The Fractalkine Receptor but Not CCR2 Is Present on from Embryonic Development throughout Adulthood

This information is current as Makiko Mizutani, Paula A. Pino, Noah Saederup, Israel F. of September 29, 2021. Charo, Richard M. Ransohoff and Astrid E. Cardona J Immunol 2012; 188:29-36; Prepublished online 11 November 2011; doi: 10.4049/jimmunol.1100421

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Fractalkine Receptor but Not CCR2 Is Present on Microglia from Embryonic Development throughout Adulthood

Makiko Mizutani,* Paula A. Pino,†,‡ Noah Saederup,x Israel F. Charo,x Richard M. Ransohoff,* and Astrid E. Cardona†,‡

Microglial cells are difficult to track during development because of the lack of specific reagents for myeloid subpopulations. To further understand how myeloid lineages differentiate during development to create microglial cells, we investigated CX3CR1 and CCR2 transcription unit activation in Cx3cr1+/GFPCCR2+/RFP knockin fluorescent reporter mice. The principal findings include: 1) CX3CR1+ cells localized to the aorta–gonad–mesonephros region, and visualized at embryonic day (E)9.0 in the yolk

sac and neuroectoderm; 2) at E10.5, CX3CR1 single-positive microglial cells were visualized penetrating the neuroepithelium; and Downloaded from 3) CX3CR1 and CCR2 distinguished infiltrating macrophages from resident surveillant or activated microglia within tissue sections and by flow cytometric analyses. Our results support the contribution of the yolk sac as a source of microglial precursors. We provide a novel model to monitor receptor expression changes in microglia and myeloid cells early (E8.0–E10.5) in development and during inflammatory conditions, which have been challenging to visualize in mammalian tissues. The Journal of Immunology, 2012, 188: 29–36. http://www.jimmunol.org/ icroglia, the resident tissue macrophages and intrinsic a transmembrane glycoprotein on neurons and peripheral, but not “immune effector” cells of the CNS, constitute ∼20% CNS, endothelial cells (5, 9). The fractalkine receptor (CX3CR1) M of the total glial population and are distributed is present on microglia and circulating , dendritic cells, throughout the brain, spinal cord, and optical tissues (1). In CNS NK cells, and T cells (10–12). Membrane-bound fractalkine on leukocyte preparations, CD45 is used to separate the CD45hi he- endothelial cells mediates adhesion of CX3CR1+ leukocytes, matogenous population from CD45lo resident microglia by flow and proteolytic cleavage releases the soluble domain that confers cytometry (2, 3). Characterization of MHC class II (MHC-II) fractalkine its chemotactic properties (5). Importantly, CX3CR1 expression and upregulation of activation markers such as distinguished a subset in peripheral blood so-called 2 2 CD40, CD44, and CD86 have provided insights into the effector resident, phenotypically identified as LFA-1+, L-Sel , Ly6C , by guest on September 29, 2021 functions of activated microglia during CNS inflammation (4). CCR22, CX3CR1hi, whereas CCR2, the MCP 1 receptor (MCP-1 However, there are still debates regarding the alterations of CD45 or CCL2), marked “inflammatory” monocytes LFA-12, L-Sel+, expression by monocytes and CNS-born microglial cells. Cur- Ly6C+,CCR2+, and CX3CR1lo (13–15). Circulating resident rently, no simple combination of markers facilitates the identifi- monocytes patrol healthy tissues through long-range crawling on cation and differentiation of resident surveillant