(A) LysM-Cre mice were crossed to R26-eYFPfl/wt reporter mice to analyze recombination
efficiency as determined by YFP expression in the indicated immune cell populations by flow
cytometry. Histograms depict YFP expression in LysM-Cre negative (shaded grey) and
LysM-Cre positive (black line) mice (n=3 per group). (B) LysM-Cre mice were crossed to
fl/fl
-ablation in myeloid cells. RT-PCR analysis of
mRNA isolated from primary microglia, CD11b+ splenic cells, peritoneal macrophages,
neutrophil granulocytes, CD11c+ splenic cells, T cells, and B cells. Cre-mediated
full-length 700bp wildtype cDNA. Mice with LysMat the expected mendelian ratio, were viable and fertile and did not exhibit any apparent
abnormalities.
CNS during EAE.
30 mg/kg body weight pioglitazone (pioglitazone) in 0.5 % carboxymethylcellulose, or
vehicle only (carboxymethylcellulose) were orally administered to C57BL/6 mice daily and
after 7d of treatment EAE was induced. (A,B) On day 35 after immunization, histological
analyses of the CNS of pioglitazone- or vehicle-treated mice were performed: (A)
Demyelination (luxol fast blue (LFB)) and axonal damage (amyloid precursor protein (APP))
(n=5 per group); (B) Quantification of immune cell infiltration (MAC-3 for
and 200
± SEM. (C,D)
Expression of (C) inflammatory mediators and (D) chemokines in the CNS of pioglitazone-
treated or vehicle-treated EAE mice was determined by quantitative real-time RT-PCR on
day 21 after immunization (n=10 per group). Data were normalized to endogenous GAPDH
expression and are expressed as n-fold induction compared to healthy controls (mean ±
SEM). *p<0.05.
todermal
origin.
fl/fl
Nestin-
-ablation in
cells of neuroectodermal origin. RT-PCR analysis of mRNA isolated from astrocytes or
microglia from neonatal mixed glial cultures. Specific Cre-mediated recombination in
astrocytes but not microglial cells is shown by the expression of a truncated 300bp cDNA
cDNA. Mice with Nestin-
t the expected mendelian
ratio, were viable and fertile and did not exhibit any apparent abnormalities.
Supplementary Figure 4 CCL2 production by stimulated T cells and astrocytes.
(A) Isolated CD4+ T cells (100.000 cells per well) were subjected to either TH1 or TH17
differentiation as described previously (Klotz et al., 2009), and CCL2 secretion was
measured in the supernatants by ELISA after 72h. As positive control of the ELISA, bone
marrow-derived macrophages (100.000 cells per we
for 24h or left untreated. (B) Primary astrocytes (40.000 cells per well) derived from LysMWT
and LysM-
KO
untreated and CCL2 secretion was measured in the supernatants by ELISA after 24h.
Supplementary Figure 5 The antibody MC-21 depletes Ly-6ChiLy-6G- monocytes in the
blood of LysM-
WT
and LysM-
KO
mice.
EAE was induced by active immunization of LysM-
WT
and LysM-
KO
mice.
At the beginning of the effector phase, on d13, mice received 5 injections (one every 24 h) of
-21; n=20 per group) or the according isotype Ig control
(n=15 per group). Depletion of monocytes was detected in the blood 8 h after the first
injection. Dotplots, gated on live CD11b+cells, depict Ly-6C and Ly-6G expression. Upper
numbers indicate the percentage of Ly-6ChiLy-6G- cells ± SEM (population within upper
gate) and lower numbers indicate the percentage of Ly-6CloLy-6G- cells ± SEM (population
within lower gate). In the graph the percentage ± SEM of Ly-6Chi and Ly-6Clo monocytes in
the blood of LysM-
WT
or LysM-
KO
KO
mice is displayed.
monocytes during EAE aggravates the
clinical disease
WT
MOG35-55-
mice. On days 12, 14, 16,
and 19 after immunization, CD115+ monocytes, isolated from the bone marrow of LysMKO
or wildtype mice, were adoptively transferred (arrows indicate timepoints);
control mice did not receive monocytes. Mice were clinically monitored and the mean
clinical score ± SEM is shown; the difference in clinical scores of the three groups did not
reach statistical significance.
Supplementary Figure 7 Clodronate liposome-treatment and subsequent FITC+ bead
injection results in labeling of circulating Ly-6Chi monocytes.
EAE was induced by active immunization of C57BL/6 mice. On day 12 of EAE, mice were
treated with either clodronate or PBS liposomes. 12 h after liposome treatment, mice received
FITC+ polystrene beads i.v. and 12 hours after bead injection, labeling of CD11b+Ly-6Gmonocytes in the blood was analyzed by flow-cytometry. Dotplots depict Ly-6C expression
and FITC labeling. Upper numbers show the mean percentage of FITC+ cells within the
population of Ly-6Chi monocytes. Lower numbers show the mean percentage of FITC+ cells
within the population of Ly-6Clo monocytes.
-activation induces development of alternatively
activated macrophages.
(A) 30 mg/kg body weight pioglitazone in 0.5 % carboxymethylcellulose (pioglitazone), or
vehicle only (carboxymethylcellulose) were orally administered to C57BL/6 mice daily and
after 7d, CD115+ monocytes were isolated from spleen or blood and expression levels of
markers associated with alternative activation of macrophages were determined by
quantitative real-time RT-PCR. Data were normalized to endogenous GAPDH expression
and displayed as n-fold induction compared to vehicle-treated control (n=3 per group). Data
are expressed as mean ± SEM. (B) Human monocytes were generated from peripheral blood
mononuclear cellss in the presence or absence of 10 µM rosiglitazone. After 7 days, mRNA
was isolated and expression of Arg-1 was determined by quantitative real-time RT-PCR.
Data were normalized to endogenous 18s expression. Graph depicts mean n-fold induction ±
SEM compared to untreated cells.
Supplementary Figure 9 PPAR
limits stimulation of human monocytes by activated
Jurkat T cells
peripheral blood mononuclear cellss were isolated from the blood of healthy donors and 10
M rosiglitazone was added to the cells during the whole isolation procedure. Then,
peripheral blood mononuclear cellss were cocultured with pre-activated Jurkat T cells at a
ratio of 1:5. Cytokine production by CD14+ monocytes was determined by intracellular
cytokine staining and flow-cytometric analysis. Contour plots show representative data from
one healthy donor out of 3. Numbers indicate mean percentage  SEM of TNF
+
and IL-6+
monocytes, respectively.
Supplementary Figure 10 Generation of human iPS cell-derived astrocytes
Astroglial differentiation of human iPS cell-derived long-term neuroepithelial stem cells. The
obtained cultures consistently expressed the glial-specific marker S100B as well as the
astrocyte intermediate filament GFAP (glial fibrillary acidic protein).
Supplementary Figure 11 PPAR
activation in human monocytes restricts induction of
astrocytic CCL2 production
rosiglitazone-treated or untreated monocytes from healthy donors or multiple sclerosispatients were pre-stimulated for 48h with LPS and IFNg, thoroughly washed, and cocultured
with U251 MG cells, a human astrocytoma cell line, at a ratio of 2:1 (monocytes : astrocytes)
for 24h. CCL2 production ± SEM was determined by ELISA. # statistical significance
between astrocytes alone and coculture group; * statistical significance within coculture
group. One representative experiment from 7 healthy donors and 5 patients with multiple
sclerosis is shown.