Primary Schwann cell cultures
Mouse SCs (mSCs) were prepared using a modified Brockes method [28]. Cells were purified and cultured as described before [29]. Briefly, sciatic nerves were dissected from neonatal (postnatal day 3 (P3)) C57BL/6 mice and digested with 0.05% collagenase (Worthington, Lakewood, NJ, USA) and 0.125% trypsin (Merck, Darmstadt, Germany). Cells were plated in poly-D-lysine (PDL)-coated (Sigma-Aldrich Corp., St. Louis, MO, USA) cell culture dishes (Greiner Bio-One GmbH, Frickenhausen, Germany) with Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen Corp., Carlsbad, CA, USA) containing 10% horse serum (HS; Invitrogen Corp., Carlsbad, CA, USA), 4 mM L-glutamine ((Glut) Invitrogen Corp., Carlsbad, CA, USA), 2 ng/mL human heregulin β-1 (Cell Sciences, Canton, MA, USA), 0.5 μM forskolin (FKL) (Sigma-Aldrich Corp., St. Louis, MO, USA) and 100 IU/mL penicillin/streptomycin (PS; Invitrogen Corp., Carlsbad, CA, USA). For complement lysis, cells were washed with Hank’s Balanced Salt Solution (HBSS; Invitrogen Corp., Carlsbad, CA, USA) containing 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES; Invitrogen Corp., Carlsbad, CA, USA) and subsequently incubated with DMEM, containing HEPES, HS, Glut, P/S and anti-thymidine 1.2 antibody (AbD Serotec, Kidlington, UK). After 15 min at 37°C, rabbit complement (Cedarlane Laboratories Inc., Burlington, NC, USA) was added and incubated for 2 h at room temperature (RT). Cells were washed twice using HBSS containing HEPES and cultured on PDL-coated culture dishes in DMEM medium, containing 10% HS (Invitrogen Corp., Carlsbad, CA, USA), 4 mM Glut (Invitrogen Corp., Carlsbad, CA, USA), 2 ng/mL human heregulin β-1 (Cell Sciences, Canton, MA, USA), 0.5 μM forskolin (Sigma-Aldrich Corp., St. Louis, MO, USA), 20 μg/mL pituitary extract bovine (PEB, Merck Millipore, Darmstadt, Germany), 10 ng/mL recombinant human fibroblast growth factor (Biomol GmbH, Hamburg, Germany), 100 IU/mL P/S (Invitrogen Corp., Carlsbad, CA, USA) and 1:4 mouse DRG supernatants; the medium was renewed every third or second day.
Preparation of rat SCs (rSCs) was performed using the modified Brockes method [28]. Sciatic nerves were dissected from neonatal (P3) Wistar rats, digested with 0.1% collagenase (Worthington, Lakewood, NJ, USA) and 0.25% trypsin (Invitrogen Corp., Carlsbad, CA, USA), and cells were finally plated with DMEM containing 10% fetal calf serum (FCS). Cultures were treated with two cycles of 10 μM cytosine arabinoside to reduce fibroblasts, followed by complement lysis with anti-thymidine 1.1 antibodies. Cultures reached a final purity of more than 95% and were maintained in DMEM Gibco 3185 (Invitrogen Corp., Carlsbad, CA, USA) with 10% FCS, 100 IU/mL P/S, 2 mM Glut, and 1 μL/mL FKL on PDL-coated culture dishes.