or activated mi- the healthy endothelium (13), playing a critical role in immune croglia from hematogenous monocytes. surveillance. In contrast, CCR2+CX3CR12Ly6C+ monocytes are The discovery of the CX3CR1 in 1994 and typically found in inflamed tissues (16). The generation of its unique ligand fractalkine in 1997 represented a major ad- CX3CR1/GFP knockin mice, in which the CX3CR1 was disrupted vancement in the understanding of microglial and monocyte by insertion of GFP, clarified the expression pattern and has function (5–8). Fractalkine is a distinct chemokine, expressed as facilitated the tracking of CX3CR1+ leukocytes in vivo. In the naive CNS of Cx3cr1+/gfp and Cx3cr1gfp/gfp mice, CX3CR1 tran- scription unit was exclusively active in microglial cells, as GFP + + *Department of Neurosciences, Neuroinflammation Research Center, Lerner Re- expression was undetectable in neurons, NG2 glia, and GFAP search Institute, Cleveland Clinic, Cleveland, OH 44195; †Department of Biology, astrocytes (12). University of Texas at San Antonio, San Antonio, TX 78249; ‡South Texas Center for Emerging Infectious Diseases, University of Texas at San Antonio, San Antonio, TX Because of the lack of appropriate anti-CCR2 Abs, most CCR2 78249; and xGladstone Institute of Cardiovascular Research, San Francisco, CA expression analyses have been limited to mRNA levels (17). It has 94158 been proposed that microglia activation during inflammatory set- Received for publication February 9, 2011. Accepted for publication September 30, tings upregulate CCR2 (18, 19). However, microglial CCR2 pro- 2011. tein expression has been challenging to prove in situ. The recent This work was supported by the National Institutes of Health (Grant R01NS32151 to R.M.R.) and the National Multiple Sclerosis Society (Grant TA3021-A1/T to A.E.C.). generation of red fluorescent protein (RFP)-CCR2 knockin mice has provided the tools to potentially fractionate CX3CR1 and Address correspondence and reprint requests to Dr. Astrid E. Cardona, University of Texas at San Antonio, Building MBT 1.216, San Antonio, TX 78249. E-mail address: CCR2 activation in microglial and peripheral monocytes and [email protected] study their trafficking, molecular signatures, and biological func- The online version of this article contains supplemental material. tions (20). CCR2-RFP mice showed an accurate correlation of Abbreviations used in this article: E, embryonic day; EAE, experimental autoimmune CCR2 and RFP expression in blood monocytes both at the protein encephalomyelitis; IB4, isolectin-B4; MHC-II, MHC class II; ON, overnight; RFP, and mRNA levels. Characterization of Ccr2rfp/rfp mice revealed red fluorescent protein; SE, surface ectoderm. that in response to thioglycolate, CCR2 was required for in vivo Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 migration of monocytes to the inflamed peritoneum. In mice with www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100421 30 IN VIVO SYSTEM VISUALIZES MICROGLIAL AND MYELOID DEVELOPMENT experimental autoimmune encephalomyelitis (EAE), CCR2 was of 20-mm-thick sections in the z-axis scanned at 1.5-mm steps. In some critical for efficient accumulation of LyC6hi/CCR2hi monocytes. experiments, brain tissues were dissected from adult mice at peak of EAE Interestingly, during CNS inflammation, monocytes that infiltrate disease and tissues sections incubated at 4˚C with 7-4 Ab (Cedarlane, Burlington, ON, Canada), and imaged at 403 magnification