Meningeal Macrophages Research Articles

Brain-Engrafted Monocyte-derived Macrophages from Blood and Skull-Bone Marrow Exhibit Distinct Identities from Microglia.

Microglia are thought to originate exclusively from primitive macrophage progenitors in the yolk sac (YS) and to persist throughout life without much contribution from definitive hematopoiesis. Here, using lineage tracing, pharmacological manipulation, and RNA-sequencing, we elucidated the presence and characteristics of monocyte-derived macrophages (MDMs) in the brain parenchyma at baseline and during microglia repopulation, and defined the core transcriptional signatures of brain-engrafted MDMs. Lineage tracing mouse models revealed that MDMs transiently express CD206 during brain engraftment as CD206 + microglia precursors in the YS. We found that brain-engrafted MDMs exhibit transcriptional and epigenetic characteristics akin to meningeal macrophages, likely due to environmental imprinting within the meningeal space. Utilizing parabiosis and skull transplantation, we demonstrated that monocytes from both peripheral blood and skull bone marrow can repopulate microglia-depleted brains. Our results reveal the heterogeneous origins and cellular dynamics of brain parenchymal macrophages at baseline and in models of microglia depletion.

Regulation of meningeal lymphatic function by APOE

AbstractBackgroundThe meningeal layers form a unique interface between the central nervous and peripheral immune systems. The lymphatic vasculature that is present in the meningeal dura and drains cerebrospinal fluid (CSF) becomes dysregulated during aging and in models of neurodegenerative disorders such as Alzheimer’s disease (AD). Apolipoprotein E (APOE), and the isoform APOE4 in particular, increases the risk for late‐onset sporadic AD. However, it is still poorly understood whether changes in meningeal lymphatic morphology and drainage are mediating the deleterious effects of APOE4 expression. In the present study, we investigate the effects of human APOE3 or APOE4 on meningeal lymphatic function and immunity.MethodWe evaluated the morphology of lymphatic vessels in dural whole mounts of age‐matched wild type, Apoe‐KO, APOE3, and APOE4 mice at 2‐3, and 11‐12 months. This assessment was performed in both male and female mice. The drainage function of meningeal lymphatics is being assessed in the same mice by in vivo imaging of CSF‐derived fluorescent tracers into the deep cervical lymph nodes. We have also performed RNAscope and protein measurements using murine meningeal tissue to investigate the main cellular source(s) of Apoe, and single‐cell RNA sequencing to explore the cell‐specific transcriptomic signatures linked to the changes in meningeal lymphatic function.ResultLymphatic vessel length is increased in 11‐12‐month‐old APOE4 male mice, when compared to their APOE3 counterparts. Interestingly, the same was not detected in female mice. Dural myeloid cells, expressing Itgam (encoding for CD11b), are the main source of Apoe mRNA transcripts, and one of the main sources of apoE protein in the brain dura mater. APOE4 expression and age markedly alter the transcriptional profile of meningeal myeloid cells, which show overexpression of lysosomal, antigen presentation, and complement genes.ConclusionAPOE4 expression leads to a sex‐dependent meningeal lymphatic vascular remodeling, which is associated with an exacerbated activation of meningeal macrophages. In future studies, we will test whether the changes in meningeal lymphatics observed in mice expressing APOE4 are mediated by myeloid cell derived APOE.

How meningeal macrophages control medulloblastoma induction.

In this issue of Developmental Cell, Pokrajac etal. report that Wnt signaling in endothelial cells maintain CXCL4 expression in meningeal macrophages, which suppresses induction of Sonic hedgehog medulloblastoma by antagonizing the pro-tumor effects of CXCL12. This work highlights how homeostatic tissue macrophages can regulate early stages of tumor induction.

Meningeal macrophages inhibit chemokine signaling in pre-tumor cells to suppress mouse medulloblastoma initiation.

The microenvironment profoundly influences tumor initiation across numerous tissues but remains understudied in brain tumors. In the cerebellum, canonical Wnt signaling controlled by Norrin/Frizzled4 (Fzd4) activation in meningeal endothelial cells is a potent inhibitor of preneoplasia and tumor progression in mouse models of Sonic hedgehog medulloblastoma (Shh-MB). Single-cell transcriptome profiling and phenotyping of the meninges indicate that Norrin/Frizzled4 sustains the activation of meningeal macrophages (mMΦs), characterized by Lyve1 and CXCL4 expression, during the critical preneoplastic period. Depleting mMΦs during this period enhances preneoplasia and tumorigenesis, phenocopying the effects of Norrin loss. The anti-tumorigenic function of mMΦs is derived from the expression of CXCL4, which counters CXCL12/CXCR4 signaling in pre-tumor cells, thereby inhibiting cell-cycle progression and promoting migration away from the pre-tumor niche. These findings identify a pivotal role for mMΦs as key mediators in chemokine-regulated anti-cancer crosstalk between the stroma and pre-tumor cells in the control of MB initiation.

