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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-1, HIF-2 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-1, HIF-2 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-1 and HIF-2 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-1, HIF-2 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 M, 24 hr), for 24 hr, and apoptotic

    thymocyte phagocytosis (left) and the expression of EPO, EPOR, HIF-1 and p-Jak2 were measured. J: Protein

    expression of HIF-1, HIF-2, 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

  • 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 m in the top panel and 15 m 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 m. 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 m. 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 m. 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 m), lung (Scale bar represents 50 m) and thymus (Scale bar represents 50 m) 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 m. 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

  • Figure S4 (related to Figure 4). EPO promotes apoptotic cell engulfment through

    Jak2-ERK-C/EBP-dependent PPAR 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 PPAR 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 PPAR 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/EBP 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-PPAR or pGL3-PPAR

    (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

  • 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 m), skin (Scale bar represents 100 m) and lungs

    (Scale bar represents 50 m) 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 m. 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 ba

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