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Immunity, Volume 44
Supplemental Information
Erythropoeitin Signaling in Macrophages Promotes
Dying Cell Clearance and Immune Tolerance
Bangwei Luo, Woting Gan, Zongwei Liu, Zigang Shen, Jinsong Wang, Rongchen Shi, YuqiLiu, Yu Liu, Man Jiang, Zhiren Zhang, and Yuzhang Wu
Figure S1 (related to Figure 1). Dying cell-derived S1P activates macrophage EPO signaling in vitro.
A: Concentrations of S1P in foetal calf serum (FCS) and conditioned media from viable, necrotic or apoptotic
thymocytes, Jurkat cells or neutrophils were measured (n = 3). B: Peritoneal macrophages were incubated with
neutrophil apoptotic cell conditioned media (ACCM) for 0, 6, 12 or 24 hr, and protein expression of EPO, EPOR,
HIF-1α, HIF-2α and p-Jak2 were measured. C: EPOR expression on cell surface of splenic macrophages from WT
or Eporloxp/loxpLyz2-cre+/+ (Epor-cKO) mice was measured by flow cytometry. D: Peritoneal macrophages were
incubated with conditioned media (CM) from viable (VCM) or necrotic (NCM) thymocytes, Jurkat cells or
neutrophils for 24 hr, and protein expression of EPO, EPOR, HIF-1α, HIF-2α and p-Jak2 and cell surface expression
of EPOR were measured. E: Sphk1-specific siRNAs reduced sphk1 expression in Jurkat cells and decreased S1P
concentration in ACCM of Jurkat cells (bottom) (n = 3). F: S1P or conditioned medium from apoptotic (ACCM),
viable (VCM) or necrotic (NCM) thymocytes were incubated with bone marrow-derived macrophages and the
expression of EPO, EPOR, p-Jak2, HIF-1α and HIF-2α were detected at indicated time points. G: Peritoneal
macrophages were incubated with rhEPO (40IU/ml), together with or without EMP-9 (0.5 mg/ml, four times at 1 h
intervals for 24 hr), for 24 hr, and protein expression of EPOR and p-Jak2 were detected. H: Peritoneal macrophages
were incubated with apoptotic, viable or necrotic thymocytes or Jurkat cells and protein expression of EPO, EPOR,
HIF-1α, HIF-2α and p-Jak2 were measured at indicated time points. I: Peritoneal macrophages were incubated with
apoptotic thymocyte, together with or without cytochalasin D (CytD, 2 μM, 24 hr), for 24 hr, and apoptotic
thymocyte phagocytosis (left) and the expression of EPO, EPOR, HIF-1α and p-Jak2 were measured. J: Protein
expression of HIF-1α, HIF-2α, EPO, EPOR and p-Jak2 were detected in viable, apoptotic or necrotic thymocytes or
Jurkat cells. Data are representative of three independent experiments. For flow cytometry data, black numbers refer
to the percentage of positive cells and red numbers refer to the mean fluorescent intensity. Error bars represent the
Figure S2 (related to Figure 2). EPOR is not expressed on blood monocytes.
A: Neither CD11b+Ly6Chigh nor CD11b+Ly6Clow monocytes from WT mice expressed EPOR. B: Apoptotic
thymocytes were given to WT mice and the EPOR expression on CD11b+Ly6Chigh or CD11b+Ly6Clow monocytes
were measured 24 hr following the administration. Data from three independent experiments are shown. For flow
cytometry data, black numbers refer to the percentage of positive cells and red numbers refer to the mean fluorescent
intensity.
Related to Figure 2.
Figure S3 (related to Figure 3). EPO signaling is important for dying cell removal by macrophages.
A: Flow cytometry analysis showing the electronic gating strategy used to identify peritoneal macrophages that had
engulfed pHrodo-labeled apoptotic cells in vitro. Alternatively, confocal microscopy was applied to score apoptotic
cell engulfment. Scale bars represent 30 μm in the top panel and 15 μm in the bottom panel. B: Following rhEPO
pre-treatment for 24 hr, peritoneal macrophages from WT mice were incubated with pHrodo-labeled apoptotic
thymocytes for 1 hr and the analyzed by fluorescent microscopy (n = 3). Scale bar represents 30 μm. C:
CFSE-labeled apoptotic thymocytes were i.v. given to mice and the deposition of ACs in spleen was detected 2 hr
later by immunostaining (n = 3). Scale bar represents 100 μm. D: Flow cytometry analysis showing the electronic
gating strategy used to identify F4/80+ splenic macrophages that had engulfed apoptotic neutrophils, T cells or B
cells in vivo. E: Apoptotic neutrophil in circulation was measured by flow cytometry in 10-week-old female WT and
Eporloxp/loxpLyz2-cre+/+ (Epor-cKO) mice. Representative spleen, thymus, lung and skin sections from 10-week-old
female WT or Epor-cKO mice were stained by TUNEL. Scale bars represent 30 μm. F: Neutrophils from WT mice
barely expressed EPOR (right). The percentages of apoptotic neutrophils in peripheral blood of 55-week-old female
Epor-cKO mice were higher than age- and gender-matched WT mice (n = 3). G: Fluorescence microscopy of kidney
(Scale bar represents 30 μm), lung (Scale bar represents 50 μm) and thymus (Scale bar represents 50 μm) sections
from 55-week old female WT or Epor-cKO mice, which show an accumulation of apoptotic cells stained by TUNEL.
