CCR2 is localized in microglia and neurons, as well as infiltrating monocytes, in the lumbar spinal cord of ALS mice

It remains controversial whether circulating monocytes expressing CCR2 infiltrate the central nervous system (CNS) and contribute to pathogenicity of amyotrophic lateral sclerosis (ALS). A previous report used conventional immunohistochemistry to show that CCR2 is exclusively expressed by astrocytes, but not infiltrating monocytes/microglia or neurons, in the spinal cords of ALS model mice. In this study, we assessed the cellular distribution of CCR2 in the CNS of ALS mice using CCR2-reporter mice (Ccr2rfp/+-Cx3cr1gfp/+-SOD1G93A Tg mice), a more sophisticated method for directly detecting the distribution of CCR2 protein. We found that infiltration of CCR2+ monocytes in the lumbar spinal cord increased over the course of disease progression. Moreover, from the middle stage of disease, CCR2 was partially distributed in microglia and neurons, but not astrocytes, in striking contrast to the previous findings. These novel observations suggested that CCR2+ monocyte infiltration leads to CNS environmental deterioration due to toxic conversion of microglia and neurons, creating a vicious cycle of neuroinflammation and leading to acceleration of ALS pathology. Our findings also show that this reporter mouse is a useful and powerful tool for obtaining new insights into the pathomechanisms of ALS.


Main text
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective loss of motor neurons in the central nervous system (CNS). The nonautonomous neuronal death hypothesis, based on recent studies, states that neuroinflammation by nonneuronal neighboring cells, such as glia and infiltrating cells, is critical for disease progression in ALS [1]. In general, tissue inflammation induces migration of circulating monocytes, especially chemokine CC motif receptor 2 (CCR2) high monocytes [2]. It remains controversial whether CCR2 high monocytes infiltrate the CNS and contribute to ALS pathogenesis [3,4]. However, Kawaguchi-Niida et al. showed using conventional immunohistochemistry, that CCR2 is exclusively expressed by astrocytes, but not infiltrating monocytes/microglia or neurons, in the spinal cords of ALS mice [5]. This variability on CCR2 + cellular distribution may partly depend on how CCR2 is detected. For immunostaining, commercially available anti-mouse CCR2 antibodies are usually suitable for flow cytometry, but less so for immunohistochemistry/immunofluorescence, as is usually the case for cell surface antigens. Accordingly, the CCR2-RFP reporter mouse was developed to circumvent this difficulty [2].
Ccr2 rfp/+ -Cx3cr1 gfp/+ -SOD1 G93A Tg mice exhibited similar disease progression to SOD1 G93A Tg mice (median survival days = 167 vs. 160, respectively; Fig. 1), and the abundance of CX3CR1 + microglia in lumbar spinal cord gradually increased as the disease progressed (Supplemental Figure 1), consistent with the previous reports [7,8]. Chronological assessments also revealed that the abundance of CCR2 + cells gradually increased in the gray matter of the lumbar spinal cord as disease progressed ( Fig.  1b and c), whereas CNS infiltration by CCR2 + cells was not detected in Ccr2 rfp/+ -Cx3cr1 gfp/+ mice. Next, we evaluated the cellular localization of CCR2 by immunofluorescence staining for NeuN (neuron marker), Iba1 (monocytes and microglia marker), and GFAP (astrocyte marker). Most of CCR2 + cells were positive for Iba1 ( Fig.  1d and h), but not for CX3CR1, until the middle stage of the disease, suggesting that most CCR2 + cells in the CNS were infiltrating monocytes at early disease stages. These findings corresponded with a previous study reporting that CCR2 + monocytes were recruited into the spinal cord of SOD1 G93A Tg mice [3]. Surprisingly, from the middle stage of the disease, CCR2 was partially distributed in CX3CR1 + microglia ( Fig. 1e and i) and neurons ( Fig. 1f and j), but not in astrocytes (Fig. 1g), in striking contrast to the previous findings by Kawaguchi-Niida et al. [5]. The proportion of each type of CNS-resident cell that was CCR2 + increased as disease progressed, whereas the percentage of CX3CR1 + or Iba1 + cells that was CCR2 + reached a plateau at the middle stage of the disease (Fig.  1i). No resident CNS cells expressed CCR2 in Ccr2 rfp/+ -Cx3cr1 gfp/+ non-Tg mice (Supplemental Figure 2). These novel observations demonstrated that CCR2 is expressed in resident CNS cells such as microglia and neurons, as well as CNS-infiltrating monocytes, in the advanced stage of ALS.
The cellular distribution and physiological role of CCR2 in the CNS have yet to be elucidated. Previous studies reported that CCR2 is present only in monocytes/macrophages and basophils, whereas Ccr2 mRNA is expressed in most leukocytes, including monocytes/ macrophages, T cells, B cells, natural killer cells, basophils, and dendritic cells [2]. However, CCR2 is constitutively expressed in neurons in murine brain, spinal cord, and dorsal root ganglia, and upregulation of CCL2 (the ligand of CCR2)-CCR2 axis in the disease state directly causes neuronal dysfunction through Akt signaling pathway [9][10][11]. Other studies reported that CCR2 is expressed in both infiltrating monocytes and microglia in a rodent model of traumatic brain injury [12]. Furthermore, another study reported CCR2 + monocyte infiltration in the perivascular areas of the primary motor cortex in ALS patients with TDP-43 pathology [13]. These data are discordant with the findings of Kawaguchi-Niida et al., who reported CCR2 expression exclusively in astrocytes [5]. Also in our ALS mice, CCR2-RFP was not detected in astrocytes, but was instead found in CNS-infiltrating monocytes, CX3CR1 + microglia, and neurons.
The other possibility is that CNS-infiltrating CCR2 + monocytes express CX3CR1. A previous study reported that chronic brain injury causes CX3CR1 upregulation in infiltrating CCR2 + monocytes, and that CCR2 + CX3CR1 + monocytes control their own inflammation via neuronal CX3CL1 signaling [15]. Therefore, CCR2 + CX3CR1 + monocytes may act as a self-limiting system of neuroinflammation in advanced ALS. Further studies are needed to elucidate the precise roles and mechanisms of CCR2 + cells in ALS pathology.
In conclusion, using a Ccr2 rfp/+ -Cx3cr1 gfp/+ mouse, we revealed that CCR2 expression expands from CNSinfiltrating monocytes to resident CNS cells such as microglia and neurons, but not astrocytes, over the course of ALS disease progression. This reporter mouse represents a useful and powerful tool that could provide new insights into ALS pathomechanisms.

Funding
This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan; grants from the Ministry of Health, Labour and Welfare of Japan; a grant for Strategic Research Promotion from Yokohama City University; and a grant from the Naito Foundation.

Availability of data and materials
The datasets used and/or analyzed in this study are available from the corresponding authors on reasonable request.

Ethics approval and consent to participate
This study was performed according to the Helsinki Declaration and approved by the ethical committee of Yokohama City University Graduate School of Medicine. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.

Consent for publication
Not applicable.