Meningeal P2X7 Signaling Mediates Migraine-Related Intracranial Mechanical Hypersensitivity.

Cortical spreading depolarization (CSD) is a key pathophysiological event that underlies visual and sensory auras in migraine. CSD is also thought to drive the headache phase in migraine by promoting the activation and mechanical sensitization of trigeminal primary afferent nociceptive neurons that innervate the cranial meninges. The factors underlying meningeal nociception in the wake of CSD remain poorly understood but potentially involve the parenchymal release of algesic mediators and damage-associated molecular patterns, particularly ATP. Here, we explored the role of ATP-P2X purinergic receptor signaling in mediating CSD-evoked meningeal afferent activation and mechanical sensitization. Male rats were subjected to a single CSD episode. In vivo, extracellular single-unit recording was used to measure meningeal afferent ongoing activity changes. Quantitative mechanical stimuli using a servomotor force-controlled stimulator assessed changes in the afferent's mechanosensitivity. Manipulation of meningeal P2X receptors was achieved via local administration of pharmacological agents. Broad-spectrum P2X receptor inhibition, selective blockade of the P2X7 receptor, and its related Pannexin 1 channel suppressed CSD-evoked afferent mechanical sensitization but did not affect the accompanying afferent activation response. Surprisingly, inhibition of the pronociceptive P2X2/3 receptor did not affect the activation or sensitization of meningeal afferents post-CSD. P2X7 signaling underlying afferent mechanosensitization was localized to the meninges and did not affect CSD susceptibility. We propose that meningeal P2X7 and Pannexin 1 signaling, potentially in meningeal macrophages or neutrophils, mediates the mechanical sensitization of meningeal afferents, which contributes to migraine pain by exacerbating the headache during normally innocuous physical activities.SIGNIFICANCE STATEMENT Activation and sensitization of meningeal afferents play a key role in migraine headache, but the underlying mechanisms remain unclear. Here, using a rat model of migraine with aura involving cortical spreading depolarization (CSD), we demonstrate that meningeal purinergic P2X7 signaling and its related Pannexin 1 pore, but not nociceptive P2X2/3 receptors, mediate prolonged meningeal afferent sensitization. Additionally, we show that meningeal P2X signaling does not contribute to the increased afferent ongoing activity in the wake of CSD. Our finding points to meningeal P2X7 signaling as a critical mechanism underlying meningeal nociception in migraine, the presence of distinct mechanisms underlying the activation and sensitization of meningeal afferents in migraine, and highlight the need to target both processes for effective migraine therapy.

Lineage tracing identifies in vitro microglia-to-neuron conversion by NeuroD1 expression.

Neuronal regeneration to replenish lost neurons after injury is critical for brain repair. Microglia, brain-resident macrophages that have the propensity to accumulate at the site of injury, can be a potential source for replenishing lost neurons through fate conversion into neurons, induced by forced expression of neuronal lineage-specific transcription factors. However, it has not been strictly demonstrated that microglia, rather than central nervous system-associated macrophages, such as meningeal macrophages, convert into neurons. Here, we show that NeuroD1-transduced microglia can be successfully converted into neurons in vitro using lineage-mapping strategies. We also found that a chemical cocktail treatment further promoted NeuroD1-induced microglia-to-neuron conversion. NeuroD1 with loss-of-function mutation, on the other hand, failed to induce the neuronal conversion. Our results indicate that microglia are indeed reprogrammed into neurons by NeuroD1 with neurogenic transcriptional activity.

Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion.

