Astrocytic phagocytosis is a compensatory mechanism for microglial dysfunction
2020; Springer Nature; Volume: 39; Issue: 22 Linguagem: Inglês
10.15252/embj.2020104464
ISSN1460-2075
AutoresHiroyuki Konishi, Takayuki Okamoto, Yuichiro Hara, Okiru Komine, Hiromi Tamada, Mitsuyo Maeda, Fumika Osako, Masaaki Kobayashi, Akira Nishiyama, Yosky Kataoka, Toshiyuki Takai, Nobuyuki Udagawa, Steffen Jung, Keiko Ozato, Tomohiko Tamura, Makoto Tsuda, Koji Yamanaka, Tomoo Ogi, Katsuaki Sato, Hiroshi Kiyama,
Tópico(s)Immune cells in cancer
ResumoArticle22 September 2020Open Access Source DataTransparent process Astrocytic phagocytosis is a compensatory mechanism for microglial dysfunction Hiroyuki Konishi Corresponding Author Hiroyuki Konishi [email protected] orcid.org/0000-0002-4321-8339 Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Takayuki Okamoto Takayuki Okamoto Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Yuichiro Hara Yuichiro Hara Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan Department of Human Genetics and Molecular Biology, Graduate School of Medicine, Nagoya University, Nagoya, Japan Search for more papers by this author Okiru Komine Okiru Komine Department of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan Search for more papers by this author Hiromi Tamada Hiromi Tamada Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Mitsuyo Maeda Mitsuyo Maeda Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Kobe, Japan Laboratory for Cellular Function Imaging, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan Search for more papers by this author Fumika Osako Fumika Osako Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Masaaki Kobayashi Masaaki Kobayashi Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Akira Nishiyama Akira Nishiyama Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan Search for more papers by this author Yosky Kataoka Yosky Kataoka Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Kobe, Japan Laboratory for Cellular Function Imaging, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan Search for more papers by this author Toshiyuki Takai Toshiyuki Takai Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan Search for more papers by this author Nobuyuki Udagawa Nobuyuki Udagawa Department of Biochemistry, Matsumoto Dental University, Shiojiri, Japan Search for more papers by this author Steffen Jung Steffen Jung orcid.org/0000-0003-4290-5716 Department of Immunology, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Keiko Ozato Keiko Ozato Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Tomohiko Tamura Tomohiko Tamura Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan Search for more papers by this author Makoto Tsuda Makoto Tsuda orcid.org/0000-0003-0585-9570 Department of Life Innovation, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan Search for more papers by this author Koji Yamanaka Koji Yamanaka orcid.org/0000-0003-4655-0035 Department of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan Search for more papers by this author Tomoo Ogi Tomoo Ogi Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan Department of Human Genetics and Molecular Biology, Graduate School of Medicine, Nagoya University, Nagoya, Japan Search for more papers by this author Katsuaki Sato Katsuaki Sato Division of Immunology, Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan Search for more papers by this author Hiroshi Kiyama Corresponding Author Hiroshi Kiyama [email protected] orcid.org/0000-0001-5963-046X Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Hiroyuki Konishi Corresponding Author Hiroyuki Konishi [email protected] orcid.org/0000-0002-4321-8339 Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Takayuki Okamoto Takayuki Okamoto Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Yuichiro Hara Yuichiro Hara Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan Department of Human Genetics and Molecular Biology, Graduate School of Medicine, Nagoya University, Nagoya, Japan Search for more papers by this author Okiru Komine Okiru Komine Department of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan Search for more papers by this author Hiromi Tamada Hiromi Tamada Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Mitsuyo Maeda Mitsuyo Maeda Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Kobe, Japan Laboratory for Cellular Function Imaging, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan Search for more papers by this