H: Flow cytometry of apoptotic splenic or peritoneal macrophages in 55-week-old female WT or Epor-cKO mice (n
= 3). I: Fluorescence microscopy of spleen sections from 55-week-old female WT or Epor-cKO mice, showing
co-localization of apoptotic cells stained by TUNEL staining (green) and B cells (B220 staining, red). Yellow
indicates co-localiztion. Scale bar represents 25 μm. J: Peritoneal macrophages from WT or Epor-cKO mice were
fed with or without apoptotic thymocytes, and macrophage apoptosis was assessed by flow cytometry stained with
annexin V and PI at indicated time points (n = 3). K: rhEPO (5000 IU/kg) was administered i.p. to WT or Epor-cKO
mice with or without apoptotic thymocytes. The cytokine concentrations in the peritoneal fluids were measured 24
hr later (n = 3). L: Apoptotic thymocytes were i.v. given to WT or Epor-cKO mice and the cytokine concentrations
in the serum were measured 2 hr later (n = 3). Data are representative of at least two independent experiments; Error
bars represent the s.e.m. *P<0.05.
Related to Figure 3.
Figure S4 (related to Figure 4). EPO promotes apoptotic cell engulfment through
Jak2-ERK-C/EBPβ-dependent PPARγ induction.
A: Peritoneal macrophages were incubated with rhEPO and the Pparg mRNA expression was measured (n = 3). B:
Peritoneal macrophages were incubated with apoptotic cell conditioned media (ACCM, left), S1P (middle) or
conditional medium (CM) from necrotic (NCM) or viable (VCM) thymocytes or Jurkat cells (right) for 24 hr, and
protein expression of PPARγ were detected. C: Following two days of rosiglitazone (RSG, 10 mg/kg/day via oral
gavage) or rhEPO (5000 IU/kg/day, i.p.) treatment, peritoneal macrophage from Eporloxp/loxpLyz2-cre+/+ (Epor-cKO)
mice were isolated for apoptotic cell (AC) phagocytosis analysis (n = 3). D: Following two days of rosiglitazone
(RSG, 10 mg/kg/day via oral gavage) or rhEPO (5000 IU/kg/day, i.p.) treatment, peritoneal macrophage from
Ppargloxp/loxpLyz2-cre+/+ (Pparg-cKO) mice were isolated for AC phagocytosis analysis (n = 3). E: RAW264.7
macrophages were incubated with rhEPO and the PPARγ protein expression was measured at indicated time points.
F: RAW264.7 macrophages (left) or primary peritoneal macrophages (right) were incubated with rhEPO and the
activation of ERK and C/EBPβ was detected at indicated time points. (n = 3) G: Schematic structure of the cloned
and mutant Pparg regulatory regions (up). RAW264.7 macrophages transfected with pGL3-PPARγ or pGL3-PPARγ
(M) were treated with or without rhEPO for 24 h, and the activation of the Pparg promoter by rhEPO was assayed
with luciferase activity. Results are shown as the fold activation over the activity of pRL-TK. Data are representative
of at least two independent experiments. Error bars represent the s.e.m. * P<0.05, *** P<0.001.
Related to Figure 4.
Figure S5 (related to Figure 6). Characterization of Eporloxp/loxpLyz2-cre+/+ mice.
