Download CpG-ODN 2006 and human parvovirus B19 genome consensus

RED CELLS, IRON, AND ERYTHROPOIESIS
CpG-ODN 2006 and human parvovirus B19 genome consensus sequences
selectively inhibit growth and development of erythroid progenitor cells
Yong-Mei Guo,1,2 Keiko Ishii,3 Makoto Hirokawa,1 Hiroyuki Tagawa,1 Hideaki Ohyagi,1 Yoshihiro Michishita,1 Kumi Ubukawa,1
Junsuke Yamashita,4 Toshiaki Ohteki,5,6 Nobuyuki Onai,5,6 Kazuyoshi Kawakami,3 Weiguo Xiao,2 and Kenichi Sawada1
1Department of Hematology, Nephrology, and Rheumatology. Akita University Graduate School of Medicine, Akita, Japan; 2Department of Rheumatology, First
Affiliated Hospital of China Medical University, Shenyang, China; 3Department of Medical Microbiology, Mycology and Immunology, Tohoku University Graduate
School of Medicine, Sendai, Miyagi, Japan; 4Bioscience Center, Radioisotope Division, Akita University School of Medicine, Akita, Japan; 5Department of
Biodefense Research, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; and 6Japan Science and Technology Agency, Core
Research for Evolutional Science and Technology, Tokyo, Japan
Recent studies have shown that anemia is
commonly observed after exposure to
pathogens or pathogen-derived products,
which are recognized via Toll-like receptor 9
(TLR9). In the current study, we demonstrate
that CpG oligodeoxynucleotide-2006, a TLR9
ligand with phosphodiester (PO; 2006PO) but not with the phosphorothioate
backbone, selectively inhibits the erythroid growth derived from human CD34ⴙ
cells. The 2006-PO was internalized by
the erythroid progenitors within 30 minutes; however, expression of TLR9 mRNA
was not detected in these cells. The
2006-PO directly inhibited burst-forming
unit-erythroid growth, resulted in the accumulation of cells in S and G2/M phases,
and increased cell size and frequency of
apoptotic cells. These features were similar to those observed in erythroid progenitors infected with human parvovirus B19
that causes pure red cell aplasia. The
consensus sequence of 2006-PO was
defined as 5ⴕ-GTTTTGT-3ⴕ, which was
located in the P6-promoter region of
B19 and inhibited erythroid growth in a
sequence-specific manner and downregulated expression of erythropoietin receptor (EPOR) mRNA and EPOR. B19
genome extracted from serum also inhibited erythroid growth and down-regulated
expression of EPOR on glycophorin Aⴙ
cells. These results provide a possible
insight into our understanding of the
mechanisms of human parvovirus B19mediated inhibition of erythropoiesis.
(Blood. 2010;115(22):4569-4579)
Introduction
Recent studies have shown that anemia is commonly observed after
exposure to pathogens or pathogen-derived products,1-4 which are
recognized via Toll-like receptor 9 (TLR9). TLR9 has evolved to
recognize unmethylated CpG (cytosine linked to a guanine by a
phosphate bound) dinucleotides that are relatively common in
bacterial and viral genomic DNA, but not in vertebral genomes.5
CpG DNA is generally most active as synthetic single-stranded (ss)
oligodeoxynucleotide (ODN) sequences 20 to 30 nucleotides long,
containing 2 to 3 CpG motifs with a modified nuclease-resistant
backbone, typically a phosphorothioate (PS) backbone in which
one of the nonbridging oxygen atoms at each of the natural (wild)
phosphodiester (PO) linkages is replaced with a sulfur.5 Of note is
that CpG ODN with different backbones and different sequence
motifs can induce dramatically different profiles and kinetics of
immune activation.6-12
Recently, several lines of evidence have demonstrated a direct
link of TLRs and hematopoiesis.13,14 Unlike the ligands for TLRs 2,
4, 7, and 8, the role of CpG-ODN on hematopoiesis has been
reported to be indirect via accessory cells. Sparwasser et al15
demonstrated that unmethylated CpG-ODN 2006 triggered extramedullary hematopoiesis by promoting expansion of granulocytemacrophage colony forming units (CFUs) and early erythroid
progenitors, and that enhanced splenic hematopoiesis is the result
of CpG-ODN–induced cytokines that mobilize bone marrow
progenitor cells to the spleen. Subsequently, Thawani et al16
showed that in vivo administration of unmethylated CpG-ODN
exerted anemia in mice. They reported that this CpG-ODN–
induced suppression was indirect and required accessory cells,
including antigen-presenting cells (APCs), which activated other
cells to produce proinflammatory cytokines, and IFN-␥ was the
major factor mediating erythropoietic suppression. However, the
presence of TLR9 on human CD34⫹ cells, including their progenies and the biologic difference of CpG-ODN backbones, has
remained unclear.
Human parvovirus B19 is a small, ssDNA virus that lacks an
envelope and is characterized by its target specificity for human
erythroid-lineage cells. This specificity is in part the result of the
distribution of its receptor known as the P antigen globoside,17,18
which, however, is present not only on erythroblasts but also on
megakaryocytes, endothelial cells, synovium, villous trophoblast
cells of placental tissues, fetal liver, and heart cells.17,19 Therefore,
the precise mechanisms underlying B19-DNA–mediated selective
inhibition of erythroid cells remain unclear. It has been reported
previously that B19-induced apoptosis and cell-cycle arrest are mediated by nonstructural protein NS1.20-22 However, the G2 arrest has been
shown to be induced by UV-inactivated B19.20 These findings suggest
that the B19-mediated G2 arrest is induced by B19 DNA in the absence
of expression of the viral genome or its proteins.
Submitted August 18, 2009; accepted March 9, 2010. Prepublished online as Blood
First Edition paper, March 26, 2010; DOI 10.1182/blood-2009-08-239202.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2010 by The American Society of Hematology
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Table 1. CpG ODN tested in this study
Oligo name
Sequence
2006-PO
TCGTCGTTTTGTCGTTTTGTCGTT
2137-PO
TGCTGCTTTTGTGCTTTTGTGCTT
2243-PO
GGGGGAGCATGCTGGGGGGG
2006-PS
TCGTCGTTTTGTCGTTTTGTCGTT
2006-PO indicates CpG ODN 2006 with PO backbone; 2137-PO, CpG ODN
2137 with PO backbone; 2243-PO, CpG ODN 2243 with PO backbone; and 2006-PS,
CpG ODN 2006 with PS backbone.
In the current study, we aimed to precisely characterize the role
of the best-characterized ligand for TLR9, CpG-ODN 2006, during
hematopoiesis focusing on different backbones. We report that
CpG-ODN 2006, containing a PO backbone rather than a PS
backbone, shares a consensus sequence with B19 genome and that
the consensus sequence selectively inhibits growth and development of human erythroid progenitor cells.