author Fumika Osako Fumika Osako Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Masaaki Kobayashi Masaaki Kobayashi Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Akira Nishiyama Akira Nishiyama Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan Search for more papers by this author Yosky Kataoka Yosky Kataoka Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Kobe, Japan Laboratory for Cellular Function Imaging, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan Search for more papers by this author Toshiyuki Takai Toshiyuki Takai Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan Search for more papers by this author Nobuyuki Udagawa Nobuyuki Udagawa Department of Biochemistry, Matsumoto Dental University, Shiojiri, Japan Search for more papers by this author Steffen Jung Steffen Jung orcid.org/0000-0003-4290-5716 Department of Immunology, Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Keiko Ozato Keiko Ozato Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA Search for more papers by this author Tomohiko Tamura Tomohiko Tamura Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan Search for more papers by this author Makoto Tsuda Makoto Tsuda orcid.org/0000-0003-0585-9570 Department of Life Innovation, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan Search for more papers by this author Koji Yamanaka Koji Yamanaka orcid.org/0000-0003-4655-0035 Department of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan Search for more papers by this author Tomoo Ogi Tomoo Ogi Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan Department of Human Genetics and Molecular Biology, Graduate School of Medicine, Nagoya University, Nagoya, Japan Search for more papers by this author Katsuaki Sato Katsuaki Sato Division of Immunology, Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan Search for more papers by this author Hiroshi Kiyama Corresponding Author Hiroshi Kiyama [email protected] orcid.org/0000-0001-5963-046X Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Author Information Hiroyuki Konishi *,1, Takayuki Okamoto1, Yuichiro Hara2,3, Okiru Komine4, Hiromi Tamada1, Mitsuyo Maeda5,6, Fumika Osako1, Masaaki Kobayashi1, Akira Nishiyama7, Yosky Kataoka5,6, Toshiyuki Takai8, Nobuyuki Udagawa9, Steffen Jung10, Keiko Ozato11, Tomohiko Tamura7, Makoto Tsuda12, Koji Yamanaka4, Tomoo Ogi2,3, Katsuaki Sato13 and Hiroshi Kiyama *,1 1Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, Nagoya, Japan 2Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan 3Department of Human Genetics and Molecular Biology, Graduate School of Medicine, Nagoya University, Nagoya, Japan 4Department of Neuroscience and Pathobiology, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan 5Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Kobe, Japan 6Laboratory for Cellular Function Imaging, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan 7Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan 8Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan 9Department of Biochemistry, Matsumoto Dental University, Shiojiri, Japan 10Department of Immunology, Weizmann Institute of Science, Rehovot, Israel 11Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA 12Department of Life Innovation, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 13Division of Immunology, Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan *Corresponding author. Tel: +81 52 744 2015; Fax: +81 52 744 2027; E-mail: [email protected] *Corresponding author. Tel: +81 52 744 2015; Fax: +81 52 744 2027; E-mail: [email protected] The EMBO Journal (2020)39:e104464https://doi.org/10.15252/embj.2020104464 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Microglia are the principal phagocytes that clear cell debris in the central nervous system (CNS). This raises the question, which cells remove cell debris when microglial phagocytic activity is impaired. We addressed this question using Siglechdtr mice, which enable highly specific ablation of microglia. Non-microglial mononuclear phagocytes, such as CNS-associated macrophages and circulating inflammatory monocytes, did not clear microglial debris. Instead, astrocytes were activated, exhibited a pro-inflammatory gene expression profile, and extended their processes to engulf microglial debris. This astrocytic phagocytosis was also observed in Irf8-deficient mice, in which microglia were present but dysfunctional. RNA-seq demonstrated that even in a healthy CNS, astrocytes