Targeting CD52 does not affect murine neuron and microglia function

European Journal of Pharmacology 871 (2020) 172923

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Targeting CD52 does not affect murine neuron and microglia function a,∗

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Erik Ellwardt , Christina Francisca Vogelaar , Carlos Maldet , Samantha Schmaul , Stefan Bittnera, Dirk Luchtmana a Focus Program Translational Neurosciences (FTN) and Immunology (FZI), Rhine Main Neuroscience Network (rmn2), Department of Neurology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany b Präventive Kardiologie und Medizinische Prävention, Zentrum für Kardiologie. Klinische Epidemiologie, Centrum für Thrombose und Hämostase (CTH), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany

A R T I C LE I N FO

A B S T R A C T

Keywords: Alemtuzumab Multiple sclerosis Neuroprotection Microglia Neurons

The humanized anti-CD52 antibody alemtuzumab is successfully used in the treatment of multiple sclerosis (MS) and is thought to exert most of its therapeutic action by depletion and repopulation of mainly B and T lymphocytes. Although neuroprotective effects of alemtuzumab have been suggested, direct effects of anti-CD52 treatment on glial cells and neurons within the CNS itself have not been investigated so far. Here, we show CD52 expression in murine neurons, astrocytes and microglia, both in vitro and in vivo. As expected, anti CD52treatment caused profound lymphopenia and improved disease symptoms in mice subjected to experimental autoimmune encephalomyelitis (EAE). CD52 blockade also had a significant effect on microglial morphology in organotypic hippocampal slice cultures but did not affect microglial functions. Furthermore, anti-CD52 neither changed baseline neuronal calcium, nor did it act neuroprotective in excitotoxicity models. Altogether, our findings argue against a functionally significant role of CD52 blockade on CNS neurons and microglia. The beneficial effects of alemtuzumab in MS may be exclusively mediated by peripheral immune mechanisms.

1. Introduction Alemtuzumab (Campath-1H) is a monoclonal antibody directed against human CD52, an antigen present at high levels on the surface of T and B lymphocytes and at lower levels on other immune cells such as natural killer cells (NK cells) (Hale et al., 1990; Rao et al., 2012). In the phase 3 clinical studies CARE-MS 1 and 2 (Cohen et al., 2012; Coles et al., 2012), the therapeutic efficacy of alemtuzumab, measured by clinical and MRI parameters in pre-treated as well as treatment-naïve relapsing remitting multiple sclerosis (RRMS) patients, was better compared to the standard treatment with IFNβ-1a. This advantage persists for up to 60 months, although concerns about side effects remain, including secondary autoimmune thyroiditis, thrombocytopenic purpura and various infectious complications appearing up to 2–3 years after treatment initiation. The antibody causes a rapid and sustained depletion of lymphoid cells, in particular T and B cells, from the circulation. This is followed by a prolonged period of lymphocyte repopulation with treatment-unaffected hematopoietic precursors, and presumably a rebalancing of immune-tolerance and restored suppressive regulatory T lymphocyte (Treg) function (Ruck et al., 2015). The precise mechanism of alemtuzumab and the physiological function of

CD52 are, however, still under investigation (Ruck et al., 2016). Recently, Turner et al. took an experimental approach to further unravel the mechanisms of alemtuzumab by generating a monoclonal antibody to mouse CD52 (anti-muCD52) and applying this to various experimental autoimmune encephalomyelitis (EAE) mouse models that mimic aspects of MS (Turner et al., 2015). Interestingly, even when applied in mice with advanced EAE disease, anti-muCD52 still ameliorated the disease course, suggesting that the therapeutic action of anti-muCD52 may extend beyond lymphocyte depletion. Similar to other immunoglobulins, however, it is heavily discussed whether antimuCD52 or alemtuzumab itself can cross the blood brain barrier (BBB) to directly act in the CNS. Nonetheless, it should be taken into account that in MS and EAE, pro-inflammatory processes may lead to compromised BBB permeability (Minagar and Alexander, 2003; Spencer et al., 2018), rendering the possibility that alemtuzumab may enter the brain parenchyma and mediate some of its effects directly there. This was already observed with Rituximab, which remained detectable in the cerebrospinal fluid (CSF) after intravenous application for up to 24 weeks, correlating with BBB integrity (Petereit and Rubbert-Roth, 2009). There are currently no reports on CD52 expression in the CNS. The