Methods
Reagents
Bovine serum albumin (BSA), Iscove modified Dulbecco medium (IMDM),
and propidium iodide were purchased from Sigma-Aldrich. Fetal bovine
serum was from HyClone. Penicillin and streptomycin were from Invitrogen. Insulin (porcine sodium, activity 28.9 U/mg) and Triton X-100 were
obtained from Wako Pure Chemical Industries. Diaminobenzidinesubstrate chromogen and fuchsin-substrate chromogen systems were from
Dako Denmark. Interleukin-3 (IL-3), stem cell factor (SCF), and thrombopoietin (TPO) were kind gifts of the Kirin Brewery Co Ltd, and erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF) were from
Chugai Pharmaceutical Co. Vitamin B12 was from Eisai Co Ltd, and folic
acid was from Takeda Pharmaceutical Co Ltd. RNase (type III-A) was from
Sigma-Aldrich. LysoTracker and carboxyfluorescein diacetate succinimidyl
ester (CFSE) were from Invitrogen. MACS MicroBeads for Indirect
Magnetic Labeling was from Miltenyi Biotec. IODO-GEN (1,3,4,6tetrachloro-3␣, 6␣-diphenylglycouril) was from Thermo Scientific, and Na
125I was from PerkinElmer Life and Analytical Sciences.
ODN
The specific sequences of ODN used in this study are presented in Table 1.
CpG-ODN 2006 (2006-PO), ODN 2137 (2137-PO), and ODN 2243
(2243-PO) all with PO backbone were commercially synthesized by
FASMAC and Hokkaido System Science Co Ltd. CpG-ODN 2006 with PS
backbone (2006-PS) and ODN 5⬘ labeled with rhodamine were commercially synthesized by Hokkaido System Science Co Ltd.
Antibodies
Fluorescein isothiocyanate (FITC)–labeled monoclonal antibodies (mAbs)
specific for CD15 (H198), CD71 (M-A712), and phycoerythrin (PE)–
labeled antibodies for CD13 (WM15) and CD71 (M-A712) were purchased
from BD Biosciences. PE-CD11c (BU15) was from Immunotech. PECDw123 (IL-3R␣; 9F5) and CD61 (VI-PL2) were from BD Biosciences
PharMingen. FITC- or PE-labeled glycophorin A (GPA; JC159), PE-CD34
(BIRMA-k3), and FITC-CD61 were from Dako Japan Co. PE-EPO
receptor (EPOR; 38409) was from R&D Systems. FITC–annexin V
apoptosis detection kit was from Sigma-Aldrich. Rabbit polyclonal antibody to erythroid Krüppel-like factor (EKLF) and mouse mAb to early
endosomes (EEA1) were from Abcam. Mouse mAb to ␣-tubulin (DM 1A)
was from Sigma-Aldrich. Alexa Fluor 488 or Alexa Fluor 546 conjugated
goat IgG directed against rabbit and mouse IgG were from Invitrogen.
Fc-blocking antibody (anti-CD16/32, clone 93) was from eBioscience.
Normal mouse and goat sera, rabbit immunoglobulins, and rabbit IgG
conjugated with horseradish peroxidase through dextran polymer to IgG
were from Cell Signaling Technology.
Cell preparation
G-CSF–mobilized human peripheral blood CD34⫹ cells were purified from
healthy volunteers and stored in liquid nitrogen until use as described
previously.23 Informed consent was obtained from each subject before the
entry into this study in accordance with the Declaration of Helsinki, and the
study was preapproved by the Akita University School of Medicine
Committee for the Protection of Human Subjects.
For the generation of erythroid progenitor cells, CD34⫹ cells were
thawed and prepared for the culture, as previously described.23 Cells were
cultured in erythroid medium (IMDM containing 20% fetal bovine serum,
10% heat-inactivated pooled human AB serum, 1% BSA, 10 ␮g/mL of
insulin, 0.5 ␮g/mL vitamin B12, 15 ␮g/mL folic acid, 50nM ␤-mercaptoethanol,
50 U/mL penicillin, and 50 ␮g/mL streptomycin, in the presence of
50 ng/mL IL-3, 50 ng/mL SCF, and 2 IU/mL EPO). Cells were maintained
at 37°C in a 5% CO2 incubator as described previously.24 The cells were
harvested at indicated days and resuspended in 2 mL of IMDM containing
0.1% BSA. The cells were incubated for 2 hours at 37°C to remove surface
EPO before fluorocytometric analysis and radiolabeled EPO-binding
measurements.
For the generation of neutrophilic and megakaryocytic progenitor cells,
the CD34⫹ cells were cultured at a density of 2 ⫻ 105 to 5 ⫻ 105 cells/mL
in the erythroid medium, with the exception that SCF, IL-3, and EPO were
replaced with either 100 ng/mL G-CSF or 100 ng/mL TPO as described
elsewhere.23,25 For the simultaneous generation of erythroid, neutrophilic,
and megakaryocytic progenitor cells, CD34⫹ cells were suspended at a
density of 2 ⫻ 104 to 3 ⫻ 104 cells/mL in the presence of 50 ng/mL IL-3,
50 ng/mL SCF, 2 IU/mL EPO, 100 ng/mL G-CSF, and 100 ng/mL TPO.
Plasmacytoid dendritic cells (pDCs, CD11c⫺ CD123⫹), conventional DCs
(cDCs, CD11c⫹ CD123⫺), and B cells (CD20⫹) were isolated from normal
human peripheral blood using MoFlo (Dako North America). The yield and
viability were measured by dye exclusion using 0.2% trypan blue dye using
a hemocytometer.
Semisolid culture of BFU-E
For the burst-forming unit-erythroid (BFU-E) colony assay, purified
CD34⫹ cells were incubated in triplicate at various concentrations, in
flat-bottom 24- or 96-well tissue culture plates (Linbro; Flow Laboratories)
containing 0.5 mL or 0.05 mL medium with fibrin clots, respectively, as
described elsewhere.26 After 14 days in culture, the clots were fixed and
stained for hemoglobin.27 Aggregates with greater than 100 hemoglobinized cells were counted as BFU-E colonies.
Flow cytometry
The cells collected from culture were washed twice with IMDM containing
0.3% BSA. The cells were then incubated with FITC- and PE-labeled
mAbs, washed twice with staining medium containing 10mM phosphatebuffered saline (PBS, pH7.4), 0.5% BSA, and 2mM EDTA, and analyzed
using a FACSCalibur (BD Biosciences), as reported elsewhere.23
Binding of 125I-EPO
Cells in 50 ␮L of IMDM at 0°C containing 0.1% BSA were incubated in the
presence of 2 U/mL of 125I-EPO prepared as reported elsewhere.27,28 After
24 hours of incubation at 0°C, the radioactivity of the cell pellets was
counted in a gamma counter (Nuclear Chicago), as reported elsewhere.27,28
Nonspecific binding was determined with a 100-fold excess of unlabeled
EPO. Specific binding was calculated by subtracting nonspecific binding
from total binding.
