Common and distinct features of diverse macrophage populations in the central nervous system

Abstract

Tissue-resident macrophages perform indispensable functions in the development, maintenance, and repair of tissues. Microglia are the primary resident immune cells in the central nervous system (CNS), functioning as intracerebral macrophages distributed throughout the brain parenchyma. In addition to microglia, there is another, less well-characterized type of macrophage known as CNS border-associated macrophages (CAMs), and the existence of these cells has been recognized for several decades. With recent advances in research technologies, an increasing number of studies have focused on CAMs, and our understanding of them has begun to improve. In this article, we review the cellular characteristics and functions of CAMs that have been elucidated thus far, with a particular focus on the similarities and differences between CAMs and microglia.

Diversity of tissue-resident macrophages in the central nervous system

The central nervous system (CNS) encompasses the brain and the spinal cord, where microglia function as the resident macrophages distributed throughout the parenchyma.^1),2) The morphology and gene expression profiles of microglia are highly conserved among animal species,³⁾ suggesting that microglia play a universally important role in the brain of each animal species from an evolutionary perspective. In contrast, there are distinct types of macrophages that are located at the border regions of the CNS, such as the meninges (comprising the dura mater, arachnoid membrane, and pia mater), perivascular spaces (also known as Virchow-Robin spaces), and the choroid plexus, which are collectively referred to as CNS border-associated macrophages (CAMs).^4)-7) CAMs are generally classified into three categories based on their anatomical localization: meningeal macrophages, perivascular macrophages, and choroid plexus macrophages. Meningeal macrophages are further subdivided into two distinct categories: dura mater macrophages and leptomeningeal macrophages. Additionally, choroid plexus macrophages are classified into two discrete populations: epiplexus Kolmer cells are situated at the epithelial apical surface, whereas choroid plexus stromal cells are located within the stroma. In contrast to microglia, which have been the subject of extensive research and are known to play crucial roles at various stages of brain development including angiogenesis, the removal of excess nerve or progenitor cells, protection of oligodendrocyte progenitors, promotion of neurogenesis, and synaptic pruning,¹⁾ the function and signature of CAMs remain largely unknown.

Origin and development of microglia and CAMs

Both microglia and CAMs are thought to originate from early erythron-myeloid progenitors (EMPs)⁸⁾ (Fig. 1), which first appear in blood islands in the yolk sac at early embryonic stages and subsequently differentiate into macrophage progenitors, a process that is dependent on transcription factors PU.1, interferon regulatory factor 8 (IRF8), and lymphoblastic leukemia-derived sequence 1 (Lyl-1).^9),10) This differentiation occurs prior to reaching the fetal brain via the fetal circulatory system at approximately the same time as the onset of neural development (in mice, it occurs after 9.5 days of gestation; in humans, it occurs after 4.5 weeks).^8),11),12) At what point do microglia and CAMs differentiate into their respective cell types? Using single-cell RNA sequencing (scRNA-seq) analysis technology, we previously conducted a comprehensive gene expression analysis of previously characterized A1 and A2 macrophage progenitor cells in the yolk sac and demonstrated the existence of a multitude of macrophage progenitor cells with disparate gene expression profiles.¹³⁾ In particular, more mature A2 progenitor cells with high expression of Ptprc and Cx3cr1 comprised not a single population, but rather a group of cells classified according to their expression levels of Mrc1, encoding mannose receptor C-type 1 (also known as CD206), and vimentin (Vim). This classification included cells with high expression of Mrc1 and Vim, as well as cells with low expression of Mrc1 and Vim. Given that Mrc1 is known to be one of the genes highly expressed in adult CAMs,^5),6) it is plausible that microglia and CAMs are already separated in the yolk sac. Utz et al. provided a comparable hypothesis, identifying two cell populations within the embryonic yolk sac that differ in their CD206 expression levels (CD206 positive and negative).¹⁴⁾ To substantiate this hypothesis, we conducted a lineage tracing analysis using the Mrc1^CreERT2 Rosa26^tdTomato mouse model, in which tamoxifen administration induces the permanent labeling of Mrc1-expressing progenitor cells with the fluorescent protein tdTomato.¹³⁾ The results demonstrated that, contrary to expectation, Mrc1-expressing progenitor cells are capable of differentiating into both microglia and CAMs within the brain.¹³⁾ In summary, despite the existence of multiple macrophage progenitor cells within the yolk sac, they are not inherently distinct progenitors with a clearly delineated subsequent cell lineage. On the other hand, these analyses do not preclude the potential involvement of alternative progenitor sources in the development of CAMs during development, such as the fetal liver and aorta-gonad-mesonephros,¹⁵⁾ necessitating further in-depth analyses.