express TAM phagocytic receptors, which were the main astrocytic phagocytic receptors for cell debris in the above experiments, indicating that astrocytes stand by in case of microglial impairment. This compensatory mechanism may be important for the maintenance or prolongation of a healthy CNS. Synopsis Microglial ablation or microglial dysfunction actuates phagocytic activity of astrocytes. Astrocytes possess phagocytic machinery and have the potential to compensate for microglia with dysfunctional phagocytic activity. Even in a healthy central nervous system, astrocytes possess phagocytic machinery, such as phagocytic receptors, Axl and Mertk. After microglia-specific ablation, activated astrocytes, rather than non-microglial mononuclear phagocytes, engulf microglial debris. Astrocytes phagocytose spontaneous apoptotic cells in Irf8-deficient mice, in which microglia are present but dysfunctional. Introduction Microglia are macrophage-related cells of the central nervous system (CNS). They originate from the yolk sac, invade the parenchyma during development, and reside in the CNS throughout life (Waisman et al, 2015). Microglia are regarded as the principal phagocytes in the CNS (Wolf et al, 2017); they engulf dying or dead cells depending on the situation. In the developing CNS, a number of unhealthy or misconnected neurons die (Oppenheim, 1991), and their debris are cleared by microglia (Rigato et al, 2011). Upon neuronal injury, a large amount of cellular debris accumulates at the injury site, which is scavenged by reactive microglia (Sierra et al, 2013). Failure to clear debris has detrimental effects on surrounding neural tissue. For instance, accumulated debris can be a barrier to growing axons (Chen et al, 2000; Tanaka et al, 2009). More importantly, some intracellular molecules can leak from dead cells and trigger inflammatory responses in surrounding cells, resulting in damage to neural tissue or activation of autoimmunity (Sierra et al, 2013). The removal of dying or dead cells by microglia is, therefore, essential for development, maintenance, and regeneration of the CNS. However, impairment of microglial phagocytosis can occur in certain conditions, such as aging and injury (Abiega et al, 2016; Pluvinage et al, 2019). Alternatively, in the event of brain ischemia or traumatic injury, a large amount of cellular debris can overwhelm microglial capacity (Ritzel et al, 2015). In this context, auxiliary or supportive clearance systems may be actuated in the CNS. To address this alternative clearance system, a microglia-specific ablation model, in which the debris of dying microglia can be tracked in the absence of microglial phagocytosis, can provide important insights. Conditional microglial ablation models using both genetic and pharmacological strategies have been established (Waisman et al, 2015; Han et al, 2019). However, previously reported ablation systems may not be suitable for identifying a compensatory phagocytosis system because of non-microglial mononuclear cells in the CNS (Prinz et al, 2017). CNS-associated macrophages reside in the CNS boundary regions, such as the meninges, perivascular space, and choroid plexus (Goldmann et al, 2016). In addition, circulating monocytes infiltrate the CNS upon inflammation or injury (Yamasaki et al, 2014). Because these non-microglial mononuclear cells have phagocytic properties (Yamasaki et al, 2014; Prinz et al, 2017), it is likely that they participate in clearance of microglial debris. However, using previously reported genetic systems, at least some of these cells are assumed to be ablated concomitantly with microglia. This is because mononuclear lineages have similar gene expression profiles, such as for Itgam (the gene encoding cluster of differentiation [CD]11b), Cx3cr1, and Aif1 (the gene encoding ionized calcium-binding adaptor molecule 1 [Iba1]; Wieghofer & Prinz, 2016). Using Cx3cr1CreER-based genetic ablation systems, CX3C chemokine receptor 1 (CX3CR1)+ monocytes circumvent death because of their short life span (Goldmann et al, 2013; Parkhurst et al, 2013; Yona et al, 2013; Bruttger et al, 2015; Cronk et al, 2018; Lund et al, 2018); however, most CNS-associated macrophages are assumed to be ablated concomitantly with microglia, given their Cx3cr1 expression and longevity (Goldmann et al, 2016). A current standard pharmacological ablation system uses colony-stimulating factor-1 receptor (CSF1R) inhibitors, PLX3397 or PLX5622 (Elmore et al, 