Confocal microscopy
Fluorescence staining was imaged using a Confocal Laser Scanning
Microscope 510 (LSM510; Carl Zeiss Microscope Systems) equipped with
a 100⫻ objective lens and a 10⫻ camera lens (Carl Zeiss Microscope
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Table 2. Primers used in this study
Primer
TLR9
EPOR
GATA-1
GATA-2
EKLF
RPS19
FOG-1
28S
Direction
Sequence
Forward
5⬘-ACC CTG GAA GAG CTA AAC C-3⬘
Reverse
5⬘-CAG TTG CCG TCC ATG AAT-3⬘
Forward
5⬘-TCA TCC TCG TGG TCA TCC T-3⬘
Reverse
5⬘-CCT TCA AAC TCG CTC TCT G-3⬘
Forward
5⬘-TGG CCT ACT ACA GGG ACG CT-3⬘
Reverse
5⬘-CTC AGC CGC TCT GTC TTC A-3⬘
Forward
5⬘-CCT CCA GCT TCA CCC CTA A-3⬘
Reverse
5⬘-CAC AGG CAT TGC ACA GGT AGT-3⬘
Forward
5⬘-CAG AGG ATC CAG GTG TGA TAG-3⬘
Reverse
5⬘-GCA GGC GTA TGG CTT CTC-3⬘
Forward
5⬘-GCT CCA TGA CCA AGA TCT AT-3⬘
Reverse
5⬘-GTC CAG ATC TCT TTG TCC CT-3⬘
Forward
5⬘-TGC ACA CGG ACA CGC TGA-3⬘
Reverse
5⬘-GTA GAT CTC ACC CTT GGA GCC A-3⬘
Forward
5⬘-TGG GTT TTA AGC AGG AGG TG-3⬘
Reverse
5⬘-CCA GCT CAC GTT CCC TAT TA-3⬘
TLR9 indicates Toll-like receptor 9; EPOR, erythropoietin receptor; EKLF,
erythroid Krüppel-like factor; RPS19, ribosomal protein S19; FOG-1, friend of
GATA-1; and 28S, 28S ribosomal gene.
Systems) at zoom 3. Fluorochromes were excited using an argon laser at
488 nm for Alexa 488. Detector slits were configured to minimize cross-talk
between channels and processed using software package (LSM510, Version
3.2) and Adobe Photoshop (Adobe Systems).
Cell cycle distribution
Cells were harvested, washed with cold PBS, and fixed in 70% ethanol. The
cells were then stored at ⫺20°C until analysis. The fixed cells were
centrifuged at 400g, washed with cold PBS twice, and RNase A added at a
final concentration of 0.5 mg/mL. The cells were then incubated for
10 minutes at 37°C. Next, 25 ␮g/mL propidium iodide was added and the
cells were incubated for 30 minutes at room temperature in the dark. The
cells were analyzed using a FACSCalibur instrument (BD Biosciences)
equipped with CellQuest, Version 3.3 software. Multicycle cell-cycle
analysis software (Beckman Coulter) was used to determine the percentage
of cells in the different cell cycle phases.
Real-time RT-PCR
Total RNA was extracted from 2 ⫻ 104 cells per sample using TRizol
reagent (Invitrogen). The extracted RNA was then reverse-transcribed using
the SuperScript III First-Strand Synthesis System for reverse transcriptasepolymerase chain reaction (RT-PCR; Invitrogen) in a 20-␮L reaction
volume. cDNA was then subjected to real-time RT-PCR using LightCycler
480 SYBR Green I Master (Roche Applied Science). The relative gene
expression levels were normalized with 28S. Primer sequences are presented in Table 2 and were purchased from Nippon Gene Research
Laboratories.
Western blot analysis
CD34⫹ cells were incubated in erythroid medium for 3 days (day 3 cells)
and the dead cells depleted using the Annexin Microbead kit (Milenyi
Biotec). Day 3 cells were then treated with or without 2006-PO for
12 hours. Western blot analysis was carried out according to the manufacturer’s protocol.
Isolation of the B19 genome
Serum samples containing B19 were collected from donated blood samples
provided by the Japanese Red Cross Saitama Blood Center. An aliquot of
the serum was used for infection and isolation of viral DNA. This aliquot
was layered on 30% sucrose in PBS and centrifuged for 24 hours at
100 000g to precipitate the B19 virion. B19 genome DNA was then
extracted from the pellet fraction using the QIAamp DNA Blood Mini Kit
SELECTIVE INHIBITION OF ERYTHROID GROWTH BY ODN
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(QIAGEN). Cellular DNA was extracted from UT7/Epo-S1 cells20 (a
generous gift of Kazuo Sugamura, Tohoku University Graduate School of
Medicine, Japan), digested with BamHI, and used as double-stranded
cellular DNA (dsDNA). ssDNA was obtained by heat denaturation of
dsDNA extracted from UT7/Epo-S1 cells.
B19 infection of erythroid progenitors
CD34⫹cells were cultured in erythroid medium for 4 days (3 ⫻ 105
cells/0.06 mL) and then either mock-infected or mixed with human serum
that contained 6 ⫻ 109 genome copies of B19. The cell-to-virus ratio was
1:20 000. This mixture was then incubated for 1 hour on ice, diluted in
erythroid medium, and further incubated at 37°C in 5% CO2 for 24, 48, or
72 hours.
Statistical analysis
Significant differences between groups were calculated using 1-factor
analysis of variance (Scheffé post-hoc test) and the paired t test. Tests were
undertaken using Stat View, Version 4.0, and significant differences were
defined as P less than .05.
Results
Selective inhibition of erythroid growth by CpG-ODN 2006-PO
To examine the effects of CpG-ODN 2006-PO (2006-PO) on the
growth of hematopoietic progenitors, human CD34⫹ cells were
cultured for 7 days in liquid medium in the presence of multiple
cytokines. Under these conditions, simultaneous differentiation of
erythroid (GPA⫹), neutrophilic (CD15⫹), and megakaryocytic
(CD61⫹) progenitors occurred. We found that 2006-PO inhibited
the growth of erythroid progenitors in a dose-dependent manner
but did not affect the growth of neutrophilic and megakaryocytic
progenitors (Figure 1A). To investigate the specificity of 2006-PO
in the inhibition of erythroid growth, we then differentiated CD34⫹
cells into each of the erythroid (80.1% ⫾ 6.7% pure GPA⫹ cells,
n ⫽ 3), neutrophilic (45.9% ⫾ 6.5% pure CD15⫹ cells, n ⫽ 3), and
megakaryocytic (90.6% ⫾ 15.9% pure CD61⫹ cells, n ⫽ 3) progenitor lineages. The inhibitory effects of 2006-PO on the growth of
erythroid progenitor cells were also evident in a dose-dependent
manner and occurred at a half-maximal dose of 1␮M (Figure 1B).
In contrast, 2006-PO exhibited no inhibitory effects on the growth
of neutrophilic (Figure 1C) and megakaryocytic (Figure 1D)
progenitors. These data indicate that 2006-PO selectively inhibited
erythroid growth. In addition, the absolute number of CD15⫹ cells
generated from 1 ⫻ 105 CD34⫹ cells without 2006-PO was
13.0 ⫻ 105 cells in the presence of multiple cytokines (Figure 1A)
and 0.6 ⫻ 105 cells in the presence of G-CSF alone (Figure 1C),
which indicates that multiple cytokines induce a 22-fold of
expansion of CD15⫹ cells compared with that with G-CSF alone.
Addition of 2006-PO did not affect the yield of CD15⫹ cells. These
data suggest that 2006-PO does not affect uncommitted CD34⫹
early progenitors.