on a Zeiss 510 the CNS maintained their signature chemokine receptor profiles as confocal microscope as previously described (20). found in circulating monocytes (20). Importantly, microglia in the 2 EAE induction adult naive and inflamed CNS were found as CX3CR1hiCCR2 using the described Cx3cr1+/gfp Ccr2+/rfp reporter mice. Active EAE was induced by s.c. immunization with myelin oligodendrocyte CNS-infiltrating monocytes appeared as CCR2hiCX3CR1lo or glycoprotein 35–55 peptides in CFA as previously described (22). Mice CCR2loCX3CR1hi. Notably, CX3CR1 expression by microglia was were weighed and examined daily for EAE symptoms and scored as fol- hi lows: 0, no signs of neurologic disease; 1, lack of tail tone; 2, abnormal significantly higher when compared with blood CX3CR1 mono- gait, hind-limb weakness; 2.5, partial hind-limb paralysis; 3, complete cytes. hind-limb paralysis; 3.5, ascending paralysis; 4, tetraplegia; and 5, death. Given the fact that CX3CR1 is predominantly expressed by Mice were sacrificed when they reached a score of 2.5–3.0 (21). Brain m “resident tissue monocytes,” we sought to investigate CX3CR1 tissues were obtained from PFA perfused mice and free-floating 30- m tissues obtained after cryoprotection and confocal images obtained. and CCR2 transcription unit activation patterns in microglial precursors as they colonize the developing CNS. For this, CCR2- Isolation of brain mononuclear cells and flow cytometry +/gfp RFP and CX3CR1-GFP mice were crossed and Cx3cr1 - For flow cytometry analysis, perfused brain and spinal cord tissues were Ccr2+/rfp embryos analyzed at various stages during embryonic dissected and mononuclear cells separated over discontinuous 70/30% development. The results indicate that CX3CR1 is active in early Percoll gradients as previously described (23), and cellular pellets were microglial precursors and sustained throughout adulthood. In resuspended in cell staining buffer (BioLegend, San Diego, CA). Blood Downloaded from was collected from the submandibular vein; RBCs were depleted by hy- contrast, RFP as reporter of activation of the CCR2 transcription potonic lysis and washed in staining buffer. Isolated cells were incubated unit was not significantly detected in developing microglia. on ice for 5 min with anti-mouse CD16/CD32 (clone 2.4G2; BD Phar- Therefore, we propose CX3CR1 and CCR2 as markers to easily mingen)toblockFcRsandthenincubatedonicefor30minwithamix differentiate infiltrating macrophages from resident surveillant of fluorochrome-conjugated anti-mouse Abs: CD45.1-Pacific blue (clone or activated microglia in tissue sections and by flow cytometric A20; eBioscience), CD45.2-allophycocyanin (clone 104; eBioscience) and CD11b-PerCP (clone M1/70; BioLegend), or CD45.1–PE-Cy7 (clone A20; analyses. Importantly, the results support the contribution of the eBioscience), CD45.2-allophycocyanin (clone 104; eBioscience), CD11b- http://www.jimmunol.org/ yolk sac as the source of microglial precursors. These results bring PerCP (clone M1/70; BioLegend), and I-A/I-E Pacific blue (clone M5/ potential applications to monitor neuronal–glial interactions through- 114.15.2). After washes, cells were resuspended in 2% paraformalde- out development and to further the understanding of microglial/ hyde and acquired in an LSR-II (BD Biosciences, Franklin Lakes, NJ). Samples were analyzed with FlowJo software (Tree Star, Ashland, OR). monocyte/macrophage and dendritic cell plasticity in vivo.

