human microglia in response to IL-4 [6].

FTL on human chromosome 19 encodes ferritin. It is an iron storage protein, and there are two subunits, heavy (H) and light (L), based on their molecular weight. The H-/L subunits ratio may be different in tissues, depending on the specific tissue and pathophysiological status. Antibodies to specifically L-ferritin selectively identify classes of microglia in human brain tissue sections [74]. Ferritin is the most abundant carrier protein for iron in the brain, and increased expression is associated with increased iron uptake. This phenotype is also associated with proinflammatory microglia responses and with reactive oxygen species production, which is dependent on iron. Ferritin has been used to describe a dystrophic and degenerating phenotype of microglia, however, their immune phenotype is unclear [11].

The protein encoded by this gene is a fructose transporter that is responsible for fructose uptake by the small intestine. The encoded protein is also necessary for the increase in blood pressure due to high dietary fructose consumption. GLUT5 is also expressed in several other tissues including microglial cells in the brain. Payne et al used immunocytochemistry and immunoblotting on formalin-fixed brain tissues to demonstrate the expression of GLUT5 on microglial cells [75]. Antibodies against human GLUT5 consistently stain microglia, including ramified, activated and amoeboid cell types, in both paraffin and cryostat sections of the human brain [76]. Increased microglial GLUT5 expression has been observed in gliomas and is suggested to promote tumor proliferation.

Also known as fractalkine receptor or GPR13, CX3CR1 is a classical Gαi-coupled 7-transmembrane receptor (GPCR) that binds to a chemokine called fractalkine (CX3CL1) and regulates the downstream signaling. In the brain, CX3CR1 is expressed exclusively on the microglia and functions as a housekeeping gene involved in microglial activation and their migration to synaptic neurons [77]. Importance of CX3CR1 in microglia had been elucidated through studies done using CX3CR1-/- mice. Depletion of fractalkine receptor in these mice causes impaired synaptic formation, together with altered behavioral responses and abnormal neurogenesis [78]. CX3CR1 deficiency also prevents activation of microglia and prevents neuroinflammation, as seen in animal models of cerebral ischemia and Alzheimer’s disease [79, 80]. In contrast, ablation of CX3CR1- CX3CL1 axis augments neurodegenerative processes in Parkinson’ disease and amyotrophic lateral sclerosis [77]. Cserép C et al, for example, used CX3CR1+/GFP mice to observe microglial processes [23].

One of the disadvantages of using above mentioned cellular markers is their insufficiency in discriminating between microglia and macrophages, as they are expressed on both cell types. However, current advancements in research methodologies such as next-generation RNA sequencing have allowed researchers to identify gene clusters that are uniquely specific to microglia. These gene clusters are collectively termed as “sensome” as they are associated with microglial ability to sense their environment, also called the sensory apparatus [81]. Microglial-specific sensome genes were first identified by Hickman et al using quantitative transcriptomics and fluorescence in situ hybridization on microglia and peritoneal macrophages isolated from healthy adult C57BL/6 mice. Of 2012 transcripts identified through sequencing, 626 genes were significantly enriched in microglia predominant among which included metabolic enzyme Hexosaminidase B (HexB), purinergic receptors P2ry12, P2ry13 and P2ry6, chemokine receptor Cx3cr1, triggering receptor Trem2 and sialic-acid binding immunoglobulin lectin Siglech [81]. Majority of these genes are known to interact with endogenous ligands, which are supposedly released as signaling molecules by other cells in response to an environmental stimulus. For this reason, sensome genes are believed to play an important role in neural development and neuroinflammation [82-84]. For example, Thion et al demonstrated a distinct in-utero embryonic sensome gene expression profile, which evolves as the mice undergo different stages of development to adulthood and aging [82]. Specificity of sensome genes on human microglia still remains debatable. However, using this gene panel as a tool to identify microglia in the brain shows promise owing to the presence of overwhelming evidence on their role in maintaining CNS homeostasis.

Nott A et al isolated microglial nuclei from human brain tissues through FANS with an antibody against PU.1/SPI1, an ETS-domain transcription factor that activates gene expression during myeloid and B-lymphoid cell development [14].

This article was derived from sections in the article "Macrophage Markers" in November 2019.