To examine the effects of different ODNs on erythroid growth,
several ODNs were synthesized. The construction of these ODNs
focused on the CpG-motif, ODN backbone, and sequence (Table 1). As
illustrated in Figure 1E, CpG-ODN (2006-PO) and non–CpG-ODN
(2137-PO; similar to 2006 but contained GC instead of CG) exhibited
similar inhibitory effects on erythroid growth, and the latter (2137-PO)
also specifically inhibited erythroid growth (supplemental Figure 1,
available on the Blood Web site; see the Supplemental Materials link at
the top of the online article). ODN-2243-PO, which contained a
different ODN sequence from that of 2006-PO and CpG-ODN with the
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B
A
Total cells
GPA+ cells
NS
*
***
Cell number (x105)
Total cells
Cell number (%)
GPA+ cells
CD15+ cells
CD61+ cells
***
0
0
1
4
2
Total cells
D
Total cells
CD15+ cells
4
4
E
Total cells
GPA+ cells
Cell number (%)
NS
Cell number (%)
Cell number (%)
0
2
1
CD61+ cells
NS
2006-PO (ȣM)
***
2006-PO (ȣM)
2006-PO (ȣM)
C
***
0
4
2006-PO (ȣM)
Control
***
***
2006
-PO
2137
-PO
PS backbone, did not exhibit any inhibitory effects on erythroid growth.
These data indicate that the inhibitory effects of 2006-PO on erythroid
growth depended on the ODN sequence and backbone but not on
the CpG motif.
Immunophenotypic analysis of the cells generated in the
presence of 2006-PO revealed that the majority of nonerythroid
progenitors (GPA⫺ cells) were CD13⫹ cells. A relatively small
population of CD4⫹, CD11c⫹, and CD123⫹ cells were also
observed (supplemental Figure 2).
Internalization of ODN by progenitors derived from CD34ⴙ cells
Purified CD34⫹ cells were cultured in erythroid medium in the
presence or absence of 2006-PO, and the TLR9 mRNA expression
was monitored at the indicated time points. B cells and pDCs were
used as positive controls, whereas cDCs were used as a negative
control.29 As illustrated in Figure 2A, pDCs and B cells were found
to express TLR9 mRNA. CD34⫹ cells harvested at days 0 to 4 also
appeared to express TLR9; however, the TLR9 expression levels
were lower than that of the cDCs. TLR9 expression at day 7 was
undetectable, and 2006-PO did not significantly affect the levels of
TLR9 mRNA expression. These data suggest that erythroid progenitors do not possess TLR9.
To examine the intracellular localization of ODNs, purified
CD34⫹ cells were cultured in erythroid medium for 2 days and
were incubated with rhodamine-labeled 2006-PO or 2243-PO
(ODN-PO with a different ODN sequence to that of 2006-PO) for
30 minutes. The cells were then incubated with anti–early endosome antibody (EEA1). The 2006- and 2243-ODN-rhodamine
were found to occasionally colocalize with the green fluorescence
of the early endosomes, indicating that ODN was internalized
within 30 minutes and was partially targeted to the early endosome
regardless of the differences in sequence and biologic activity
(Figure 2B). When LysoTracker Green was used as a lysosome
tracker in day 2 cells, 2006-PO (Figure 2C) and 2243-PO (Figure
2D) were partially colocalized with the lysosomes after a 2-hour
NS
NS
2243
-PO
2006
-PS
Figure 1. Effects of ODN on the growth of hematopoietic
progenitors derived from CD34ⴙ cells. (A) CD34⫹ cells were
cultured with IL-3, SCF, EPO, G-CSF, TPO, and various
concentrations of 2006-PO ranging from 0 to 4␮M. Seven days
later, the total cell yield was counted and GPA, CD15, and
CD61 expression examined using fluorocytometry. Data are
mean ⫾ minus SD of 3 independent experiments. (B-D) CD34⫹
cells were cultured with IL-3 and SCF in the presence of EPO
at a cell density of 2 ⫻ 104 cells/mL (B), with G-CSF at a cell
density of 3 ⫻ 105 cells/mL (C), or with TPO at a cell density of
3 ⫻ 105 cells/mL (D) with or without 2006-PO. Seven days
later, the cell yields were calculated as a percentage relative to
the total number of cells without 2006-PO. Data are mean ⫾ SD
of 3 independent experiments. The absolute number of
cells generated from CD34⫹ cells without 2006-PO was
7.6 ⫾ 2.2 ⫻ 105 cells (A), 8.6 ⫾ 3.6 ⫻ 105 cells (B),
4.0 ⫾ 0.6 ⫻ 105 cells (C), and 4.0 ⫾ 0.6 ⫻ 105 cells (D).
(E) CD34⫹ cells were cultured in erythroid medium in the
presence or absence of various forms of ODN at 4␮M. The
total cell yield was then calculated as a percentage relative to
the total number of cells without ODN (5.3 ⫾ 1.4 ⫻ 105 cells/
2 ⫻ 104 CD34⫹ cells cultured; n ⫽ 4). Data are mean ⫾ SD of
4 independent experiments. *P ⬍ .05. ***P ⬍ .001. NS indicates no significance.
incubation. At 4 hours after incubation with ODN-rhodamine,
2243-PO and 2006-PO were partially colocalized with the vesicular LysoTracker Green, suggesting that a portion of the ODN had
escaped degradation in the lysosomes. In addition, the half-life of
2006-PO in serum was estimated at 4 hours, whereas that of
2006-PS exceeded more than 24 hours (supplemental Figure 3).
The PS backbone appeared to increase the nonspecific ODN
binding to a wide variety of serum proteins, as reported previously.30
2006-PO inhibits the early stages of erythroid growth
To further understand the kinetics of erythroid inhibition by
2006-PO, purified CD34⫹ cells were cultured in erythroid medium
in the presence or absence of 2006-PO and the surface phenotypes
observed at different time points (Figure 3A). The total number of
cells recovered in both the absence and presence of 2006-PO was
comparable after 3 days in culture. The cell yield substantially
increased in the cultures that did not contain 2006-PO and was
decreased in the cultures that did contain 2006-PO after 4 days in
culture. We also found that the main cell population that was
decreased after treatment with 2006-PO was the GPA⫹ cell
population. These findings indicated that 2006-PO affected CD34⫹
cell development by inhibiting erythroid growth in the early stage
of development. When purified CD34⫹ cells were cultured for
7 days in erythroid medium and 2006-PO was added at the
indicated time points (Figure 3B), addition of 2006-PO to the
medium resulted in significant inhibitory effects on the generation
of erythroid progenitors as late as 4 days after treatment. In
contrast, 2006-PO did not affect the growth of more mature
erythroid progenitor cells in the stage of CFU-erythroid (CFU-E)27
(Figure 3C). These findings suggest that 2006-PO inhibits the
erythroid progenitor cells in the stage of BFU-E.
To investigate whether the inhibitory effects of 2006-PO
mediated inhibition of the clonal development of BFU-E, purified
CD34⫹ cells were cultured in semisolid erythroid medium for
14 days, with or without different concentrations of 2006-PO or
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SELECTIVE INHIBITION OF ERYTHROID GROWTH BY ODN
A
C
1.0
0.9
TLR9/28s
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DIC
Lysotracker 2006-PO
Green
-rhodamine
Merge
DIC
Lysotracker 2243-PO
Green
-rhodamine
Merge
Control
2006-PO
0.020
2h
0.015
0.010
4h
0.005
0
Day0 Day1 Day2 Day4 Day7 cDC B cells pDC
B
DIC
Early
endosome
ODNrhodamine
D
Merge
2006
-PO
2243
-PO
2h
4h
Figure 2. Internalization of ODN by hematopoietic progenitors derived from CD34ⴙ cells. (A) TLR9 expression in pDCs, B cells, cDCs, and hematopoietic progenitors
induced erythroid differentiation. CD34⫹ cells (day 0) were cultured in the liquid phase in erythroid medium with or without 4␮M 2006-PO for 1 to 7 days. The relative gene
expression levels of TLR9 were normalized with 28S transcripts. The inset represents amplification of the y-axis. The results are representative of 3 independent experiments.
