Mul;plets& 4.14%& 4.75%& Singlets& SSC2A& Ctrl& DTR… · Supplementary Figure 7 d g Short-term DT PBS Clod Ctrl DTR 0 5 10 Liposomes CD169-DTR *** n.s. Splenic RPM (x10 5) a b c
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Ter119&
F4/80&
7.89%& 2.19%&
SSC2H&
SSC2A&
Ctrl& DTR&
Mul;plets&4.14%&
Ctrl& DTR&
Mul;plets&4.75%&
Singlets&96.4%&
Singlets&95.7%&
a
b
Supplementary Figure 1 Nature Medicine doi:10.1038/nm.3057
Ctrl
DTR
Gate i
x106
Ctrl DTR0.00
0.05
0.10
0.15
0.20
0.25
**
Gate ii
x106
Ctrl DTR0.0
0.5
1.0
1.5
**
Gate iii
x106
Ctrl DTR0.0
0.5
1.0
1.5
**
Gate iv
x106
Ctrl DTR0.0
0.5
1.0
1.5
2.0
*
Gate v
x106
Ctrl DTR0.0
0.2
0.4
0.6
*
a
e
b
BM
CFU
-E (x
103 )
Ctrl DTR0
2
4
6
*
BM
BFU
-E (x
103 )
Ctrl DTR0
1
2
3
4n.s.
Supplementary Figure 2
f g h i
Gate I
CtrlDTR0
1
*
Pro
eryt
hrob
last
s (x
105 )
Gate I
CtrlDTR0
1
*P
roer
ythr
obla
sts
(x10
5 )
Gate II
CtrlDTR0
1
2
3
4
**
Bas
ophi
lic E
B (x
106 )
Gate II
CtrlDTR0
1
2
3
***
Bas
ophi
lic E
B (x
106 )
Gate III
CtrlDTR0.0
0.2
0.4
0.6
0.8
**
Pol
ychr
omat
ic E
B (x
105 )Gate III
CtrlDTR0.0
0.1
0.2
0.3
***
Pol
ychr
omat
ic E
B (x
105 ) Gate IV
CtrlDTR0.0
0.1
0.2
0.3
0.4
0.5
*
Orth
ochr
omat
ic E
B (x
106 ) Gate IV
CtrlDTR0.0
0.1
**
Orth
ochr
omat
ic E
B (x
106 )c d
4 hr
8 hr12
hr24
hr36
hr48
hr 4 wk
6 wk
0
50
100********** ***
Time after 1st DT injection
CtrlDTR
BM
ery
thro
blas
ts (%
of
ctrl)
WT
WT+D
TDTR
DTR+DT
WT
WT+D
TDTR
DTR+DT
0
20
40
60
80
100
n.s.
24 hr 48 hr
Viab
le (%
)
WT
WT+D
TDTR
DTR+DT
WT
WT+D
TDTR
DTR+DT
0
10
20
30
40
50
n.s.
x103
24 hr 48 hrNature Medicine doi:10.1038/nm.3057
50.6% 51.2%
n.s. n.s. n.s.
b
d e i
n.s.
n.s. n.s. n.s. n.s.
j
a f
c g hAnnexin V
DA
PI
91.0%
Ctrl DTR
90.9%
BrdU
SS
C
Ctrl DTR
Supplementary Figure 3
– 24 hr0
20
40
60
80
100
Time after DT
Viab
le (%
)
– 36 hr 48 hr0
20
40
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80
100
Time after DT
Viab
le (%
)
– 12 hr0
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40
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100
Viab
le (%
)
Time after DT – 4 hr 8 hr
0
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100
Viab
le (%
)
Time after DT
– 36 hr 48 hr0
10
20
30
40
Time after DT
BrdU
+ (%
)
– 12 hr0
20
40
60
Time after DT
BrdU
+ (%
)
– 4 hr 8 hr0
20
40
60
Time after DT
BrdU
+ (%
)
– 24 hr0
20
40
Time after DT
BrdU
+ (%
)
Nature Medicine doi:10.1038/nm.3057
c d
Supplementary Figure 4
e
a
0.043% 0.094%
Ctrl DTR
Ter119
Hoe
scht
333
42
b
Ctrl DTR0
1
2
3
Pro
eryt
hrob
last
s (x
103 ) **
Ctrl DTR0
2
4
6
8
10*
Bas
ophi
lic E
B (x
104 )
Ctrl DTR0
2
4
6
8
Pol
ychr
omat
ic E
B (x
103 )
**
Ctrl DTR0
5
10
15O
rthoc
hrom
atic
EB
(x10
4 )f g h i
Ctrl DTR0
2
4
6
8 *
PB
ery
thro
blas
ts (x
104 )
Ctrl 1 d 4 wk0
1
2
3
4 * *
PB
ery
thro
blas
ts (x
104 )
BM PB BM PB0
20
40
60
Ctrl
** ***
DTR
BrdU
(%)
BM PB BM PB0
5
10
15
20
25
Ctrl DTR
n.s. n.s.
