RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation

Brain, Behavior, and Immunity xxx (2016) xxx–xxx

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RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation Mehdi Djelloul 1,2, Natalia Popa 2, Florence Pelletier, Gilda Raguénez, José Boucraut ⇑ Aix Marseille Université, CRN2M, CNRS UMR 7286, 13344 Marseille Cedex 15, France

a r t i c l e

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Article history: Received 6 April 2016 Received in revised form 30 June 2016 Accepted 16 July 2016 Available online xxxx Keywords: RAE-1 Microglia EAE Neuroinflammation Cell proliferation M-CSF Cell sorting

a b s t r a c t Retinoic acid early induced transcript-1 (RAE-1) glycoproteins are ligands of the activating immune receptor NKG2D. They are known as stress molecules induced in pathological conditions. We previously reported that progenitor cells express RAE-1 in physiological conditions and we described a correlation between RAE-1 expression and cell proliferation. In addition, we showed that Raet1 transcripts are induced in the spinal cord of experimental autoimmune encephalomyelitis (EAE) mice. EAE is a model for multiple sclerosis which is accompanied by microglia proliferation and activation, recruitment of immune cells and neurogenesis. We herein studied the time course expression of the two members of the Raet1 gene family present in C57BL/6 mice, namely Raet1d and Raet1e, in the spinal cord during EAE. We report that Raet1d and Raet1e genes are induced early upon EAE onset and reach a maximal expression at the peak of the pathology. We show that myeloid cells, i.e. macrophages as well as microglia, are cellular sources of Raet1 transcripts. We also demonstrate that only Raet1d expression is induced in microglia, whereas macrophages expressed both Raet1d and Raet1e. Furthermore, we investigated the dynamics of RAE-1 expression in microglia cultures. RAE-1 induction correlated with cell proliferation but not with M1/M2 phenotypic orientation. We finally demonstrate that macrophage colonystimulating factor (M-CSF) is a major factor controlling RAE-1 expression in microglia. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Retinoic acid early induced transcript-1 (RAE-1) a-e glycoproteins are ligands of the activating receptor NKG2D expressed by Natural Killer (NK), NKT, cd T cells and some CD8+ T cells (Cerwenka et al., 2001). In C57BL/6 mice, NKG2D recognizes several ligands: MULT-1, H60 a, b, c, RAE-1d and RAE-1e. The two RAE-1 proteins are encoded by two distinct genes: Raet1d and Raet1e. Three other Raet1 genes (Raet1a-c) were described in other mouse strains but are absent in C57BL/6. RAE-1 molecules are stress markers. Overexpression of RAE-1 and other NKG2D ligands has been observed in tumor cell lines, following viral infection and upon inflammatory lesions, and has been linked to immunosurveillance and graft rejection (Ogasawara et al., 2004; Backstrom et al., 2007; Chen et al., 2007; Ito et al., 2008; Guerra et al., 2013). Moreover, DNA damage is responsible for NKG2D ligands induction, including RAE-1, in ⇑ Corresponding author. E-mail address: [email protected] (J. Boucraut). Present address: Karolinska Institute, CMB, Box 285SE-171 77, Stockholm, Sweden. 2 These authors contributed equally to this study. 1

numerous tumor cell lines and after genotoxic drugs treatment (Gasser et al., 2005; Cerboni et al., 2007). Interestingly, RAE-1 expression is also linked to cell proliferation. Raet1 genes are expressed in the embryonic central nervous system (CNS) (Nomura et al., 1996). We have previously demonstrated that RAE-1 expression persists in the subventricular zone (SVZ) of the adult mice brain (Popa et al., 2011), a neurogenic niche that contains resident neural stem/progenitor cells (NSPCs). In vitro, NSPCs (Popa et al., 2011) and embryonic stem cells (Bonde and Zavazava, 2006) express RAE-1. Furthermore, we have correlated RAE-1 expression with NSPCs proliferation (Popa et al., 2011). Using a wound healing model, the Raulet laboratory has demonstrated that RAE-1 expression, but not MULT-1 or H60b, is induced in vivo in fibroblasts undergoing proliferation (Jung et al., 2012). In proliferating primary fibroblasts in vitro, the induction of expression was greater for Raet1e than Raet1d transcripts (Jung et al., 2012). By contrast, in our NSPCs primary culture model, we observed a dominant expression of Raet1d transcripts and its corresponding surface protein (Popa et al., 2011). Similarly, we have also reported that experimental autoimmune encephalomyelitis (EAE) induction in mice results in a higher increase of Raet1d transcripts expression compared to Raet1e in the spinal cord (Cedile et al., 2010; Popa et al., 2011).