Fig. 1

(Color online) Origin, development, and maintenance of diverse central nervous system macrophages. During embryogenesis, erythro-myeloid progenitors (EMPs) arise in the blood island of the yolk sac and give rise to A2 pre-macrophage progenitors via immature A1 progenitors in a PU.1- and interferon regulatory factor 8 (IRF8)-dependent manner. The A2 progenitors include cells with high expression of Mrc1 (Mrc1^＋) and a Mrc1-negative population (Mrc1^－), both of which travel to the brain and give rise to either microglia or CAMs. Microglia reside in the CNS parenchyma, where they undergo maturation in response to transforming growth factor-beta (TGF-β) signaling. After perivascular spaces are established in the perinatal stages, leptomeningeal macrophages give rise to perivascular macrophages. Microglia, leptomeningeal macrophages, and perivascular macrophages are maintained by self-renewal in a manner dependent on colony stimulating factor 1 receptor (CSF1R) signaling, with no contribution from bone marrow (BM)-derived cells. In the dura mater and choroid plexus, a fraction of macrophages appears to be continuously replaced by BM cells. The CSF1R ligands involved in CAM survival (CSF1 vs. interleukin 34) are unknown.

Maturation and maintenance of microglia and CAMs

After entering the brain parenchyma,^8),16) macrophage progenitor cells originally from yolk sac receive transforming growth factor-beta signaling (Fig. 1),¹⁷⁾ which is produced and released by neurons, astrocytes, oligodendrocyte progenitors, vascular cells, and microglia. This signaling activates the SMAD pathway, which leads to differentiation and maturation. Once established in the CNS, the cell density and function of microglia are tightly regulated by a colony-stimulating factor 1 receptor (CSF1R) signaling-dependent cycle of infrequent proliferation and cell death,^18),19) without replacement by bone marrow (BM)-derived peripheral blood cells. Furthermore, microglia and CAMs exist independently from each other without any cellular exchange in homeostatic conditions or during depletion-induced repopulation.²⁰⁾

Conversely, both meningeal and perivascular macrophages display distinctive behaviors within the developing brain.⁷⁾ Perivascular macrophages reside in the perivascular space, a region flanked by the basement membrane formed by vascular endothelial cells and mural cells, and the basement membrane formed by the astrocyte endfeet.⁷⁾ However, it should be noted that the perivascular space is not present in the cortical region of the embryonic brain. Instead, a narrow perivascular space is first formed at the perinatal stage.¹³⁾ Following a period of maturation, during which a sufficiently large perivascular space is formed, macrophages (or their progenitors) in the meninges migrate to and settle in the perivascular space, thereby becoming perivascular macrophages.¹³⁾ In other words, the developing brain meninges serve an important function as a niche for the maintenance of perivascular macrophage precursor cells. Moreover, the presence of vascular smooth muscle cells is indispensable for the formation of perivascular macrophages. The number of perivascular macrophages is markedly reduced in the brains of genetically modified mice with aberrant vascular smooth muscle cells.¹³⁾ The signaling molecules that mediate the interaction between vascular smooth muscle cells and perivascular macrophages remain unclear. However, it is plausible that perivascular macrophages play an active role in regulating vascular function, which would need further detailed analysis to confirm. The localization of the majority of perivascular macrophages to the perivascular space surrounding arterioles may also provide a significant insight into the understanding of cellular functions.

