All experimental procedures were carried out at the Neuroscience Institute Cavalieri Ottolenghi (NICO, Orbassano), approved by the Ethical Committee of the University of Torino and authorized by the Italian Ministry of Health (authorization number: 59/2016-PR). The experiments have been carried out in accordance with the European Communities Parliament and Council Directives of 24 November 1986 (86/609/EEC) and 22 September 2010 (2010/63/EU). Mice were housed with a 12 hours light/dark cycle and free access to food/water. Adequate measures were taken to minimize pain and discomfort.
TNFAIP3flox/flox mice not commercially available, were obtained from the no-profit RIKEN BioResource Center (Japan). The mice were deposited from Dr. H. Honda (http://www.brc.riken.jp/inf/en/index.shtml, RBRC05494) (19). The transgenic mice carrying the CRE recombinase under the control of the myeloid-specific chemokine C-X3-C motif receptor 1 (cx3cr1-CRE+/-) promoter element were purchase from The Jackson Laboratories (Bar Harbor, ME, US) (B6J.B6N(Cg)-cx3cr1tm1.1(CRE)Jung/J; stock No: 025524) (20). 3 months-old female and male TNFAIP3flox/flox::cx3cr1-CRE+/- (TNFAIP3cx3cr1-KO, homozygous knock-out), and TNFAIP3flox/flox::cx3cr1-CRE-/- (WT, wild-type) mice were used for all experimental paradigms. Their genotype were confirmed by means of polymerase chain reaction (PCR) according to the manufacturer’s protocol.
3 months-old TNFAIP3cx3cr1-KO (n=4) and WT (n=8) littermate mice were euthanized by inhalation of isoflurane. Spleen, axillary and inguinal lymph nodes and femoral bone were removed and immersed in fresh phosphate buffered saline (PBS). To collect bone marrow the femurs were cut at both ends and with a 23G (gauge) needle and a 10 ml syringe filled with ice-cold PBS the bone marrow was flushed out. Tissues (i.e. spleen, axillary and inguinal lymph nodes) were manually homogenized, and rinsed with PBS. The suspended cells obtained from spleen, lymph nodes and bone marrow were centrifuged at 1500 RPM for 10 minutes. The supernatant was discarded and the pellet was suspended in 3mL of red blood cells lysis solution (Z3141, Promega, Madison, WI, US) and incubate at 4°C for 10 minutes. After adding 10 ml of PBS, the cells were centrifuged at 1500 RPM for 10 minutes. The supernatant was discarded and the cells were cryopreserved at−80 °C until use in Roswell Park Memorial Institute (RPMI) 1640 (Invitrogen Life Technologies, Grand Island, NY, US), supplemented with 30% heat-inactivated fetal bovine serum (FBS, Invitrogen Life Technologies) and 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich, St Louis, MO, US).
After gentle thawing at 37 °C, the cryopreserved cells obtained from spleen, lymph nodes and bone marrow were immediately added to 5 ml RPMI 1640 (Invitrogen Life Technologies), supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies) and centrifuged to remove DMSO. Samples were re-suspended in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and counted for flow cytometry experiments. Macrophages (i.e CD11b+F4/80+), monocytes (i.e CD11b+Ly-6C+Ly-6G+), dendritic cells (DCs) (i.e CD11c+CD86+), NK (i.e. CD49b+), NK T (CD3+CD49b+), T (i.e. CD3+, CD3+CD4+, CD3+CD8+), and B (ie. CD45R-B220+) cells were evaluated in the same spleen and lymph nodes specimen of 3 months-old TNFAIP3cx3cr1-KO and WT littermates mice. The common myeloid precursors (CMPs, Lin-Sca1-c-kit+CD34+CD16/CD32-) and the common monocyte and granulocyte precursors cells (CMGPs, Lin-Sca1-c-kit+CD34+CD16/CD32+) were evaluated in the same bone marrow specimen of 3 months-old TNFAIP3cx3cr1-KO and WT littermate mice. Non-specific sites of 1 × 106 cells were blocked with rabbit immunoglobulins G (IgG, Sigma-Aldrich), and cells were then incubated with fluorochrome-conjugated monoclonal Ab (mAb) and isotype–matched negative controls for 10 min at 4 °C.