http://dx.doi.org/10.1016/j.bbi.2016.07.147 0889-1591/Ó 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: Djelloul, M., et al. RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation. Brain Behav. Immun. (2016), http://dx.doi.org/10.1016/j.bbi.2016.07.147

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Finally, RAE-1 expression was also described in cells of the myeloid lineage. In peritoneal macrophages, Toll-like receptor (TLR) signaling through the MyD88 adaptor up-regulates the transcription and surface expression of RAE-1, but not the transcription of H60 or MULT-1 (Hamerman et al., 2004). Moreover, myeloid derived suppressor cells (MDSCs) from tumor-bearing mice express RAE-1 (Nausch et al., 2008). Interestingly, MDSCs are found in the spinal cord of EAE mice where they are able to suppress antigen-specific CD8+ and CD4+ T-cell activation (MolineVelazquez et al., 2011). They also cooperate with activated iNKT cells to protect mice against the development of EAE (Parekh et al., 2013). Recruitment of monocytes from the circulation and proliferation/activation of local microglia are hallmarks of EAE and several other experimental models of CNS injury where these cells play either detrimental or protective functions according to their phenotypic orientation M1/M2a/M2b/M2c (Soulet and Rivest, 2008; Henkel et al., 2009; Polazzi and Monti, 2010; Chhor et al., 2013). Recruitment of myeloid cells could thus explain the induction of expression of RAE-1 in spinal cord during EAE. Nevertheless, whether RAE-1 overexpression could originate from microglia has not been evaluated yet. We herein report that Raet1d and Raet1e expression is induced early during the course of EAE, reaching a maximum at the peak of the disease, and show that myeloid cells, i.e. macrophages as well as microglia, are cellular sources of Raet1 transcripts. Microglia expressed higher levels of Raet1d than Raet1e transcripts and RAE-1 protein expression correlated with cell proliferation in vitro. Finally, we demonstrate that cell proliferation under the control of macrophage colony-stimulating factor (M-CSF) is the main factor controlling RAE-1 expression in microglia. 2. Material and methods 2.1. Induction and evaluation of EAE All mice were maintained under pathogen-free conditions in our animal facility (Agreement number 00056.02). All experiments were performed according to the guidelines of the local ethical committee on animal research (Marseille, France). EAE was induced in C57BL/6J mice and transgenic CX3CR1+/gfp mice (MGI:2670351) (Charles River, L’Arbresle, France) using 100 lg myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide (Bio-Synthesis Inc., Lewisville, Texas, USA) in complete Freund adjuvant (BD Diagnostics, Le Pont de Claix, France). Just after immunization as well as 48 h later, mice received intravenous injection of 200 ng pertussis toxin (Sigma Aldrich, Lyon, France). Mice clinical symptoms were assessed according to the following scale: 0, no symptoms; 1, decreased tail tone; 2, mild monoparesis or paraparesis; 3, severe paraparesis; 4, paraplegia; 5 quadriplegia and moribund.

2.3. Flow cytometry and cell sorting 3x105 cells were stained for 30 min on ice with phycoerythrincyanine (PE-Cy)7 labeled anti-CD11b (BD Biosciences, Le Pont de Claix, France; AB_394491), allophycocyanin (APC)-labeled rat anti-RAE-1 (pan-specific) (clone 186107, R&D systems, Lille, France; AB_357086) and labeled isotype controls (BD Biosciences). The cells were washed and fixed with 2% paraformaldehyde (Sigma Aldrich) in PBS containing 5 mM EDTA and analyzed on a FACSCanto cytometer (Becton Dickinson, Le Pont de Claix, France). The data were processed with the FlowJo software (FlowJo, LLC, Ashland, Oregon, USA). For cell cycle analysis, 2x105 cells were incubated in cold buffer containing 25 mM HEPES, 150 mM NaCl, 10 mM EDTA and 10 mM EGTA (Sigma Aldrich). Plasma membrane was lysed by addition of Triton X-100 and nuclei were then incubated with 50 lg/ml of propidium iodide (BD Biosciences) before analysis on a FACSCanto cytometer. For the analysis and cell sorting of EAE mononuclear CNS cells, staining was performed using CD3-APC (AB_10896663), CD11bPE-Cy7 (AB_394491), Ly6C-PE antibodies (AB_1727556) (BD Biosciences), and major histocompatibility class II (MHC-II)-APC-Cy7 (AB_2069377) (Biolegend), and sorted on a FACSAria III cell sorter (BD Biosciences).

