Bone marrow (BM)-derived macrophages in vitro culture
Bone marrow (BM) cells were flushed from femur and tibia of C57BL/6 mice and cultured
with macrophage colony-stimulating factor, M-CSF, (100 ng ml
?1
, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in ?-minimum essential medium
(MEM) (Invitrogen) for 7 days. The purity of BM-macrophage cultures was confirmed
by FACS using CD11b (1:100, BD Biosciences, Mountain View, CA, USA) and F4/80 (1:100,
BioLegend, San Diego, CA, USA) antibodies (average purity 80–90 %, not shown). After
7 days, BM-derived macrophages were cultured in ?-MEM medium (Invitrogen) and differentiated
to M1 and M2, respectively, with rIFN? (20 ng/ml, Peprotech, USA) and rIL4 (20 ng/ml,
RD System, Minneapolis, MN, USA) for 48 h.
Peritoneal macrophage cultures
Sterile 3 % thioglycolate (TG) was i.p. injected in C57BL/6 mice. After 5 days, mice
were sacrificed, and peritoneal cells (Pec) were recovered by lavage with saline (S.A.L.F,
Bergamo, Italy). Pec were centrifuged at 1200 rpm for 10 min at 4 °C. Supernatants
were eliminated and pellets re-suspended in RPMI 1640 medium supplemented with penicillin
(100 U/ml), streptomicin (100 U/ml), and ultra-glutamine (100 U/ml) (Lonza, Milano,
Italy). Cells were counted with Turk’s solution (Merck Chemicals), plated at 5?×?10
6
in 60-mm petri dishes (Becton Dickinson) and then incubated at 37 °C and 5 % CO
2
for 1 h. Finally, cells were washed twice with saline and RPMI 1640 medium supplemented
with 10 % FBS, penicillin (100 U/ml), streptomicin (100 U/ml), and ultra-glutamine
(100 U/ml) was added. One hour after incubation at 37 °C and 5 % CO
2
, Pec polarization into M1 or M2 was performed by stimulation with rIFN? (20 ng/ml,
Peprotech, USA) plus LPS (100 ng/ml) for 4 h or rIL4 (20 ng/ml, RD System, Minneapolis,
MN, USA) for 18 h.
RT-PCR analysis
RT-PCR analyses were performed on in vitro BM-derived macrophage assay and for IL4
gene therapy studies of the EAE model. Briefly, RNA was extracted using Trizol (Invitrogen)
according to manufacturer’s instructions. Residual DNA was removed by treatment with
1 U DNase per 1 ?g RNA (RQ1 RNase-free DNase, Promega) at 37 °C for 30 min. Complementary
DNA (cDNA) synthesis from 3 to 5 ?g total RNA was performed using Ready-To-Go You-Prime
First-Strand Beads (Amersham) and Random Hexamer (New England Biolabs, Ipswich, MA,
USA) according to the manufacturer’s instructions. Arg-1 (Mm00475988_m1), CCL17 (Mm005161
36_m1), CD206 (Mm00485148_m1), IFN? (Mm01168134_m1), IL-1? (Mm01336189_m1), IL4 (Mm00445259_m1),
IL6 (Mm00446190_m1), iNOS (Mm00440502_m1), TNF-? (Mm00443258_m1), and Ym1 (Mm00657889_mH).
Messenger (mRNA) levels were measured by real-time RT-PCR (Applied Biosystems, Invitrogen).
The 2???CT method was used to calculate relative changes in gene expression 20].
Fluorescence microscopy
BM-derived and Pec macrophages, differentiated as above, were stained for CD206 FITC
(RD) and, IL6 PE (Becton, Dickinson) for 5 min. Cells were then fixed in PFA 4 %
for 10 min and incubated in 5 % FBS, 0.1 % Triton X-100, and PBS for 1 h to block
any nonspecific binding site. Nuclei were stained with DAPI. A Leica SP5 (Leica Microsystems,
Milano, Italy) confocal microscope and a GE Healthcare Delta Vision were used for
image acquisitions.