Preparation of dorsal root ganglia
DRGs were prepared from embryonic day 15 (E15) C57BL/6 mice (BL6) by opening the cutis and subcutis along the spine and removing the spinal cord. DRG were collected, centrifuged and resuspended [29, 30]. Twenty-four well plates (Greiner Bio-One AG, Frickenhausen, Germany) were pre-coated twice with collagen type I (Becton Dickinson AG, Franklin Lakes, New Jersey, USA) and 0.02 N acetic acid (1:6) (Carl Roth GmbH, Karlsruhe, Germany), before plating the ganglia cells. The DRG cultures were kept in neurobasal medium for 7 days, DMEM medium containing (BioWhittacker, Lonza Group AG, Basel, Switzerland), 2 mM Glut (Invitrogen Corp., Carlsbad, CA, USA) 10% HS (Invitrogen Corp., Carlsbad, CA, USA), 100 IU/L P/S (Invitrogen Corp., Carlsbad, CA, USA), 100 ng/mL nerve growth factor (NGF; Sigma-Aldrich corp., St. Louis, MO, USA), and 4 g/L glucose (Sigma-Aldrich Corp., St. Louis, MO, USA). After 1 week of culture, neurobasal medium was exchanged for myelination media containing minimal essential media (MEM, Invitrogen Corp., Carlsbad, CA, USA), 20 μg/mL PEB (Merck Millipore, Darmstadt, Germany), 50 mg/L L-ascorbic acid (AA, Sigma-Aldrich Corp., St. Louis, MO, USA), 0.5 μM FKL (Sigma-Aldrich Corp., St. Louis, MO, USA), 2 mM L-glut (Invitrogen Corp., Carlsbad, CA, USA), 5% HS (Invitrogen Corp., Carlsbad, CA, USA), 1× N2-supplement (N2; Invitrogen Corp., Carlsbad, CA, USA), 4 g/L D-(+)-glucose 10% (Sigma-Aldrich Corp., St. Louis, MO, USA), and 50 ng/mL NGF (Sigma-Aldrich Corp., St. Louis, MO, USA). The culture myelination medium was renewed every 3 to 4 days. Cultures were kept for 28 days in vitro and treated as indicated from the sixth day after explantation until fixation, followed by staining.
Immunocytochemistry
For immunocytochemistry, cells grown on glass cover slips were initially washed with phosphate-buffered saline solution (PBS) and fixed with 4% paraformaldehyde (PFA; Merck, Darmstadt, Germany) for 30 min for NF-L (neurofilament L) and 10 min for IL-17 receptor (IL-17R), following another washing step with PBS containing 1% bovine serum albumin (BSA; Sigma-Aldrich Corp., St. Louis, MO, USA). Samples were blocked using PBS-based blocking solution containing 10% (NF-L) or 4% (IL-17R) natural goat serum (NGS, DAKO, Hamburg, Germany) and 0.1% (NF-L) or 0.2% (IL-17R) Triton X-100 (Merck, Darmstadt, Germany) for 30 min at RT. We used primary antibodies against IL-17 receptor A (IL-17R A; Abcam, Cambridge, UK), IL-17 receptor B (IL-17R B; Abcam, Cambridge, UK), and rabbit anti-NF-L (Millipore, Billerica, MA, USA), each diluted 1:400. Furthermore, antibodies against MHCI (1:750, mouse monoclonal antibody; Novus Biologicals, Littleton, CO, USA), MHCII (1:50, mouse monoclonal antibody; AbD Serotec Kidlington, UK) and transporter associated with antigen presentation (TAP) II (1:200, rabbit polyclonal; Bioss, Woburn, MA, USA) were used.
Primary antibodies were diluted in PBS, containing 0.1% Triton (0.05% for the MHCI antibody), 10% NGS, and for MHCII, an additional 0.25% BSA. Cells were incubated for 1 hour at 37°C (overnight at 4°C for NF-L). After three washing cycles with PBS, the secondary antibody was applied for 1 hour at RT. The following secondary antibodies were used: Alexa Fluor™ 594 goat anti-rabbit, Alexa Fluor™ 594 mouse anti-rabbit, Alexa Fluor™ 594 goat anti-rabbit (Invitrogen Corp., Carlsbad, CA, USA), 1:200 diluted in PBS and 1% BSA (Sigma-Aldrich Corp., St. Louis, MO, USA) and for NF-L 1:400 diluted in antibody diluent, followed by three washing cycles with PBS. Samples were embedded in 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) containing mounting medium (Vectashield™, Vector Laboratories Inc., Burlingame, CA, USA) and analyzed with an upright fluorescence microscope (Nikon Eclipse TE200, Nikon AG, Tokyo, Japan and Axioplan 2 Imaging, Zeiss, Oberkochen, Germany).
