Cells, reagents and antibodies
BV2 microglia were kindly provided by Gerd Bicker, University of Veterinary Medicine, Hannover, Germany. If not indicated otherwise, BV2 cells were cultured in DMEM with 4.5-g/l glucose containing 100-µg/ml penicillin/streptomycin and supplemented with 2.5% fetal bovine serum (all from Gibco, Rockville, MD, USA). Passaging was performed at about 80% confluency and cells were detached mechanically with a cell scraper. All experiments performed in this study were obtained between passages 6 and 20. The plasmid pX330A-1 × 2 for CRISPR/spCas9-D10A-mediated knockout [18] was a kind gift from Takashi Yamamoto (Addgene, Watertown, MA, USA, plasmid #58766; https://n2t.net/addgene:58766; RRID:Addgene_58766). The plasmid DHC#4731 containing a P2A element followed by eGFP [19] was kindly provided by Dirk Heckl, Hannover Medical School, Hannover, Germany. Endosialidase was produced as described before [20]. Trisialic acid, i.e., α2,8-linked sialic acid with a degree of polymerization (DP) of 3, and tetrasialic acid (DP4) were from Nacalai Tesque (Kyoto, Japan); α2,8-linked polySia (colominic acid from E. coli) was from Sigma-Aldrich [St Louis, MO, USA, catalog no. (cat. #) C5762, lot no. 110M1383]. Streptavidin and biotin were from Vector Laboratories (Burlingame, CA, USA), Cy3-conjugated streptavidin from Rockland (Limerick, PA, USA). Lipopolysaccharide (LPS) extracted from E. coli serotype O127:B8, 4-chloro-m-cresol (4-CmC), TAK-242, 1,1′‐diheptyl‐4,4′‐bipyridinium dibromide (DHBP), genistein, and biotinyl tyramide were from Merck, Darmstadt, Germany.
The following monoclonal (mAb) or polyclonal (pAb) antibodies were used: Siglec-E-specific sheep pAb (kindly provided by Paul Crocker, University of Dundee, Scotland, 3.2 µg/ml), ESL-1-specific rabbit pAb (kindly provided by Martin Wild, Münster, Germany, 1:3000), polySia-specific mouse mAb 735 ([21]; produced in-house as described by Werneburg et al. [4] and used at 2 µg/ml for immunofluorescence or 1 µg/ml for immunoblotting), CD11b‐specific rat mAb (AbD Serotec, Raleigh, NC, USA, cat. #MCA74GA, 1:250), Iba1‐specific rabbit pAb (Wako Chemicals, Neuss, Germany, cat. #019‐19741, 1:300), giantin‐specific rabbit pAb (Covance, Denver, PA, USA, cat. #PRB‐114C, 1:10,000), NRP2‐specific rabbit mAb D39A5 (Cell Signaling, Beverly, MA, USA, cat. #3366S, 1:1000), EEA1 (early endosomal antigen 1)-specific antibody rabbit pAb (Santa Cruz Biotechnology, Santa Cruz, CA, cat. #sc-33585, 1:100).
Secondary antibodies were HRP-conjugated donkey anti-sheep IgG (Sigma-Aldrich, cat. #A-3415, 1:1000), Cy3-conjugated donkey anti-rat IgG (Merck, cat. #AP189C, 1:500), Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA, cat. #A-31573, 1:500), Alexa Fluor 488-conjugated donkey anti-mouse IgG (Thermo Fisher Scientific, cat. #A21020, 1:500), Alexa Fluor 488-cojugated goat anti-mouse IgG (Thermo Fisher Scientific, cat. #A11029, 1:500), HRP-conjugated goat anti-rabbit IgG (Sigma-Aldrich cat. #6157, 1:15,000), and HRP-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA, cat. #1010–05, 1:20,000).
Immunocytochemistry
For immunocytochemistry, cells were seeded on glass coverslips in 24-well plates at a density of 20,000 cells per well. After a given treatment, as specified for each experiment separately, cells were washed once in PBS, fixed with 4% paraformaldehyde in PBS for 20 min at room temperature (RT) and stored in PBS at 4 °C. Immunofluorescence staining (IF) and embedding was performed as described by Werneburg et al. [4] with minor modifications by permeabilizing with 0.4% Triton X-100 in PBS for 15 min, blocking for 2 h at 37 °C, and washing three times with 0.1% Triton in PBS and once with water, before mounting.