A: Genetic identification of Eporloxp/loxpLyz2-cre+/+ (Epor-cKO) mice by PCR of DNA from tails. B, C: In peritoneal
macrophages from Epor-cKO mice, the mRNA (B) and protein (C) expression of EPOR was significantly reduced
compared to WT control. D: Comparison of the cell surface EPOR expression of different spleen immune cells
between Epor-cKO and WT mice. E: Comparison of the concentration of haemoglobin and red blood cells between
10-week-old femal Epor-cKO mice and WT mice (n=3). F: Flow cytometry analysis of splenocytes and
lymphocytes from 10- or 55-week-old WT and Epor-cKO female mice (n = 3). The weight of spleens and cell count
of lymph nodes was measured (n=3). G: Representative images of hematoxylin-and-eosin (HE) staining or Periodic
acid Schiff staining (PAS) of the kidney (Scale bars represent 30 μm), skin (Scale bar represents 100 μm) and lungs
(Scale bar represents 50 μm) from 55-week-old female WT and Epor-cKO mice (n = 6 per group). Furthermore,
glomerular damage was evaluated on a scale 0-4. Circles indicate glomeruli within the kidney. Arrows indicate the
inflammatory cells. H: Fluorescence microscopy of kidney sections from 55-week-old WT and Epor-cKO female
mice, showing B220+, F4/80+ or CD4+ infiltrating cells. Circles indicate glomeruli within the kidney. Scale bars
represent 30 μm. Moreover, the infiltrated immune cells were determined by flow cytometry (bottom). I:
Fluorescence microscopy of skin sections from 55-week-old female WT and Epor-cKO mice, showing IgG
deposition (n = 6 per group). Arrows indicate IgG deposition. Scale bar represents 50 μm. J: Representative images
of immunostaining of lung sections from 55-week-old female WT and Epor-cKO mice, showing B220+ or F4/80+
cells (n = 6 per group). Scale bars represent 50 μm. For flow cytometry data from D, black numbers refer to the
percentage of positive cells and red numbers refer to the mean fluorescent intensity. Error bars represent the s.e.m. *
P<0.05, ** P<0.01.
Related to Figure 6.
Figure S6 (related to Figure 7). Characterization of pristane–induced lupus-like mice.
A-I: For induction of lupus-like disease in mice, female C57BL/6 mice at 10-12 weeks of age were given a single
intraperitoneal injection of 0.5 ml pristane or PBS, and mice were analysed for lupus-related symptoms six months
later (n = 6). Six months after pristane treatment, the serum EPO concentration (A) and spleen macrophage EPOR
expression (B) were greatly reduced. Pristane administration reduced the ability of macrophages to engulf apoptotic
cells (C), increased apoptotic cell accumulation in the blood (D) and spleen (E, TUNEL staining, scale bar
represents 30 μm), increased the concentrations of ADA and ANA in the serum (F), enhanced the deposition of IgG
in the kidney (G, scale bar represents 50 μm), impaired kidney function (H) and altered the cytokine profile (I). For
flow cytometry data from B, black numbers refer to the percentage of positive cells and red numbers refer to the
mean fluorescent intensity. Error bars represent the s.e.m. *; P<0.05.
Related to Figure 7.
Supplemental Experimental Procedures
Animals. All mice were housed under pathogen-free conditions in the animal facility of Third Military Medical
University. Experiments were performed according to local ethical guidelines and were approved by the local
Administration District Official Committee of Third Military Medical University, Chongqing, China. C57BL/6 mice
were purchased from Vital River Laboratories. Mice bearing the lox-P-targeted Epor (Eporloxp/loxp) allele on mixed
Sv129/C57Bl/6 backgrounds were generated as previously described (Wu et al., 1995) and were kindly provided by
Hong Wu. These mice were backcrossed onto the C57Bl/6 strain for more than eight generations. Mice carrying the
lox-P-targeted Pparg (Ppargloxp/loxp) allele and the lysozyme-M Cre (Lyz2Cre) recombinase transgene were purchased
from the Jackson Laboratory. Eporloxp/loxp and Lyz2Cre mice were crossed to generate myeloid-specific Epor genetic
deletion mice lacking macrophage EPOR expression. Ppargloxp/loxp and Lyz2Cre mice were crossed to generate
offspring with myeloid-specific loss of the Pparg gene, as described (Chen et al., 2015). We refer to
Eporloxp/loxpLyz2Cre+/+ or Ppargloxp/loxpLyz2Cre+/+ littermates as Epor-cKO and Pparg-cKO mice, respectively. We
assessed spontaneous autoimmunity development in Epor-cKO femal mice at 10, 40 and 55 weeks of age. For the
PPARγ rescue experiment, female Epor-cKO mice that were 30 weeks old were fed a standard chow diet with
rosiglitazone (10 mg/kg/day, Sigma-Aldrich, St Louis, MO, USA) or unsupplemented chow (Vehicle) for four
months. For the EPO rescue experiment, female Pparg-cKO mice that were 30 weeks old were treated daily with
intraperitoneal injections of recombinant human EPO (rhEPO, 5000 IU/kg, three times per week, Sunshine
Pharmaceutical, Shenyang, China) or PBS (Vehicle) for four months. For pristane SLE model induction, female
C57BL/6 mice, at 10-12 weeks of age, were given a single intraperitoneal injection of 0.5 ml of pristane
(Sigma-Aldrich) to induce SLE-like autoimmunity. Two months after pristane injection, mice were treated with
intraperitoneal injections of rhEPO (5000 IU/kg, three times per week) for four months, and the same volume of
PBS was given to the control group.