Real-time polymerase chain reaction
Total cellular RNA was extracted using an RNeasy™ Mini Kit (Qiagen, Hilden, Germany) and quantified by NanoDrop-1000 (PEQLAB, Erlangen, Germany). Cells were washed twice with PBS and detached with buffer RLT. Total RNA (400 ng) was applied as matrix for cDNA synthesis using TaqMan™ Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA) and High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s protocol (10 min at 25°C, 120 min at 37°C, and 5 min at 85°C). For subsequent real-time polymerase chain reaction (rtPCR) the thermal cycler (AbiPrism7000, Foster City, CA, USA) was set to run for 2 min at 50°C, 10 min at 95°C, 40 cycles at 95°C for 15 sec, and 1 min at 60°C. Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and TaqMan™ Universal PCR Mastermix (Applied Biosystems, Foster City, CA, USA) were used. cDNA was inserted for amplification at a final concentration of 0.6 μM for each primer. rtPCR was followed by a melting curve analysis. Overall, the experiments were performed with the housekeeping genes 18S (rRNA probe dye, VIC-MGB, Applied Biosystems, Foster City, CA, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to calculate ∆∆ct and shown as expression correlated to housekeeping gene and control expression [31]. cDNA was amplified with the following primers: for IL-17A, 5′-TGG GAT CTG TCA TCG TGC T-3′ and 5′-ATC ACC ATG TTT CTC TTG ATC G-3′; for IL-17B: 5′-GGA CAG CCC TTC TTT GTC TG-3′ and 5′-TGC TTT TTA TAT TTC ATT ACG TGG TT-3′; for IL-17C, 5′-CCA CCC CAA CCT CTG TGT-3′ and 5′-CAA GGA GTC AGC CCA CGA-3′; for P0, 5′-ACC TTC AAG GAG CGC ATC C-3′ and 5′- GCC ATC CTT CCA GCT AGG GT -3′; for KROX-20, 5′-CTG GGC AAA GGA CCT TGA TG-3′ and 5′-GTC CGT GAG AAG GTG GGA CA-3′. Four independent experiments were performed, and for each experiment, three PCR runs, each in triplicate, were analyzed.
Sudan staining
Cultures were stained with Sudan black dye to assess in vitro myelination [32]. Sudan staining has been approved before as an efficient and reliable method for myelin quantification in DRG co-cultures [29]. After washing twice with PBS, cells were fixed for 1 hour using 4% PFA (Sigma-Aldrich Corp., St. Louis, MO, USA), then washed twice again with PBS and treated with 0.1% osmium tetroxide (Sigma-Aldrich corp., St. Louis, MO, USA) for 1 hour. After sequential ethanol treatment (25%, 50%, 70%, each for 5 min), myelin was stained using 0.5% Sudan black (Flukan, Zurich, Switzerland) dissolved in 70% ethanol for 1 hour, followed by treatment with ethanol at decreasing concentrations (70%, 50%, 25% each for 1 min). Myelin was analyzed after Sudan-Black staining, using an upright microscope (Nikon Eclipse TE200, Nikon AG, Tokyo, Japan). The complete cell layer was recorded using a 20x objective, and individual pictures were reconstructed using Adobe Photoshop (version 8.0, Adobe Systems Incorporated, Delaware, USA). Quantification was performed by counting the number of internodes and correlating them to the number of neurons within the cultures. Counting was performed using ImageJ (version 1.41o, National Institutes of Health, USA). To account for inter-experimental variability of the quotient, data are shown on an ordinal scale and normalized to 100%.