2.4. Glial cell cultures Primary mixed glial cultures were established from the forebrains of C57BL/6 newborn mice. Forebrains were carefully freed of meninges, chopped into small sections and dissociated by mild trypsinization and mechanical disruption. The cells were seeded onto poly-L-lysine (10 lg/ml) coated 25 cm2 flasks at the density of 5  105 cells/cm2 in DMEM containing 10% FCS, 1% L-glutamine (Life Technologies) and 1% Penicillin/Streptomycin (Life Technologies). The medium was changed at day 4. Amoeboid microglia floating cells were detached from the astroglial monolayer by short and gentle manual shaking of the culture flasks at different time of the culture. The purity of the microglia thus collected was assessed by staining an aliquot of the cell suspension with CD11b-PE-Cy7 antibody (AB_394491). We regularly obtained 98% of CD11b positive microglial cells with negligible CD11b negative astrocytic contamination. These floating cells were centrifuged and either used for RT-qPCR analysis, flow cytometry staining, cell cycle analysis, or seeded into culture flasks. Glial mixed or microglia cultures were analyzed after exposure to lipopolysaccharide (LPS) (100 ng/ml, Sigma Aldrich), interferonc (IFNc) (100 U/ml), interleukin-4 (IL-4) (20 ng/ml), IL-10 (20 ng/ml), M-CSF (20 ng/ml) (Preprotech, Neuilly-sur-Seine, France) or blocking goat anti-mouse M-CSF antiserum (0.4 lg/ml, AbD Serotec, Oxford, UK) for 24 h.

2.2. Tissues preparation For the analysis of mouse CNS cells and for cell sorting, spinal cords from 4 to 8 animals were flushed from the vertebral columns, crushed into small pieces on a sieve and treated with TrypLE Select (Life Technologies, Carlsbad, California, USA) for 15 min at 37 °C and then DNase I (Roche Diagnostics, Meylan, France) for 5 min at room temperature. Tissues were mechanically dissociated, washed with HBSS supplemented with 3% fetal calf serum (FCS) (PAA Laboratories, Les Mureaux, France), and cells were separated on a Percoll (GE Healthcare, Uppsala, Sweden) gradient. Infiltrating cells and microglia were collected from the interlayer between the 75% and 35% Percoll fractions, and further washed with HBSS 3% FCS.

2.5. RNA extraction, quantitative PCR Total RNA extractions were performed with the RNeasy Mini Kit (Qiagen, Courtaboeuf, France). Complementary DNA (cDNA) was generated by reverse transcription with M-MLV reverse transcriptase (Life Technologies) and random primers. Quantitative PCR (qPCR) was performed on a 7500 Fast Real Time PCR System (Applied Biosystem, Foster City, California, USA) with Power SYBR Green PCR Master Mix (Applied Biosystem). All primers are reported in Supplementary Table 1. The amplification procedure included a 10 min-step at 95 °C followed by 40 cycles with 30 s at 95 °C and 1 min at 62 °C prior to final Melting Curve analysis.

Please cite this article in press as: Djelloul, M., et al. RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation. Brain Behav. Immun. (2016), http://dx.doi.org/10.1016/j.bbi.2016.07.147

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symptoms appear, then the clinical score worsens within a short period (5–6 days) until reaching a peak. Then the clinical score decreases within 5–8 days (remission) (Supplementary Fig. 1). In order to characterize the inflammation during the course of the pathology, we sacrificed mice at different time points after immunization i.e. before disease onset (day 7 and day 10), after onset (day 13), at the peak of the disease (day 16), after the peak (day 20) and after remission (day 28). For each selected time point, we analyzed the expression levels of Raet1 genes in the spinal cord along with other genes indicative of cell lineage and cell proliferative state. Raet1d and Raet1e transcripts were almost undetectable in the spinal cord of control mice and before the disease onset whereas their expression increased from the beginning of the pathology and reached a maximum at the peak of the disease, then decreased rapidly until day 20 and remained constant until day 28, following a similar pattern of expression (Fig. 1A). We also compared their relative expression levels and observed that Raet1d was the most highly induced and its expression level was higher than Raet1e at all analyzed time points (Fig. 1B). NSPCs express RAE-1 and this expression is correlated with cell proliferation (Popa et al., 2011). Consequently, we wondered whether the increase of expression for Raet1 genes that occurs in the spinal cord of EAE mice could be due to resident/proliferative NSPCs. To this end, we analyzed the cell proliferation marker Ki67 and observed a significant upregulation of Ki67 expression during the early stages of the EAE pathology, followed by a