(B) ODN-rhodamine and early endosome expression. CD34⫹ cells were cultured in erythroid medium for 2 days and incubated with rhodamine-conjugated 2006-PO or
2243-PO. Results are representative of 3 independent experiments. (C-D) ODN-rhodamine and lysosome expression. CD34⫹ cells were cultured in erythroid medium for
2 days, incubated with LysoTracker Green and rhodamine-conjugated 2006-PO (C) or 2243-PO (D) and analyzed using confocal microscopy. Results are representative of
3 independent experiments.
2243-PO. 2006-PO administered at a concentration of 4␮M
completely abolished the formation of visible BFU-E colonies,
whereas 2243-PO promoted it (Figure 3D top panel). Microscopic
observation of the clots stained for hemoglobin (Figure 3D bottom
panel) showed that the number of BFU-E colonies was greatly
reduced compared with that formed in the control treated cells
(Figure 3E).
2006-PO directly inhibits erythroid growth, produces large
erythroblasts, retards cell division, and induces G2/M
accumulation and apoptosis of erythroblasts
To exclude the possibility that 2006-PO may inhibit BFU-E growth
by nonerythroid cells, we investigated the clonal basis of BFU-E
growth, in both the presence and absence of 0.5␮M 2006-PO, using
limiting dilution analysis.31 Purified CD34⫹ cells were plated at a
variety of concentrations in fibrin clots as indicated in Figure 4A,
and the percentage of nonresponder wells plotted against the
number of cells per well. Straight lines through the origin for
BFU-E colonies could be constructed, indicating that 2006-PO
directly inhibited BFU-E during clonal development.
After 7 days in culture, the size of the erythroid cells generated
in the presence of 2006-PO appeared to be larger than those
generated without 2006-PO when observed using light microscopy
(Figure 4B left and middle panels). Size distribution analysis of
these GPA⫹ cells revealed that 2006-PO treatment promoted larger
erythroid progenitors compared with control cells (Figure 4B right
panel). Phenotypic analysis of CD34⫹ cells cultured in the
erythroid medium showed that 2006-PO treatment decreased
CD71⫹ GPA⫹ cells after 4 days in culture (Figure 4C top panel).
When CD34⫹ cells were stained with CFSE and cultured in
erythroid medium with or without 2006-PO, the GPA⫹ cells
exhibiting a high CFSE intensity were observed in the presence of
2006-PO after 4 days in culture (Figure 4C bottom panel). These
findings suggested that 2006-PO retarded erythroid progenitor cell
proliferation. When purified CD34⫹ cells were cultured with
2006-PO for 7 days, 2006-PO resulted in the accumulation of cells
in the S and G2/M phase. This result was accompanied by an
increase in both the apoptotic fraction (Figure 4D) and the number
of apoptotic cells that were annexin V–positive (Figure 4E).
Taken together, 2006-PO both directly and selectively inhibits
erythroid cell development by promoting the growth of large
erythroid progenitors that demonstrate reduced cell division and
accumulation in the S and G2/M phase. These results were
accompanied by an increased frequency of apoptosis. In combination, the majority of these features were similar to those observed in
erythroid progenitor cells infected with B19,20-22 a virus that causes
pure red cell aplasia (PRCA) with giant erythroblasts. Thus, we
subsequently searched for the 2006-PO consensus sequence within
the B19 genome.
B19 (453 5ⴕ-GTTTTGT-3ⴕ 459) consensus sequence is shared
with 2006-PO and inhibits erythroid growth
We found that the 2006-PO consensus sequence 5⬘-GTTTTGT-3⬘
was located within the P6-promoter region of B19 (Figure 5A). To
examine the effects of synthetic ODN that shared the same
nucleotide length as 2006-PO (24 nucleotides) and the identical
sequence to B19, purified CD34⫹ cells were cultured in erythroid
medium for 7 days with or without 4␮M synthetic ODN. The
synthetic ODN-436, -441, -451, and -453 that contained the
consensus sequence inhibited erythroid growth. These ODNs were
found to inhibit growth by 86% plus or minus 2% (P ⬍ .001), 79%
4574
BLOOD, 3 JUNE 2010 䡠 VOLUME 115, NUMBER 22
GUO et al
Tot al cells
CD71+ cells
*
***
Cell number (%)
A
***
Days of culture
B
GPA+ cells
Total cells
GPA+ cells
Days of culture
C
Total cell s
GPA+ cells
D
Days of culture
E
Control
NS
NS
*** *** ***
2006
-PO
2243
-PO
BFU-E
colony
Days of 2006-PO-addition
BFU-E colonies
*** ***
Cell number (x105)
Cell number (%)
***
Control 0.5 4.0
2006-PO
0.5 4.0 (ȣM)
2243-PO
2006-PO (ȣM)
Figure 3. 2006-PO inhibits erythroid growth in the early stages of development. (A) Kinetics of erythroid growth. CD34⫹ cells were cultured in erythroid medium with (F) or
without (E) 2006-PO. At the indicated days, the cells were collected, washed, and counted. The total cell yields are represented as a percentage relative to the total number of
cells without 2006-PO on the seventh day (4.9 ⫾ 0.2 ⫻ 105 cells, n ⫽ 3). Insets: Amplification of the y-axis. (B) CD34⫹ cells were cultured for 7 days in erythroid medium. At the
indicated days, 2006-PO was added to the medium. The total cell yields are represented as a percentage relative to the total number of cells without 2006-PO
(7.4 ⫾ 3.8 ⫻ 105 cells, n ⫽ 3). (C) Effects of 2006-PO on CFU-E. CD34⫹ cells were cultured in erythroid medium. After 7 days in culture, the cells were collected and washed,
and then cultured for a further 5 days (until day 12) in the presence of EPO, with or without 2006-PO. (A-C) Data are mean ⫾ SD of 3 independent experiments. (D) Purified
CD34⫹ cells were cultured in semisolid medium with IL-3, SCF, and EPO, with or without ODN. After 14 days in culture, the clots were observed directly (top panel) and then
fixed and stained for hemoglobin (bottom panel). (E) BFU-E and small erythroid colonies were then differentially counted under light microscopy. The number of colonies is a
representative of 2 independent experiments and the mean ⫾ SD of triplicate culture. *P ⬍ .05. ***P ⬍ .001. NS indicates no significance.
plus or minus 3% (P ⬍ .001), 69% plus or minus 10% (P ⬍ .001),
and 75% plus or minus 7% (P ⬍ .001) compared with control
growth, respectively (Figure 5B). ODN-461 that lacked the consensus sequence did not show any significant inhibitory effects on
erythroid growth (23% ⫾ 24% compared with control growth,
P ⬎ .05). These results suggested that the 24 nucleotide sequences
containing the 5⬘-GTTTTGT-3⬘ consensus sequence of the B19
DNA inhibited erythroid progenitor growth in a sequencedependent manner. To confirm the minimum consensus sequence
length required for erythroid growth, purified CD34⫹ cells were
cultured in erythroid medium for 7 days with or without 4␮M
2006-PO, 5⬘-GTTTTGT-3⬘ or 5⬘-ACAAAAC-3⬘, the latter being
the complimentary sequence of 5⬘-GTTTTGT-3⬘ (Figure 5C top
panel). ODN containing the GTTTTGT inhibited erythroid growth
in a similar fashion to that observed by 2006-PO. ODNs containing
ACAAAAC also inhibited erythroid growth, but only to a minimal
degree. A similar effect was also observed with 5⬘-TTTTGT-3⬘
(Figure 5C bottom panel). These results indicated that the consensus DNA sequence of the human parvovirus B19 was shared with
2006-PO and inhibited erythroid growth.
