NG2 Plays a Role in Neuroinflammation but Is Not Expressed by Immune Cells

Kitic et al.

NG2 plays a role in neuroinflammation but is not expressed by immune cells

Supplementary information

Materials and Methods

Mice

NG2-EYFP knock-in mice used in experiments were obtained from heterozygous breeding and maintained under specific pathogen-free conditions at the central animal facility in Mainz.Genotyping procedure was performed as described [2].Experiments were performed in accordance with guidelines of the central animal facility institution (TARC, University of Mainz) and in accordance with relevant laws and guidelines and with permission of the state Rhineland-Palatinate.

Active EAE induction and clinical evaluation

Mice were immunized according to a previously described protocol[1].Eight to 12-week old mice were subcutaneously injected with 100 μl of emulsion containing 50 μg MOG35-55 (GenScript) and550 μg of heat-inactivatedM. tuberculosis H37 Ra in CFA (BD). On days0 and 2 following MOG/ CFA administration, mice received intraperitoneal (i.p.) injections of 100 or 200 ng of pertussis toxin (List Biological Laboratories, Inc.). According to the supplier's certificate of analysis, minimal concentration of pertussis toxin for inductionof positive response in CHO cell assay was 0,1 ng/ml forthe lot #180217A1, used in initial experiments shown in Fig. 1, and 0,002 ng/ml for the lot #180232A1, used in the experiment shown in Supplementary Fig. 1.Adenylatecyclase activity in the presence of 1 μM calmodulinwas 15,5 and 3 pmol cAMP/min/μg toxin for the lot #180217A1 and #180232A1, respectively. Mice were scored daily according to the following criteria: 0, healthy; 1, limp tail; 2, impairment of righting reflex and hind limbs weakness; 3, partial paralysis of hind limbs; 4, complete paralysis of hind limbs and fore limb weakness; 5, moribund.

Immunohistochemistry

Deeply anesthetizedmice wereperfused with saline and 4% paraformaldehyde (PFA) solution. CNS tissue was fixed overnight in 4% PFA and embedded in paraffin before sectioning. Active EAE samples were processed and stained by Histology Core Facility, University Medical Center of the Johannes Gutenberg University Mainz. Luxol Fast Blue (LFB) staining was performed to visualize the extent of myelin loss. T cell infiltrates were immunolabelledwith rabbit anti-CD3 (1:250, Abcam), followed by application of HRP-coupled anti-rabbit secondary antibody (1:200, Vector Laboratories) and signal development using 3, 3 -diaminobenzidine (DAB) as a substrate for HRP.

EYFP-reporter expression analysis

CNS pericytes were isolated as previously described [5]. Cell suspension was then mixed with following dyes/ antibodies: viability dye eFluor®780 (1:1000, eBioscience), mouse anti-mouse CD45.2V500 (1:200, BD Bioscience),rat anti-mouse CD31 APC (1:75, BD Bioscience)and rat anti-mouse CD140b PE (PDGFRb, 1:100, eBioscience), followed by a fixation in 2% formaldehyde solution (Roti®-Histofix, Carl Roth GmbH). Pericytes were definedas CD45-/CD31-/PDGFRB+population. Myeloid cells were obtained according to a previously published procedure [3]. Briefly, spleen tissue was subjected to digestion in PBS solution (+ Ca2+, Mg2+) containing 2 mg/ml collagenase type II (Gibco) and 20 U DNase I (Sigma-Aldrich) for 20 min at 37°C, further homogenized with a 18-gauge needle and passed through a 70-μm cell strainer. This was followed by removal of erythrocytes by incubation in ACK lysis buffer. Finally, the following dyes/ antibodies were applied: viability dye eFluor®780,mouse anti-mouse CD45 V500, rat anti-mouse/ humanCD11b APC (1:1000, Biolegend) andarmenian hamster anti-mouseCD11c PE-Cy7 (1:1000, Biolegend). Samples were analysed by FACSCanto II system (BD Biosciences) and FlowJo software (FlowJo, LLC).

