hep26670-sup-0001-suppinfo01

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Supporting Materials and Methods
Histology and Immunofluorescence Analysis. Liver specimens were fixed in 10%
neutral buffered formalin and paraffin-embedded. Deparaffinized sections (5-10 m)
were stained with hematoxylin and eosin. For immunofluorescence staining, frozen
sections (8 m) from liver specimens were incubated with rat anti-mouse CD4 (BD
PharMingen, San Diego, CA, USA) and were then labeled with Alexa Fluor
555-conjugated goat anti-rat IgG (Invitrogen). Nuclei were stained using DAPI
(Roche Applied Science, Indianapolis, IN, USA).
Induction of ConA-induced Hepatitis. C57BL/6 mice were injected i.v. with a single
dose of ConA (15 mg/kg of body weight) suspended in 100 l of PBS. For the
indicated experiments, a total of 1×106 MSCs or vehicle was i.v. injected 0.5 hour
after ConA injection. Serum and liver tissues were harvest and analyzed at 24 hour
after ConA injection.
In vivo Tumorigenesis Assays. Female nude mice were inoculated s.c. with 1×106
MSCs at 5th passage or 15th passage in the lower back region. Mice inoculated with
5×105 or 1×106 HCT 116 colon cancer cells were used as positive controls. The in
vivo tumorigenesis was analyzed 1 month after inoculation.
Cytokine Analysis. The levels of TNF-, IL-1, IL-12, TGF-,and IL-10 in
supernatants, and IL-17 in serum, were assessed with enzyme-linked immunosorbent
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assay kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s
instructions. The serum levels of TNF-, IFN-, IL-4, and IL-5 were performed by
Cytometric Bead Array Mouse Th1/Th2 Cytokine Kit (BD PharMingen).
Cell Preparations. MSCs were generated from bone marrow of tibia and femur of 6to 10-week-old mice. Cells were cultured in DMEM medium supplemented with 10%
fetal bovine serum, 2 mM glutamine, and antibiotics (all from Invitrogen, Carlsbad,
CA, USA). Nonadherent cells were removed after 24 hours, and adherent cells were
maintained with medium replenishment every 3 days. To obtain MSC clones, cells at
confluence were harvested and seeded into 96-well plates by limited dilution.
Individual clones were then picked and expanded. Cells were used at 5th to 15th
passage.
MNCs of spleens and livers were obtained by Ficoll density gradient
centrifugation. Cont-DCs, MSC-DCs, CD4+ T cells, and CD4+CD25- T cells were
magnetically purified from liver or spleen MNCs of C57BL/6 or BALB/c mice
according to the manufacturer’s recommendations (Miltenyi Biotec, Auburn, CA,
USA). The purity of sorted cells in this study was consistently more than 95%.
We prepared CD11c+B220- DC precursors as previously described.14 In brief,
peripheral blood was obtained by cardiac puncture under ether anesthesia. After
density gradient centrifugation, CD11c+B220- cells were sorted out by flow cytometry.
The purity of sorted cells in this study was consistently more than 99%.
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Flow Cytometric Analysis. Monoclonal antibodies with the following specificities
were obtained from BD Biosciences, eBioscience, or BioLegend (San Diego, CA,
USA): CD4 (GK1.5), CXCR3 (CXCR3-173), CCR5 (C34-3448), CCR7 (4B12),
CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD25 (PC61), TNF- (MP6-XT22),
IFN- (XMG1.2), Foxp3 (FJK-16s using eBioscience Foxp3 staining kit), F4/80
(BM8), CD11c (HL3), MHCII (M5/114.15.2), CD80 (16-10A1), CD86 (GL1), and
DEC205 (205yekta). Multiple-color flow cytometric analysis was performed using
FACS Aria (BD Biosciences).
Induction of Tregs in Vitro. CD4+CD25- T cells (3×105) from C57BL/6 mice were
incubated with or without syngeneic cont-DCs or MSC-DCs (3×104) in RPMI 1640
supplemented with 10% fetal bovine serum and antibiotics in the presence of
plate-coated anti-CD3 (5 g/ml) and soluble anti-CD28 (2 g/ml) (both from
eBioscience). In some experiments, anti-TGF-, anti-IL-10 neutralizing antibody, or
control IgG was added into the coculture system. After 5 days, cells were harvested
for surface staining of CD4 and CD25, and intracellular staining of Foxp3 for flow
cytometric analysis.
Treatment with becteria-induced liver injury with DCs. In some experiments, mice
were infused i.v. with 5×105 cont-DCs or MSC-DCs on days 2 and 4 after P. acnes
priming. In some experiments, mice injected with MSC-DCs were also treated with
anti-TGF-, anti-IL-10 neutralizing antibody, or control IgG (7 mg/kg) (all from
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PeproTech).