Materials and Methods Results CX3CR1-GFP reporter expression colocalizes to early Mice microglial cells that colonize the developing CNS Ccr2RFP/RFP and Cx3cr1GFP/GFP mice were crossed at the Biological by guest on September 29, 2021 Resources Unit, Cleveland Clinic and the Laboratory Animal Resources Whole mounted embryos during E8.5–E10.5 embryonic stages Unit at the University of Texas at San Antonio. Timed pregnancies were were dissected as they easily displayed the overall mental map of set up to isolate embryos at embryonic stages 8.5–13.5. All experiments the embryo and allowed grossly localization of CX3CR1-green/ were performed in accordance with National Institutes of Health guidelines CCR2-red cells based on landmark histological structures. and approved by the Cleveland Clinic and the University of Texas at San Antonio Institutional Animal Care and Use Committees. Unturned embryos at E8.5 stage showed clusters of double- positive CX3CR1GFP/CCR2RFP cells colocalizing to the gut re- Tissue processing gion and sparsely visualized throughout the embryo (Fig. 1A–C).

Female mice were sacrificed by CO2 asphyxiation, and embryos dissected At stage E8.5–E9.0 (turned embryo), cells revealing fluorescence and kept in cold PBS. A small tissue (, 1 mm) biopsy was taken from the intensity appeared as double positive within tracks in the devel- caudal part of the embryo for genotyping purposes. Embryos (embryonic oping somites (Fig. 1D–F) and also present in neural tissues, day [E]8.5–E9.5) were fixed in 4% PFA overnight (ON), and whole em- within the telencephalic vesicle (Fig. 1G–I). IB4 (Fig. 1; purple) bryos were permeabilized ON in 3% Triton X-100. Alternatively, after fixation, embryos (.E10.5) were transferred sequentially to 15, 20, and was used to highlight the vasculature but also allowed the visu- 25% sucrose solutions and 10-mm cryosections were obtained, or 60-mm alization of cells with features of developing microglia that free-floating sections were collected by sectioning embryos on a sliding colonize the neural tissue appearing as CX3CR1GFP+ (Fig. 1G, microtome (12). asterisks). The exact origin of these cells is not clear. However, Generation of bone marrow chimeric mice visualization of yolk sac tissues at E9.5 revealed the presence of + +/GFP +/RFP sparsely located GFP cells with large cell bodies, suggesting that Recipient Cx3cr1 /Ccr2 CD45.2 mice (5–6 wk old) irradiated with + a dose of 900 rad were reconstituted with 20 3 106 cells isolated from a population of CX3CR1 cells develops in the embryonic cir- C57BL/6 CD45.1 donor mice as previously described (21). Six weeks after culation (Supplemental Fig.1). Double-positive cells were also reconstitution, mice were bled via the submandibular vein, heparinized blood found in foci at mid and caudal regions of the embryo in close was lysed, and efficiency of reconstitution was determined by flow cytom- proximity to the peritoneal cavity colocalizing to the midline etry using anti-CD45.2 and -CD45.1 Abs. Chimeric mice were allowed dorsal aorta with tracks of cells distributing throughout the de- to reconstitute for 10 wk after bone marrow transfer before EAE induction. veloping embryo (Supplemental Fig. 1). Microscopic analysis To closely monitor the morphology and neural colonization + Whole embryos or selected sections were rinsed in 13 PBS, covered with of CX3CR1 cells, we analyzed cross sections (Fig. 2) and sagittal 2 ml of a solution containing 450 nM DAPI in PBS, and incubated for slices (Supplemental Fig. 2) from E9.5–E10.5 tissues. Double- 10 min at room temperature. Excess DAPI was absorbed on paper towels positive cells were found in both posterior and anterior ends of the and slides rinsed in PBS. To visualize the vasculature, tissues were per- neural tube (Fig. 2), mostly confined to the surface ectoderm (SE) meabilized ON in 3% Triton in PBS and incubated at 4˚C for 2 d with and tightly opposed to the outer layer of the neuroepithelium. A dis- biotin-conjugated isolectin-B4 (IB4). Streptavidin-Cy5 was used to detect bright dim the bound biotin-IB4. DAPI-counterstained tissues were visualized under tinct population of CX3CR1-GFP and CCR2-RFP cells was a fluorescent microscope. Confocal images were obtained as projections visualized in the junction of the SE and neuroepithelium (Fig. 2). The Journal of Immunology 31 Downloaded from http://www.jimmunol.org/

FIGURE 2. Ccr2+/RFP/Cx3cr1+/GFP cells are localized in the neuro- ectoderm (NE) and visualized in developing E9.5 tissues. A, The plane of section in schematic form. An E9.5 tissue shows that both receptors are expressed by myeloid precursors localized between the developing neu- roepithelium and SE. Tissues were stained with DAPI to visualize the nuclei (blue). B, Distribution of CCR2-RFP+ cells and (C) CX3CR1-GFP+ cells. D, Merged image shows a dense band of myeloid precursors that appear mostly double positive. CX3CR1 single-positive microglial pre- cursor cells were clearly detected in the SE (arrows). The schematic inset by guest on September 29, 2021 on A was published in The Atlas of Mouse Development, M. H. Kauffman, “Plate 20,” p 83, Copyright Elsevier (1994).

