Mice

All mice used were on a C57BL/6 background and housed under specific pathogen-free conditions. Rosa26-Cyld-tdTomato mice were generated by Taconic Biosciences. The genotypes of the generated Rosa26-Cyld-tdTomato mice were assessed with the following primers: forward_1 5’-TTGGGTCCACTCAGTAGATGC-3’; forward_2 5’- CTCTTCCCTCGTGATCTGCAACTCC-3’; reverse 5’-CATGTCTTTAATCTACCTCGATGG-3’. A PCR with all 3 primers detects the Rosa26 wildtype allele (299 bp; forward_2 and reverse primer) and the Rosa26-Cyld-tdTomato knock-in allele (744 bp; forward_1 and reverse primer). Rosa26-Cyld-tdTomato mice were crossed to Cx3cr1CreER mice [23] to obtain R26-Cyld-OECx3 mice with the genotype Cx3Cr1CreER/WT; Rosa26Cyld−OE−tdTom/WT. Rosa26-tdRFP reporter mice [24] were crossed to R26-Cyld-OECx3 mice to obtain littermate control animals (R26RFP/WT) with the genotype Cx3Cr1CreER/WT; Rosa26RFP/WT. For the EAE experiment, heterozygous Cyld+/- mice [25] were crossed to obtain Cyld−/− mice and heterozygous (Cyld+/-) and homozygous (Cyld+/+) littermate controls.

Tamoxifen treatment

Tamoxifen (Sigma-Aldrich) at a concentration of 20 mg/ml was dissolved in olive oil (Sigma-Aldrich) containing 5% ethanol by rotation overnight at 4 °C. Mice were injected subcutaneously with 100 μl (= 2 mg tamoxifen) at the age of 3–4 weeks on two days with a one-day break in between. R26RFP/WT littermate controls were treated equally.

Immunofluorescence stainings and Sholl analysis

Brains were isolated from mice perfused with 0.9% NaCl (Sigma-Aldrich), were fixed in 4% PFA for 6 h at 4 °C and cryoprotected in 30% sucrose. The tissue was embedded in TissueTek® O.C.T™ compound (Sakura Finetek) and 50 μm free-floating sections were cut at a cryostat.

Endogenous peroxidase was blocked in methanol containing 0.6% H2O2 for 20 min. at room temperature. Endogenous biotin and avidin binding sites were blocked with the Avidin/Biotin Blocking Kit (Vector Laboratories) for 15 min. each at room temperature. Sections were blocked with 10% ROTI®ImmunoBlock (Carl Roth) and 2% bovine serum albumin (BSA; PAN-Biotech) diluted in TBST (Tris Buffer Saline containing 1% TWEEN® 20) for 15 min. at room temperature. Sections were incubated with primary antibodies against Iba-1 (goat polyclonal, 1:500, FUJIFILM Wako Chemicals) and RFP (rabbit polyclonal, 1:1000, Rockland Immunochemicals) diluted in 2% BSA/TBST overnight at 4 °C. Sections were washed and incubated with secondary antibodies against goat IgG (donkey polyclonal, Alexa Fluor 647, 1:400, Invitrogen) and against rabbit IgG (donkey polyclonal, Biotin, 1:500, Invitrogen) in TBST for 1.5 h at room temperature. After washing, sections were treated with the TSA® Cyanine 3 detection kit (Akoya Biosciences) following manufacturer’s instructions. Sections were washed and mounted with Vectashield® Antifade Mounting Medium with DAPI (Vector Laboratories). For microglia quantification, images were acquired at a DMi8 widefield microscope (Leica) with an objective magnification of 20 × and only microglia with a clear cell body in-focus were manually counted. For Sholl analysis, images were acquired at a TCS SP8 multiphoton microscope (Leica) with an objective magnification of 20 × with 3 × optical zoom and 1.0 µm increments. Sholl analysis was performed in Fiji (ImageJ). First, Iba1 stacks were collapsed using maximum projection. The threshold was set to the same level for all images and only microglia with the cell body at the center of the stack analyzed. The cell body was defined and Sholl analysis was performed with a starting radius of 1 μm from the center and in increments of 1 μm.

Cell isolation from the CNS

Brains were dissected from mice transcardially perfused with 0.9% NaCl solution (Sigma-Aldrich) and digested with 1 mg/ml Papain (Sigma-Aldrich) and 100 µg/ml DNase I (Roche) in HBSS with calcium and magnesium with mechanical dissociation with the GentleMACS homogenizer (Miltenyi, Bergisch Gladbach Germany) at 37 °C for 30 min. CNS homogenates were passed through a 70 μm cell strainer and cells were separated using a 70/30% Percoll (Sigma-Aldrich) gradient centrifugation for 45 min, 500 × g at 18 °C without brakes. Myelin was discarded and cells at the 70/30% interphase were carefully collected for further processing.

Microglia isolation by magnetic-activated cell sorting (MACS)

Cells obtained from the CNS cell isolation were resuspended in 90 µl MACS buffer and 10 µl anti-CD11b MicroBeads (Miltenyi Biotec) and incubated for 15 min. at 4 °C. After washing, cells were applied to a MS column in an OctoMACS separator (Miltenyi Biotec), washed 3 × with MACS buffer and CD11b+ cells were eluted.