∗ Corresponding author. Focus Program Translational Neurosciences (FTN), Rhine Main Neuroscience Network (rmn2), Department of Neurology, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstraße 1, 55131, Mainz, Germany. E-mail address: [email protected] (E. Ellwardt).

https://doi.org/10.1016/j.ejphar.2020.172923 Received 22 October 2019; Received in revised form 9 January 2020; Accepted 13 January 2020 Available online 18 January 2020 0014-2999/ © 2020 Elsevier B.V. All rights reserved.

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2.4. Primary cell cultures and treatment with LPS

most likely candidates for CD52 expression in the CNS are microglia, due to their fundamental role in brain inflammation as well as their demonstrated role in EAE pathogenesis (Bauer et al., 1995; Yamasaki et al., 2014). Alternatively, CD52 expression on neurons would indicate putative direct neuroprotective effects. Here, we sought to investigate CD52 expression in CNS cells by gene expression analysis and histology of murine dissociated CNS cells and brain sections. Furthermore, we performed two-photon imaging to assess microglia structure and function and neuronal calcium levels following application of anti-muCD52 under inflammatory conditions in vivo and in vitro.

Culture plates, including 24 and 96-well plates (Fisher Scientific GmbH, Germany), were coated with sterile 0.01% poly-d-lysine (PDL) (Sigma Aldrich GmbH, Germany) the day prior to culture. For 24 well plates, 10 mm cleaned and sterile round cover glasses (Thermo Scientific, Germany) were coated with PDL and inserted into the wells. Coated material was left in 4 °C overnight and washed with sterile H2O. For neuronal cultures, cerebral cortices were dissected from embryonic day 16 fetuses and maintained on ice-cold Hanks' balanced salt solution with Ca2+ and Mg2+ (HBSS+, Life Technologies GmbH, Germany), then washed 3 x in HBBS without Ca2+ and Mg2+ (HBSS−), followed by incubation with 5% trypsin (Life Technologies GmbH, Germany), effective dilution 0,3%, for 10–15 min at 37 °C. Trypsin action was then neutralized by addition of 2 ml fetal calf serum (FCS) (Life Technologies GmbH, Germany) and tissue washed and triturated in sterile filtered neurobasal media (NBM) (Life Technologies GmbH, Germany) containing 1% Glutamin, 1% Penicilin/Streptomycin and 2% B27. Cells were seeded in the PDL-coated 96 or 24-well plates at a density of 25,000 or 250,000 cells/well respectively. After 24 h culture in an incubator with humidified 5% CO2/95% air at 37 °C, half of the media was refreshed and the cycle repeated every three days. At approximately 7–14 days in vitro (DIV), neurons were used for experimental treatment. For astrocyte and microglia cultures, P0–P1 B6 pups (Envigo) were decapitated and brains removed from the skull. The brains were prepared in ice-cold HBSS (Gibco, 24020-133). The olfactory bulb and the meninges were removed from the cortex. The hippocampus was stripped from the cortex and all cortices were collected in ice-cold HBSS. Cortices from up to three animals were pooled. The tissue was washed once with ice-cold HBSS and digested in HBSS with 1% DNase (Roche, 04,536,282 001) and 0.5% trypsin (Gibco, 15400054) for 10 min at 37 °C. For homogenizing, tissue was sucked through two small glass pipettes and finally poured over a 70 μm mesh (Greiner BioOne, 89508-344). 75,000–150,000 cells/well were seeded in DMEMC (DMEM (Gibco, 41966-052) with 1% Pen/Strep (Gibco, 15140122), 10% fetal bovine serum (Gibco, 10270106), 2 mM L-Glutamine (Gibco, 25030081)) on a 24 well plate (Starlab, CC7672-7524) with glass cover slips coated with poly-L-lysine (Sigma Aldrich, P1274). The next day, cells were washed with DMEMC. The cultures were washed every two to three days with DMEMC. Neuron and astrocyte-microglia cultures were inflamed with LPS 100 ng/ml and LPS 1 μg/ml respectively at the last day of culture. Cultures were then either fixed 24h later with 5 min 2% paraformaldehyde (PFA, Roth, P087.1) and 20 min 4% PFA, for histological stainings, or cells were collected for RNA isolation.