DC Precursor-MSC Cocultures. Cocultures were initiated by plating MSCs in 6-well
plates overnight at a concentration of 1×105 cells/well. At the indicated time points, a
total of 1×106 purified DC precursors were seeded onto MSCs monolayers at a 10:1
DC precursor/MSC ratio in the presence of appropriate cytokines as described in
“Induction of DC Maturation”. In some experiments, NS398 (5 M), L-NMMA (1
mM), AH6890 (10 M), AH23848 (10 M) (all from Sigma-Aldrich), or DMSO was
added into the coculture system in the presence of MSCs. CD11c+ DCs were sorted
out by flow cytometry at the indicated time points for flow cytometric analysis and
western blot.
MLR. Cont-DCs, MSC-DCs, or DCs generated from DC precursors, which were
treated with MSCs at different induction phases, were lethally irradiated (30 Gy).
Then, DCs were cultured in graded doses with CD4+ T cells (3×105 cells/well)
isolated from naive BALB/c mice in RPMI 1640 medium for 5 days. In some
MSC-treated experiments, NS398, L-NMMA, or DMSO was included in the culture.
[3H]Thymidine (1 Ci/well, Shanghai Institute of Applied physics, Chinese Academy
of Sciences, China) was added 18 hours before the end of culture. Cells were then
harvested onto glass fiber mats for measurement of [3H]thymidine incorporation.
Endocytosis Assay. Cont-DCs or MSC-DCs (3×105) were collected and incubated in
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300 l of medium containing 500 g/ml FITC-Dextran (40 kDa) for 120 minutes at
37 °C. Cells incubated at 4 °C were set as negative controls. Then, cells were washed
three times with cold PBS. Endocytosis was analyzed by flow cytometry and the
fraction of FITC-positive cells was calculated.
Quantitative Real-time PCR. Total RNA was extracted from tissues or cells at
indicated time points and was subsequently reverse-transcribed using AMV Reverse
Transcription System (Takara, Shiga, Japan). Quantitative real-time PCR was
performed using SYBR Green PCR mix on an ABI Prism 7900HT (Applied
Biosystems, Foster City, CA, USA). Thermocycler conditions included 2-minute
incubation at 50 °C, then 95 °C for 10 minutes; this was followed by a 2-step PCR
program, as follows: 95 °C for 15 seconds and 60° C for 60 seconds for 40 cycles.
-actin was used as an internal control to normalize differences in the amount of total
RNA in each sample. Primers are listed in Table 1.
Total Genomic DNA Extraction and P. acnes Specific 16S rDNA Amplication. Mice
liver homogenating samples were digested with 20 mg/ml of lysozyme at 37 °C for
60-minute lysis. Total genomic DNA was extracted using TRIzol (Invitrogen,
Carlsbad, CA, USA) according to the manufacturer’s instructions. Then, 100 ng of
total genomic DNA of each sample was used as the template in PCR. The PCR
amplification was conducted using Takara Premix Taq (Takara) for 30 cycles of 94 °C
for 1 minutes, 60 °C for 30 seconds, and 72 °C for 30 seconds. The primers were as
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following: 5'-CTG GTA GTC CAC GCT GTA AAC G-3' (the forward primer for 16S
rDNA of P. acnes) and 5'-TCC ATG TCA AAC CCA GGT AAG G-3' (the reverse
primer of 16S rDNA for P. acnes). PCR products were electrophoresed on 1.5%
agarose gels and subsequently stained with ethidium bromide.
Western Blot. Cells were directly lysed with RIPA containing protease and
phosphatase inhibitors (Roche) and proteins were separated by 10% SDS-PAGE after
denaturation. Immunoblot analysis was performed by initial transfer of proteins onto
polyvinylidenefluoride membranes using Mini Trans-Blot (Bio-Rad Laboratories,
Richmond, VA, USA) and followed by a blocking step with 5% nonfat dried milk plus
0.1% Tween 20 for 2 hours at room temperature and exposed to primary antibodies
diluted 1,000-fold that recognized PI3K, p-ERK1/2, ERK1/2, or Vinculin overnight at
4 °C and subsequently washed. The blots were then incubated with a secondary
antibody conjugated with Horse Radish Peroxidase diluted 5,000-fold for 1 hour at
room temperature. Signals were detected by FluorChem E system (Alpha Innotech
Corp, Santa Clara, CA, USA).
Statistical Analysis. Significant differences were evaluated using an
independent-samples t test or Wilcoxon rank test, except that multiple treatment
groups were compared within individual experiments by ANOVA or the
Kruskal-Wallis test. Kaplan-Meier analysis was performed to estimate cumulative
survival rates of mice. Values of p less than 0.05 were considered significant. All
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values were presented as mean±S.E.M.
Supporting Figure Legends
Supporting Figure S1. MSC treatment cures granulomatous hepatitis and prevents
FHF. Mice were injected with P. acnes (P.ac). PBS or MSCs were administrated i.v.
on days 3, 5, and 7 after P. acnes priming. (A) LPS was injected 12 hours after MSC
injection on day 7 to induce FHF. Cumulative survival rates of mice were analyzed.
Data from two independent experiments are combined (n=10 mice per group). (B)
Serum were sampled on day 7 and levels of ALT and AST were measured (n=8 mice
per group). Results are mean±S.E.M. from three independent experiments. (C) Liver
tissues were sectioned for histological examination. Scale bar, 100 m.