2006-PO inhibits the expression of EPOR mRNA, EPOR, and
EKLF protein
It is well established that several transcription factors and genes are
required for commitment to the erythroid lineage. We therefore
examined the effects of 2006-PO on the expression of GATA-2,32
GATA-1,33,34 friend of GATA-1 (FOG-1),35 EKLF,36 ribosomal
protein S19 (RPS19),37 and EPOR. For this purpose, CD34⫹ cells
were incubated for 3 days in erythroid medium (day 3 cells) and
CD71⫹ cells purified using MiniMACS. The CD71⫹ cells were
then incubated with or without 2006-PO in erythroid medium for
12 hours, with the exception of the EPOR gene that required
several days in culture to be activated. We found that 2006-PO
inhibits EPOR mRNA expression but did not affect the expression
of any of other genes examined (Figure 6A,E; supplemental Figure
4). Fluorocytometric analysis showed that 2006-PO significantly
decreased the expression of EPOR in the GPA⫹ cells (Figure 6B-C
left panel). A similar effect was also observed with ODNGTTTTGT (Figure 6C right panel). 125I-EPO binding assay showed
a decrease of the specific binding of 125I-EPO to the cells treated
BLOOD, 3 JUNE 2010 䡠 VOLUME 115, NUMBER 22
A
SELECTIVE INHIBITION OF ERYTHROID GROWTH BY ODN
B
CD34+ cells/well
Control
100
2006-PO
90
Control
80
Cell number
BFU-E negative well (%)
4575
70
60
2006-PO
50
Control
2006-PO
FSC
40
C
Day 1
Day 3
Day 2
Day 4
Day 5
Day 6
Day 7
0.0
0.3 0.2
3.0
0.0
9.0
0.0
28.9 0.0
51.0
0.0
70.4
0.1
76.3
64.8
34.9 34.8
62.0
9.6
81.4
4.6
66.6 8.4
40.6
5.2
24.4
9.5
12.2
0.6
0.3 0.1
2.7
0.8
10.2
0.9
19.3 0.4
31.6
0.0
37.7
0.4
39.0
31.9 36.5
60.6
12.0
67.8 25.2
42.9 24.1
38.1 41.8
18.9
D
Control
Cell number
Control
GPA
2006-PO
67.3
14.3 74.8
CD71
0.0
0.8
1.4
98.3 21.7
76.1
1.3
3.3
3.2
18.6
5.3
40.2
8.7
56.7
9.0
64.6
11.2
25.9 67.6
39.4
36.7 32.8
18.3
22.4
11.9 12.9
11.3
8.7
9.9
14.6 24.4
18.7 28.6
14.8
18.5
62.9 25.6
19.2
37.7 22.6
34.0
E
0.6
0.4
1.0
0.3
99.1
5.2
93.4
1.9
2.5
21.5
2006-PO
0.5
7.3
3.8
21.3
74.3
38.3
22.1
PI
0.0
G0/1 : 27.4%
S
: 64.3%
G2/M: 8.3%
Control
0.5
2006-PO
GPA
G0/1 : 48.2%
: 47.9%
S
G2/M: 3.9%
DNA content
Control
0.4
2006-PO
7.4
Annexin V
CFSE
Figure 4. 2006-PO directly inhibits BFU-E growth. (A) Limiting dilution analysis of BFU-E growth from purified CD34⫹ cells in semisolid medium. CD34⫹ cells were cultured
in fibrin clots in 96-well flat-bottom plates in the presence of IL-3, SCF, and EPO, with (F) or without (E) 2006-PO. After 14 days, the clots were fixed, stained for hemoglobin,
and the number of clots in which BFU-E colonies did not form counted as BFU-E colony-negative wells. These data were plotted against the number of CD34⫹ cells plated into
the wells originally. Each point represents the values obtained from 60 wells. The results are representative of 3 independent experiments. (B) Morphology and size distribution
of the generated cells. Purified CD34⫹ cells were cultured in erythroid medium, with or without 2006-PO for 7 days and subjected to May-Grünwald-Giemsa staining (⫻1000).
Results are representative of 5 independent experiments. Scale bars represent 10 ␮m. (C) Top panel: Purified CD34⫹ cells were cultured in erythroid medium. At the indicated
days, the cells were harvested and analyzed by fluorocytometry. Results are representative of 3 independent experiments. Bottom panel: CFSE dilution assay. Purified CD34⫹
cells were incubated with CFSE and cultured in erythroid medium. At the indicated days, the cells were harvested and analyzed by fluorocytometry. Results are representative
of 2 independent experiments. (D-E) Purified CD34⫹ cells were cultured in erythroid medium with or without 2006-PO for 7 days. Results are representative of 2 independent
experiments. (D) Cell-cycle analysis of the generated cells. (E) The cells were labeled with propidium iodide and annexin V and analyzed using fluorocytometry.
with 2006-PO (Figure 6D). Western blot analysis revealed that
2006-PO resulted in a significant decrease in EKLF protein levels
(Figure 6F-G).
B19 down-regulates the expression of EPOR
To examine the effects of B19 genome DNA on erythroid
growth, B19 genome was extracted from the sera of patients
with acute B19 infection (Figure 7A). When CD34⫹ cells were
differentiated to the erythroid lineage for 7 days in either the
presence or absence of native single-stranded B19 genome, B19
genome significantly inhibited erythroid growth to a level
similar to that observed with 2006-PO (Figure 7B). dsDNA
derived from UT7/Epo-S1 cells exhibited no effects on erythroid growth, whereas ssDNA demonstrated a mild inhibition.
These data indicated that nonsynthetic B19 genome inhibited
erythroid growth.
Several lines of evidence have demonstrated that the most
permissive target cells for B19 are erythroid progenitors, BFU-E
and CFU-E, and erythroblasts.38-40 We therefore used cells derived
from CD34⫹ cells that had been cultured in erythroid medium for
4 days as the target cells for B19. After incubating cells in serum
containing B19 for 1 hour, the cells were further cultured in
erythroid medium for various periods. The expression of EPOR in
GPA⫹ cells was found to be down-regulated in cells cultured with
B19 compared with that observed in mock-infected cells (Figure
7C-D). These data suggested that B19 infection in erythroid
progenitor cells down-regulated EPOR expression, a result that is
consistent with that indentified in the erythroid cells exposed to
2006-PO.