Generation of apoptotic and necrotic cells and their conditioned media. Thymocytes were harvested from 4- to
6-week-old female C57BL/6 mice and were cultured in RPMI supplemented with 10% foetal calf serum (FCS,
Gibco). To generate apoptotic thymocytes, cells were treated with 1 mM dexamethasone (Sigma-Aldrich) for 6 hr at
37°C. Afterwards, cells were washed three times with PBS and resuspended in 10% FCS in RPMI. This method
results in 80% thymocyte apoptosis, as measured with FITC-AnnexinV/propidium iodide staining (Sungene Biotech,
Tianjin, China). To generate apoptotic Jurkat cells cells were cultured in RPMI without FCS, and apoptosis was
induced with 0.5 μg/mL staurosporine (Sigma-Aldrich) for 3 hr. Afterwards, cells were washed three times with PBS
and resuspended in 10% FCS in RPMI. Staurosporine treatment yielded a population with 90% apoptotic cells. For
the generation of apoptotic neutrophils, peritoneal neutrophils were collected from C57/Bl6 mice after 4 hr
zymA-induced peritonitis, pooled together and aged for 24 hr in culture in complete RPMI 1640 (Gibco, Grand
Island, NY, USA). This method results in 90% neutrophil apoptosis, as measured with FITC-AnnexinV/propidium
iodide staining (Sungene Biotech, Tianjin, China). Necrotic thymocytes, Jurkat cells and neutrophils were induced
by incubation at 56°C for 20 minutes. Efficient induction of cell death was confirmed by flow cytometry with
annexin V/propidium iodide staining.
The apoptotic cell conditioned medium was generated as previously reported (Weigert et al., 2006). Briefly,
apoptotic cells were washed three times with PBS, which was followed by incubation for another 2 or 4 hr in RPMI
with 10% FCS. Afterward, the cell medium was obtained by centrifugation at 13,000 g for 10 minutes and then
filtered through 0.2 μm pore filters to remove apoptotic bodies. Necrotic or viable cell conditioned media were
prepared accordingly. RPMI with 10% FCS was used as a vehicle control for conditioned medium.
Generation of bone marrow-derived macrophages. Tibias and femurs of 6-to 8-week-old C57/BL6 mice were
aseptically removed, and bone marrow cells were flushed out with sterile sterile RPMI 1640 (Gibco, Grand Island,
NY, USA). Cells were then incubated for 7 days in bone marrow differentiation medium, which was composed of
RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100
mg/mL streptomycin, 100 U/mL penicillin (Gibco), and 10 ng/mL macrophage colony-stimulating factor (M-CSF,
Peprotech Asia, Rehovot, Israel). The medium was replenished on the 4th day and the non-adherent cells were
purged. Seven days later, the cells were detached from the dishes, counted, reseeded, and cultivated in tissue culture
plates overnight before any further experimental procedures.The population of BMDMs, as assessed by expression
of F4/80 and CD11b surface molecule by FACS analysis, was typically 97% pure (Data not shown).
In Vitro Phagocytosis Assays. In vitro phagocytosis assays with mouse peritoneal macrophages were performed as
previously described, with some modifications (N et al., 2009). Briefly, 10-12-week old female C57/Bl6 mice were
intraperitoneally injected with 3% Brewer’s thioglycollate (Sigma-Aldrich). After 72 hr, primary peritoneal
macrophages were isolated from peritonitis exudates by peritoneal lavage with 5 ml ice-cold DMEM. Macrophages
were plated in 6-well plates in DMEM with 10% FCS and were allowed to rest overnight at 37°C with 5% CO2
before the start of experiments. Before being fed to macrophages, apoptotic cells were labeled with pHrodo™ Green
(pHrodo, Molecular Probes), a pH-sensitive phagocytosis-dependent indicator that requires no wash steps or
quenchers, according to the manufacturer’s instructions. Unless indicated, peritoneal macrophages were incubated
with pHrodo-labeled apoptotic cells at a ratio of 1:5 (macrophages: apoptosis cells) and cultured at 4°C (negative
control) or 37°C for 60 min in RPMI supplemented with 10% FBS. In some cases, macrophages were pretreated
with rhEPO, rosiglitazone, S1P or cell conditioned media in the presence or absence of VPC23019 (Avanti Polar
Lipids, AL, USA) or JTE-013 (Avanti Polar Lipids, AL, USA) for the indicated times before co-incubation with
pHrodo-labeled apoptotic cells. After incubation, cells were resuspended and stained with fluorescence-labeled
anti-F4/80 (BM8; Sungene Biotech) and anti-CD11b (M1/70; Sungene Biotech); they were later analysed by flow
cytometry. Doublet discrimination was used to distinguish internalized from externally bound apoptotic cells and the
proportion of macrophages containing ingested apoptotic cells was determined.