It is thought that CAMs, similar to microglia, are long-lived cells.²¹⁾ In addition, they are not replaced by BM cells in the normal brain, with the exception of those in the dura mater and choroid plexus, where a portion of CAMs appear to undergo a constant replacement with BM cells.⁵⁾ Treatment with a CSF1R inhibitor results in a depletion of CAMs across all regions,²⁰⁾ suggesting that CAMs, similar to microglia, exist in the brain border regions in a CSF1R signaling-dependent manner. However, the precise CSF1R ligands involved in CAM survival (CSF1 vs. interleukin 34)²²⁾ and the extent to which they resemble microglia,²³⁾ as well as their regional dependence, remain to be elucidated. In light of the observed repopulation of CAMs following the cessation of CSF1R inhibitor administration,^6),20) it has been postulated that residual CAMs possess the capacity for self-renewal and redistribution in each brain border region, like microglia. However, a recent study showed that CCR2-expressing peripheral monocytes can partially occupy the CNS borders after treatment with a CSF1R inhibitor and in the diseased brain.²⁴⁾ BM CAM-like cells, as well as microglia-like cells, have been demonstrated to be present in the brains of humans who have a history of intersex transfusions.²⁵⁾ The occurrence of similar cellular engraftment in healthy individuals remains uncertain. Nevertheless, in certain conditions, BM cells have the potential to infiltrate the brain and perform functions. Although no significant phenotypic differences were observed between BM and resident cells, further analysis is required to ascertain the functional differences between them.

Transcriptional maturation and segregation of microglia and CAMs

Once established in each territory, both microglia and CAMs undergo stepwise developmental and specification processes, accompanied by a dynamic shift in gene expression during the course of development.^6),13),26) The transcriptional and epigenetic mechanisms that regulate the state of microglia have been the subject of long-standing investigation, and the importance of key transcription factors, such as IRF8, SALL1, BATF3, SMAD3 and SMAD4, has become increasingly evident.^27)-31) However, our understanding of the transcriptional machinery that determines the fate of CAMs remains poorly understood. We recently showed that, in addition to microglia, IRF8 deficiency causes a substantial change in gene expression in adult CAMs,³²⁾ suggesting the significance of IRF8 for the maintenance of CAMs in homeostatic conditions. On the other hand, SMAD4 deficiency in CAMs elicited only a slight change in gene expression, compared with SMAD4-sufficient controls.³⁰⁾ The roles of SALL1, BATF3, and SMAD3 in CAMs remain to be thoroughly examined. Brioschi and colleagues recently identified binding motifs of transcription factors differentially enriched in cells exhibiting a CAM-like signature, which exhibited an abundance of binding motifs for PU.1, IRF1, and several members of the MAF bZIP transcription factor (MAF) families, including MAFB.³⁰⁾ However, the absence of MAFB in CAMs had a negligible impact on their gene expression profile.³²⁾ Further detailed analysis is needed to gain a deeper understanding of the regulatory mechanisms of CAMs.