Ann
exin
V+
(%)
Nature Medicine doi:10.1038/nm.3057
Supplementary Figure 5
4 weeks DT
WT DTR0
1
2
3
4
***
Spl
enic
RP
M (x
106 )
g h i
Ter119
CD
71
Ctrl
I. 0.006% II. 1.79%
III. 1.02%
IV. 59.0%
DTR
I. 0.052% II. 1.79%
III. 1.58%
IV. 41.2%
e
f
d
cb
a
Ter119
CD
71
DTR
I. 0.43%
II. 35.7%
III. 2.5%
IV. 10.8% Ctrl
I. 0.32%
II. 14.4%
III. 1.4%
IV. 18.2%
4 weeks DT
WT DTR0
2
4
6
8
**
Live
r Kup
ffer
cells
(x10
4 )4 weeks DT
WT DTR0
2
4
6
8
***
BM
mac
roph
ages
(x10
5 )
0 2 4 60
10
20
30
40
50
Ctrl - ShamDTR - ShamCtrl - SplenectomyDTR - Splenectomy
*
Time of depletion (weeks)
Hem
atoc
rit (%
)
Ctrl DTR0.0
0.5
1.0
1.5
2.0
2.5n.s.
Hep
atic
ery
thro
blas
ts (x
106 )
Ctrl DTR Ctrl DTR0
2
4
6
8
10
4 weeks 6 weeks
* **S
plen
ic e
ryth
robl
asts
(x10
6 )
Ctrl DTR DTR0
200
400
600
800
1 d 4 wk
n.s. n.s.
EPO
(pg
per m
l)
Nature Medicine doi:10.1038/nm.3057
![#$]!& = [(1[*ø+][#$,] + .(2((1[*ø+][#$,]) .+ .(3((2(((1[*ø+][#$,]))).. . . ] .− .(4[#$]
[*ø+] = concentration of macrophages important in production. Rate of erythroblast accumulation = [EB] = [(1[*ø+][#$,] + .(2((1[*ø+][#$,]) .+ .(3((2(((1[*ø+][#$,]))).. . . ] Rate of erythroblast conversion to RBC = (4[#$] k1 = constant by which presence of Møp promotes erythroblast precursors (EBP) conversion to mature erythroblasts. Assumption: Møp are not required for formation of EBP, which is substantiated by no change in BFU-E (Fig. S2) and literature evidence that MФ are important for the erythroblast stages starting with the pro-erythroblasts/CFU-E1,2. k2 = constant by which (1[*ø+][#$,] divide. k3 = constant by which (2((1[*øp][#$,]) at next maturation stage divide. ... = additional terms of the same form to indicate erythroblasts that accumulate from dividing erythroblasts. k4 = constant by which erythroblasts are converting to RBC. ![7$8]!& = .(4[#$] .− .(5[*ø:][7$8]
[*ø:] = concentration of macrophages important in clearance. k5 = constant by which RBCs are being cleared by macrophages. At steady-state equilibrium in Mø-sufficient and Mø-depleted mice, ![#$]!& = . ![7$8]!& = .0
since the lifespan of RBC (>45-55 days) is substantially longer than EB development (hour to days). [(1[*ø+][#$,] + .(2((1[*ø+][#$,]) .+ .(3((2(((1[*ø+][#$,]))).. . . ] .− .(4[#$] = 0 (4[#$] .= . [(1[*ø+][#$,] + .(2((1[*ø+][#$,]) .+ .(3((2(((1[*ø+][#$,]))).. . . ] (4[#$] − .(5[*ø:][7$8] = .0 (4[#$] .= .(5[*ø:][7$8] Thus, [(1[*ø+][#$,] + .(2((1[*ø+][#$,]) .+ .(3((2(((1[*ø+][#$,]))).. . . ]= (5[*ø:][7$8] ((1 + (1(2 + (1(2(3.+.. . . )[*ø+][#$,] = (5[*ø:][7$8] [7$8] = [((1 + (1(2 + (1(2(3.+.. . . )/(5] * [#$,] [[*ø+]/[*ø:]] Since we know that Mø depletion affects both *ø+ and *ø: (Supplementary Fig. 5) even if not exactly 1:1, [*ø+][*ø:] = .(6
.[7$8] = [((1 + (1(2 + (1(2(3.+.. . . )/(5] *.(6 [#$,] Therefore, [7$8] is independent of [*ø].