In some studies, phagocytosis was confirmed by confocal fluorescent microscopy or immunofluorescence
microscopy. Alternatively, macrophages were plated on sterile glass coverslips in 24-well culture plates in RPMI
supplemented with 10% FBS. Apoptotic thymocytes were stained with CFSE and were added to peritoneal
macrophages at a 5:1 ratio (thymocytes:macrophages), for 60 min, at 37°C. After incubation, macrophages were
washed several times with cold PBS and Cell Dissociation buffer, enzyme free PBS-based buffer (Invitrogen), to
remove free apoptotic cells. Macrophages were then fixed with 2% paraformaldehyde and analysed by confocal
fluorescent microscopy or immunofluorescence microscopy. Phagocytosis was expressed as phagocytic index (PI):
number of cells ingested per total number of macrophages × 100.
In vivo phagocytosis assay. For in vivo peritoneal macrophage phagocytosis assays, 2x107 pHrodo-labeled
apoptotic thymocytes were injected into the peritoneal cavity of 10- to 12-week-old female mice 3 days after the
induction of peritonitis with thioglycollate. In some cases (Figure 3D), 1x107 pHrodo-labeled apoptotic thymocytes
were injected intraperitoneally into 10- to 12-week-old female mice. Mice were sacrificed after 1 hr, and peritoneal
lavage was performed with 5 ml ice-cold PBS that was subsequently transferred into plastic tubes. Cells were
washed twice, and a single-cell suspension of peritoneal flush was stained with anti-F4/80 (BM8, Sungene Biotech)
and CD11b (M1/70, Sungene Biotech) to identify macrophages. Cells were analysed by flow cytometry, and the
number of peritoneal macrophages ingesting apoptotic thymocytes was determined (Uderhardt et al., 2012). In some
experiments, mice were pre-treated with rhEPO (5000 IU/kg/day, i.p.) or rosigitazone (10 mg/kg/day via oral gavage)
for 2 days before apoptotic cell injection. Also, the same volume of PBS served as the respective vehicle control.
For in vivo splenic macrophage uptake assays, 8x107 pHrodo-labeled apoptotic thymocytes were intravenously
injected into 10- to 12-week-old female mice; two hours after the injection, the mice were killed and their spleens
were collected. The single-cell splenocyte suspensions were stained with anti-F4/80-APC (BM8, Sungene Biotech)
to identify the macrophage population after lysis of red blood cells. Cells were analysed by flow cytometry and the
number of spleen macrophages that took up pHrodo-labeled apoptotic thymocytes by phagocytosis was quantified
(Nakaya et al., 2013). In some experiments, mice were pre-treated with rhEPO (5000 IU/kg/day, i.p.) or the same
volume of PBS for 2 days before apoptotic cell injection.
To assay the remain of injected apoptotic in spleen in vivo, 8x107 CFSE-labeled apoptotic thymocytes were
intravenously injected into 10- to 12-week-old female mice; two hours after the injection, the mice were killed and
their spleens were collected for immunostaining. The spleen sections were incubated with anti-F4/80 antibdy (BM8,
ab16911, Abcam Inc.) with subsequent corresponding secondary antibodies to identify the macrophage population.
The macrophages and free CFSE-labeled apoptotic thymocytes in sections were viewed using a fluorescence
microscope.
For in vivo thymic phagocytosis assays, 4-week-old mice were injected i.p. with 0.2 mg of dexamethasone (Dex) in
PBS or PBS alone (Control). After 24 hr, mice were sacrificed and thymi were extracted and mechanically
dissociated. The percentage of Annexin V+ apoptotic cells and recruitment of F4/80+ macrophages and EPOR on
F4/80+ macrophages was evaluated in cell suspensions by flow cytometry (N et al., 2009). S1P, EPO and certain
cytokines concentrations in thymus were measured by ELISA or flow cytometry. For some cases, dexamethasone
(0.2 mg) was given together with VPC23019 (0.5 mg/kg ) or PBS (Control).
To assay the basal macrophage clearance of apoptotic cells in vivo, spleens from WT mice were collected. The
single-cell splenocyte suspensions were stained with anti-F4/80-APC (BM8, Sungene Biotech) to identify the
macrophage population after lysis of red blood cells. Subsequently, cells were permeabilized to enable the labeling
of ingested neutrophils, T cells or B cells with PE-conjugated anti-Ly6G, anti-CD3 or anti-B220 antibody,
respectively. Cells were analysed by flow cytometry and the number of spleen macrophages that took up apoptotic
neutrophils, T cells or B cells by phagocytosis was quantified (Schwab et al., 2007).