Functions of CAMs in health and disease

To date, multiple pieces of evidence have emerged to support the physiological roles of CAMs, such as regulation of the hypothalamo-pituitary-adrenal axis,³³⁾ drainage of the CNS,³⁴⁾ and their pathophysiological roles during viral infections and in neurodegenerative disorders, such as Alzheimer’s disease (AD) and cerebral amyloid angiopathy, as have been comprehensively reviewed elsewhere.²¹⁾ For instance, in the TgCRND8 transgenic AD mouse model, depletion of pvMΦ by intraventricular injection of clodronate liposomes, a method that does not directly affect microglia or other CNS cells, resulted in a drastic increase in pathological Aβ deposition along the vasculature.³⁵⁾ Furthermore, a recent study by Schepper and colleagues identified perivascular macrophages and fibroblasts, as major sources of SPP1, which functions as an extrinsic modulator of microglial phagocytosis and contributes to microglial synaptic engulfment and upregulation of phagocytic markers, in an AD mouse model.³⁶⁾ However, this study did not demonstrate the functional relevance of perivascular macrophages on the progression of Aβ plaque deposition, which needs further investigation. Apolipoprotein E4 (APOE4), the leading genetic risk factor in sporadic AD, is known to be associated with the neurovascular alterations. Of particular interest is the recent finding that CAMs function as the source and effector of APOE4 mediating deleterious vascular effects through reactive oxygen species, thus enhancing vulnerability to oligemic white matter damage.³⁷⁾ Another recent study by Schonhoff et al. revealed previously unappreciated roles of CAMs in the pathogenesis of Parkinson’s disease. Using an animal model of Parkinson’s disease, they showed that α-syn expression leads to an increase in the number of CAMs with a unique activation state, which was identified as being responsible for CD4^＋ T cell antigen recruitment and restimulation necessary for α-syn-mediated neuroinflammation.³⁸⁾ Despite the existence of published data suggesting the involvement of CAMs in various CNS diseases, many studies rely on cell depletion methods, including injection of clodronate liposomes, which are not specific to the resident CAM population; they also target other cells including infiltrating monocytes, although microglia are not affected.³⁹⁾ Thus, novel technologies that allow for the functional segregation of CAMs from other cell types will provide new insights into the nature of CAMs.

Tools to study microglia and CAMs

Groundbreaking discoveries with respect to the ontogeny, kinetics, gene expression profiles, diversity, and functions of microglia and CAMs in health and disease have been achieved with genetic tools that allow precise manipulation and in-depth analysis.^1),40) For example, the Cx3cr1^Cre mouse line allows Cre-loxP-mediated gene targeting of myeloid cells including microglia and CAMs.⁴¹⁾ In addition, recently established Crybb1^Cre mice exhibited high recombination efficiency in microglia with limited recombination in peripheral myeloid cells.³⁰⁾ Onset of Cre activity was detectable in the embryonic brain at around embryonic E13.5, reaching nearly 100％ microglia recombination in the perinatal window, with no recombination detected in yolk sac macrophages or their progeny.³⁰⁾ Furthermore, the tamoxifen-inducing Cre-mediated gene targeting tools, such as Cx3cr1^CreERT2 and Csf1r^Mer-iCre-Mer mouse lines, are the most widely used mouse models to target microglia and CAMs at all developmental time points.^41),42) However, all the results obtained with these mice encompass the characteristics and functions of both microglia and CAMs, making it impossible to explore cell type-specific features of CAMs including their ontogeny and the specific functions during homeostatic development and disease. To address these challenges, researchers including our group have employed single-cell RNA-sequencing to identify a panel of genes with distinct expression patterns in microglia and CAMs and generated novel genetic mouse tools to separate the two CNS macrophages.^20),43) Microglia are characterized by the expression of specific genes, including P2ry12, Tmem119, Hexb, Slc2a5, and Sall1. In contrast, CAMs exhibit higher levels of gene expression, particularly for Mrc1, Pf4, Lyve1, and Msr1. Sall1 is a transcription factor that plays a crucial role in microglia development,²⁸⁾ and genetic engineering of mice to express the CreERT2 transgene under the control of the Sall1 promoter (Sall1^CreERT2)⁴⁴⁾ was expected to provide a valuable tool for microglia-specific manipulation of cell functions. Indeed, CAMs were largely eliminated and functional segregation from microglia was achievable. However, Sall1 expression was also present in astrocytes.¹⁷⁾ Thus, Sall1^CreERT2 mice not only target microglia but also other cell types such as astrocytes in the CNS.²⁰⁾ Conversely, genetically engineered mice such as P2ry12^CreERT2, Tmem119^CreERT2, and Hexb^CreERT2 allow selective manipulation of microglia-specific genes with some limitations,^20),45),46) which are expected to facilitate the elucidation of microglia-specific functions. On the other hand, there are several transgenic mouse lines available for specifically studying the biology of CAMs. For instance, the use of Lyve1^EGFP/cre mice, in which the expression of Cre recombinase with enhanced green fluorescent protein (EGFP) in these knock-in mice is driven by the Lyve1 promoter.⁴⁷⁾ Additionally, Pf4^iCre mice, in which an improved Cre recombinase (iCre) is induced under the control of the Pf4 promoter,⁴⁶⁾ allows for cell type-specific gene targeting of CAMs. In these mice, CAMs are nicely segregated from microglia, while lymphatic endothelial cells in the Lyve1^EGFP/cre mouse line⁴⁷⁾ and megakaryocytes in the Pf4^iCre line⁴⁸⁾ can also be targeted. Furthermore, to study the definition and dissection of the precise ontogeny and the specific functions of CAMs, we and others have recently developed novel mouse lines, which enable the targeted manipulation of CAMs in a time-controlled and cell type-specific manner (e.g., Mrc1^CreERT2) without affecting other CNS cells or circulating blood cells.¹³⁾ When evaluating the systemic effects (e.g., mouse behavior) using this mouse line, it is essential to consider the potential involvement of other tissue-resident macrophages in the periphery that express Mrc1. Nevertheless, these newly developed mouse models offer a valuable avenue for elucidating the functions and the characteristics of CAMs during development, homeostasis, and diseases of the CNS.