2. Materials and methods 2.1. Mice All animal experiments were approved by local authorities and performed in accordance with German Animal Protection Laws (approval number for mice experiments by the authorities of RhinelandPalatinate: G-14-1-038). All animals were kept under pathogen-free conditions in individually ventilated cages with ad lib access to food and water at all times. B6.CX3CR1+/EGFP (locally bred) mice were used for in vivo imaging in EAE and as well for ex vivo imaging of microglial cells in brain slices. B6.Thy1.TN-XXL mice (locally bred) were exclusively used for ex vivo imaging of neurons in brain slices. C57BL6 mice (Janvier, Labs, France) were used for primary cultures of neurons, astrocytes and microglia. B6.2D2.CFP and B6.2d2.RFP mice (locally bred), in which all CD4+ T cells are MOG35–55 specific and express CFP and RFP respectively, were used for co-cultures with B6.CX3CR1+/EGFP and B6.Thy1.TN-XXL hippocampal slices. 2.2. Experimental autoimmune encephalomyelitis (EAE) and mouse treatments EAE was induced by immunizing B6.CX3CR1+/EGFP or C57Bl6 mice subcutaneously with 200 μg of myelin oligodendrocyte protein MOG35–55 in 200 μl complete Freund adjuvant (CFA) emulsion, followed by intraperitoneal injection of 200 ng pertussis toxin (Hooke kit, Hooke laboratories, USA) at day 0 and 1. Mice were evaluated for clinical symptoms daily as follows: 0, no detectable signs; 0.5, tail weakness; 1, complete tail paralysis; 2, partial hind limb paralysis; 2.5, unilateral complete hind limb paralysis; 3, complete bilateral hind limb paralysis; 3.5, complete hind limb paralysis and partial forelimb paralysis; 4, total paralysis of forelimbs and hind limbs; 5, moribund/ death. Treatment was performed from clinical score 1 onward by intraperitoneal injection with either anti-muCD52, obtained from Sanofi Genzyme (Cambridge, MA, USA), at a daily dose of 10 mg/kg body weight, or with vehicle (PBS), for five consecutive days (Turner et al., 2015). In addition, some mice were given anti-muCD52 or PBS when they reached a score of 2.

2.5. Quantitative real-time PCR For analysis of CD52 expression, RNA was isolated using the RNeasy Mini Kit ©(Quiagen) according to the manufacturer's protocol; quality and integrity of total RNA preparation was confirmed using a NanoDropTM 2000c Spectrophotometer (Thermo Scientific). Complementary DNA (cDNA) synthesis was performed by reverse transcription of total RNA using the SuperScript©III First Strand Synthesis System and random hexamer primers (Invitrogen) following the manufacturer's instructions. Amplification primers for real-time PCR analysis were designed using Beacon Designer 8 Software (PREMIER Biosoft International) according to the manufacturer's guidelines and subsequently tested for amplification efficiency and specificity. Sequences for primers are listed in Supplementary Table 1. Real-time PCR was performed using iQ SYBR Green supermix (BioRad Laboratories) in a CFX ConnectTM Real Time Detection System (BioRad). Relative changes in gene expression were determined using the Ct method (Livak and Schmittgen, 2001) with RPS29 as the reference gene.

2.3. T cell culture T cells were isolated as previously described (Siffrin et al., 2009, 2010). Briefly, cells from B6.2D2.CFP and B6.2D2.RFP mice were harvested from spleen and lymph nodes and magnetic bead-based cell sorts (MACS®, Miltenyi, Germany) performed according to the manufacturer's instructions to obtain naive CD4+CD62Lhi cells. These cells were then stimulated with 2 μg/ml CD3 (BD Bioscience, USA) in the presence of irradiated CD90-depleted C57BL/6 splenocytes at a 1:5 ratio and culture medium enriched with the following cytokines for Th17 differentiation: 3 ng/ml TGF-beta, 20 ng/ml IL-23 and 20 ng/ml IL-6 (all R&D Systems, USA). The Th17 cultures were maintained with 50 U/ml IL-2 and 10 ng/ml IL-23 (all R&D Systems, USA) at full confluence. At day 5 in culture, the Th17 cells were used for co-culture with brain slices. 2