For Siglec-E immunostaining in combination with other primary antibodies, biotin-tyramide signal amplification was used. Briefly, after permeabilization, peroxidase activity was blocked by incubation with 0.3% H2O2 in PBS for 15 min at RT. After washing, endogenous biotin was blocked by adding streptavidin and, after further washing, biotin (1:10 in PBS, 15 min at RT, each). After another washing step, cells were incubated with primary antibodies as described above, and for 45 min with HRP-conjugated anti-sheep IgG for the detection of Siglec-E-specific antibody, followed by washing with TBS pH 7.6 and incubation in TBS with 0.1-M imidazole, 0.001% H2O2, and 2.5-µg/ml biotinyl tyramide for 10 min at RT. After three washes with PBS, enzymatically produced biotin precipitate was detected by Cy3-conjugated streptavidin (1:1.000 in PBS with 0.1% Triton X-100 and 2% BSA, 45-min RT), before fluorescently labeled secondary antibodies were added and cells were embedded, as described above.
Specificity of double- and triple-immunostaining procedures was controlled by omitting one of the primary or secondary antibodies at a time. For polySia immunoreactivity, specificity was additionally controlled by the loss of immunoreactivity after degradation of polySia with 6-µg/ml endosialidase applied during blocking. This enzyme degrades polySia with high specificity [20].
Immunoaffinity chromatography
Stationary phase consisting of polySia-specific mAb 735 coupled to protein A sepharose beads (GE Healthcare, Amersham, UK) was prepared using standard protocols [22]. A C16/20 column (GE Healthcare) was packed with 3.2-ml beads coupled with 2.1-mg antibody per ml beads (bed volume) and chromatography was performed at RT on an Äkta Pure 25 protein purification system (GE Healthcare) at a flow rate of 0.4 ml/min with 20-mM Tris–HCl, pH 8.0, 10-mM MgCl2 containing 90-mM NaCl for loading and 2-M NaCl for elution. Conductivity and absorption at 214 and 280 nm were recorded.
Supernatant of LPS-induced BV cells cultured in serum-free medium was passed through a 0.22-µm syringe filter prior to loading and following loading to the column elution was started after a stable baseline was obtained. For subsequent analysis by immunoblotting (see below), fractions of 0.4 ml were collected and 1.6-ml propanone per fraction was added to precipitate proteins overnight at -20 °C. After centrifugation at 16,000×g for 15 min, pellets were air dried and reconstituted in 20-µl Laemmli buffer.
Immunoprecipitation and immunoblotting
Immunoprecipitation (IP) of polySia from cell lysates using M-280 tosyl-activated Dynabeads (Thermo Fisher Scientific) covalently coupled to mAb 735, SDS-PAGE and analyses of IP fractions by immunoblotting and enhanced chemiluminescence detection were performed as described by Werneburg et al. [5]. Fractions obtained by immunoaffinity chromatography were blotted on nitrocellulose membrane (GE Healthcare) and analyzed with the Odyssey Infrared Imaging System (LI-COR Biosciences, Bad Homburg, Germany) as described previously [23]. For enzymatic removal of polySia prior to SDS-PAGE, samples in Laemmli buffer were treated with 6-µg/ml endosialidase for 45 min at 37 °C.
Anion exchange chromatography
Polysialic acid was separated on the Äkta Pure 25 system equipped with a 22 × 50 guard column followed by a DNAPac PA-100 column 22 × 250 (Thermo Fisher Scientific). Chromatography was performed at RT with a flow rate of 3.5 ml/min using 10-mM Tris–HCl pH 8.0 during loading. Elution was performed in 10-mM Tris–HCl pH 8.0 with consecutive steps of linear NaCl gradients and steady concentrations as follows: 4-ml 0–70 mM, 44-ml 70–180 mM, 0.5-ml 180 mM, 16-ml 180–220 mM, 48-ml 220–280%, 40-ml 280–320 mM, 80-ml 320–355 mM, and 40-ml 355–385 mM, 40-ml 385–410 mM, 44-ml 410–432 mM, 40-ml 432–447 mM, 40 ml 447–458 mM, 40-ml 458–466 mM, 40-ml 466–470 mM, 40-ml 470–472 mM and 45-ml 1 M NaCl. Conductivity and absorption at 214 and 280 nm were recorded.