Co-culture of DCs-CD4
+
T cells with M2 macrophages
BM cells were differentiated in dendritic cells (DCs) for 6 days in RPMI complete
medium with GM-CSF (25 ng/ml) and rIL4 (25 ng/ml) (RD Systems) and were activated
with LPS (1 ?g/ml) for 4 h. T cells were obtained from the spleens from TCR transgenic
mice (2D2) specific for the myelin oligodendrocyte glycoprotein peptide, MOG 35–55
(Espikem, Florence, Italy), on the C57Bl/6 background 21] by preparing a cell suspension by mechanical dispersion using a cell strainer, followed
by positive selection of CD4
+
cell using Miltenyi columns (Miltenyi Italy). Fifty thousand activated DCs were co-cultured
with 2?×?10
5
CD4
+
T cells and with the following concentration of MOG peptide: 0.3, 1, 3, 10 ?M in triplicates.
These cultures were added 2?×?10
5
non stimulated (NS) or M2 macrophages, as indicated, and then incubated at 37 ° C
and 5 % CO
2
for 48 h.
ELISA assay for mIL4, IL-2, and IL6
mIL2 and IL6 were measured from supernatants of DCs-CD4
+
cells co-cultures with NS and M2 macrophages, by using a validated mouse-specific
mouse IL2 and IL6 ELISA (RD Systems). mIL4 was measured in supernatants and lysates
of infected cells or CSFs (withdrawn from mice cisterna magna by capillarity) from
IL4-injected EAE mice, using mouse IL4 ELISA (RD Systems).
Generation of mouse IL4-expressing lentivirus
Murine interleukin 4 (mIL4) was obtained from a plasmid expressing mIL4 in an adenoviral
vector (Ad-G/IL4) 18]. The coding sequence of mIL4 was extracted with forward and reverse primers specifically
designed to contain BamHI and SalI digestion sites, both synthesized by Primm S.r.l.
(Milano, Italy). A third-generation lentivirus expressing mIL4 was generated cloning
the mIL4 cDNA in the backbone of a p277 lentiviral transfer vector and producing a
lentivirus as previously described 22]. A GFP-expressing lentivirus was also produced to be used as negative control in
all the experiments.
Mice
Six- to 8-week-old C57Bl/6 female mice were purchased from Charles River Laboratories
(Calco, Italy). All mice were housed in specific pathogen-free conditions, in roomy
cages, allowing free access to food and water. All efforts were made to minimize animal
suffering and to reduce the number of mice used, in accordance with the European Communities
Council Directive of November 24, 1986 (86/609/EEC). All procedures involving animals
were performed according to the animal protocol guidelines prescribed by the Institutional
Animal Care and Use Committee (IACUC # 449) at San Raffaele Scientific Institute (Milan,
Italy). Because of the use of lentiviral vectors, animals were housed in isolated
cages in the Biosafety Level 2 room of the Animal Care facility at San Raffaele Scientific
Institute.
IL4 gene therapy of EAE mouse model
Chronic EAE was induced in female C57BL/6 mice, by subcutaneous with 300 ?l of 200 ?g
per mouse of MOG35–55 in Freund’s Adjuvant Incomplete liquid, IFA, (Sigma) supplemented
with 8 mg ml
?1Mycobacterium tuberculosis (strain H37Ra; Difco, Lawrence, KS, USA). Pertussis toxin (500 ng, List Biological
Laboratories, Campbell, CA, USA) was injected i.v. on the day of the immunization
and again 2 days later. IL4-expressing lentivirus or GFP-expressing lentivirus were
injected in the cisterna magna (i.c.) of the mice at 12 d.p.i. A 30-gauge needle attached
to a Hamilton syringe was inserted into the intrathecal space of the cisterna magna
of anesthetized mice 23]. IL4-expressing or GFP-expressing lentiviruses (10 ?l) in sterile phosphate-buffered
saline (10
9
PFU ml
?1
) were injected over 10 s. Mice were weighed and scored for clinical signs daily up
to the day of culling. Clinical assessment of EAE was performed according to the following
scoring criteria: 0?=?healthy, 1?=?limp tail, 2?=?ataxia and/or paresis of hindlimbs,
3?=?paralysis of hindlimbs and/or paresis of forelimbs, 4?=?tetraparalysis, and 5?=?moribund
or death. EAE mice were killed at 34 d.p.i for real-time PCR and histological analysis.