developing cortical tissue without direct association with vascu- lature. These CX3CR1+ cells displayed amoeboid morphology typical of early microglial precursors (Fig. 3). At stage E11.5, the neural tissue revealed thicker neopallial cortex regions, and mixed populations of cells showing various degrees of CX3CR1-GFP and CCR2-RFP intensities were visualized. Clusters of small/ Dim bright FIGURE 1. Ccr2+/RFP/Cx3cr1+/GFP-positive cells are visualized at E8.5 round cells appearing as CX3CR1-GFP /CCR2-RFP were stage of development. Whole embryos were incubated with IB4-Cy5 to detected in the SE or meningeal membranes (Figs. 3, 4). Inter- bright visualize the vasculature (magenta), and tissues (A–F)werecounterstained estingly, single-positive CX3CR1-GFP cells appeared local- with DAPI (blue). Merged image from an unturned embryo (A) at the level ized within the neuroepithelium, migrating from the SE reaching of gut region (A,arrowsandinset) shows clusters of CX3CR1+ (B, green) the inner part of the developing cortex (Fig. 4). The number of + and CCR2 (C, red) double-positive cells. Images from a turned embryo at CX3CR1-GFP+ cells expands dramatically at E11.5 stage, and the level of the somites (D–F) and telencephalic vesicle (G–I)showabun- + + they colonize not only the developing cortex but the optic vesicles dant numbers of CX3CR1 (E, H, green) and CCR2 cells (F, I,red)present and developing spinal cord (Supplemental Fig. 2). throughout the tissues. Notably, a population consisting of CX3CR1hi single- + Detailed visualization of E13.5 embryos revealed a significant positive cells was detected in the developing brain that appear IB4 (G–I, + asterisks). A, D, G, Insets show a schematic of the embryo with a small expansion of CX3CR1 cells detected throughout the whole em- square highlighting the source of the image. The schematic insets on bryo. E13.5 neural tissue showed a similar pattern to E10.5 stage, + A, D,andG were published in The Atlas of Mouse Development,M.H. with more single GFP cells populating the developing CNS. The Kauffman, “Plate 16,” p 65, Copyright Elsevier (1994). developing spinal cord is easily visualized at this stage, and neural CX3CR1+ cells showed the characteristic microglial phenotype with bigger cell bodies and extended processes, IBA-1 immuno- CX3CR1, but not CCR2, is expressed by developing microglial reactivity, and lack of CCR2-RFP reporter expression (Supple- cells mental Fig. 2). Fig. 3 shows a sagittal section at stage E11.5 highlighting IB4+ The previous observations suggest a scenario in which the cells vasculature, with presence of double-positive cells inside blood that first colonize neural tissues are double-positive CX3CR1/ vessels. Scarce CX3CR1 single-positive cells localized to the CCR2 and accumulate at the SE. Cells that remain in the SE, 32 IN VIVO SYSTEM VISUALIZES MICROGLIAL AND MYELOID DEVELOPMENT

FIGURE 3. Developing microglial cells appear as CX3CR1bright and CCR22. A, Low power of the whole embryo is shown in the inset to locate the origin of the image (red square). E11.5 tissues show that CX3CR1, but