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phosphate-buffered saline (PBS). This was followed by blocking in PBS containing 0.1% Triton X-100 (Life Technologies GmbH, Germany) and 5% goat serum (Life Technologies GmbH, Germany) for 1 h at RT. For in vivo studies mice were perfused with PBS and 4% PFA. The following primary antibodies were used in this study: mouse anti-muCD52 (1:1000, Sanofi Genzyme), rabbit anti-NeuN (1:1000, Millipore), rabbit anti-Iba-1 (1:500, Wako) and rabbit anti-GFAP (1:1000, Millipore). The next day, the stained cells were washed 3 times in PBS, followed by incubation with the secondary antibodies such as goat anti-mouseAlexa-Fluor 488 (1:1000, Life technologies) or goat anti-rabbit-AlexaFluor 568 (1:1000, Life technologies). Then, cells were washed again and stained with VECTASHIELD Mounting Medium (Vector Laboratories, USA).

2.6. Organotypic hippocampal slice cultures, transfection and slice culture treatments OHSCs were prepared using the interface method introduced by Stoppini et al. (1991). Briefly, brains from B6.CX3CR1+/GFP or B6.Thy1.TN-XXL pups (P3-5) were transferred to ice-cold preparation medium (MEM, Minimum Essential Medium), containing 2 mM glutamine. Hippocampi were isolated with enthorinal cortex attached and coronally sliced (300 μm thickness) using a McILWAIN tissue chopper. Slices were cultivated on Millicell cell culture inserts (Merck Millipore) in the presence of medium containing 48% MEM, 24% Basal Medium Eagle, 24% heat inactivated horse serum, 2 mM glutamine and 0.6% glucose and incubated at 37 °C, 95%/5% O2/CO2. The medium was exchanged 24 h after plating and then every other day. After 7 to 14 DIV, hippocampal slices were used for experiments. For co-cultures, 1 × 105 Th17 cells (in 10 μl) were added directly on top of the hippocampal slices and incubated at 37 °C, with or without pre-treatment of the slices with anti-muCD52 (50–200 μg/ml) for 24–48 h. To distinguish viable and apoptotic T cells, co-cultures were stained with Image-iT® LIVE Red Caspase-3 and -7 Detection Kit (Thermofisher, Weltham, MA) 30–60 min prior to imaging. After 48 h of Th17 co-culture, slices were either imaged with two-photon microscopy for microglial morphology (B6.CX3CR1+/GFP) or neuronal calcium (B6.Thy1.TN-XXL). Furthermore, slices from B6.Thy1.TN-XXL mice were treated with 2–5 μM NMDA (Sigma-Aldrich, Germany) for 24 h, with or without anti-muCD52, directly dissolved in the slice culture medium.

2.10. Confocal imaging All sections were counterstained with DAPI before being transferred and embedded onto object plates. Sections were analyzed using a Leica confocal microscope (Leica TCS SP8, DM 6000CS), and sequential scans with a 63 × objective (Leica, NA 1.4) and a resolution of 1024 × 1024 pixels were performed. 2.11. Statistical analysis All data were analyzed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). Mean group differences were investigated by two-way ANOVA with repeated measures (for EAE curve analyses), oneway ANOVA followed by Tukey's multiple comparison test, or independent-sample two-tailed-t-tests. Significance level was set at 0.05. Data are plotted as mean ± S.E.M. unless differently stated.

