A Guide to Microglia Markers

Microglia are the resident macrophage population of the central nervous system (CNS). These highly plastic cells constantly survey the brain microenvironment with their long, ramified processes, scavenging for debris and infectious agents. This scavenging role also extends to the development and maintenance of neuronal circuits via synaptic pruning (the removal of neuronal connections). Microglia react to pathological insults by retracting their processes and taking on a more amoeboid morphology that is more conducive to movement, immune activation, and debris clearance, a process classically referred to as microglial activation. Under these circumstances, however, peripherally derived leukocytes can infiltrate the CNS and acquire microglia-like phenotypes, complicating the identification of bona fide microglia in such contexts.   

Microglia-specific markers have been discovered with the help of omics technologies like RNA-seq. This article reviews the general and discriminating markers of microglia as described in recent literature.

Figure: This diagram highlights general markers for microglia, including those that are unique and those that are shared with macrophages and other leukocytes. 

General Markers of Microglia

Traditionally, microglia have been identified using nonspecific macrophage markers. CD45 and CD11b are used to detect microglia in single-cell suspensions of brain tissue by flow cytometry; Iba1 is a popular marker of microglia for immunohistochemistry; and CX3CR1-GFP mice, in which Cx3cr1⁺ cells are labeled green, have been an indispensable tool for in vivo studies of microglia using two-photon microscopy. These  markers, however, are not microglia specific: CD45 and CD11b are expressed by many leukocytes, and Iba1 and Cx3cr1 by many macrophages inside and outside the CNS. When using these markers, additional features such as the level of marker expression (microglia are CD45^{lo/intermediate} when compared to other CD11b⁺ leukocytes) and morphological features (long, thin processes extending from the cell body) are required to more definitively (but not conclusively) discriminate microglia from other macrophages in flow cytometry and microscopy, respectively. And even then, these markers should be used only with the caveat that they do not exclusively label microglia.

Additional general microglial (and, indeed, macrophage) markers include CD14, CD80, CD115, CD206, HLA-DR, F4/80, CSF-1R, and CD163, as well as the transcription factors PU.1 (SPI1) and MafB. CD206 and PU.1 can be useful for discriminating between yolk sac-derived macrophages (like microglia) and bone marrow-derived macrophages (such as those that infiltrate the CNS during pathology). Microglia are PU.1-dependent and CD206^lo; by contrast, bone marrow-derived macrophages are Myb-dependent and CD206^int/hi. CD206 is also highly expressed by the perivascular macrophages that reside at the CNS border, enabling them to be discriminated from microglia in the CNS parenchyma. 

Discriminating Markers of Microglia

The application of high-dimensional technologies — especially RNA-seq — to immunophenotyping has shed more light on the uniqueness of microglia, enabling the identification of highly specific markers of this macrophage population. Here, specific refers to specificity among macrophage populations. Indeed, many so-called microglia-specific markers are expressed by non-macrophage cell types. 

Discriminating, or microglia-specific, markers include Tmem119, P2ry12, Hexb, Fcrls, Sall1, C1q, Gpr34, Olfml3, Mertk, Pros1, Tyro3, and Tgfbr1. 

Tmem119 has been proposed to be a highly specific marker of mature microglia. In mice, microglia begin expressing this marker at around postnatal day 14, a time at which microglia stop proliferating and after which their transcriptome changes very little. Whereas many microglia-specific markers become downregulated during microglial activation, Tmem119 immunoreactivity remains stable during this phenotypic switch, making it useful for discriminating reactive microglia from CNS-infiltrating macrophages. Although Tmem119 is also expressed by non-macrophage cell types such as osteoblasts outside of the central nervous system (CNS), its expression in the mature CNS is limited to microglia. 

Like Tmem119, P2ry12 is a microglia-specific marker, at least in the healthy, unperturbed brain. However, not only is this marker downregulated on reactive microglia, it can also be found on peripherally derived macrophages that immigrate to the CNS. 

Fcrls, while microglia-specific, exclusively marks mouse microglia, as it has no human ortholog.  

Finally, among bone marrow-derived and tissue macrophage populations, the transcription factors Sall1 and Mef2a are limited to microglia. However, Sall1 is also expressed by select neuronal populations and glial cells in the CNS.   

Markers of Microglial Activation

When homeostasis is disturbed, microglia react by becoming “activated”. The morphological changes observed during this transformation — from ramified to amoeboid — are accompanied by transcriptomic and proteomic changes. Efforts to delineate these changes have revealed many markers of microglial activation. 

Some of the most well-established markers of microglial activation are actually the general microglial markers Iba1, CD68, CD206, and CD45. Not only do microglia express these markers at baseline, some at low levels (CD206 and CD45 in particular), they upregulate these markers during activation. 

Microglia typically downregulate so-called “homeostatic” markers during activation. These include P2ry12, Fcrls (in mice), and Sall1, among others. Tmem119, as mentioned above, is a notable exception: although Tmem119 is transcriptionally downregulated in activated microglia, microglia retain Tmem119 immunoreactivity (protein).   

Markers of microglial activation depend on the activating stimulus. The classical model of macrophage activation (the M1/M2 paradigm) does not adequately capture the spectrum of microglial activation phenotypes. The M1/M2 phenotypes are in vitro phenotypes that occur in response to pro-inflammatory (M1) or anti-inflammatory (M2) stimulation. Their in vivo relevance is dubious.  

Perhaps one the best characterized activated microglial phenotypes is that observed in Alzheimer’s disease. These microglia, called disease-associated microglia (DAM), were initially characterized in mouse models of Alzheimer's disease. In DAM, homeostatic microglial markers are downregulated and Trem2, Apoe, Itgax, Clec7a, Axl, and Lpl, among other genes, are upregulated. Since their discovery, these DAM markers have been observed in other neurodegenerative contexts, too.

Markers of Microglial Function

Arguably, all microglial markers serve some function or other. However, the functions of some markers remain poorly understood. Tmem119 is a notable example of a marker whose function remains elusive despite its prominence as a microglial marker.

Other markers are better characterized. To provide a few examples: the complement protein C1q participates in synaptic pruning during development and disease; P2ry12 mediates the chemotactic movement of microglial processes; the Tyro/Axl/Mertk (TAM) receptors are used during efferocytosis, or the clearance of apoptotic bodies; CSF-1R is critical for self-renewal; and IGF-1 plays roles in myelin formation. Of course, as macrophages, microglia also express a wide array of cytokines and chemokines in support of inflammatory processes (not summarized here).

Table of Microglial Markers

The table below lists human and mouse proteins characterizing various subsets of microglia as described by a recent review literature. The majority of proteins listed are membrane markers expressed on the cell surface, but also included are other defining proteins, including transcription factors and signaling proteins, such as cytokines. Accompanying each marker are links to relevant antibodies and ELISA kits, as these immunodetection tools are routinely used in cell characterization studies via flow cytometry and immunostaining. The associated products are offered by a variety of manufacturers and can serve as a useful reference for microglial immunophenotyping.

Note: *Some markers are protein isoforms, multi-subunit protein complexes, or protein families composed of several distinct genes. Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org (for human genes only). 

References

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