not CCR2, is expressed by developing microglial cells (A, merged image). Downloaded from Free-floating sagittal sections, 30 mm in thickness, were obtained and in- cubated with IB4 (blue) to visualize the vasculature. Confocal image of the neopallial cortex shows CX3CR1+–(A, B, green) and CCR2+ (A, C, red)– expressing cells. Developing amoeboid microglia appear as CX3CR1bright (asterisks) and CCR22 (asterisks), whereas circulating mononuclear phagocytes exhibit a CX3CR1loCCR2bright phenotype and appear within blood vessels (BV, arrows). http://www.jimmunol.org/ meningeal areas, downregulate CX3CR1 and appear predomi- nantly as CCR2+. Because of the fact that most cells in the SE are double positive, it is likely that cells entering the deep layers of the neuroepithelium downregulate CCR2 appearing as single- positive CX3CR1+. However, we do not rule out the possibility of discrete single-positive cells that reach the SE, proliferate, and create the population that colonizes and expands within the de- +/RFP +/GFP veloping CNS tissue. Therefore, early stages of development FIGURE 4. Distribution of cells in Ccr2 /Cx3cr1 E11.5 tis- by guest on September 29, 2021 (E8.5) showed: 1) cells with the capacity of becoming peripheral sues. A, The plane of section in schematic form is shown. B–G, Confocal m monocytes, and 2) cells that colonize the perivascular and cortical images of free-floating, 30- m cross sections of E11.5 tissues counter- stained with DAPI (B, E, merged image, DAPI, blue) show CX3CR1+/ regions as CNS microglia. Both populations appeared distinct CCR2+ double-positive cells in the SE (C, F, CX3CR1-GFP, green) and based on the activity of the CCR2 and CX3CR1 transcription (D, G, CCR2-RFP, red). Developing amoeboid microglia appear as units, respectively. CX3CR1bright and localized in deep layers of neuroepithelium (arrows). At postnatal day 0, CCR2 expression remained confined to The schematic inset on A was published in The Atlas of Mouse Develop- a population of cells that localize to meningeal membranes and ment, M. H. Kauffman, “Plate 26,” p 137, Copyright Elsevier (1994). also visualized within blood vessels. All CCR2+ cells appeared as CX3CR1Dim. As the cortical tissue developed, migrating micro- glial cells that seeded parenchymal tissues exhibited a rapid ex- CCR2 expression was analyzed by flow cytometry in brain leu- pansion and appeared as single CX3CR1+ (Fig. 5). In the devel- kocyte preparations. Results reveal that brain inflammatory lesions oped CNS, RFP+ cells appeared confined to meningeal areas and contain cells with distinct levels of CX3CR1 and CCR2 expres- absent in brain parenchyma, indicating that neither astrocytic, sion. Notably, microglia did not upregulate RFP expression, flow microglial cells, oligodendroglial, nor neuronal cell types express cytometry analyses distinctly showed CCR2-RFP expression in significant amounts of CCR2 protein to be visualized by expres- hematogenous CD45hi leukocytes, and CCR2-RFP signal was sion of the RFP reporter (Fig. 5). absent in the CD45lo microglial population (Fig. 6A,6B). Two These observations suggest that CX3CR1 and CCR2 expression populations of CCR2-RFP+ cells are visualized, and we have is timely orchestrated during development. It has been established previously characterized them by flow cytometry (20) and shown that neither CX3CR1 nor CCR2 are required for the development that CCR2 deficiency depletes inflammatory Ly6Chi monocytes. 2 of microglia as knockout model, for both receptors show normal Importantly, absence of CCR2hiCX3CR1 Ly6Chi monocytes microglia in the healthy adult CNS. However, the tight regulation of delays EAE progression. To have a better understanding of both receptors provides a key phenotypic approach to characterize CCR2 and CX3CR1 expression in myeloid cells, brain tissues and distinguish monocytes/macrophages from resident microglia. were imaged, and CCR2-RFP and CX3CR1-GFP cells were

+ 2 visualized (Supplemental Fig. 3). In addition, we implemented Adult microglia exhibit a CX3CR1-GFP CCR2-RFP – a three-color imaging using the 7-4 marker that identifies expressing pattern, and microglia do not show significant hi RFP Ly6C monocytes and neutrophils, but not NK or T cells. In the CCR2 expression upon inflammation lesions, we observed activated microglial cells with shorter and To get insights into the CCR2 expression pattern in microglial cells thicker processes, and these cells clearly appear as CX3CR1 during inflammation, we immunized Cx3cr1+/GFP/Ccr2+/RFP mice single positive (Supplemental Fig. 3), whereas classical infil- with myelin oligodendrocyte glycoprotein 35–55. CX3CR1 and trating monocytes appear CX3CR1lo or negative and CCR2+. The Journal of Immunology 33

∼9.3 6 3.5% of the inflammatory infiltrated. Comparison of the CX3CR1-GFP and CCR2-RFP fluorescence intensities (Fig. 6H) shows that the CD45.2+ myeloid population from recipient mice is negative for CCR2-RFP. Mean fluorescent intensities from the resident microglial population were 9860 6 632.6 for GFP and 120.7 6 18.38 for RFP (n = 5 mice). Furthermore, the CD11b population, although activated based on MHC-II upregulation expression (Fig. 6I), appears with a CD45.2lo phenotype in flow cytometric analysis. Confocal images of brain tissues of chimeric mice confirmed that the activated microglial population as noted by the shorter processes and bigger cell bodies appear as CX3CR1-GFP single positive (Fig. 6J, arrows).