2.6. Statistics Data are from 2 to 6 independent samples for each time point or treatment (EAE or control mice, cultured microglia) and presented as mean ± SEM. Data were assessed for normality with the ShapiroWilk test. When compared to a control condition, gene expression was assessed using a multiple t-test and statistical significance was determined using the Holm-Sidak method. For the comparison of multiple time points, gene expression was analyzed using ANOVA and a Bonferroni’s post-test or a Kruskal-Wallis test with a Dunn’s post-test. For the comparison of 2 genes expression at different time points or of 1 gene expression at different time point and in different tissues, two-way ANOVA was used with a Bonferroni’s post-test. The Pearson’s test was used for correlation analysis. All ANOVA test values, degrees of freedom (df) and significance are summarized in the Supplementary Table 2. For all tests, significance of the post-tests is shown on the graphs as follows: * p < 0.05; **p < 0.01; ***p < 0.001. The statistical test used for each data is indicated in the figure legends. Analyzes were performed with the GraphPad Prism 6.0 software. 3. Results 3.1. Raet1 expression is induced in the spinal cord of EAE mice In C57BL/6 mice, EAE pathology induced by MOG35-55 injection evolves in 3 steps. Typically, 12 days after immunization, the first

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Fig. 1. Time course of gene expression in the lumbar spinal cord of EAE mice along disease evolution. Data are represented as mean of 3–6 mice per time point ±SEM. Relative expression values of Raet1d and Raet1e (A), Ki67 and Sox2 (C) and Ccl2, Iba1 and H2eb1 (D) genes are illustrated as a percentage of the maximal mean value for each gene. Data were analyzed with ANOVA and a Bonferroni’s post-test or with a Kruskal-Wallis test and a Dunn’s post-test (*p < 0.05; **p < 0.01; ***p < 0.001). In order to compare the relative expression of each Raet1 gene, the values are shown in panel B relatively to Gapdh (glyceraldehyde-3-phosphate dehydrogenase) as 2 DCt  106. The differences between gene expression levels at each time point were analyzed with a two-way ANOVA followed by a Bonferroni’s post-test (***p < 0.001).

Please cite this article in press as: Djelloul, M., et al. RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation. Brain Behav. Immun. (2016), http://dx.doi.org/10.1016/j.bbi.2016.07.147

M. Djelloul et al. / Brain, Behavior, and Immunity xxx (2016) xxx–xxx

to be involved in the recruitment of monocytes, was found rapidly and strongly overexpressed at disease onset, which could correlate with the early recruitment of monocytes (Fig.1D). Monocytederived macrophages and activated microglia express MHC-II molecules (H2eb1). Strikingly, we observed that H2eb1 expression followed an expression pattern similar to that of Raet1 genes, reaching a maximum at the peak of the disease, and then decreasing (Fig. 1A and D). Finally, Iba1 is a hallmark of monocyte recruitment and microglia activation. Noteworthy, we found that Iba1 expression is upregulated to reach a maximum of expression at the peak of disease prior to decrease, in a similar way to what we observed with Raet1 genes and H2eb1 (Fig. 1A and D). Supporting further the important role of myeloid cells in the overexpression of Raet1 observed during EAE, we identified a gradient of expression along the caudo-rostral axis of the spinal cord. Indeed, we determined that Raet1 expression is the strongest in the lumbar spinal cord compared to the thoracic/cervical parts of the spinal cord and to the cerebral trunk. This is in agreement with

decrease and a stabilization phase of its expression (Fig. 1C), which indicates a pattern of expression similar to the one observed with Raet1. However, the stem cell marker Sox2 displayed a different profile of expression during the course of the pathology (Fig. 1C). Taken together, these data suggest that Raet1 upregulation upon EAE induction, at least within the spinal cord, is unlikely linked to the proliferation of resident NSPCs. EAE is characterized by a massive infiltration of monocytes, as well as an important proliferation of microglia (Ajami et al., 2011). Using FACS analysis of Ki67 expression in microglia from EAE nervous tissue, we confirmed the induction microglia proliferation (Supplementary Fig. 3). Accordingly, we hypothesized that myeloid cells could be responsible for the strong upregulation of Raet1 genes observed in the spinal cord of EAE mice. To test our hypothesis, we established the expression profiles of three genes associated to macrophages/microglia, namely Ccl2 (MCP-1), H2eb1 and Iba1, in the spinal cord of EAE mice and at different time points. Interestingly, the chemokine Ccl2 that is well known

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Fig. 2. Gene expression of selected phenotypic markers (A) and Raet1 (B) in microglia (MG) and macrophages (MP) sorted from dissociated spinal cords of EAE mice at the peak of the disease. In EAE mice, Ly6C microglia and Ly6C+ macrophages were separated using cell sorting into MHC-II positive and negative sub-populations. Control CD11b+ CX3CR1high microglia (Ctrl MG) were sorted from non-EAE mice. Genes relative expression levels were determined by qPCR relatively to Gapdh (2 DCt  103) and represented as mean of 4 experiments ±SEM. The data were analyzed with ANOVA and the results of a Bonferroni’s post-test are shown (*p < 0.05; **p < 0.01; ***p < 0.001).