Western blot

Thirty milligrams of brain tissue were harvested from naive NG2EYFP/EYFP and NG2wt/wt mice and homogenized in 1X RIPA buffer (Cell Signaling Technology) containing protease inhibitors cocktail (Roche). For isolation of CD11b+ and CD90+ splenocytes from naive and immunized mice, spleen tissue was processed as described in a previous section, without erythrocyte lysing step.Cell suspension was subjected to magnetic-activated cell sorting (MACS) procedure using mouse/ human CD11b and mouse CD90.2 MicroBeads, according to manufacturer's instructions (Miltenyi Biotec). Obtained cells were lysed with 1X RIPA buffercontaining protease inhibitors. Protein concentration was determined usingBCA Protein Assay (Thermo Fisher Scientific). Fifty (brain) or 60 μg (immune cells) of protein samples were loaded into NuPage™ 4-12% Bis-Trisgels (Invitrogen™,Thermo Fisher Scientific) and run for 1 h at 150 V in 1X NuPage® MOPS SDS Running buffer (Novex™, Thermo Fisher Scientific). Proteins were transferred onto PVDF membrane (Amersham Hybond P, GE Healthcare Life Sciences) for 2 h at 300 mA in 1X Tris-Glycine Transfer Buffer. The membrane was then incubated with a blocking solution containing 4% milk and 0,1% Tween-20 in 1X PBS for 1 h at room temperature. Afterwards, the membrane was incubated with a rat monoclonal anti-mouse NG2 antibody (dilution 1:100,[4]) overnight at 4°C. HRP-conjugated goat anti-rat secondary antibody (dilution 1:10000, Dianova) was applied for 1 h at room temperature.HRP-conjugated anti-mouse actin antibody (1:10000, Santa Cruz Biotechnology) was incubated for 2 h at room temperature. Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) was used for signal development, which was then analysed by Bio-Rad ChemiDoc MP system (Bio-Rad Laboratories).

Quantitative Real-time PCR

PDGFRB+ pericytes were isolated from a wild type brain tissue as described in a previous section and sorted usingFACSAriaII system (BD Biosciences). 1 × 105 pericytes were used for subsequent RNA isolation using RNeasy Micro Kit (Qiagen). RNA from 3 - 5 × 106MACSed CD11b+ and CD90.2+ cells was extracted using peqGOLD Total RNA Kit (VWR Peqlab). 50-100 ng pericyte-derived RNA and 750 ng RNA from immune cellsweresubjected tosubsequentcDNAsynthesis using SuperScript reverse transcriptase (Invitrogen). Quantitative real-time PCR analysis of Cspg4 and Hprt1 genes was done using QuantiTectPrimer Assay (Qiagen) and StepOnePlus Real-Time PCR System (Applied Biosystems™, Thermo Fisher Scientific), according to manufacturers' instructions.

Intracellular cytokine detection

For analysis of cytokine production from MOG-specific Th cells, NG2wt/wt, NG2EYFP/wt and NG2EYFP/EYFP mice were immunized as described for active EAE, with omission of pertussis toxin. Skin-draining lymph nodes were harvested 10 days after immunization, homogenized in 1X PBS/ 2% FCS solution and passed through a 40-μm cell strainer (Falcon®, Corning). 3 × 106 cells were re-stimulated with 20 μg/ml MOG for 6 h at 37°C. Brefeldin A (5 mg/ml) was included for the last 3 h of incubation.Immediately after that, cell suspensionwas labelled with viability dye eFluor®780 and rat anti-mouse CD4 PerCP (1:1000, Biolegend). Fixation/ permeabilization and intracellular immunostaining was then performed using BD Cytofix/Cytoperm™ kit (BD Biosciences), according to the manufacturer's instructions. The following antibodies were used: rat anti-mouse GM-CSF PE (1:200, eBioscience), rat anti-mouse IFN-γ PE-Cy7 (1:1000, eBioscience), anti-mouse/ rat IL-17A eFluor®450 (1:300, eBioscience) and armenian hamster anti-mouse CD154 (CD40L) APC (1:300, Biolegend). Samples were analysed by FACSCanto II system (BD Biosciences) and FlowJo software (FlowJo, LLC).

Statistical analysis

Experimental values were represented as mean ± SEM and p values were calculated using one-way analysis of variance (ANOVA) test with Bonferroni'stest for multiple comparisons. All statistical calculations were performed by Prism GraphPad software (GraphPad Software, Inc.).

Supplemental references

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2Karram K, Goebbels S, Schwab M, Jennissen K, Seifert G, Steinhauser C et al (2008) NG2-expressing cells in the nervous system revealed by the NG2-EYFP-knockin mouse. Genesis 46: 743-757

3Lukas D, Yogev N, Kel JM, Regen T, Mufazalov IA, Tang Y et al (2017) TGF-β inhibitor Smad7 regulates dendritic cell-induced autoimmunity. Proceedings of the National Academy of Sciences 114: E1480-E1489

4Niehaus A, Stegmuller J, Diers-Fenger M, Trotter J (1999) Cell-surface glycoprotein of oligodendrocyte progenitors involved in migration. J Neurosci 19: 4948-4961

5Tigges U, Welser-Alves JV, Boroujerdi A, Milner R (2012) A novel and simple method for culturing pericytes from mouse brain. Microvasc Res 84: 74-80