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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, Yuqi Liu, Yu Liu, Man Jiang, Zhiren Zhang, and Yuzhang Wu

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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

s.e.m. *; P<0.05, **; P<0.01.

Related to Figure 1.

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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