Discussion Surveillant parenchymal microglial cells are extremely plastic and provide the first line of defense within the CNS. Resident micro- glial cells are morphologically and functionally distinct from other mononuclear CNS populations, such as perivascular macro- FIGURE 5. CX3CR1 is confined to microglial cells during develop- phages, supraependymal macrophages, epiplexus cells of the choroids Downloaded from ment, and expression is sustained throughout adulthood. Confocal images plexus, and meningeal macrophages (24, 25). In the naive brain, were obtained from brain tissues at P0 and counterstained with DAPI microglia display small cell bodies with thin, long, and branched (blue). Cortical (A–C) and hippocampal (D–F) images show that paren- processes (1). Although microglial functions are intended to be bright chymal cells are mostly CX3CR1 (A, C, D, F; green) and fewer protective, it is documented that dysregulated microglial responses + CCR2 cells (A, B, D, E; red) remain localized in the meningeal region. lead to neurotoxicity in vitro and in vivo (12). Recent data have

clearly shown that activation of microglia might be beneficial http://www.jimmunol.org/ Analyses of skin and cardiac tissues (Supplemental Fig. 4) in some pathological settings. More specifically, absence of show that in naive tissues, long-lived macrophage populations CX3CR1 in two different models of Alzheimer’s disease corre- appear CX3CR1hi/CCR2-RFPneg similar to the phenotype of latedtoreducedb-amyloid deposition because of enhanced resident microglial cells. phagocytosis by activated microglia (26, 27). Upon activation, During EAE, microglial cells become activated, and markers because of inflammation of neuronal damage, microglial cells become indistinguishable from infiltrating myeloid cells using undergo programmed molecular and morphological changes that conventional monocytes. Therefore, we sought to clarify the re- include cytoskeleton rearrangements, that is, increases in cell lationship between CCR2, CX3CR1, and microglial cells using body area exhibiting shorter and thicker processes that ultimately congenic bone marrow transfer in CX3CR1-GFP/CCR2-RFP re- make amoeboid microglia phenotypically indistinguishable from by guest on September 29, 2021 cipient mice. For this, Cx3cr1+/GFP/Ccr2+/RFP double-heterozygous peripheral macrophages. mice (in a CD45.2 congenic background) were reconstituted with Another challenging area regarding microglial biology relates congenic wild type CD45.1 bone marrow cells. Upon EAE in- to their origin, which has been a subject of controversy for many duction, these chimeric mice would allow us to distinguish the years. It is now well accepted that microglia arise from mesodermal CD45.1 infiltrating population from radioresistant CNS resident progenitors that colonize the nervous tissues during embryonic CD45.2+ cells, among them microglial cells carrying CX3CR1- development and fetal periods, and become fully established dur- GFP/CCR2-RFP reporter . Upregulation of the CCR2 ing postnatal life (28–31). However, the exact mechanisms that transcription unit measured by virtue of red fluorescence will then control the accumulation of microglial progenitors to the devel- be analyzed. First, we determined the efficiency of bone marrow oping CNS are still enigmatic. Del Rio-Hortega in the 1910s reconstitution; PBMCs from chimeric mice were analyzed 6 wk classified microglia into two morphological distinct types, which after bone marrow transfer for CD45.1 and CD45.2 expression. are still current: ramified and amoeboid microglia (1). Ramified The protocol produced 96.8 6 0.75% reconstitution (Fig. 6C). At microglial cells are fully established postnatally, persist in the peak of EAE disease, brain leukocytes were isolated and stained adult, and represent the surveying population that constantly scans with Abs against CD45.1, CD45.2, and CD11b to distinguish the surrounding environment via extension and retraction of their peripherally derived myeloid cells (CD45.1) from resident processes to perceive activating or inhibitory signals. It is hypoth- microglial cells (CD45.2). Single-positive controls (Fig. 6D,6E) esized that during development, amoeboid microglia are initially for CX3CR1-GFP and CCR2-RFP expression were used to com- highly proliferative cells. Subsequently with a decrease in plas- pensate data acquired from chimeric mice and to compare levels ticity, microglial proliferation slows and the remaining cells dif- of expression in the infiltrating versus recipient myeloid pop- ferentiate into ramified microglia (32, 33). Supporting these initial ulations. Brain leukocytes were initially analyzed based on ex- findings, Juba in 1933 carried out the first comprehensive study pression of CD45.1 and CD11b to identify myeloid cells of of microglia in a series of human embryos 23–280 crown-rump peripheral origin (Fig. 6F, R1 gate). Disease severity is evident length (measurement of the length of human embryos and by the massive infiltration of peripherally derived leukocytes fetuses from the top of the head [crown] to the bottom of the (CD45.1+ cells; Fig. 6F), which accounted for 90 6 3.5% of the buttocks [rump]) (34, 35). He identified the various cell forms with total CD45hi population. Next, the CD45.12 population (Fig. 6F, developmental progression, ranging from amoeboid to mature R2 gate) was analyzed for CD45.2 expression to identify the branched varieties. Juba considered microglia to be derived from population of resident microglial cells (Fig. 6G, R3 gate). Even invading mesodermal elements because microglia-like cells were though bone marrow reconstitution was .95% efficient, periph- also detected within connective tissue of the head, which were eral CD45.2 recipient cells that remained created a CD45.2hi closely related to embryonic vascular elements. Since the early population that infiltrated the CNS tissue and accounted for 1930s, it has been maintained that microglia were “transformed 34 IN VIVO SYSTEM VISUALIZES MICROGLIAL AND MYELOID DEVELOPMENT