The following anti-mouse mAbs were used: CD3-allophycocyanin (APC, 130-109-838), CD45R-B220- phycoerythrin (PE, 130-102-292), CD49b- fluorescein isothiocyanate (FITC, 130-102-258), CD11c-APC (130-102-493), F4/80-APC (130-102-379), CD86-PE (130-102-604), c-kit-(CD117)-PE (130-111-693), CD16/CD32-PE (130-102-429) (Miltenyi Biotec, Bergisch Gladbach, Germany), and CD4-phycoerythrin-cy7 (PECy7, BMS25-0041-82), CD8-fluorescein isothiocyanate (FITC, BMS11-0081-82), CD11b-PE (BMS12-0112-82), Ly6-C-AlexaFluo488 (BMS53-5932-82), Ly6-G-APC (BMS17-5931-82), Lineage cocktail (Lin)-PerCP-Cy5.5 (BD Pharmingen, 51-9006964, San Jose, CA, US), Sca1-APC (130-106-425), CD34-FITC (130-105-831) (eBioscience, ThermoFisher Scientific, San Diego, CA, US). Living cells identified by propidium iodide (Sigma-Aldrich) exclusion were gated according to their light-scatter properties to exclude cell debris. Samples were analyzed using a CyAn ADP, running Summit 4.3 software (Beckman Coulter, Brea, CA, US).
3 months-old TNFAIP3cx3cr1-KO (n=3) and WT (n=6) littermates mice were deeply anesthetized (ketamine 200mg/kg, xylazine 50mg/kg) and trans-cardially perfused with 4% paraformaldehyde in 0.12 M phosphate buffer, pH 7.2–7.4. Brain and spinal cord were removed and immersed in the same fixative at 4°C for 24 h and then cryo-protected in 30% sucrose in 0.12 M phosphate buffer as reported in (21). Brains and spinal cords were frozen and serially cut by a cryostat in 30 µm-thick coronal sections collected in PBS. In order to detect microglia, slices were incubated overnight at 4°C with the polyclonal anti-rabbit Ionized Calcium Binding Adaptor molecule 1 (Iba1, 019-19741, WAKO, Japan) diluted 1:1000 in PBS 1X with 0.25% Triton X-100 and 1.5% normal donkey serum (NDS). The secondary biotinylated anti-rabbit antibody (715 065 152, Jakson Immuno Research, Philadelphia, PA, US) diluted 1:100 in a solution of PBS containing 0,2% Triton-X100 and 1,5% of NDS were used. Immunohistochemical reactions were performed with the avidin–biotin–peroxidase method (Vectastain ABC Elite kit; Vector Laboratories, Burlingame, CA, US) and revealed using 3,3’-diaminobenzidine (3% in Tris–HCl) as chromogen. Images for morphometric analysis were acquired by means of the Nikon Eclipse E600 microscope and analyzed by means of the Cell Counter Tools of ImageJ software (http://rsbweb.nih.gov/ij/index.html). Iba1+ positive (+) cell number (cell number/mm2) in the corpus callosum (Bregma 1.10 mm, Interaural 4.90; Bregma -0.82 mm, Interaural 2.98) and in the white and grey matter of the cervical level of spinal cord were quantified. At least 3 sections for each animal were evaluated.
Statistical analysis was performed using GraphPad Prism(GraphPad Software, version 5; San Diego, CA,US). Continuous data were presented as medians and ranges and categorical data were given as counts and percentages. Normality of distribution was assessed by the Shapiro-Wilk test. The differences between groups were calculated with Mann-Whitney U test. p values < 0.05 were considered significant.