Flow Cytometry. Single-cell suspensions of the cultured cells, peritoneal lavage, lymph nodes, spleens and kidneys
were generated from mice, washed twice in staining buffer, resuspended, and incubated with anti-CD16/32
antibodies (Sungene Biotech) to block Fc receptors (0.5 μg/million cells). Then, the cells were subjected to surface
antibody staining with labeled antibodies diluted in staining buffer for 20 min at 4°C. After incubation, cells were
washed in staining buffer and analysed immediately. Intracellular staining of FoxP3 was performed according to the
manufacturer’s instructions (eBiosciences). For all staining, isotype controls were used. Following staining, cells
were washed and suspended in PBS and then analysed on CANTO II (Becton Dickinson). The following labeled
Abs were used to detect different leukocyte subpopulations: CD3 (145-2C11), CD4 (GK1.5), CD8 (YTS169.4),
CD8α (53.6.7), CD11b (M1/70), CD11c (N418), CD19 (1D3), CD45 (30-F11), CD68 (FA-11), B220 (RA3-6B2),
F4/80 (BM8), GR-1 (RB6-8C5), Ly6G (1A8), Ly6C (HK 1.4), Foxp3 (150D) (all from Sungene Biotech), and
EPOR (Santa Cruz). In some experiments, flow cytometry was applied with a Mouse Inflammation CBA Kit (BD
PharMingen) to evaluate the inflammatory cytokines, including IL-6, IL-10, IL-12, MCP-1, IFN-γ and TNF-α in cell
culture supernatants or peritoneal fluid according to the manufacturer’s instructions. Flow data were collected with
CellQuest Software and analysed with FlowJo software for windows (Treestar, Inc.)
ELISA and biochemical parameters. Mouse anti-dsDNA antibody kit (Alpha Diagnostic, San Antonio, TX),
anti-nuclear Abs ELISA kits (Alpha Diagnostic, San Antonio, TX), anti-smooth muscle antibody kit (Guduo
Biotechnology, Shanghai) and anti-Smith antibody kit (Guduo Biotechnology, Shanghai) were used to detect the
serologic titres of anti-dsDNA, anti-nuclear, anti-smith and anti-smooth muscle Abs, respectively. Murine serum was
prepared at a 1:100 dilution in PBS, and 100 μl of the diluted serum was added to the 96-well ELISA plates
according to the manufacturer’s instructions. The absorbance at 450 nm was measured using a Paradigm
Multi-Mode Plate Reader (Beckman Coulter, Fullerton, CA, USA). Similarly, the presence of S1P, EPO, TGF-β,
IFN-α and IFN-β in the collected cell culture supernatants or mice sera was examined using a Mouse S1P ELISA Kit
(Westang, Shanghai), Quantikine Mouse Epo Immunoassay Kit (R&D Systems), mouse TGF-β ELISA kit (Catalog:
MB100B, R&D Systems, Minneapolis, MN, USA), Mouse IFN-α ELISA Kit (Guduo Biotechnology, Shanghai) and
Mouse IFN-β ELISA Kit (Guduo Biotechnology, Shanghai) according to the manufacturer’s instructions,
respectively. Theconcentration of haemoglobin and the red blood cells in mice peripheral blood were measured in
Southwest hospital, Chongqing, China.
For the assessment of urine, mice were individually kept in sterilized metabolic cages and urine samples were
pooled over 24 hours. The total urinary albumin was determined with an Albumin Assay Kit (Jiancheng, Nanjing).
The total protein in uria was determined by Bradford method using BCA Assay kit (Beyotime Biotechnology,
Shanghai, China). The concentrations of creatinine and urea in the urine and serum samples were measured with the
Creatinine Assay Kit and Urea Assay Kit (Jiancheng, Nanjing) according to the manufacturer’s instructions.