Discussion
It is well established that unmethylated CpG-ODN induces APCs
to produce cytokines, such as IL-12, IL-6, IL-1, and TNF-␣.41 It has
been previously reported that the biologic effect of unmethylated
CpG-ODN 2006 on hematopoietic progenitor cells is indirect and
is exerted by accessory cells, including APCs that produce
proinflammatory cytokines.15,16 Unfortunately, the precise backbone expressed by unmethylated CpG-ODN was not described in
these reports. In our study, the limiting dilution analysis of BFU-E
growth indicated that 2006-PO directly inhibited clonal development and proliferation of BFU-E. This finding appears to be
4576
BLOOD, 3 JUNE 2010 䡠 VOLUME 115, NUMBER 22
GUO et al
A
2006-PO : 5’-TCGTCGTTTTGTCGTTTTGTCGTT-3’
ODN436 5’-TATAATTTTATTGGT CAGTTTTGT-3'
ODN441
5’-TTTTATTGGT CAGTTTTGTAACGG-3’
ODN451
5’-CAGTTTTGTAACGGTTAAAATGG G-3’
ODN453
5’-GTTTTGTAACGGTTAAAATGGG CG-3’
ODN461
5’-ACGGTTAAAATGGGCGGAGCGTAG-3’
431 TTCACTATAATTTTATTGGTCAGTTTTGTAACGGTTAAAATGGGCGGAGCGTAG 484
INT
INT
0
1000
2000
3000
4000
P6 promoter
Total cells
NS1
B
5000
Total cells
GPA+ cells
VP1
VP2
GPA+ cells
C
***
***
***
*
***
***
***
***
***
***
NS
***
Cell number (%)
Cell number (%)
Figure 5. Consensus sequence with 2006-PO in human parvovirus B19 genome (453 5ⴕ-GTTTTGT-3ⴕ 459) inhibits erythroid growth. (A) Map of the B19 genome,
highlighting the region that includes the consensus sequence (5⬘-GTTTTGT-3⬘) and the sequences of synthetic ODN. (B) Effects of synthetic B19 ODN with PO backbone on
erythroid growth. Purified CD34⫹ cells were cultured in erythroid medium, with or without ODN. After 7 days in culture, the generated cells were collected, washed, and counted.
The cell yields are presented as a percentage relative to the total number of cells without ODN. Data are mean ⫾ SD of 3 independent experiments. The total cells generated
from 2 ⫻ 104 CD34⫹ cells were 4.3 ⫾ 0.8 ⫻ 105 cells. (C) Effects of ODN 5⬘-GTTTTGT-3⬘ and its complementary ODN 5⬘-ACAAAAC-3⬘ (top panel), and 5⬘-TTTTGT-3⬘
(bottom panel) with PO backbone on erythroid growth. Purified CD34⫹ cells were cultured in erythroid medium, with or without ODN. After 7 days in culture, the generated cells
were collected, washed, and counted. The total cell yields are presented as a percentage relative to the total number of cells without ODN. Data are mean ⫾ SD of
3 independent experiments. The absolute number of cells generated from 2 ⫻ 104 CD34⫹ cells was 5.4 ⫾ 2.1 ⫻ 105 cells (top panel) and 6.8 ⫾ 0.8 ⫻ 105 cells (bottom panel).
*P ⬍ .05. ***P ⬍ .001. NS indicates no significance.
inconsistent with that reported by other investigators. These
differing biologic effects were probably the result of the differences
in both the sequence and ODN backbone used in this study.
Depending on the precise DNA sequence, ODNs may function
as a decoy or as siDNA and result in the reduction of target gene
mRNA levels.5 The EPOR DNA sequence has been shown to
possess 5⬘-ACAAAAC-3⬘, a sequence complimentary to 5⬘GTTTTGT-3⬘. This sequence is located upstream of the first
TATA-box in EPOR (4713-4716). The 5⬘-GTTTTGT-3⬘ consensus
sequence is not identified in the EPOR DNA. Therefore, the
inhibition of EPOR mRNA expression suggests that the sequence,
5⬘-GTTTTGT-3⬘, acts as a siDNA for EPOR gene, and results in
the down-regulation of EPOR expression and the decrease of EPO
binding. EPO represents the main and the specific cytokine
involved in the control of erythropoiesis. It is most probable that
2006-PO selectively inhibits erythroid growth through the inhibition of EPOR mRNA expression. A modest inhibition of the
expression of EPOR mRNA suggests that other unknown mechanisms may also exist.
Among the erythroid lineage-related genes examined in this
study, only FOG-1 possesses 5⬘-ACAAAAC-3⬘, but GATA-2,
GATA-1, FOG-1, EKLF, and RPS19 do not. The reason why
2006-PO did not inhibit FOG-1 mRNA transcription remains
unclear; however, it might be explained by the possibility that the
antigene effect of ODN depends on multiple factors, including
optimal length, hybridization capacity, and secondary conformations.42 In addition, 2137-PO containing the GpC-motif, rather than
the CpG-motif, also inhibited erythroid growth, which led us to
confirm that the 5⬘-TTTTGT-3⬘ motif also inhibits erythroid
growth. A sequence complimentary to 5⬘-TTTTGT-3⬘ (5⬘ACAAAA-3⬘) is included in the same site as that of 5⬘ACAAAAC-3⬘ in the EPOR gene. ODNs also act as an antisense,
forming a duplex with the mRNA and inhibiting its translation or
processing, consequently inhibiting protein biosynthesis.42 However, mRNA of the erythroid lineage-related genes examined in this
study do not possess 5⬘-ACAAAAC-3⬘, a sequence complimentary
to 5⬘-GTTTTGT-3⬘. Two sites of 5⬘-ACAAAA-3⬘, a sequence
complimentary to 5⬘-TTTTGT-3⬘, are present in GATA-2 mRNA,
but 2006-PO treatment did not affect the transcriptional level of
GATA-2 mRNA. The mechanism of reduced expression of EKLF
protein by 2006-PO remains unclear. Down-regulation of EPOR
may cause instability of EKLF protein through the differentiation
arrest of the erythroid lineage.43
Additional human DNA viruses also express the consensus
sequence 5⬘-GTTTTGT-3⬘ and include the Epstein-Barr virus
(EBV, 10 sites), cytomegalovirus (27 sites), and adenovirus
(19 sites). Among these viruses, EBV has been reported to be
associated with PRCA, although the frequency of PRCA is
extremely low. Socinski et al reported a patient who developed
PRCA after EBV infection.44 In their case, depletion of bone
C
Control
2006-PO
Day0 Day3 Day5 Day6 Day7
EPOR mRNA
Control
2.4
Day5
12.8
0.1
0.0
1.4
4.5
32.9
10.0
4.6
0.1
22.7
1.6
52.5
3.0
0.1
40.6
0.2
4.5
69.7
0.0
1.0
0.0
EPOR
0.9
14.3
IgG2
0.1
EPOR
IgG2
Day6
30.8
2.9
63.7
0.1
IgG1
E
16.6
GPA
0.1
IgG1
Day 3 CD71+ (12h)
0
Day 3 CD71+
+ 2006-PO (12 h)
20
0
GPA+ cells
NS
NS
*
Control
2006-PO
Day5 Day6 Day7
G
Control
EKLF
Day 3 CD71+
1
60
40
Control
2006-PO
GPA
Day 0 CD34+
2
80
GPA+ cells
20.1
F
NS
3
*
Day5 Day6 Day7
125
Day7
D
100
Day5 Day6 Day7
28.3
0.3
12.5
0.1
*
2006-PO
Tubulin
EKLF mRNA
4577
Control
GTTTTGT
*
***
2006-PO
Relative expression
B
Control
2006-PO
*
*
EPOR+ cells (%)
*
*
I-EPO binding (cpm)
NS
1.2
1.0
0.8
0.6
0.4
0.2
0.0
SELECTIVE INHIBITION OF ERYTHROID GROWTH BY ODN
EPOR+ cells (%)
A
Relative expression
BLOOD, 3 JUNE 2010 䡠 VOLUME 115, NUMBER 22
1.2
***
1
0.8
0.6
0.4
0.2
0
EKLF/Tubulin
Figure 6. 2006-PO down-regulates the expression of EPOR and EKLF. (A) Quantitative analysis of EPOR using real time RT-PCR. Purified CD34ⴙ cells were cultured for
3 days in erythroid medium and the cells enriched as CD71ⴙ cells. The CD71ⴙ cells were then cultured in erythroid medium, with or without 2006-PO. At the indicated days, the
cells were harvested and mRNA extracted from the cells and subjected to real-time RT-PCR. (B-C) Fluorocytometric analysis of EPOR expression. At the indicated days, the
cells were harvested and EPOR expression on GPAⴙ cells analyzed using fluorocytometry. Data are representative of 3 independent experiments (B) and are mean ⫾ SD
(C left panel). (C right panel) Parallel experiments in which 2006-PO was replaced with ODN 5⬘-GTTTTGT-3⬘. (D) Specific binding of 125I-EPO. Purified CD34ⴙ cells were
cultured for 3 days in erythroid medium and the cells were then cultured in erythroid medium, with or without 2006-PO. At the indicated days, the cells were harvested and
subjected to 125I-EPO binding. The cells were collected in IMDM at 0°C containing 0.1% BSA, and 4 ⫻ 105 cells in 50 ␮L were incubated in the presence of 2 U/mL 125I-EPO.