Flow cytometry

Cells obtained from the CNS cell isolation were resuspended in anti-mouse CD16/CD32 (5 µg/ml, BioXCell,) in FACS buffer (PBS containing 2% fetal calf serum) for 10 min. at 4 °C to block Fc receptors. Cells were stained for 30 min at 4 °C on the cell surface with antibodies against CD4 BV421 (GK1.5, rat monoclonal, 1:400, BioLegend), CD45 BV510 (30-F11, rat monoclonal, 1:300, BioLegend), TCR-β FITC (H57-597, hamster monoclonal, 1:300, BioLegend), CD11b PECy7 (M1/70, rat monoclonal, 1:1000, eBioscience™, Thermo Fisher Scientific), CD8a PerCP (53–6.7, rat monoclonal, 1:500, BioLegend), MHC-II eFluor™450 (M5/114.15.2, rat monoclonal, 1:4000, eBioscience™, Thermo Fisher Scientific). For dead cell exclusion, fixable viability dye eFluor™780 (eBioscience™, Thermo Fisher Scientific) was added to the surface staining. Afterwards, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Bioscience) and stained overnight at 4 °C with anti-CD68 APC antibody (FA-11, 1:500, rat monoclonal, BioLegend). Samples were acquired at a FACSCanto II using FACS Diva software (BD Bioscience) and analyzed with FlowJoTM v10 software (BD Bioscience). For all analyses, doublets (FSC and SSC properties) and dead cells were excluded.

RNA isolation, reverse transcription and real-time PCR

RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System (Promega) following manufacturer’s guidelines. RNA concentrations were determined by measuring absorbance at 260 nm and 280 nm using the NanoQuant Plate™ (Tecan) at an Infinite M200 pro plate reader (Tecan). cDNA was synthesized with the QuantiTect® Reverse Transcription Kit (Qiagen) using all RNA obtained following manufacturer’s guidelines. Real-time PCR (RT-PCR) was performed with the StepOnePlus™ Real-Time PCR System (Life Technologies) using SYBR Green reagent (Promega). Fold enrichment was calculated using the Delta-Delta CT method normalized to hypoxanthin-guanin-phosphoribosyltransferase (Hprt) as house-keeping gene reference. Hprt primers were self-designed: forward 5’-CGTCGTGATTAGCGATGATG-3’, reverse 5’- TCCAAATCCTCGGCATAATG-3’. Cyld primers were ordered as QuantiTect Primer Assay (QT00103768, Qiagen).

RNA isolation and RNA sequencing

For RNA sequencing, RNA was isolated with the RNeasy Micro Kit (Qiagen). RNA quality was assessed with the Qubit RNA High Sensitivity Assay Kit on a Qubit 3 Fluorometer (Invitrogen) and with the High Sensitivity RNA ScreenTape on a 4200 TapeStation system (Agilent). 100 ng RNA were used for library preparation with the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina using NEBNext Multiplex Oligos for Illumina (New England Biolabs). cDNA quality was assessed with Agilent’s High Sensitivity DNA kit on an Agilent 2100 Bioanalyzer. Quantity of the library was assessed with the Qubit 1X dsDNA assay kit on a Qubit 4 Fluorometer (Invitrogen) and library size was determined with a D1000 ScreenTape analysis on a 4200 TapeStation system (Agilent). All libraries were sequenced in paired-end mode (2 × 50 nt) on an Illumina NovaSeq 6000 instrument resulting in an average of 20 million distinct sequencing reads per library.

RNA sequencing data analysis

Quality control of the sequencing data was performed with the FastQC tool (v0.12.1). RNA sequencing reads were mapped to the Mus_musculus.GRCm38 reference genome (ENSEMBL v84) using kallisto (v0.46.0). Alignments were processed with kallisto to obtain gene transcripts per million (TPM) values and gene counts. Differential gene expression analysis was performed with DESeq2 (v1.34.0) and apeglm (v1.16.0) was applied for shrinkage of log-fold changes.

Experimental autoimmune encephalomyelitis (EAE)

Mice were immunized 12 weeks after tamoxifen administration at the age of 15–16 weeks. MOG35-55/CFA was prepared by mixing 1 mg/ml MOG35-55 (GenScript) in PBS with Complete Freund’s Adjuvants (CFA, BD Biosciences) containing mycobacterium tuberculosis H37RA (BD Biosciences). 50 μg of the MOG35-55/CFA emulsion were injected subcutaneously into the tail base. Mice were also injected intraperitoneally with 150 ng pertussis toxin (List Biological Laboratories) in PBS at the day of immunization and two days later.

Mice were weighed daily and clinical scores were documented as follows: 0 - no disease; 0.5 - limb tail; 1 - paralyzed tail; 1.5 - weakened righting reflex; 2 - no righting reflex; 3 - partial paralysis of hind legs; 3.5 - paralysis of one hind leg; 4 - paralysis of both hind legs.

LPS-induced neuroinflammation

LPS (from Salmonella typhimurium, Sigma-Aldrich) was solved in PBS at a concentration of 0.2 mg/ml. Mice were injected 12 weeks after tamoxifen administration at the age of 15–16 weeks with 1 mg/kg LPS intraperitoneally and were sacrificed 24 h later.

Statistics

Statistical analyses were performed with Prism v10 software (GraphPad). Values are shown as mean ± standard error of the mean (SEM) unless indicated otherwise. P values were considered significant with * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.