Please cite this article in press as: Djelloul, M., et al. RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation. Brain Behav. Immun. (2016), http://dx.doi.org/10.1016/j.bbi.2016.07.147

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We confirmed a much higher expression of Cx3cr1 in microglia compared to macrophages. Of note, Cx3cr1 expression level was significantly decreased in microglia sorted from EAE tissue compared to control microglia. As expected, Ccr2 was found expressed in macrophages whereas its expression was almost undetectable in microglia. Moreover, the profile of H2eb1 expression validated the purity of the cell sorting on the basis of MHC-II surface expression (Fig. 2A). Following up on the characterization of the sorted cells, we found that Iba1 was highly expressed in microglia with no significant difference between MHC-II , MHC-II+ or control microglial cells whereas macrophages expressed lower levels of Iba1 transcripts (Fig. 2A). Moreover, higher expression of Ki67 was observed in microglia sorted from EAE mice compared to macrophages and control microglia, which confirmed that microglial cells are proliferating in EAE spinal cord (Ponomarev et al., 2005). In addition, macrophages expressed more Il1b indicating that they may be more pro-inflammatory than microglia (Fig. 2A), as expected (Vainchtein et al., 2014).

the caudo-rostral evolution of the pathology and the degree of neuroinflammation as shown by H2eb1 and Iba1 expression profiles (Supplementary Fig. 2). Altogether, our data indicate that the induction of Raet1 expression early after EAE onset correlates with cell proliferation and the early recruitment of myeloid cells. 3.2. Infiltrating myeloid cells and microglia in EAE spinal cord express Raet1 We analyzed ex vivo myeloid cells subsets sorted at the peak of EAE disease. To this end, microglia (CD11b+ CX3CR1high Ly-6C ) and macrophages (CD11b+ CX3CR1low Ly-6C+) were further subdivided into MHC-II negative and MHC-II positive cells. Almost 50% of microglial cells and 85% of macrophages were MHC-II+. As control, we also sorted microglia from the CNS of non-EAE mice (Supplementary Fig. 3). Sorted cells were analyzed by qPCR for the expression of selected markers and Raet1 genes.

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Fig. 3. Analysis of mixed glial cultures and floating microglia by flow cytometry and qPCR. A, The expression of CD11b (PE-Cy7) allowed the discrimination of positive microglia from negative astrocytes isolated from the adherent cell layer (left histogram). Expression of RAE-1 was analyzed on CD11b+ microglia (middle) and CD11bastrocytes (right histogram) after co-staining with APC-labeled anti-RAE-1 pan-specific antibody. In all figures, isotype controls are represented with a thin line. B–E, Floating microglia were harvested from mixed glial cultures at different days in vitro (DIV) after culture seeding. B, Surface expression of RAE-1 protein on CD11b+ microglia. C, Raet1 and Ki67 genes relative expression determined by qPCR as 2 DCt  103 relatively to Gapdh. Data from 2 independent cultures are represented as mean ± SEM. They were analyzed with a multiple t-test and significance was corrected with the Holm-Sidak method (*p < 0.05; **p < 0.01). D, Cell cycle was analyzed by flow cytometry after staining with propidium iodide. E, Correlation between the percentage of cells in S and G2/M phases of the cell cycle and Raet1 gene expression relatively to Gapdh (2 DCt  103) in 16 different cultures harvested at different DIV.

Please cite this article in press as: Djelloul, M., et al. RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation. Brain Behav. Immun. (2016), http://dx.doi.org/10.1016/j.bbi.2016.07.147

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cells. Notably, the addition of anti-M-CSF antiserum was able to reduce Raet1 and Ki67 expression as well as the percentage of S + G2 M cycling cells. These data indicate that, at this time point of analysis, the high level of microglia proliferation is probably under the influence of M-CSF released by the astrocytes present in the mixed culture. Adding recombinant M-CSF at day 13 of culture led to a significant increase of Ki67 and Raet1 expression along with an increase in the percentage of S + G2 M cells. Conversely, the M-CSF blocking antibodies decreased cell proliferation and Raet1 expression (Fig. 4). Of note, Csf1r expression level is inversely correlated with both the proliferative state of microglia and Raet1 gene expression level (Supplementary Fig. 4A). This suggests that the less microglia proliferate, the more they are able to respond to the mitotic signal of M-CSF. Taken together, our data demonstrate that Raet1 expression by proliferating microglia is, at least partly, under the control of MCSF.