Data are mean ⫾ SD of triplicate measurement; representative data of 2 experiments are shown. (E) Quantitative analysis of EKLF transcripts by real-time RT-PCR. CD71ⴙ
cells were incubated in erythroid medium with or without 2006-PO, the cells harvested, and mRNA extracted and quantified by real-time RT-PCR. (F) Western blot analysis of
EKLF. The cells were harvested from the parallel experiments shown in panel E, and the lysates separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and
transferred to nylon membranes. Western blot analyses were performed using an anti-EKLF antibody. (G) Relative expression of EKLF. Data are the mean ⫾ SD of
3 independent experiments. *P ⬍ .05. ***P ⬍ .001. NS indicates no significance.
marrow T cells by E-rosetting resulted in an increase in CFU-E in
the patient’s bone marrow and addition of bone marrow T cells
significantly suppressed autologous CFU-E from T cell–depleted
bone marrow. These results suggested that the PRCA associated
with chronic EBV infection may be mediated by cytotoxic
T lymphocytes.44 The role of the 5⬘-GTTTTGT-3⬘ sequence present
in these dsDNA viruses during hematopoiesis is unclear. In
addition to the target specificity of B19 for human erythroid cells,
the major difference between these viruses and B19 is that the
former represents a dsDNA virus, whereas the latter (B19) is an
example of a ssDNA virus. These differences may result in the
pathophysiologic functions. dsDNA but not ssDNA derived from
either pathogens or the host activates both immune cells (macrophages and DCs) and nonimmune cells when introduced into the
cytoplasm by transfection.45,46 In addition, dsDNA activates a set of
genes, including those encoding major histocompatibility complex,
costimulatory molecules, and immunoproteasome subunits, as well
as the transcription factors STAT1.45,46 Evidence suggests that the
induction of interferon-inducible genes and up-regulation of costimulatory molecules by dsDNA are mediated in part by a
TLR9-independent pathway47 and that double-stranded B-form
DNA but not Z-form DNA stimulated mouse and human stromal
and DCs, resulting in the production of type I interferon and
chemokines by a TLR9-independent pathway.48 These findings
suggest that these dsDNA viruses may be processed by a different
pathway than that of ssDNA viruses.
In conclusion, the data presented in the current study describe,
for the first time, the presence of a ssDNA consensus sequence in
both synthetic ODN-2006 and the B19 genome and that this site
selectively inhibits erythroid growth. The inhibition of erythroid
growth by the consensus sequence was also accompanied by the
inhibition of EPOR mRNA expression, suggesting a potential
mechanism for lineage-specific inhibition of erythroid progenitor
cells by B19 DNA. Further work is necessary to understand the
complete mechanism of ODN with PO backbone in association
with the pathology of B19.
BLOOD, 3 JUNE 2010 䡠 VOLUME 115, NUMBER 22
GUO et al
Mock
C
B19
0.1
kb
Day5
10
5.6
5
0.1
IgG2
Day6
B
0.2
11.2 25.8
0.2
11.8
6.9
0.1
0.1
0.2
B19
0.1
59.9
8.0
2.4
0.2
75.0
0.2
0.3
0.2
9.0 13.2
0.1
11.0
0.1
10.1 41.5
0.0
9.4
0.1
EPOR
M
IgG2
A
EPOR
4578
2.8
64.3
Day7
0.2
Total cell s
9.5
0.1
14.5
GPA+ cells
IgG1
GPA
IgG1
D
GPA
*
*
Day6
Day7
***
NS
EPOR+ cells (%)
***
*
*
Day5
Cell number (x105)
GPA+ cells
Figure 7. Native single-stranded B19 genome inhibits erythroid growth and down-regulates EPOR expression. (A) Purification of the B19 genome. B19 genome DNA
was extracted from the virions. An equal molar ratio of plus and minus strands were then annealed and analyzed by agarose gel electrophoresis (arrow) and compared with
molecular size markers (M). (B) Effects of B19 genome on erythroid growth. Purified CD34⫹ cells were cultured in erythroid medium with or without native single-stranded B19
genome DNA, dsDNA, and ssDNA derived from UT7/Epo-S1 cells. After 7 days in culture, the generated cells were collected, washed, and counted. Data are mean ⫾ SD of 3
triplicates. (C-D) B19 down-regulated EPOR expression. Purified CD34ⴙ cells were cultured for 4 days in erythroid medium, the cells harvested, and then incubated with serum
containing B19. The cells were then cultured in erythroid medium. At the indicated days, the cells were harvested and EPOR expression on GPAⴙ cells analyzed using
fluorocytometry. Data are representative of 3 independent experiments (C) and are mean ⫾ SD (D). *P ⬍ .05. ***P ⬍ .001. NS indicates no significance.
Acknowledgments
Authorship
The authors thank Dr Sanford B. Krantz for helpful discussions and
comments and Keiko Iwamoto, Hiromi Kataho, and Etsuko
Kobayashi (Department of Hematology, Nephrology, and Rheumatology, Akita University Graduate School of Medicine) for their
valuable technical assistance.
This study was supported in part by the Global Center of
Excellence Program of the Ministry of Education, Science, Technology, Sports, and Culture of Japan (Grants-in-Aid 20591144) and
the Idiopathic Disorders of Hematopoietic Organs Research Committee of the Ministry of Health, Labor and Welfare of Japan
(research grant).
Contribution: Y.-M.G., K.I., and K.S. designed and performed the
experiments, analyzed data, and prepared the manuscript; M.H.,
H.T., H.O., Y.M., J.Y., and K.U. performed experiments and helped
write the manuscript; and T.O., N.O., W.X., and K.K. analyzed and
interpreted data and helped write manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Kenichi Sawada, Department of Hematology,
Nephrology, and Rheumatology, Akita University Graduate School
of Medicine, Hondo 1-1-1, Akita 010-8543, Japan; e-mail:
[email protected].
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