Brain sections
To conserve animals, sections of mouse brains with cortical lesions were drawn from a control group of animals used in a prior study, which was designed to examine drug effects on epileptogenesis induced by intrahippocampal kainate injection [24]. Sections were kindly provided by Wolfgang Löscher, University of Veterinary Medicine, Hanover, Germany. The animal experiments were performed according to the EU council directive 2010/63/EU and the German Law on Animal Protection. Ethical approval was granted by the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES, project number 14/1659). As described in detail elsewhere [24], 8- to 10-week-old male NMRI mice were anesthetized with chloral hydrate (500 mg/kg i.p) and received a unilateral stereotactic injection of kainate (1 nM in 50-nl saline) through the cortex into the CA1 sector of the dorsal hippocampus. Injections were carried out with a 0.5-μl microsyringe over 60 s and after injection, the syringe was kept in place for another 2 min to limit reflux along the wound channel. In addition, mice received daily i.p. injections of saline. Seven days after the brain injection, and before the onset of clinical seizures [25], mice were perfused and paraffin-embedded brains were sectioned at 3 µm for use in immunohistochemistry.
Immunohistochemistry
Paraffin sections were rehydrated and antigen retrieval was performed in 400-mM Tris–HCl pH 9.0 with 1-mM EDTA and 0.05% Tween-20 at 90 °C for 10 min. After permeabilization with 0.4% Triton X-100 in PBS for 30 min and blocking with 2% BSA, 0.1% Triton in PBS for 3 h at 37 °C, primary antibodies were incubated in blocking buffer at 4 °C overnight. All other steps and specificity controls were carried out as described for immunocytochemistry, except for the endosialidase treatment, which was performed with 10 µg/ml.
Microscopy, image acquisition, cell counting and densitometry
Microscopy was performed with Axio Observer.Z1 equipped with an ApoTome module for structured illumination, AxioCam MRm digital camera, and Zen 2012 (blue edition) software (Carl Zeiss Microscopy). A 20 × Plan‐Apochromat objective with a numerical aperture of 0.8 was used to acquire optical sections of 1.62-μm (488 channels), 1.75-μm (568 channels) or 2.09-µm thickness (647 channels), and five optical sections were merged to a maximal intensity projection, respectively. For 3D reconstruction, stacks of 40 optical sections were obtained using a 63 × Plan-Apochromat oil immersion objective with a numerical aperture of 1.4. Identical settings were used for all samples within one experimental setting. Images were arranged using PowerPoint (Microsoft Office 2010). For the evaluation of immunostained cells in cultures, cell counts were performed on randomly chosen frames as specified in the respective figure legends. For the evaluation of Siglec-E immunoreactivity, cells were encircled manually and signal intensities were determined using the polygon contour measurement tool of the ZEN software. For the evaluation of Iba-1- and polySia-positive cells on brain sections, immuno-positive cells were counted in bins of 0–50, 50–200 and 200–400 µm away from the wound channel.
Generation of Siglec-E knockout cells
The CRISPR/spCas9-D10A strategy [18, 26] was used to generate Siglec-E knockout cells. The all-in-one CRSIPR/spCas9-D10A vector pX330A-1 × 2 (Addgene plasmid # 58766) was modified by insertion of a P2A element followed by eGFP in C-terminal orientation. The latter was obtained from plasmid DHC#4731 as a template by overlap extension PCR with the primers 5′- GGAAGAGAATGCTGGCCTCT -3′ and 5′- AATCCAGAATTCGATTATCGATTTAACGC -3′ containing recognition sequences for BsmI and EcoRI. In addition, the following guide RNAs (gRNA) targeting the sialic acid-binding V-domain of Siglec-E located within the first exon were inserted: 5′- CACCGCAGACGCAAAGATTCCATCG -3′ (leading strand gRNA1), 5′- AAACCGATGGAATCTTTGCGTCTGC -3′ (reverse strand gRNA1), 5′- CACCGTGTACCAGAATCCATGAACT -3′ (leading strand gRNA2), 5′- AAACAGTTCATGGATTCTGGTACAC -3′ (reverse strand gRNA2).