2.7. Two-photon imaging Operation procedures and two-photon laser scanning microscopy were performed as previously described (Luchtman et al., 2016; Siffrin et al., 2009, 2010). In brief, the anesthetized animal was transferred to a custom-built microscopy table and the head inclined to allow access to brainstem regions. Dual near-infrared and infrared (IR) excitation at 850 nm was applied by an automatically tunable Ti:Sa laser (Mai Tai HP, Spectra Physics, Santa Clara, CA, USA) and 1110 nm by an optical parametric oscillator (OPO, APE, Berlin, Germany) pumped by the Ti:Sa laser. Using the TriMScope I from LaVision Biotec, volumes of 300 × 300 × 72 μm3 were acquired over time. For the ex vivo slice experiments, slices were transferred onto the stage of a Leica TCS-MP5 multi-photon system with heated and gassed (95%O2/5%CO2) Ludin enclosure and areas of 300 × 300 μm2 at variable depth (max 80 μm) acquired at 1000 nm to visualize CX3CR1EGFP microglia morphology, or at 850 nm to image both the CFP and YFP channels of B6.Thy1.TN-XXL slices. For imaging the interactions between CX3CR1GFP microglia and B6.2d2.CFP Th17 cells, we used the TriMScope I from LaVision Biotec with the Bold-Line series of stage top incubators from Okolab (Pozzuoli, NA, Italy).

3. Results 3.1. Anti-murine CD52 causes lymphopenia and modestly affects microglia morphology in EAE

For microglial morphology and interaction studies, TIFF and LIF files were imported into 3-D reconstruction software Imaris (Bitplane, version 8.1.2). T cell tracks were created using the tracking tool implemented in Imaris software. Contact types and durations were determined by 3-D rotation and surface analysis to verify contacts. Morphological parameters were generated by using the filament function. LIF files from the experiment with Thy1.TN-XXL slices were imported into Volocity Software (Improvision, Forchheim, Germany) for ratiometric analyses of calcium.

To first determine whether anti-muCD52 treatment may affect microglia activity and morphology in EAE, we performed a classic MOG3555-peptide-induced active EAE in B6.Cx3Cr1-GFP mice, which express enhanced green fluorescent protein (EGFP) in microglia, and treated these mice from clinical score 1 or 2 with anti-muCD52 (10 mg/kg) for 5 days. Compared to vehicle treated controls, anti-muCD52-treated mice from score 1 onward showed improvement in EAE disease symptoms soon after injection (Fig. 1A). Mice treated with antimuCD52 from score 2 displayed a mild yet non-significant improvement in clinical score (Fig. 1B). The treatment from clinical score 2 still led to a significant depletion of CD4+ and CD8+ splenocytes, whereas numbers of natural killer (NK) cells and CD11b+ microglia/macrophages remained unchanged (Fig. 1C and D). One day following the last injection in late anti-muCD52 treated animals (from score 2 onward), the dorsal brainstems (including dorsal column nuclei) of mice were imaged with two-photon microscopy and image sequences 3-D reconstructed for microglial morphological parameters. Microglia in EAE mice treated with anti-muCD52 lost their ramified morphology, as evident by reduced process (dendritic) length and an overall decreased ramification index, compared to healthy controls but not significantly compared to vehicle-treated EAE mice (Fig. 1E and F, Movie 1-3). These data suggest that microglia were significantly more activated under EAE conditions in combination with anti-muCD52 treatment. Supplementary video related to this article can be found at https:// doi.org/10.1016/j.ejphar.2020.172923.

2.9. Immunohistochemistry

3.2. Positive CD52 expression in CNS cell populations

Primary cell cultures were fixed by 4% paraformaldehyde for 20 min at room temperature (RT), and then washed 3 times in

To assess the CNS expression of CD52, we first quantified the CD52 mRNA expression via qPCR in murine primary cultures of microglia,

2.8. Analysis of two-photon imaging results

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Fig. 1. Anti-murine CD52 ameliorates EAE diseases and causes lymphopenia. A) Clinical score of MOG35-55-peptide-induced EAE mice treated with PBS or antimuCD52 (10 mg/kg) between days 12 and 16 (from clinical score 1 onward) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to PBS controls; n = 10 mice for anti-muCD52 and n = 10 for PBS. B) Clinical score in EAE mice treated with PBS or anti-muCD52 from score 2; the last 5 days before FACS analysis and microglia imaging are shown, during which the mice were injected daily; n = 10 mice per group. C) Representative FACS scatterplots showing CD4+ and CD8+ splenocytes isolated from PBS- (left panel) and anti-muCD52 treated EAE mice from clinical score 2 (right panel). D) Quantification of CD4+ and CD8+ splenocytes as well as CD11b+ and natural killer (NK) cells obtained from the spleen of PBS- and anti-muCD52 treated EAE mice from clinical score 2. *P < 0.05, **P < 0,01 compared to PBS groups, n = 10 mice per group. E) Representative 3-D reconstructions (scale bar 20 μm, soma in red, dendrites in green) of microglia morphology in healthy controls (left panel), PBS-treated (middle panel) and anti-muCD52-treated (right panel) EAE mice from clinical score 2. Imaging was performed in the dorsal brainstem. F) Quantification of microglial morphological aspects of mice treated from clinical score 2 including dendrite length (left panel), soma volume (middle panel) and ramification index (dendrite length/volume, right panel). *P < 0.05 compared to healthy controls; n = 4 mice per group.