Raet1 expression was almost absent in control microglial cells. Raet1d was expressed in all 4 sorted cell populations from EAE mice, whereas Raet1e expression was undetectable in microglia sorted from EAE tissues. This may explain the lower expression of Raet1e in the EAE spinal cord (Fig. 1B). Interestingly, Raet1d seems to be more expressed in MHC-II microglia than in MHCII+ microglia. On the contrary, no difference was observed in Raet1d expression between MHC-II+ and MHC-II macrophages, whereas Raet1e expression was higher in MHC-II macrophages (Fig. 2B). In summary, combining cell sorting together with a cell-specific gene expression signature allowed us to identify both microglia and macrophages as cells expressing Raet1 genes in the spinal cord of EAE mice. 3.3. RAE-1 expression is correlated with cell proliferation We next investigated the expression of RAE-1 in mixed glial cell cultures containing both astrocytes (CD11b-negative) and microglia (CD11b-positive). Floating microglial cells but not astrocytes expressed RAE-1 (Fig. 3A). We then analyzed the expression of RAE-1 protein (Fig. 3B) and Raet1 transcripts (Fig. 3C) in floating microglial cells at different time points after seeding. RAE-1 and Raet1 expression were maximal at day 9, the first analyzed time point, and decreased progressively up to day 15 (Fig. 3B and C). Decrease of Raet1 expression was accompanied by a reduced cell proliferation as shown by the decrease of Ki67 expression (Fig. 3C) and the reduced percentage of (S + G2/M) cycling cells (Fig. 3D and E). Of note, a Pearson’s correlation analysis has confirmed that the expression levels of Raet1d transcripts were correlated with both the expression of Ki67 and the percentage of (S + G2/M) cycling cells (respective coefficients 0.602, p < 0.001 and 0.791, p < 0.001) whereas Raet1e expression correlated only to a weaker extent with the percentage of S + G2/M (coefficient 0.696, p < 0.01) (Supplementary Fig. 4A and B). This indicates that microglial cell proliferation is better correlated to the expression level of Raet1d than Raet1e. Because M-CSF is a mitogenic factor for microglia (Kondo and Duncan, 2009), we analyzed the proliferation of floating microglia and Raet1 expression after treatment of mixed glial cultures with either recombinant M-CSF or blocking anti-M-CSF antibodies (Fig. 4). At day 10 of culture, M-CSF treatment enhanced the expression of Raet1 but not of Ki67 nor the percentage of dividing

3.4. RAE-1 expression is not associated to M1/M2a/b polarization of microglia In response to insult or injury, microglia and macrophages are capable of acquiring diverse and complex phenotypes that have been precisely described across the literature (Mosser and Edwards, 2008; Ransohoff and Perry, 2009; Weinstein et al., 2010). These studies were performed in vitro using purified adherent microglial cells polarized by various inducers. Thus, it was demonstrated that factors such as LPS, IFNc, IL-4 or IL-10 can direct the microglial phenotype into three main states, i.e. classically activated M1 with cytotoxic properties, M2a with an alternate phenotype (repair and regeneration) and M2b with an immunoregulatory phenotype. Recently, a profile of phenotypic markers allowing to assess the functional outcome of polarized microglia has been indexed (Chhor et al., 2013). In our study, we used these phenotypic markers to assess the polarization of microglia. The orientation of microglia towards M1 in response to LPS or IFNc was confirmed by the increased expression of Cd86 and/or Il1b (Supplementary Fig. 5A). The polarization towards M2a in response to IL-4 is illustrated by the increased expression of Arg1 and Lgals3 (Supplementary Fig. 5B) and towards M2b in response to LPS and IL-10 by the increased expression of Il10 or Socs3 (Supplementary Fig. 5C).

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Fig. 4. Analysis of microglia from mixed glial cultures at day 10 and day 13 after addition of either 40 ng/ml M-CSF or 0.4 lg/ml blocking anti-M-CSF antibodies. The percentage of nuclei in S and G2/M phases was determined by flow cytometry and the expression of Raet1 and Ki67 transcripts by qPCR. Gene relative expression is represented as fold change relatively to untreated cells (Ctrl) at day 10 and as mean of two experiments ±SEM for each condition. The data were analyzed with multiple t-test and significance was corrected with the Holm-Sidak method (*p < 0.05; **p < 0.01).

Please cite this article in press as: Djelloul, M., et al. RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation. Brain Behav. Immun. (2016), http://dx.doi.org/10.1016/j.bbi.2016.07.147

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M. Djelloul et al. / Brain, Behavior, and Immunity xxx (2016) xxx–xxx

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Fig. 5. Gene expression of Raet1 (A), Ki67 (B) and phenotypic markers (C) in cultured microglia exposed to M1 or M2 inducing conditions or to M-CSF. Primary microglia isolated from mixed glial cultures was exposed to 100 ng/ml LPS (M1/M2a), 100 U/ml IFNc (M1), 20 ng/ml IL-4 (M2a), 20 ng/ml IL-10 (M2b) or 20 ng/ml M-CSF for 24 h. Relative expression values are represented as fold change relative to the untreated control (dashed line), as mean of 3–6 independent cultures ±SEM, and analyzed with ANOVA followed by a Bonferroni’s post-test (*p < 0.05; **p < 0.01; ***p < 0.001).