To transfect BV2 cells, electroporation with the Neon Nucleofector (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. 1 × 105 cells were treated with a 30-ms pulse of 1325 V followed by incubation for 12 h in conditioned medium consisting of supernatant from 1 × 105 BV2 cells per ml, cultured for 24 h in the absence of penicillin and streptomycin, which was filtered through a 0.22-µm syringe filter and supplemented with FCS to a final concentration of 7.5%. Subsequently, cells were cultured in conditioned medium with penicillin and streptomycin supplemented with 5% FCS for 3 days and 2.5% FCS for 7 days, followed by standard cultivation conditions. GFP-positive cells were identified, clones were isolated by limited dilution and Siglec-E mutations were assessed by sequencing of genomic PCR products obtained with primers flanking the targeted site (forward: 5′- CAGTTTTAGCTGGACATGCTG -3′, reverse: 5′- CGGGTTTCCTTCACTGCTT -3′).
Nitrite Assay
Nitric oxide (NO) production by LPS treatment was evaluated by the colorimetric Griess assay, detecting the stable NO breakdown product nitrite in cell culture supernatants. The assay was performed as described before [7], with the following modifications: 5 × 104 cells were seeded in 96-well plates. After adherence for 4 h, the medium was changed and cells were cultured for 2 h in the presence of 60-µM minocycline, where indicated. After another medium change, cells were kept for 24 h in 200-µl medium for further treatment (see results), before 150 µl of cell culture supernatant were collected and centrifuged at 3000×g for 5 min. Nitrite concentrations were assessed by reacting 70 µl of the supernatant for 20 min with 70-µl Griess reagent consisting of 1% (4-[(4-aminobenzene)sulfonyl]aniline), 0.05% N-1-napthylethylenediamine dihydrochloride and 2.5% HCl.
RNA isolation and quantitative real-time RT-PCR
Total RNA was isolated using TRIzol (Thermo Fisher Scientific), and cDNA was generated as described [4] using RevertAid H Minus reverse transcriptase (Thermo Fisher Scientific). Quantitative real-time PCR of mouse Siglec-E, TNF and IL-6 was performed in 10-µl 1:2 diluted BIO SyGreen Lo-ROX mix (PCR Biosystems, London, UK) with ImageQuantQ3 and ImageQuant software (Thermo Fisher Scientific) using the comparative threshold cycle (ΔCT) method as described elsewhere [27] with HPRT and PPIA as reference genes [28]. The following primers were used: HPRT, 5′- TTCCTCATGGACTGATTATGGACA-3′ (forward) and 5′- AGAGGGCCACAATGTGATGG -3′ (reverse); PPIA, 5′- CCACAGTCGGAAATGGTGAT-3′ (forward) and 5′- TGCACTGCCAAGACTGAATG-3′ (reverse); TNF, 5′- CTGTAGCCCACGTCGTAGC-3′ (forward) and 5′- TTGAGATCCATGCCGTTG-3′ (reverse) [29]; IL-6, 5′-GGCCTTCCCTACTTCACAAG -3′ (forward) and 5′-ATTTCCACGATTTCCCAGAG -3′ (reverse) [30]; Siglec-E 5′-TGGTACAGGGAAGGAACCGA -3′ (forward) and 5′-GTGAGGGCTGTTACAACCAGA -3′ (reverse). The NCBI primer designing tool with Primer3 version 4.1 [31, 32] was used to select Siglec-E-specific primers.
Statistics
Statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA). Unpaired Student’s t-test, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test, and mixed two-way ANOVA followed by Holm–Sidak post hoc test were applied as indicated. Normality and equality of variances were assessed by the Shapiro–Wilk and the Brown–Forsythe test, respectively.