Preparation of CNS mononuclear cells
Mice were deeply anesthetized and perfused transcardially with cold phosphate-buffered
saline at the indicated time point. The brains and the spinal cords were dissected
out at the desired time point, removed, and homogenized through a 70-?m cell strainer
in HBSS. Mononuclear cells were isolated using a neural dissociation kit (Milteny
Biotech) and by 30/37/70 % Percoll (GE Healthcare) gradient centrifugation and collection
of mononuclear cells from the 37/70 % interphase.
CD11b
+
cell separation
CNS mononuclear cells were spun at 300g for 10 min and then re-suspended in cold MACS buffer (1× PBS, 0.5 % BSA, 2 mM EDTA).
Cells were incubated with biotin-conjugated monoclonal antibodies against CD11b (Mac-1,
Rat IgG2b) (Myltenyi Biotech) at a concentration of 10 ?l/10
7
total cells in 40 ?l MACS buffer for 10 min at 4 °C. Anti-biotin microbeads (20 ?l/10
7
cells in 80 ?l MACS buffer) (Myltenyi Biotech) were added to the cells and incubated
for 15 min at 4 °C. Finally, the cells were loaded on the MS-columns and the column-bound
CD11b
+
fraction isolated (Myltenyi Biotech). Cells were then centrifuged at 300g for 10 min, and re-suspended in 500 ?l of TRizol (Invitrogen) and frozen at –80 °C.
Histological evaluation
At 34 d.p.i., at least three mice per group were perfused through the left cardiac
ventricle with saline plus EDTA 0.5 mM for 10 min followed by fixation with cold 4 %
paraformaldehyde, PFA, (Sigma) in 0.1 M phosphate buffer (pH 7.4). Subsequently, the
spinal cords and brains from EAE mice were carefully dissected out and post-fixed
in 4 % PFA overnight and processed for cryogenic embedding. The quantification of
neurological damage in EAE mice was performed via histological analysis of 10-?m frozen
CNS sections of control or IL4-injected or GFP-injected EAE mice. Three different
stainings were used to detect inflammatory infiltrates (hematoxylin and eosin), demyelination
(Kluver Barrera), and axonal damage (Bielshowsky). Neuropathological findings were
quantified on an average of 10 complete cross sections of spinal cord per mouse taken
at eight different levels of the spinal cord. The number of perivascular inflammatory
infiltrates were calculated and expressed as the numbers of inflammatory infiltrates
per square millimeter, and demyelinated areas and axonal loss were expressed as percentage
of damaged area.
Immunohistochemistry and immunofluorescence
T cells were stained using a rat anti-CD3 (pan-T cell marker, Cat.No. MCA1477, Serotec,
Oxford, UK), CD206 positive cells were stained with rat anti-mouse CD206 (Cat.No.
MCA2235, Serotec, Oxford, UK), revealed with a biotin-labeled secondary anti-rat antibody
(Cat.No. BA9400, Vector Laboratories, Burlingame, CA). Macrophages were stained with
biotin-labeled BS-I isolectin B4 and IB4 (Cat.No. L2140-0, Sigma-Aldrich, St. Louis,
MO). Biotin-labeled antibodies were developed with the ABC kit (Cat. No. PK-6200,
Vector Laboratories, Burlingame, CA) followed by liquid DAB
+
Substrate Chromogen System (Cat. No. K3467, Dako, Carpinteria, CA). We also stained
sections with the same rat anti-mouse CD206 as above with a rabbit anti-mouse Iba1
(Dako, Carpinteria, CA) using fluorescent secondary antibodies as indicated. Nuclei
were stained with DAPI. A Leica SP5 (Leica Microsystems, Milano, Italy) confocal microscope
and a GE Healthcare Delta Vision were used for image acquisitions.
Statistical analysis
Statistical evaluations of RT-PCR data, ELISA assay, EAE score, and immunohistochemical
analyses results were expressed as mean?±?s.d. or mean?±?s.e.m, as appropriate. Results
were analyzed using unpaired Student’s t test and Mann–Whitney U test for samples with unknown and potentially disparate variances. Analyses were
performed using the Prism V5.0a software (Graph-Pad, San Diego, CA, USA). Statistical
significance was ranked *P??0.05, **P??0.01, and ***P??0.001.