In these experimental conditions, the expression of both Raet1 genes was reduced after LPS, IFNc and IL-10 treatment and did not change in response to IL-4 (Fig. 5A). On the contrary, M-CSF exposure induced Raet1d and Ki67 expression (Fig. 5A and B) but was not able to induce M1/M2 phenotypic orientation (Fig. 5C). This suggests that, similarly to what we observed in floating microglial cell culture, Raet1 expression also correlates with cell proliferation in adherent condition. Interestingly, Raet1d expression is more highly induced than Raet1e, matching with the previous observation obtained with microglial cells sorted from EAE spinal cords. Furthermore, immunostaining on adherent CD11b+ microglia showed that the membrane expression of RAE-1 was faint and punctiform. As expected, RAE-1 expression was up-regulated by M-CSF which also increased the number of Ki67+ cells. Conversely, RAE-1 expression was down-regulated by IFNc and abolished by LPS (Supplementary Fig. 6). Finally, we observed the lack of correlation between the expression of Raet1 transcripts and the M1/M2a/b phenotypic markers in floating microglia in vitro (Supplementary Fig. 7A and B) as well as in microglial cell subsets sorted from EAE spinal cords (Supplementary Fig. 7C).

Overall, these data obtained in vitro indicate that RAE-1 expression is correlated with the proliferative state of microglial cells but not with their phenotypic M1/M2 orientation.

4. Discussion Our data indicate that Raet1 expression is early induced in the spinal cord of EAE mice, and reaches its maximal level at the peak of the disease concomitantly with the early recruitment of myeloid cells and the surge of both cell proliferation and inflammatory markers. Using cell sorting, we indeed identified macrophages and microglia as cells expressing Raet1 genes in the spinal cord at the peak of EAE. Raet1 expression by murine microglia had never been reported before, although it was suspected (Walsh et al., 2008). We additionally show that RAE-1 expression correlated with cell proliferation and was controlled by M-CSF. In this study, we focused our interest on myeloid cells but we cannot rule out the possibility that other proliferating cell types could also express Raet1 genes in EAE spinal cord. Data regarding neurogenesis in the spinal cord of EAE mice are controversial.

Please cite this article in press as: Djelloul, M., et al. RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation. Brain Behav. Immun. (2016), http://dx.doi.org/10.1016/j.bbi.2016.07.147

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M. Djelloul et al. / Brain, Behavior, and Immunity xxx (2016) xxx–xxx

Reduced proliferation of central canal ependymal cells (Gao et al., 2015) as well as increased proliferation of subpial radial glia (Bannerman et al., 2007) were described. Raet1 could thus be expressed by a subset of proliferating radial glia. Angiogenesis was also described in the spinal cord of EAE mice (Roscoe et al., 2009). Interestingly, RAE-1 expression was linked to angiogenesis in tumoral vasculature (Zhang and Sentman, 2013). Proliferating angiogenic precursors could thus express RAE-1 in EAE mice. Primary cultures of human adult oligodendrocytes expressed ligands for NKG2D in vitro and NKG2D-L expression co-localized with an oligodendrocyte marker in white matter sections obtained from multiple sclerosis lesions (Saikali et al., 2007). In mice, there is no clue of RAE-1 expression by oligodendrocyte progenitors cells. Here we show that in the brain of EAE mice, the difference of expression level of each Raet1 gene allows to discriminate microglia from macrophages. Indeed, microglia only express Raet1d while macrophages express both Raet1d and Raet1e. This is relevant because it was shown that proliferating resident microglia and infiltrating circulatory monocytes represent distinct populations with differential function. Genome-wide transcriptional profiles demonstrate that microglial cells are unique and clearly distinct from other macrophage cell types (Beutner et al., 2013). Indeed, during EAE, monocyte derived macrophages are highly phagocytic and inflammatory, whereas those arising from microglia demonstrate an unexpected signature of globally suppressed cellular metabolism at disease onset (Yamasaki et al., 2014). Moreover, inhibition of chemokine receptor–dependent recruitment of monocytes to the CNS blocked EAE progression, suggesting that these infiltrating cells are essential for pathogenesis (Ajami et al., 2011). Our observation that the level of expression of Raet1d and Raet1e allow to differentiate microglia from infiltrating macrophages is noteworthy since we (Cedile et al., 2010) and others (Carayannopoulos et al., 2002) demonstrated that RAE-1d is a weaker NKG2D ligand compared to RAE-1e. NKG2D-expressing cells could thus respond differently to microglia and macrophages. NKG2D positive cells infiltrate the CNS during EAE (Supplementary Fig. 8). NKG2D KO mice show no clear difference in EAE evolution (Guerra et al., 2013) probably because the interactions between myeloid cells expressing NKG2D ligands, and NKG2D positive infiltrating immune cells have both deleterious or neuroprotective consequences, as it was described for NK cells (Poli et al., 2013). Studying the role of NKG2D expressing cells i.e. NK cells, invariant NKT cells, cd T cells, CD8+ T cells and subsets of CD4+ T cells (reviewed in (Lanier, 2015)) is complex and should take into account the other NKG2D ligands, i.e. MULT-1 and H60. We detected an induction of Mult1 expression in the spinal cord of EAE mice of intensity and time-course comparable to Raet1d. H60b was expressed at lower levels and it expression was slightly and lately induced during EAE (Supplementary Fig. 8). Mult1, was expressed by sorted MHC-II and MHC-II+ microglia and macrophages while H60b was undetectable in these cells (data not shown). We also observed a dominant expression of Raet1d gene in microglia in vitro which correlate with cell proliferation. We identified M-CSF as a factor inducing Raet1d expression and microglial proliferation, while maintaining microglia in a resting state different from M1/M2 phenotype. We additionally tested the effect of astrocytes conditioned medium on microglia culture and observed an increase of Raet1 expression (Supplementary Fig. 9). M-CSF is a major mitogen stimulus of microglia in vivo, since op/op mice invalidated for the Csf1 gene have reduced numbers of microglia (Kondo and Duncan, 2009). Extensive treatment with selective CSF-1R inhibitors in adult mice results in elimination of 99% of all microglia brain-wide, showing that microglia in the adult brain are physiologically dependent upon CSF-1R signaling (Elmore et al.,