2014; Dagher et al, 2015). A very recent study showed that PLX3397 almost completely depletes all types of CNS-associated macrophages (Van Hove et al, 2019). To improve the specificity of cell ablation, we recently produced a new mouse model utilizing the Siglech locus. Studies by ourselves and others demonstrate that sialic acid-binding immunoglobulin-like lectin H (Siglec-H) expression is almost entirely confined to microglia in the CNS; its expression is absent in circulating monocytes and CNS-associated macrophages, except for a fraction of choroid plexus macrophages (Konishi et al, 2017; Van Hove et al, 2019). Accordingly, Siglechdtr mice, in which diphtheria toxin (DT) receptor (DTR) is knocked into the 3′-untranslated region of the Siglech gene, enable highly specific ablation of microglia without affecting most other mononuclear populations (Konishi et al, 2017). In this study, we demonstrate that astrocytes, rather than non-microglial mononuclear cells, readily phagocytose microglial debris in our microglia ablation model. Furthermore, astrocytes are capable of engulfing spontaneous apoptotic cells in mutant mice with phagocytosis-impaired microglia, but not in wild-type (WT) mice, indicating that astrocytes have the potential to compensate for microglia with dysfunctional phagocytic activity. Results Microglial-independent clearance of microglial debris after ablation of microglia Siglec-H expression is almost entirely confined to microglia among CNS-related mononuclear cells (Fig EV1), and ablation using Siglechdtr mice is nearly specific for microglia (Konishi et al, 2017). Here, we focused on the hippocampal CA1 region because it has a high ablation rate for microglia. Intraperitoneal administration of DT induced apoptosis of Iba1+ microglia, which were identified by pyknotic or fragmented nuclei, in the hippocampal CA1 of Siglechdtr/dtr mice (Fig 1A and B). The number of live microglia with normal nuclei was significantly reduced by ~85% in the 2–4 days post-DT injection (Fig 1A and C). The number then recovered to normal levels by day 7, although the repopulated microglia exhibited less ramified morphology (Appendix Fig S1). Thereafter, by day 28, the morphology became identical to that before DT administration. Click here to expand this figure. Figure EV1. CNS-related mononuclear cells and their marker moleculesSiglec-H expression is almost confined to parenchyma-resident microglia. CCR2 is a near-specific marker for infiltrating monocytes. CD206 is specifically expressed by CNS-associated macrophages residing in the CNS boundary region (i.e., leptomeningeal macrophages, choroid plexus macrophages, and perivascular macrophages). Download figure Download PowerPoint Figure 1. Clearance of microglial debris after microglial ablation Time-course images of the hippocampal CA1 region of Siglechdtr/dtr mice after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). Arrow: live microglia with normal nucleus. Double arrow: apoptotic microglia with pyknotic nucleus. Arrowhead: microglial debris with no nucleus. Scale bar, 50 μm. Number of apoptotic microglia with pyknotic or fragmented nucleus (n = 5 animals per group, Kruskal–Wallis test with post hoc Dunn's test). Number of live microglia with normal nucleus (n = 5 animals per group, one-way ANOVA with post hoc Tukey's test). Number of pieces of microglial debris (Iba1+ spheres with a diameter > 2 μm and no nucleus) (n = 5 animals per group, Kruskal–Wallis test with post hoc Dunn's test). A representative image of a survived microglial cell in the hippocampal CA1 region of Siglechdtr/dtr mice 2 days after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). A 3D image (rightmost panel) was reconstructed using Imaris software. Arrow: live microglia. Arrowhead: microglial debris. Scale bar, 10 μm. Data information: Values show the mean ± SEM. N.S.: no significance; ***P < 0.001. Download figure Download PowerPoint In previously reported microglial ablation models, the origin of repopulated microglia is controversial (Waisman et al, 2015; Han et al, 2019); therefore, we first addressed this issue (Fig EV2). Parabiotic coupling of Siglechdtr/dtr mice with CAG-EGFP mice, in which green fluorescent protein (GFP) is expressed in all cells under the control of a β-actin-based CAG promotor, resulted in 54.8 ± 3.5% (mean ± SEM, n = 5) GFP+ peripheral blood cells 3 weeks after parabiotic surgery (Fig EV2A and B). Seven days after DT administration, lymph nodes, as a