Representative images from one experiment out of three are shown.**, p<0.01.
Supporting Figure S2. Amelioration of liver injury by MSC treatment. Mice were
injected with P. acnes (P.ac). PBS or MSCs were administrated i.v. on days 0, 2, and 4.
(A) Liver tissues were isolated and photographed. Representative images from one
experiment out of three are shown. (B) Liver weight from 7 to 9 mice per group was
measured. Results are mean±S.E.M. from three independent experiments. **, p<0.01.
Supporting Figure S3. Allogeneic MSCs (allo-MSCs) ameliorate bacteria-induced
liver injury. C57BL/6 mice were injected with P. acnes (P.ac). PBS or MSCs derived
from BALB/c mice were administrated i.v. on days 0, 2, and 4, and LPS was injected
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on day 7 to induce FHF. (A) Cumulative survival rates of mice were analyzed. Data
from two independent experiments are combined (n=10 mice per group). (B-E) Serum
and liver tissues from PBS or allo-MSC-treated mice were sampled on day 7 after P.
acnes priming. Serum levels of ALT and AST (B; n=8 mice per group) on day 7 were
measured. (C) Levels of MHCII, CD80, and CD86 on CD11c+ DCs of livers were
analyzed by flow cytometry. Gray shading and solid line indicate
immunofluorescence intensity of cells for the control and test antibodies, respectively.
(D) CD4+ T cells (3×105 cells/well) from the spleen of naive BALB/c mice were
incubated with or without lethally irradiated (30 Gy) cont-DCs (3×104 cells/well)
from C57BL/6 mice. In the indicated experiments, MSC-DCs from C57BL/6 mice
were added to the coculture system in graded doses. Five days later, T cell
proliferation in response to the indicated treatments was measured by incorporation of
[3H]thymidine. Data are mean±S.E.M. from three independent experiments (n=3). (E)
CD4+CD25- T cells from C57BL/6 mice were cultured with syngeneic cont-DCs or
MSC-DCs in the presence of plate-coated anti-CD3 antibody (5 g/ml) and soluble
anti-CD28 antibody (2 g/ml). Five days later, cells were stained for surface markers
and intracellular expression of Foxp3, and analyzed by flow cytometry.
Representative dot plots are shown on the left and summarized results are shown as
mean±S.E.M. from three independent experiments (n=6 mice per group) on the right.
*, p<0.05; **, p<0.01.
Supporting Figure S4. Treatment effect of MSCs on ConA-induced hepatitis. 1×106
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MSCs or vehicle was administered i.v. 0.5 hour after ConA (15 mg/kg) injection. (A)
Serum levels of ALT and AST (n=8 mice per group) 24 hours after ConA injection
were measured. Results are mean±S.E.M. from three independent experiments. (B)
Liver tissues were sectioned for histological examination. Scale bar, 100 m.
Representative images from one experiment out of three are shown. **, p<0.01.
Supporting Figure S5. In vivo tumorigenesis assay of MSCs. Female nude mice were
inoculated s.c. with 1×106 MSCs at 5th passage or 15th passage or HCT 116 colon
cancer cells in the lower back region (n=5 mice per group). One month later, the
representative image of tumorigenesis from one experiment out of three are shown.
Supporting Figure S6. Long-term effect of MSC treatment on bacteria-induced liver
injury. Mice were injected with P. acnes (P.ac). PBS or MSCs were administrated i.v.
on days 0, 2, and 4. (A) DNA copies of P. acnes 16S rDNA in the liver of mice at
different time after P. acnes injection are shown. (B) Liver tissues on day 28 were
sectioned for histological examination. Scale bar, 100 m. (C) Serum ALT and AST
levels on day 28 (n=8 mice per group) were measured. (D) Serum IFN- and TNF-
levels on day 28 were measured by cytometric bead array (n=8 mice per group). (A,B)
Representative images from one experiment out of three are shown (C,D) Results are
mean±S.E.M. from three independent experiments. **, p<0.01.
Supporting Figure S7. Amelioration of bacteria-induced liver injury by MSC-DCs.
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Mice were injected with P. acnes (P.ac). C57BL/6 mice were infused i.v. with 5×105
cont-DCs or MSC-DCs on days 2 and 4 after P. acnes priming. Liver tissues and
serum were sampled on day 7 after P. acnes priming. (A) Serum ALT and AST levels
(n=8 mice per group) were measured. Results are mean±S.E.M. from three
independent experiments. (B) Liver tissues were sectioned for histological
examination. Bars, 100 m. Representative images from one experiment out of three
are shown. *, p<0.05; **, p<0.01.
Supporting Figure S8. mRNA levels of inhibitory factors expressed in DCs. Mice
were injected with P. acnes (P.ac). PBS or MSCs were administrated i.v. on days 0, 2,
and 4 after P. acnes injection. mRNA levels of CD200R3 and Aldh1a in cont-DCs or
MSC-DCs were measured by quantitative real-time PCR. Data are normalized to
expression levels of cont-DCs and results are mean±S.E.M. (n=6).
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