Real-time quantitative PCR. Real-time PCR was performed as described. In brief, total RNA was harvested using
Trizol LS Reagent (Invitrogen, Carlsbad, CA, USA), and 1 μg total RNA was reverse transcribed into first-strand
cDNA using QuantScript RT Kit (Tiangen Biotech, Beijing, China) and oligo(dT) primers. The cDNA was used to
measure the relative expression of genes using RealMasterMix (SYBR green I) according to the manufacturer’s
protocol (Tiangen Biotech). Real-time quantitative PCR assays of gene expression were performed in 25 μl reactions
with DNA Engine Opticon 2 Real-Time Cycler PCR detector (Bio-Rad Lab., Richmond, CA, USA) using the
standard 2-ΔΔCT method as described. The mRNA expression were normalized to β-actin as an internal control. The
gene expression values were expressed as the relative mRNA expression (fold change) compared to untreated
controls, whose expression was set as 1. The primers used to measure the gene expression are listed as follows: Actb
(forward, 5′-TGGAATCCTGTGGCATCCATGAAA-3′; reverse, 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′),
Ppara (forward, 5′-TCAGGGTACCACTACGGAGT-3′; reverse, 5′-CTTGGCATTCTTCCAAAGCG-3′), Ppard
(forward, 5′-CTCCTGCTGACTGACAGATG-3′; reverse, 5′-TCTCCTCCTGTGGCTGTTC-3′), Pparg (forward,
5′-TATCACTGGAGATCTCCGCCAACAGC-3′; reverse, 5′-GTCACGTTCTGACAGGACTGTGTGAC-3′), Lxra
(forward, 5′-TCCATCAACCACCCCCACGAC-3′; reverse, 5′-CAGCCAGAAAACACCCAACCT-3′), Lxrb
(forward, 5′-TCGCCATCAACATCTTCTCAG-3′; reverse, 5′-GTGTGGTAGGCTGAGGTGTAA-3′), Hif1a
(forward, 5′-GAAATGGCCCAGTGAGAAAA-3′; reverse, 5′-CTTCCACGTTGCTGACTTGA-3′), Hif2a
(forward, 5′-CCTGCAGCCTCAGTGTATCA-3′; reverse, 5′-GTGTGGCTTGAACAGGGATT-3′), Cd36 (forward,
5′-TCGGAACTGTGGGCTCATTG-3′; reverse, 5′-CCTCGGGGTCCTGAGTTATATTTTC-3′), Mertk (forward,
5′-GTGGCAGTGAAGACCATGAAGTTG-3′; reverse, 5′-GAACTCCGGGATAGGGAGTCAT-3′), Gas6 (forward,
5′-TCTTCTCACACTGTGCTGTTGCG-3′; reverse, 5′-GGTCAGGCAAGTTCTGAACACAT-3′), Mfge8 (forward,
5′-GGACATCTTCACCGAATACATCTGC-3′; reverse, 5′-TGATACCCGCATCTTCCGCAG-3′), C1qa (forward,
5′-AAAGGCAATCCAGGCAATATCA-3′; reverse, 5′-TGGTTCTGGTATGGACTCTCC-3′), C1qb (forward,
5′-AACGCGAACGAGAACTATGA-3′; reverse, 5′-ACGAGATTCACACACACAGGTTG-3′), and C1qc (forward,
5′-CAACGCCCTCGTCAGGTT-3′; reverse, 5′-ACAACCCAAGCACAGGGAAGT-3′).
Western blot analysis. Western blot analysis was performed as described. Samples were homogenized in ice-cold
RIPA buffer with 1 mg/mL protease inhibitor cocktail (Beyotime Institute of Biotechnology, Haimen, China). The
homogenates were centrifuged at 13,500 rpm for 5 min at 4°C and the supernatants were collected. BCA assays
were subsequently performed to determine the protein concentration of the supernatants. Cell extracts were resolved
by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto
PVDF membranes (Immobilon-P, Millipore). The following antibodies were used: HIF-1α (1:1000, NB100-479,
Novus Biologicals, Littleton, CO), HIF-2α (1:1000, NB100-122, Novus Biologicals), EPO (1:500, sc-7956, Santa
Cruz, CA), EPOR (1:500, sc-697, Santa Cruz,CA), PPARγ (1:500, ab19481, Abcam), C/EBPβ (1:500, sc-150, Santa
Cruz, CA), ERK (1:500, #9102s, Cell signaling), p-Jak2 (1:500, sc-16566-R, Santa Cruz, CA), Sphk-1 (1:500,
sc-48825, Santa Cruz, CA), p-ERK (1:500, #4370s, Cell Signaling), p-Akt (1:500, sc-33437, Santa Cruz, CA),
p-C/EBPβ (1:500, #3084s, Cell Signaling), GAPDH (1:1000, AB-P-R001, Goodhere Biotechnology, Hangzhou,
China) and goat antirabbit IgG-HRP secondary antibody (1:1000, NBP1-75297, Novus Biologicals, Littleton, CO).
Proteins were visualized with an enhanced chemiluminescence system (BeyoECL Plus, Beyotime Institute of
Biotechnology). Results are representative of at least 3 independent experiments.