The meninges are densely innervated by nociceptive sensory neurons that mediate pain and headache1,2. Bacterial meningitis causes life-threatening infections of the meninges and central nervous system, affecting more than 2.5 million people a year3-5. How pain and neuroimmune interactions impact meningeal antibacterial host defences are unclear. Here we show that Nav1.8+ nociceptors signal to immune cells in the meninges through the neuropeptide calcitonin gene-related peptide (CGRP) during infection. This neuroimmune axis inhibits host defences and exacerbates bacterial meningitis. Nociceptor neuron ablation reduced meningeal and brain invasion by two bacterial pathogens: Streptococcus pneumoniae and Streptococcus agalactiae. S. pneumoniae activated nociceptors through its pore-forming toxin pneumolysin to release CGRP from nerve terminals. CGRP acted through receptor activity modifying protein 1 (RAMP1) on meningeal macrophages to polarize their transcriptional responses, suppressing macrophage chemokine expression, neutrophil recruitment and dural antimicrobial defences. Macrophage-specific RAMP1 deficiency or pharmacological blockade of RAMP1 enhanced immune responses and bacterial clearance in the meninges and brain. Therefore, bacteria hijack CGRP-RAMP1 signalling in meningeal macrophages to facilitate brain invasion. Targeting this neuroimmune axis in the meninges can enhance host defences and potentially produce treatments for bacterial meningitis.

Microglia and meningeal macrophages depletion delays the onset of experimental autoimmune encephalomyelitis

In multiple sclerosis and the experimental autoimmune encephalomyelitis (EAE) model, both resident microglia and infiltrating macrophages contribute to demyelination as well as spontaneous remyelination. Nevertheless, the specific roles of microglia versus macrophages are unknown. We investigated the influence of microglia in EAE using the colony stimulating factor 1 receptor (CSF-1R) inhibitor, PLX5622, to deplete microglial population and Ccr2RFP/+fmsEGFP/+ mice, to distinguish blood-derived macrophages from microglia. PLX5622 treatment depleted microglia and meningeal macrophages, and provoked a massive infiltration of CCR2+ macrophages into demyelinating lesions and spinal cord parenchyma, albeit it did not alter EAE chronic phase. In contrast, microglia and meningeal macrophages depletion reduced the expression of major histocompatibility complex II and CD80 co-stimulatory molecule in dendritic cells, macrophages and microglia. In addition, it diminished T cell reactivation and proliferation in the spinal cord parenchyma, inducing a significant delay in EAE onset. Altogether, these data point to a specific role of CNS microglia and meningeal macrophages in antigen presentation and T cell reactivation at initial stages of EAE.

A single-cell atlas reveals the heterogeneity of meningeal immunity in a mouse model of Methyl CpG binding protein 2 deficiency.

Methyl CpG binding protein 2 (MeCP2) is a DNA methylation reader protein. Mutations in MeCP2 are the major cause of Rett syndrome (RTT). Increasing evidence has shown that dysregulated immunity and chronic subclinical inflammation are linked to MeCP2 deficiency and contribute to RTT development and deterioration. The meninges surrounding the central nervous system (CNS) contain a wide repertoire of immune cells that participate in immune surveillance within the CNS and influence various brain functions; however, the characterization and role of meningeal immunity in CNS with MeCP2 deficiency remain poorly addressed. Here, we used single-cell sequencing to profile Mecp2-deficient meningeal immune cells from the dura mater, which has been reported to contain the most meningeal immune cells during homeostasis. Data showed that the meninges of Mecp2-null mice contained the same diverse immune cell populations as control mice and showed an up-regulation of immune-related processes. B cell populations were greater in Mecp2-null mice than in control mice, and the expression of genes encoding for immunoglobulins was remarkably higher. Mecp2-deficient meninges also contained more cytotoxic CD8+ T cells than control meninges. With increased interferon-γ transcription in T and natural killer cells, meningeal macrophages showed decreased suppression and increased activity in Mecp2-deficienct mice. Together, these findings provide novel insights into meningeal immunity, which is a less studied aspect of neuroimmune interactions in Mecp2-mutated diseases, and offer an essential resource for comparative analyses and data exploration to better understand the functional role of meningeal immunity in RTT.

Infiltration of meningeal macrophages into the Virchow-Robin space after ischemic stroke in rats: Correlation with activated PDGFR-β-positive adventitial fibroblasts.