3.3. Anti-muCD52 affects microglial morphology but not function

astrocytes and neurons that were unstimulated or stimulated with lipopolysaccharide (LPS). Cultured Th17 lymphocytes served as positive controls. We detected CD52 mRNA in all cell types, in particular in microglia (Fig. 2A, black bars). Treatment with LPS significantly increased this CD52 expression in microglia, whereas the other cells types showed a slight but non-significant increase (Fig. 2A, grey bars). We next performed immunocytochemistry for CD52 with NeuN, GFAP and Iba1, corresponding to neurons, astrocytes and microglia, from LPS-treated dissociated CNS cells (Fig. 2B–D). To our surprise, we found CD52 protein expression to be strongest in neurons, whereas a weak signal was found in primary astrocytes and microglia. Performing anti-CD52 immunohistochemistry in murine brain sections from C57Bl6 mice also revealed a positive signal, in particular in neurons and to a lesser extent in microglia, but not clearly in astrocytes (Fig. 3A–C). CD52 staining in splenocytes was used as a positive control (Fig. 3D). Murine brain sections of C57Bl6 mice, which underwent active EAE, showed a similar expression pattern of CD52 compared to the baseline non-inflamed condition with a clear signal for neurons and microglia but not for astrocytes (Fig. s1).

Due to the apparent neural CD52 expression in vitro and in vivo, we next focused on a possible functional role of CD52 in the CNS. For this, we treated organotypic hippocampal slice cultures from B6.Cx3Cr1 pups (P3–P5) with the anti-muCD52 antibody. First, we assessed microglial morphology with two-photon imaging at 24 and 48 h after treatment with 50–200 μg/ml of the antibody. Although no effect of CD52 blockage was found after 24 h, a significant decrease in the ramification index, similar to the in vivo EAE experiments, was detected at the highest concentration of anti-muCD52 after 48 h (Fig. 4A and B). We then determined whether this change in morphology and presumably activation state influenced the interaction between microglia and co-cultured Th17 lymphocytes (B6.2d2.CFP Th17 cells). In order to minimize treatment effects on lymphocyte motility, the maximum concentration of anti-CD52 used here was 100 μg/ml. Furthermore, to ensure that we were imaging live T cells (or microglia), the T cells were stained with a red caspase-3 and -7 dye prior to addition to the slices. We observed that with 50 and 100 μg/ml anti-muCD52, there was no 4

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Fig. 2. Positive in vitro CD52 expression in primary neurons, astrocytes and microglia. A) qPCR quantification of CD52 mRNA expression relative to housekeeping gene RPS29 in murine primary cultures of neurons, astrocytes and microglia, unstimulated or stimulated with LPS. In vitro differentiated Th17 cells were used as positive controls. *P < 0.05. n = 4 independent experiments for each condition (unstimulated and LPS stimulated). B-D) Immunocytochemistry for CD52 protein co-stained with NeuN (B), Iba1 (C), GFAP (D) and DAPI in primary cultures of neurons and mixed astrocyte-microglia cultures (scale bars upper panels: 30 μm and lower panels: 5 μm). n ≥ 3 mice per condition for culture experiments.

Fig. 3. In vivo CD52 expression in neurons, astrocytes and microglia. A-C) Immunohistochemistry for CD52 protein (green) co-stained with NeuN (A), GFAP (B), Iba1 (C) and DAPI in brain sections from C57Bl6 mice (scale bars upper panels: 30 μm and lower panels: 5 μm). D) Positive control staining of CD52 (green) and DAPI in the spleen of C57Bl6 mice (scale bars upper panels: 30 μm and lower panels and spleen: 5 μm). n = 3 animals.