FIGURE 6. Analyses of monocyte subsets in brain lesions of EAE CX3CR1+/GFP CCR2+/RFP mice at peak disease. Flow cytometry analysis of mononu- clear cells isolated from adult brain at peak EAE disease shows that the CD45lo population contains Downloaded from CX3CR1bright microglial cells (A), contrasting their lack of CCR2-RFP expression (B). Degree of bone marrow reconstitution was evaluated by staining PBMCs with CD45.1 and CD45.2 Abs (C). CX3CR1- GFP+ (D) and CCR2-RFP+ (E) controls were obtained from Cx3cr1GFP/+/CCR2+/+ or Cx3cr1+/+/CCR2RFP/+

mice, respectively. Brain leukocytes were analyzed http://www.jimmunol.org/ for CD45.1 and CD11b expression (F), and periph- erally derived myeloid cells (R1) were distinguished from resident CD45.2+ CD11b+ cells via expression of the congenic markers (G,R3).Analysesof CX3CR1 and CCR2 based on activation of the tran- scription unit and expression of the GFP and RFP reporters (H) shows that resident microglial cells appear as CX3CR1 single positive. I, activated resi- dent cells upregulated MHC-II expression upon EAE induction as compared with naive brains, and (J) by guest on September 29, 2021 confocal imaging from diseased mouse brains shows morphologically activated microglia (arrows) as CX3CR1-GFP bright cells (original magnification 340, scale bar, 15 mm). Results show one representative set of data from one mouse out of eight analyzed by flow cytometry and histology.

blood elements” or “wandering mesenchymal elements” that mi- important structure defined as the birthplace of hematopoietic grated into the nervous tissue from the lumen of blood vessels. cells in mammals where primitive macrophages and erythrocytes Although recent studies have elegantly identified microglia as an originate, and therefore likely to harbor microglial progenitors. It ontogenically distinct population in the mononuclear phagocyte was proposed that microglia have a dual origin: first coming from system (36), we still lack a precise lineage-restricted marker for the yolk sac macrophages during the nonvascularized prenatal microglial progenitors that is a key to clarify the debates as to the stage, followed by a second engraftment from a population that transformation of these cells in vivo. represents a developmental and transitory form of fetal macro- The presence of CX3CR1+ cells in the yolk sac at E9.5–E11.3 phage (derived from blood-borne precursors, possibly monocytes), stages of development is of great interest. The yolk sac is an which may be related to the amoeboid microglial population seen The Journal of Immunology 35 in the postnatal period in rodents (34). This hypothesis has been 5. Bazan, J. F., K. B. Bacon, G. Hardiman, W. Wang, K. Soo, D. Rossi, D. R. Greaves, A. Zlotnik, and T. J. Schall. 1997. 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