Macrophages play a crucial role in wound healing and fibrosis progression after brain injury. However, a detailed analysis of their initial infiltration and interaction with fibroblasts is yet to be conducted. This study aimed to investigate the possible route for migration of meningeal macrophages into the ischemic brain and whether these macrophages closely interact with neighboring platelet-derived growth factor beta receptor (PDGFR-β)-positive adventitial fibroblasts during this process. A rat model of ischemic stroke induced by middle cerebral artery occlusion (MCAO) was developed. In sham-operated rats, CD206-positive meningeal macrophages were confined to the leptomeninges and the perivascular spaces, and they were not found in the cortical parenchyma. In MCAO rats, the number of CD206-positive meningeal macrophages increased both at the leptomeninges and along the vessels penetrating the cortex 1 day after reperfusion and increased progressively in the extravascular area of the cortical parenchyma by 3 days. Immunoelectron microscopy and correlative light and electron microscopy showed that in the ischemic brain, macrophages were frequently located in the Virchow-Robin space around the penetrating arterioles and ascending venules at the pial surface. This was identified by cells expressing PDGFR-β, a novel biomarker of leptomeningeal cells. Macrophages within penetrating vessels were localized in the perivascular space between smooth muscle cells and PDGFR-β-positive adventitial fibroblasts. In addition, these PDGFR-β-positive fibroblasts showed morphological and molecular characteristics similar to those of leptomeningeal cells: they had large euchromatic nuclei with prominent nucleoli and well-developed rough endoplasmic reticulum; expressed nestin, vimentin, and type I collagen; and were frequently surrounded by collagen fibrils, indicating active collagen synthesis. In conclusion, the perivascular Virchow-Robin space surrounding the penetrating vessels could be an entry route of meningeal macrophages from the subarachnoid space into the ischemic cortical parenchyma, implying that activated PDGFR-β-positive adventitial fibroblasts could be involved in this process.

Meningeal macrophages protect against viral neuroinfection

Meningeal macrophages protect against viral neuroinfection

Meningeal macrophages protect against viral neuroinfection.

The surface of the central nervous system (CNS) is protected by the meninges, which contain a dense network of meningeal macrophages (MMs). Here, we examined the role of tissue-resident MM in viral infection. MHC-II- MM were abundant neonatally, whereas MHC-II+ MM appeared over time. These barrier macrophages differentially responded to invivo peripheral challenges such as LPS, SARS-CoV-2, and lymphocytic choriomeningitis virus (LCMV). Peripheral LCMV infection, which was asymptomatic, led to a transient infection and activation of the meninges. Mice lacking macrophages but conserving brain microglia, or mice bearing macrophage-specific deletion of Stat1 or Ifnar, exhibited extensive viral spread into the CNS. Transcranial pharmacological depletion strategies targeting MM locally resulted in several areas of the meninges becoming infected and fatal meningitis. Low numbers of MHC-II+ MM, which is seen upon LPS challenge or in neonates, corelated with higher viral load upon infection. Thus, MMs protect against viral infection and may present targets for therapeutic manipulation.

BSCI-08 NEURONAL MIMICRY PROMOTES BREAST CANCER LEPTOMENINGEAL METASTASIS FROM BONE MARROW

Breast cancer (BC) patients diagnosed with leptomeningeal disease (LMD) have a median survival of less than six months. There has been limited therapeutic innovation in treating LMD due to our poor understanding of the molecular mechanisms governing breast cancer cell (BCC) invasion and survival within the leptomeninges (LM). Here we show that BCCs can invade the LM by migrating along the outer surface of emissary vessels that passage from the skull and vertebral bone marrow through cortical bone fenestrations, emerging as LM vasculature in the sub-arachnoid space. This process requires BCC integrin α6 engaging laminin on the vascular basement membrane of emissary vessels, mimicking an α6 integrin-dependent mechanism used by neural progenitors to migrate to the olfactory bulb. Once in the LM, BCCs co-localize with perivascular CSF1R+ meningeal macrophages which support BCC survival through the secretion of the protective neurotrophin, GDNF. Pharmacologic depletion of these meningeal macrophages causes a marked reduction of GDNF concurrent with a decrease in LMD progression, which is rescued by intraventricular delivery of recombinant GDNF. Together these data suggest that BCCs hijack neural migratory pathways and leptomeningeal macrophages to invade and survive in the LM niche. Finally, analysis of craniotomy samples from patients with breast cancer revealed a correlation between BCC integrin α6 expression and meningeal metastasis suggesting the potential of integrin α6 as a novel target to predict or treat LMD.