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Fig. 4. Anti-muCD52 affects microglial morphology but not function. A) Representative 3-D reconstructions of microglia morphology in organotypic hippocampal slice cultures, obtained from P4 B6.Cx3Cr1.EGFP pups, treated with anti-muCD52 (50, 100 and 200 μg/ml) for 48h (scale bar: 20 μm). B) Quantification of microglial morphological aspects. *P < 0.05 compared to the untreated (control) condition. n ≥ 5 slices per condition except for 200 μg/ml anti-muCD52: n = 3 slices. C) Quantification of lymphocyte motility parameters, including displacement length (μm), displacement speed (μm/sec), and track straightness. n ≥ 8 slices per condition. D) Representative images of temporary (less than 10 min) and stable (more than 10 min) contacts between EGFP-expressing microglia (green) and B6.2d2.Th17 cells (blue) in hippocampal slice cultures from P4 B6.Cx3Cr1 pups (scale bar: 10 μm) and quantification of contact index (number of contacts with microglia/number of T cells), temporary and stable contacts between microglia and Th17 cells in untreated slices and slices pre-treated with 50 and 100 μg/ml antimuCD52. n ≥ 9 slices per condition.

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Fig. 5. Anti-muCD52 does not affect Th17 or NMDA-induced excitotoxicity in neurons. A) Representative two-photon images (CFP-YFP overlay) of B6.TN-XXL slices after 24 h of NMDA (2 and 5 μM), with or without anti-muCD52 pre-treatment (50–200 μg/ml). Increased neuronal calcium levels lead to higher intensity of the green signal due to increased Förster-resonance-energy-transfer (FRET)-activation of the YFP channel relative to the CFP channel. (scale bar: 20 μm). Right panel: Quantification of calcium levels. *P < 0.05, ****P < 0.0001 compared to control. n ≥ 4 slices per condition. B) Left panels: Representative two-photon images (CFP-YFP overlay) of TNXXL slices after 48 h co-culture with B6.2d2.RFP Th17 cells (red), with or without anti-muCD52 pre-treatment (50–100 μg/ml), scale bar: 20 μm. Right panel: Quantification of calcium levels. *P < 0.05 compared to control. n ≥ 12 slices per condition.

4. Discussion

caspase 3–7 staining of the T cells or the microglia and microglia appeared intact, showing normal motility (Movie 4). There was also no significant effect of these concentrations on T cell motility measured via displacement length, velocity and track straightness (Fig. 4C). The interactions between Th17 cells and microglia were then monitored after 48 h of co-culture using two-photon microscopy. As shown in Fig. 4D and E, there was no change in the contact index of Th17 cells (number of contacts with microglia/number of T cells) or in the number of temporary (less than 10 min contact) or stable (more than 10 min contact) contacts between Th17 cells and microglia. Supplementary video related to this article can be found at https:// doi.org/10.1016/j.ejphar.2020.172923.

Alemtuzumab has been successfully used in the treatment of RRMS, but so far, the mechanisms by which it exerts its therapeutic effects in MS are not fully elucidated. Particular uncertainty surrounds the neuroprotective potential of alemtuzumab relative to its anti-inflammatory and immune-modulatory effects (Ruck et al., 2016). To date, there are no reports on CD52 expression in the CNS, but since alemtuzumab may enter the CNS in the case of compromised BBB integrity (Minagar and Alexander, 2003; Spencer et al., 2018), it is of relevance to study CD52 expression and direct effects of CD52 blockade on CNS cells. Remarkably, we detected CD52 mRNA expression in dissociated cells, especially in microglia, which further increased upon inflammatory LPS stimulation, but CD52 protein was most clearly expressed in neurons. In line with this, we found neuronal and to a lesser extent microglial CD52 protein to be consistently detectable in murine brain sections, whereas little to no expression was found in astrocytes. To assess the functional relevance of this expression, we proceeded by treating organotypic hippocampal slice cultures with the anti-muCD52 blocking antibody. Organotypic hippocampal slice cultures are particularly useful, as they allow the monitoring of defined populations of cells over time and preserve the gross in vivo tissue architecture, while allowing precise experimental control (De Simoni and Yu, 2006; Gahwiler et al., 1997; Noraberg et al., 2005; Stoppini et al., 1991).