Conclusions and future perspectives

Since the discovery of microglia over a century ago, converging evidence from human and animal studies has significantly advanced our understanding of macrophage biology in the CNS. Recent studies have shed new light on the diversity of macrophages in the CNS, elucidating the shared and distinctive characteristics of CAMs and microglia and their important roles in intracerebral processes. Nevertheless, there are still unanswered questions that require further investigation. In addition, given that the majority of analyses aimed at elucidating the function of CAMs have been derived from the removal of CAMs using clodronate liposomes, there is a need to re-evaluate the interpretation of the role of CAMs using novel technologies including the aforementioned mouse models. Further elucidation of microglia- and CAM-specific functions and cellular characteristics will facilitate a deeper understanding of the nature of CNS-resident macrophages in health and disease.

Acknowledgements

T.M. is supported by the MEXT Cooperative Research Project Program, Medical Research Center Initiative for High Depth Omics, and CURE:JPMXP1323015486 for MIB, Kyushu University, AMED JP20gm6310016, JP21wm0425001, JP23gm1910004, JP23jf0126004, and JP24zf0127012, JSPS KAKENHI JP21H02752, JP22H05062, The Mitsubishi Foundation, Daiichi Sankyo Foundation of Life Science, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Astellas Foundation for Research on Metabolic disorders, Ono Pharmaceutical Foundation for Oncology, Immunology and Neurology, The Nakajima Foundation, The Uehara Memorial Foundation and Takeda Science Foundation.

Declarations

The authors declare that they have no competing interests.

Author contributions

T.M. has contributed to the work and approved it for publication.

Notes

Edited by Shizuo AKIRA, M.J.A.

Correspondence should be addressed to: T. Masuda, Division of Molecular Neuroimmunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan (e-mail: [e-mail]).

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Takahiro Masuda was born in 1983 in Yamaguchi Prefecture, Japan. He graduated from Kyushu University in 2006 and received his Ph.D. degree in 2011 from the Graduate School of Pharmaceutical Sciences, Kyushu University. He became an assistant professor at Kyushu University in 2012 and studied the role of microglia in the pathogenesis of neuropathic pain. From 2015 to 2020, he studied the nature of brain macrophages with Prof. Marco Prinz at the University of Freiburg, Germany. He then returned to Kyushu University as an assistant professor and was then promoted to associate professor in 2021 He then received his current professorial position in the Medical Institute of Bioregulation at Kyushu University in 2023. He is now a Distinguished Professor at Kyushu University. His research interest is to elucidate how brain macrophages develop and function in health and disease.