In vivo tracking of [89Zr]Zr-labeled engineered extracellular vesicles by PET reveals organ-specific biodistribution based upon the route of administration

Extracellular vesicles (EVs) have garnered increasing interest as delivery vehicles for multiple classes of therapeutics based on their role as mediators in an important, natural intercellular communication system. We recently described a platform to allow the design, production and in vivo study of human EVs with specific properties (drug or tropism modifiers). This article seeks to compare and expand upon historical biodistribution and kinetic data by comparing systemically and compartmentally administered labeled engineered EVs using in vivo and ex vivo techniques. MethodsEVs were surface-labeled to high radiochemical purity and specific activity with 89Zirconium deferoxamine ([89Zr]Zr-DFO) and/or cy7-scrambled antisense oligonucleotide (Cy7-ExoASOscr), or luminally loaded with GFP for in vivo tracking in rodents and non-human primates (NHPs). Positron Emission Tomography (PET) and subsequent immunohistochemistry (IHC) and autoradiography (ARG) cross-validation enabled assessment of the anatomical and cellular distribution of labeled EVs both spatially and temporally. ResultsOver time, systemic administration of engineered EVs distributed preferentially to the liver and spleen (Intravenous, IV), gastrointestinal tract and lymph nodes (Intraperitoneal, IP) and local/regional lymph nodes (Subcutaneous, SC). Immunostaining of dissected organs displaying PET signal revealed co-localization of an EV marker (PTGFRN) with a subset of macrophage markers (CD206, F4/80, IBA1). Compartmental dosing into NHP cerebrospinal fluid (CSF) resulted in a heterogenous distribution of labeled EVs depending upon whether the route was intrathecal (ITH), intracisterna magna (ICM) or intracerebroventricular (ICV), compared to the homogeneous distribution observed in rodents. Thus anatomically, ITH administration in NHP revealed meningeal distribution along the neuraxis to the base of the skull. In contrast ICM and ICV dosing resulted in meningeal distribution around the skull and to the cervical and thoracic spinal column. Further characterization using IHC shows uptake in a subset of meningeal macrophages. ConclusionsThe present studies provide a comprehensive assessment of the fate of robustly and reproducibly labeled engineered EVs across several mammalian species. The in vivo distribution was observed to be both spatially and temporally dependent upon the route of administration providing insight into potential targeting opportunities for engineered EVs carrying a therapeutic payload.

The Role of Meningeal Immune Cells in the Efficacy of CGRP-based Migraine Therapies

Migraine is a painful, chronic neurological disorder that represents the second most disabling illness worldwide. There are two prominent mechanisms thought to contribute to migraine pain: immune cell activation and calcitonin gene-related peptide (CGRP) signaling. Cortical spreading depression (CSD), thought to underlie the aura that precedes migraine attack in a subpopulation of migraineurs, is associated with increased meningeal macrophage activation. Macrophages can activate meningeal primary afferent neurons and contribute to migraine pain through release of pro-inflammatory cytokines. CSD also increases CGRP synthesis and release, and single-cell RNA sequencing data support the expression of CGRP receptor subunit mRNA in mouse meningeal immune cells. The efficacy of CGRP receptor antagonists as migraine therapies support the critical role of CGRP in the development of migraine pain. However, the mechanism of action of these drugs is incompletely understood. Given the effect of CSD on immune cell activation and CGRP release, the presence of CGRP receptor on immune cells, and the use of CGRP receptor antagonists to treat migraine, the following study uses anatomical, functional, and behavioral approaches to assess the role of meningeal immune cells in the efficacy of CGRP-based migraine therapies. This is done in the context of a minimally invasive model of migraine with aura (optogenetic spreading depression, OSD). I plan to characterize OSD-dependent changes in CGRP receptor expression in meningeal immune cells, assess the ability of CGRP receptor antagonists to prevent OSD-induced changes in macrophage activation, and determine the contribution of macrophage CGRP receptors to OSD-induced pain. The outcomes of these experiments may not only reveal a mechanism underlying the therapeutic efficacy of CGRP antagonists but potentially novel targets and mechanisms to improve existing migraine therapies. Grant support from R01 NS122784.

Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophagesubsetsin the CNS.

Neuroprotective Potential of Dendritic Cells and Sirtuins in Multiple Sclerosis.

Myeloid cells, including parenchymal microglia, perivascular and meningeal macrophages, and dendritic cells (DCs), are present in the central nervous system (CNS) and establish an intricate relationship with other cells, playing a crucial role both in health and in neurological diseases. In this context, DCs are critical to orchestrating the immune response linking the innate and adaptive immune systems. Under steady-state conditions, DCs patrol the CNS, sampling their local environment and acting as sentinels. During neuroinflammation, the resulting activation of DCs is a critical step that drives the inflammatory response or the resolution of inflammation with the participation of different cell types of the immune system (macrophages, mast cells, T and B lymphocytes), resident cells of the CNS and soluble factors. Although the importance of DCs is clearly recognized, their exact function in CNS disease is still debated. In this review, we will discuss modern concepts of DC biology in steady-state and during autoimmune neuroinflammation. Here, we will also address some key aspects involving DCs in CNS patrolling, highlighting the neuroprotective nature of DCs and emphasizing their therapeutic potential for the treatment of neurological conditions. Recently, inhibition of the NAD+-dependent deac(et)ylase sirtuin 6 was demonstrated to delay the onset of experimental autoimmune encephalomyelitis, by dampening DC trafficking towards inflamed LNs. Thus, a special focus will be dedicated to sirtuins’ role in DCs functions.

Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease

The immune cells of the central nervous system (CNS) comprise parenchymal microglia and at the CNS border regions meningeal, perivascular, and choroid plexus macrophages (collectively called CNS‐associated macrophages, CAMs). While previous work has shown that microglial properties depend on environmental signals from the commensal microbiota, the effects of microbiota on CAMs are unknown. By combining several microbiota manipulation approaches, genetic mouse models, and single‐cell RNA‐sequencing, we have characterized CNS myeloid cell composition and function. Under steady‐state conditions, the transcriptional profiles and numbers of choroid plexus macrophages were found to be tightly regulated by complex microbiota. In contrast, perivascular and meningeal macrophages were affected to a lesser extent. An acute perturbation through viral infection evoked an attenuated immune response of all CAMs in germ‐free mice. We further assessed CAMs in a more chronic pathological state in 5xFAD mice, a model for Alzheimer’s disease, and found enhanced amyloid beta uptake exclusively by perivascular macrophages in germ‐free 5xFAD mice. Our results aid the understanding of distinct microbiota–CNS macrophage interactions during homeostasis and disease, which could potentially be targeted therapeutically.

Annexin A3 as a Marker Protein for Microglia in the Central Nervous System of Rats.

The parenchymal microglia possess different morphological characteristics in cerebral physiological and pathological conditions; thus, visualizing these cells is useful as a means of further investigating parenchymal microglial function. Annexin A3 (ANXA3) is expressed in microglia, but it is unknown whether it can be used as a marker protein for microglia and its physiological function. Here, we compared the distribution and morphology of parenchymal microglia labeled by ANXA3, cluster of differentiation 11b (CD11b), and ionized calcium-binding adaptor molecule 1 (Iba1) and measured the expression of ANXA3 in nonparenchymal macrophages (meningeal and perivascular macrophages). We also investigated the spatiotemporal expression of ANXA3, CD11b, and Iba1 in vivo and in vitro and the cellular function of ANXA3 in microglia. We demonstrated that ANXA3-positive cells were abundant and evenly distributed throughout the whole brain tissue and spinal cord of adult rats. The morphology and distribution of ANXA3-labeled microglia were quite similar to those labeled by the microglial-specific markers CD11b and Iba1 in the central nervous system (CNS). ANXA3 was expressed in the cytoplasm of microglia, and its expression was significantly increased in activated microglia. ANXA3 was almost undetectable in the nonparenchymal macrophages. Meanwhile, the protein and mRNA expression levels of ANXA3 in different regions of the CNS were different from those of CD11b and Iba1. Moreover, knockdown of ANXA3 inhibited the proliferation and migration of microglia, while overexpression of ANXA3 enhanced these activities. This study confirms that ANXA3 may be a novel marker for parenchymal microglia in the CNS of adult rats and enriches our understanding of ANXA3 from expression patterns to physiological function.

Distinct Features of Brain-Resident Macrophages: Microglia and Non-Parenchymal Brain Macrophages.

Tissue-resident macrophages play an important role in maintaining tissue homeostasis and innate immune defense against invading microbial pathogens. Brain-resident macrophages can be classified into microglia in the brain parenchyma and non-parenchymal brain macrophages, also known as central nervous system-associated or border-associated macrophages, in the brain-circulation interface. Microglia and non-parenchymal brain macrophages, including meningeal, perivascular, and choroid plexus macrophages, are mostly produced during embryonic development, and maintained their population by self-renewal. Microglia have gained much attention for their dual roles in the maintenance of brain homeostasis and the induction of neuroinflammation. In particular, diverse phenotypes of microglia have been increasingly identified under pathological conditions. Single-cell phenotypic analysis revealed that microglia are highly heterogenous and plastic, thus it is difficult to define the status of microglia as M1/M2 or resting/activated state due to complex nature of microglia. Meanwhile, physiological function of non-parenchymal brain macrophages remain to be fully demonstrated. In this review, we have summarized the origin and signatures of brain-resident macrophages and discussed the unique features of microglia, particularly, their phenotypic polarization, diversity of subtypes, and inflammasome responses related to neurodegenerative diseases.