RNA Interference. The silencing of Sphk1, C/EBPβ or EPOR was performed by Sphk1-specific siRNA (sense:
5’-GGGCAAGGCUCUGCAGCUCdTT-3’; antisense: 5’-GAGCUGCAGAGCCUUGCCCdTT-3’),
C/EBPβ-specific siRNA (5’-CCCUGCGGAACUUGUUCAAGCAGCU-3’) or EPOR-specific siRNA (sc-39959,
Santa Cruz), respectively. The scramble siRNA (sc-37007, Santa Cruz) was used as a control oligonucleotide.
SiRNAs were transfected with Lipofectamine2000 (Invitrogen) according to a standard protocol, and cells were
collected for Western blot analysis to examine the efficiency of gene knockdown 48 hours after transfection.
Plasmids and luciferase assays. The mouse Pparg promoter (bp –378 to +97) was cloned by PCR from mouse
genomic DNA, and the amplified fragment was sequenced to confirm its integrity. The Pparg promoter fragment
was cloned into the pGL3-basic luciferase expression vector (Promega) to generate pGL3-PPARγ. The C/EBPβ site
mutant construct pGL3-PPARγ mutC/EBP, in which the C/EBPβ elements at bps –327 and –340
(5′-ctgcaattttaaaaagcaatc-3′) were mutated to 5′-ctagtattttaaaaatacctc-3′, was generated using PCR. For transfection,
RAW264.7 cells were plated at 20,000 cells/cm2 in 12-well plates 1 day before transfection. When RAW264.7 cells
reached 60–80% confluence, they were transiently transfected with plasmids using Polyfect transfection reagent
(QIAGEN) and then harvested 48 h later. The luciferase activities in whole cell lysates were measured using a
luciferase assay system (Promega). The luciferase activity was normalized to the protein concentration of each cell
lysate (Oishi et al., 2005).
Histological assessment. Histopathologic examination of the skin, lung, spleen and kidney samples was performed
as previously reported. Briefly, tissues were harvested, fixed in 4% paraformaldehyde, dehydrated, bisected,
mounted in paraffin and sectioned for H&E and periodic acid-Schiff (PAS) staining according to the manufacturer’s
protocol (Sigma-Aldrich).
For immunohistochemistry staining, the paraffin-embedded tissue blocks were cut into 4 μm sections. After
dewaxing, sections were boiled (in a 600 W microwave oven) for 15 min in citrate buffer (2.1 g sodium citrate/L, pH
6). The sections were cooled to room temperature, and endogenous peroxidase was inhibited with 1% hydrogen
peroxidase (H2O2) in methanol for 15 minutes. To block non-specific binding of immunoglobulins, sections were
incubated with 3% albumin bovine V. Thereafter, the sections were incubated with primary antibodies overnight at 4
°C. The following antibodies were used: F4/80 (BM8, ab16911, Abcam Inc.), C3 (11H9, ab11862, Abcam Inc.),
CD4 (sc-7219, Santa Cruz, CA), B220 (RA3-6B2, sc19597, Santa Cruz, CA), goat Anti-Mouse IgA alpha chain
(FITC) (ab97234, Abcam Inc), goat anti-mouse IgG biotin secondary antibody (ab97033, Abcam Inc.), goat anti-rat
IgG biotin secondary antibody (ab150158, Abcam Inc.) and goat anti-rabbit IgG-biotin secondary antibody (ab6012,
Abcam Inc.). After washing, the sections were incubated with corresponding secondary antibodies for 30 min.
Subsequently, the Vecta-stain ABC kit (Vector Laboratories, San Diego, CA, USA) was used for the avidin–biotin
complex method according to the manufacturer’s protocol. Peroxidase activity was visualized with a DAB Elite kit
(K3465, DAKO, Copenhagen, Denmark). The sections were lightly counterstained with hematoxylin and dehydrated
through an ethanol series to xylene and mounted. TUNEL staining (Derma TACS, Trevigen Inc., Gaithersburg, MD,
USA) was performed to identify apoptotic cells in sections from the spleen, thymus, lung, and kidneys with the
following procedure: 4 μm sections were fixed with 2% PFA, permeabilized with 0.1% Triton X-100 in 0.1%
sodium citrate, and stained with the TUNEL reaction. All sections were viewed using a light or fluorescence
microscope.
Renal histopathologic alterations were scored by observers unaware of the mouse type according to a
semiquantitative scale ranging from 0 to 4: 0, normal; 1, a small increase of cells within the mesangium of the
glomerulus; 2, more pronounced increase in the number of mesangial cells and perivascular lymphocytic infiltration
in the cortex and medulla; 3, lobular formation ofthe glomerulus, thickening of basement membrane, and prominent
numbers of lymphocytes surrounding vessels; 4, glomerular crescent formation, some sclerotic glomeruli, tubular
atrophy, and casts and/or vasculitis.
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