Electron microscopy
DRG co-cultures were grown on collagen-coated plastic dishes, washed with PBS, and fixed for 12 h with 1% glutaraldehyde (Serva GmbH, Heidelberg, Germany) and 6% tannin (Merck KGaA, Darmstadt, Germany) in 0.1 M cacodylate buffer (Merck KGaA, Darmstadt, Germany), followed by postfixation in 1% osmium tetroxide (Sigma-Aldrich Corp., St. Louis, MO, USA) for 1 h, dehydrated using a series of alcohol, and finally embedded in Epon. Ultrathin sections were prepared from regions of interest and analyzed by electron microscopy (EM 910; Carl Zeiss, Oberkochen, Germany).
Viability assay
A CellTiter-Blue™ Assay (Promega, Madison, WI, USA) was performed to measure cell viability after IL-17 stimulation for 10 days in vitro. Analysis was performed with cells grown in 96-well plates (Greiner Bio-One AG, Frickenhausen, Germany), 12 samples per concentration and three independent experiments were performed. The CellTiter-Blue™ reagent was added directly to cells cultured in serum-supplemented medium. After an incubation period of 1 to 3 h, spectral data (579Ex/584Em) were recorded using a GENios-Pro reader (LI-COR Biosciences, Bad Homburg, Germany) and analyzed statistically.
MMP-9 and MMP-2 activity
Gelatinase activity was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) as described before [32, 33]. A 12 μL amount of culture supernatants was incubated with Novex™ Tris-glycine sodium dodecyl sulfate sample buffer and applied to a 10% polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA, USA) containing 0.1% sodium dodecyl sulfate (Sigma-Aldrich Corp., St. Louis, MO, USA) and 0.1% gelatin from porcine skin (Sigma-Aldrich Corp., St. Louis, MO, USA); the stacking gels were 5% polyacrylamide gels. After electrophoresis and washing, incubation in Novex™ Zymogram developing buffer (Invitrogen Corp., Carlsbad, CA, USA) for 20 h at RT was followed by staining for 3 h in 30% methanol (Merck, Darmstadt, Germany), 10% acetic acid (Carl Roth GmbH, Karlsruhe, Germany) containing 0.5% Coomassie brilliant blue (Sigma-Aldrich Corp., St. Louis, MO, USA). Finally, gels were destained using staining buffer without dye. Gelatinase activity was quantified using densitometry of unstained bands representing the areas of gelatin digestion. Densitometry was performed using ImageJ, a public domain image processing program (version 1.41o, National Institutes of Health, USA).
Statistical analysis
Four independent experiments were performed for rtPCR, and for each experiment, three PCR runs, each in triplicate, were analyzed. For the Sudan staining three independent experiments were performed, each analyzing 10 wells for each condition and for each 10.000 internodes were counted. Statistical myelin quantification comprises three independent experiments, internodal length is displayed as one representative experiment; an analysis of variance between the three independent experiments revealed no significant difference. For immunocytochemistry (IL-17R, MHCI, TAPII, MHCII) three independent experiments were performed, each with 12 wells for each condition. The graphs depict one representative experiment with the analysis of 50 sectors of the cell layer in each well. An analysis of variance between the three independent experiments revealed no significant difference.
Immunocytochemistry of neurofilament is graphed for three independent experiments.
Electron Microscopy was performed in at least three independent experiments; graph for viability analysis shows results of three independent experiments in a 96-well plate (24 samples per condition). Gelatinase zymography graphs show densitometry of one representative experiment out of four; due to the difficulties in comparing different zymography-runs (for example, background), we present the results of one experiment.
Statistical analysis (mean, standard deviation, and P values) was performed with GraphPad Prism software version 4.0 (GraphPad Software, Inc., La Jolla, CA, USA). Calculations involved unpaired t-tests respectively at 95% confidence interval, one-way ANOVA with Bonferroni and Dunnett’s test (Figure 1G-I, Figure 2E-G, Figure 3A, Figure 4B-C, Figure 5A-C), with P <0.05 considered as statistically significant (P values: * <0.05, ** <0.01, *** <0.001).