2015). Of note, CSF-1R has another ligand, IL-34. Studying EAE evolution in mice lacking IL-34 would be of particular interest since these mice displayed a decrease of microglia without overgrowth or osteopetrotic phenotype, and little effect on other myeloid populations (Greter et al., 2012). It was shown that cooperation between CSF-1R and DAP-12 is essential for the proliferation and survival of macrophages. Through nuclear accumulation of betacatenin and its interaction with a co-activator of the LEF or TCF family of transcription factors, they mediate the transcription of cell cycle molecules such as cyclin D1 and c-Myc (Otero et al., 2009). Jung et al. have shown direct transcriptional activation of Raet1 genes by E2F family transcription factors in proliferating cells (Jung et al., 2012). Whether CSF-1R signaling involves E2F transcription factors has not been addressed. Analysis of microglia in vitro allowed us to establish a direct correlation between microglia proliferative state and the level of RAE-1 expression, in terms of both transcript and protein. We observed that microglia but not macrophages proliferate in the CNS of EAE mice. Microglial proliferation has already been described during the course of EAE (Ponomarev et al., 2005). The frequency of proliferating microglia correlates to disease score. Microglia enters the cell cycle early in EAE pathogenesis and returns to quiescence following remission (Ajami et al., 2011). Considering our observations in vitro and ex vivo, we conclude that RAE-1 is a marker of microglial proliferation and postulate that proliferative microglia in the spinal cord of EAE mice express RAE-1. To confirm this hypothesis, specific analysis of proliferating microglia is necessary. RAE-1 protein observation in vivo is extremely challenging because of its weak, mainly extracellular expression and its sensitivity to enzymatic treatment (Ladeby et al., 2005). Finally, we showed for the first time that proliferation of microglia is correlated with the expression of RAE-1. The neuroprotective potential of microglia in some models is shared by proliferative microglia, expressing RAE-1, which serves as an endogenous producer of neurotrophic and anti-apoptotic molecules such as IGF-1 (Lalancette-Hebert et al., 2007). Characterization of the molecular and cellular environment of proliferating microglia is thus of particular interest in the field of therapeutic targeting of microglia.

Author’s contribution MD, NP, FP, GR performed the in vitro experiments. NP, FP, GR performed the in vivo experiments. FP bread the animals. MD, NP, GR and JB participated in experimental design and prepared the manuscript.

Conflict of interest All authors declare that there are no conflicts of interest.

Acknowledgments We thank Dr El Chérif Ibrahim and Emmanuel Nivet for critical reading of the manuscript. The research was supported by fundings of Aix-Marseille Université and CNRS.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbi.2016.07.147.

Please cite this article in press as: Djelloul, M., et al. RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation. Brain Behav. Immun. (2016), http://dx.doi.org/10.1016/j.bbi.2016.07.147

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Please cite this article in press as: Djelloul, M., et al. RAE-1 expression is induced during experimental autoimmune encephalomyelitis and is correlated with microglia cell proliferation. Brain Behav. Immun. (2016), http://dx.doi.org/10.1016/j.bbi.2016.07.147