3.4. Anti-muCD52 does not affect Th17 or NMDA-induced excitotoxicity in neurons To investigate putative direct effects of anti-muCD52 on neurons, we performed hippocampal slice cultures from B6.thy1.TN-XXL mice, which express a ratiometric calcium reporter in neurons. Treating the slices for 24 h with 2–5 μM NMDA significantly increased excitotoxic calcium levels in the neurons (Fig. 5A). Pre-treatment of the slices with 50–200 μg/ml anti-muCD52 did not affect NMDA-induced calcium levels (Fig. 5A). In line with these results, anti-muCD52 50–100 μg/ml was also unable to reduce Th17-induced calcium levels (Fig. 5B). 7

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Appendix A. Supplementary data

Since we found the anti-muCD52 blocking antibody effective at ameliorating EAE disease and causing lymphopenia, as reported also by others (Turner et al., 2015), we deemed it appropriate for testing the functionality of CD52 in slice cultures. In slices from B6.Cx3Cr1 pups, all observed microglia appeared intact following antibody treatment thus excluding the possibility that the antibody depleted the microglia. Similar to what we found in EAE mice, in particular in those mice treated with anti-muCD52, microglia morphology appeared less ramified, suggestive of a more activated state. Since microglia avidly interact with their environment (Nimmerjahn et al., 2005), we were interested to determine whether this altered microglial morphology and presumably activation state, might impact microglial functions. As previously reported (Nitsch et al., 2004), the co-cultured lymphocytes readily entered the slice tissue and two-photon imaging indeed revealed vivid interactions between microglia and these cells. However, anti-muCD52-treatment did not alter these interactions. We also investigated the effect of the antibody on neuronal function in slices. We previously used B6.Thy1.TN-XXL mice to reliably detect calcium fluctuations in neurons upon Th17 cell contact (Siffrin et al., 2010), using two photon imaging. This was mediated through the formation of an immune synapse or T-cell – neuron contact. In a recent study we have shown, that T cells themselves are capable of releasing glutamate after T-cell-neuron contacting via a beta1-integrin – VCAM-1 interaction (Birkner et al., 2019). Calcium elevations measured by ratiometric TN-XXL have been shown to precede neuronal cell death (Luchtman et al., 2016; Siffrin et al., 2010). Here, we incubated hippocampal slice cultures from these mice with NMDA and Th17 cells to induce excitotoxicity mediated elevations in neuronal calcium. Even in the highest concentrations, anti-muCD52 was unable to protect neurons from excitotoxicity, and also did not affect baseline calcium levels in control conditions. Altogether, we can confidently state that although CD52 is expressed on murine neurons and microglia, this appears to have no functional relevance regarding the working mechanisms of the antimuCD52. Therefore, it is more likely that the positive effects of antimuCD52 that we observed in EAE are solely due to peripheral immune effects, and rather indirectly lead to neuroprotection. This is supported by our observation that only early anti-muCD52 treatment (from clinical score 1 onward) leads to an ameliorated disease course, mediated via direct immunomodulatory mechanisms, whereas late treatment, when the neuronal damage has taken place, does not. The hypothesis that anti-CD52 treatment rather indirectly acts neuroprotective, is further corroborated by the observation that peripheral blood mononuclear cells from alemtuzumab-treated patients produce brain-derived neurotrophic factor (BDNF), which is able to promote the survival of neurons and oligodendrocyte precursors (Jones et al., 2010). Thus, in MS, the beneficial effects of alemtuzumab are likely to be mediated exclusively by immune mechanisms.

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Author agreement All authors approved the revised version of the manuscript. Declaration of competing interest The authors declare no conflict of interest with relevance to the project. Acknowledgements This project was supported by Sanofi Genzyme and by the German Research Council (DFG, CRC-TR-128). We would like to thank Rosalind Gilchrist for proofreading the manuscript.

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