positive control, contained a significant number of GFP+ cells (Fig EV2C). By contrast, there were no GFP+ microglia in hippocampal CA1, indicating that repopulated microglia are not derived from circulating blood cells, including any myeloid lineage, which are reported to enter the brain parenchyma and differentiate into microglia-like cells in some microglial ablation models (Lund et al, 2018; Shemer et al, 2018). On day 4, the onset of the recovery phase, ~90% of microglia expressed proliferation marker Ki-67 in their nucleus (Fig EV2D and E), indicating that survived microglia actively proliferate to replenish the brain, which is similar to other microglial ablation models, such as using Cx3cr1CreER:Rosa26dtr mice (Bruttger et al, 2015) or CSF1R inhibitor PLX compounds (Huang et al, 2018; Van Hove et al, 2019). Click here to expand this figure. Figure EV2. Survived microglia proliferate to replenish the brain A schematic drawing of parabiotic coupling of Siglechdtr/dtr and CAG-EGFP mice. Flow cytometric analysis of blood chimerism of the parabiont. Representative data are shown. Images of hippocampal CA1 and lymph node of parabiont WT mice 7 days after DT administration. GFP signal (green), Iba1 immunoreactivity (red), and DAPI signal (cyan) are shown. Images were acquired using the same laser power and sensitivity, and image processing was the same for GFP signals (green). Scale bar: 200 μm. Time-course images of Ki-67 immunoreactivity in hippocampal CA1 of Siglechdtr/dtr mice after DT administration. Sections were stained with anti-Iba1 (green) and anti-Ki-67 (red) antibodies, and DAPI (cyan). Arrows indicate Ki-67+ proliferating microglia. Scale bar, 50 μm. Percentage of Ki-67+ microglia after DT administration (n = 5 animals per group, Kruskal–Wallis test with post hoc Dunn's test). Values show the mean ± SEM. N.S.: no significance; ***P < 0.001. Download figure Download PowerPoint In our ablation model, we found a large amount of microglial debris (Iba1+ spheres with a diameter > 2 μm and no nuclei) on day 2; however, the debris was diminished by day 4 (Fig 1A and D). During this time, most of residual microglia exhibited abnormal morphology with fewer ramified processes (Appendix Fig S1), and did not interact with the debris (Fig. 1E), indicating that a microglia-independent clearance system worked in the absence of functional microglia. Non-microglial mononuclear populations do not participate in debris clearance Most non-microglial mononuclear cells are not expected to be ablated in this ablation system (Fig EV1); therefore, we explored the possibility that microglial debris was cleared by such cells around day 2. We first considered the involvement of CC chemokine receptor 2 (CCR2)+Ly6Chigh inflammatory monocytes (Fig EV1; Serbina & Pamer, 2006), which conditionally infiltrate diseased or inflamed CNS and act as phagocytes (Yamasaki et al, 2014; Ritzel et al, 2015). As a positive control, we crossed Siglechdtr with Ccr2RFP mice to label CCR2+ cells with red fluorescent protein (RFP) and confirmed that a large number of RFP+ cells infiltrated the spinal cord of Siglechdtr/dtr:Ccr2RFP/+ mice after induction of experimental autoimmune encephalomyelitis (EAE; Fig 2A). As expected, 2 days after DT administration to Siglechdtr/dtr:Ccr2RFP/+ mice, RFP+Ly6Chigh inflammatory monocytes were present in peripheral blood, and their number was slightly increased by an unknown mechanism (Fig 2B). Even in the presence of circulating inflammatory monocytes after DT injection, we found no infiltrated RFP+ cells in hippocampal CA1 2–4 days after DT administration (Fig 2A), eliminating the possibility that inflammatory monocytes infiltrated the hippocampal parenchyma and cleared microglial debris. Figure 2. Non-microglial mononuclear populations do not act as scavengers for microglial debris Histological analysis of RFP+ cells in Siglechdtr/dtr:Ccr2RFP/+ mice. Lumbar spinal cord after EAE induction and hippocampal CA1 2 or 4 days after PBS or DT injection were analyzed. Iba1 immunoreactivity (green) and RFP fluorescence (red) are shown. Images were acquired using the same laser power and sensitivity, and image processing was the same for all RFP signals (red). Scale bar, 50 μm. Flow cytometric analysis of RFP+Ly6Chigh inflammatory monocytes in peripheral blood of Siglechdtr/dtr:Ccr2RFP/+ mice 2 days after PBS or DT administration. Representative data and a quantification graph (n = 5 animals per group, two-tailed unpaired Student's t-test) are shown. Immunohistochemical localization of CD206+ perivascular macrophages (arrows) in hippocampal CA1 of Siglechdtr/dtr mice. Sections prepared 2 days after administration of PBS or DT were stained with anti-Iba1 (green), anti-CD206 (red), and anti-CD31 (blue) antibodies. Scale bar, 50 μm. Number of perivascular macrophages in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals per group, two-tailed unpaired Student's t-test). Data information: Values show the mean ± SEM. N.S.: no significance; *P < 0.05. Download figure Download PowerPoint We next focused on CNS-associated macrophages, which are located at boundary regions of the CNS and specifically express CD206 in contrast to microglia (Fig EV1) (Goldmann et al, 2016). Among these macrophage cell types, the involvement of leptomeningeal macrophages and choroid plexus macrophages was excluded, given the distance of the leptomeninges and choroid plexus from the hippocampal parenchyma. In contrast to these two cell types, perivascular macrophages were found in association with medium- or large-sized CD31+ vessels in the hippocampal CA1 of control mice (Fig 2C). Consistent with our previous result obtained in the cerebral cortex (Konishi et al, 2017), perivascular macrophages remained intact even 2 days after DT administration in hippocampal CA1 (Fig 2D). They still associated with vessels, similarly to the PBS-administrated mice, and did not infiltrate hippocampal parenchyma for debris clearance (Fig. 2C). Together, these results suggest that brain-resident non-professional phagocytes play roles in debris clearance. Activated astrocytes engulf microglial debris To investigate the microglia-independent clearance system, we analyzed mRNA levels of marker molecules for various CNS cell types in the hippocampus 2 days after DT treatment (Fig 3A). Expression of Aif1 (the gene encoding Iba1) was significantly decreased, confirming microglial ablation. Expression of Map2 (a neuron marker), Mbp (an oligodendrocyte marker), and Cspg4 (the gene encoding oligodendrocyte precursor cell marker, neuron-glial antigen 2 [NG2]) was not altered. By contrast, the expression of astrocyte marker Gfap was significantly increased after microglial ablation. Immunohistochemical staining of glial fibrillary acidic protein (GFAP) demonstrated that astrocytes exhibited hypertrophic morphology (Fig 3B), which is a hallmark of astrocytic activation (Sun & Jakobs, 2012), although the number of cells was unchanged (Fig 3C). Figure 3. Astrocyte activation after microglial ablation qPCR analysis of marker molecules for CNS cell types. The hippocampus of WT and Siglechdtr/dtr mice 2 days after PBS or DT administration was analyzed (n = 3 animals per group, one-way ANOVA with post hoc Tukey's test). Results are normalized to Gapdh and are shown as ratios to the value of WT mice injected with PBS. Immunohistochemical detection of astrocytes in Siglechdtr/dtr mice. Hippocampal CA1 sections were prepared 2 days after PBS or DT administration, and stained with an anti-GFAP antibody. Images were acquired using the same laser power and sensitivity, and image processing was the same. Scale bar, 30 μm. Astrocyte number in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals, two-tailed unpaired Student's t-test). Data information: Values show the mean ± SEM. N.S.: no significance; ***P < 0.001. Download figure Download PowerPoint These results prompted us to look for interactions between astrocytes and microglial debris. For this purpose, we used an antibody against CD11b, instead of Iba1, because microglial debris was detected more clearly by staining for CD11b than Iba1 (Appendix Fig S2). In contrast to PBS-injected control mice, we frequently observed that astrocytes extended their processes to contact microglial debris 2 days after DT administration (Fig 4A). We also occasionally found that a single piece of microglial debris was surrounded by several processes from multiple astrocytes. The interaction between activated astrocytes and microglial debris was observed throughout the brain, including in the cerebral cortex, thalamus, and medulla (Fig EV3). Figure 4. Astrocytes engulf microglial debris after microglial ablation A. Representative images of microglial debris surrounded by astrocyte processes in hippocampal CA1. Sections were prepared from Siglechdtr/dtr mice 2 days after PBS or DT administration, and stained with an
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