Supplementary Figure 6 Nature Medicine doi:10.1038/nm.3057
Supplementary Figure 7
d gShort-term DT
PBS Clod Ctrl DTR0
5
10
Liposomes CD169-DTR
*** n.s.
Spl
enic
RP
M (x
105 )
b ca e
PBSClod
0
2
4
6
8
*Spl
enic
RP
M (x
106 )
f
PBSClod
0.0
0.5
1.0
1.5
2.0
**Spl
enic
BF
U-E
(x10
5 )
F4/80
Naive PHZ + Lip PBS PHZ + Lip Clod
BMP4
DAPI
Merged
PBSClod
0.0
0.5
1.0
1.5
**
BM
mac
roph
ages
(x10
5 )
PBSClod
0
2
4
6
8
*
BM
ery
thro
blas
ts (x
106 )
PBSClod
0
100
200
300
*
Spl
enic
ery
thro
blas
ts (x
106 )
Nature Medicine doi:10.1038/nm.3057
Supplementary Figure 8
d e f g
a b c
h
0 2 4 6 80
2
4
6
8
10
CtrlDTR
******
Time after BMT (d)
Bio
tin+
RB
Cs
(x10
9 )
0 5 10 15 200
20
40
60
CtrlDTR
*
*
Time after 5FU (d)
Hem
atoc
rit (%
)
0 5 10 15 200
10
20
30
40
50
CtrlDTR
*
Time after 5FU (d)
Ret
icul
ocyt
e (%
)
0
2
4
6
5FU – + +
***
*
Ctrl DTRCtrl
BM
ery
thro
blas
ts (x
106 )
0
1
2
3
4
5
5FU – + +Ctrl DTRCtrl
*
**
BM
mac
roph
ages
(x10
5 )
0
20
40
60
80****
5FU – + +Ctrl DTRCtrl
Spl
enic
ery
thro
blas
ts (x
106 )
0
1
2
3
5FU - + +Ctrl DTRCtrl
**
Spl
enic
RP
M (x
106 )
Nature Medicine doi:10.1038/nm.3057
Supplementary Figure 9
i j k
a b c d
f e g h
Host Donor0.0
0.5
1.0
1.5
2.0
Sple
nic
RPM
(x10
5 )
***
l
Ctrl DTR DTR0.0
0.5
1.0
1.5
2.0
n.s.
+ Iron
Spl
enic
ery
thro
blas
ts (x
105 )
Ctrl DTR DTR0
5
10
15
n.s.
+ Iron
BM
ery
thro
blas
ts (x
105 )
0 10 20 300
5
10
15
CtrlDTR
***
Time after BMT (d)
MC
H (p
g)
0 10 20 300
5
10
15
20
CtrlDTR
**********
CH
r (pg
)
Time after BMT (d)
0 2 4 60
5
10
15
CtrlDTR
***
Time of depletion (wk)
CH
r (pg
)
0 2 4 60
5
10
15 * * *
CtrlDTR
Time of depletion (wk)
MC
H (p
g)
Ctrl DTR DTR0
20
40
60
80
1d 4wk
*n.s.
Tra
nsfe
rrin
sat
urat
ion
(%)
Ctrl DTR DTR0
200
400
600
800
n.s. *
1d 4wk
Ser
um ir
on (µ
g pe
r dl)
– Ctrl DTR0
100
200
300
400 n.s.
Ser
um ir
on (µ
g pe
r dl)
– Ctrl DTR0
10
20
30
40
50 n.s.
Tra
nsfe
rrin
sat
urat
ion
(%)
WT!
WT
DTR!W
T
WT!
DTR
DTR!DTR
0
1
2
3
4
5
** **
n.s.
Spl
enic
RP
M (x
105 )
Nature Medicine doi:10.1038/nm.3057
Supplementary Figure 10
Vcam1
(rela
tive
units
)
Gr1hiGr1lo Mφ0
200
400
600
800
1000
0 103 104 105CD15
0
103
104
105
CD163
31
7.24
58.6
0 103 104 105CD14
0
50K
100K
150K
200K
250K
SSC-A 24.6
68.4
0 103 104 105CD169
0
103
104
105
VCAM1
6.27
0 103 104 105CD169
0
103
104
105
VCAM1
0.0225
0 103 104 105CD169
0
103
104
105
VCAM1
0.0417
0 103 104 105CD169
0
103
104
105
VCAM1
0.0801
0 103 104 105CD169
0
103
104
105
VCAM1
0.294
0 103 104 105CD169
0
103
104
105
VCAM1
0.983
Without CD169 Ab Without VCAM1 Ab
0 103 104 105CD15
0
103
104
105
CD163
31
7.24
58.6
0 103 104 105CD14
0
50K
100K
150K
200K
250K
SSC-A 24.6
68.4
0 103 104 105CD169
0
103
104
105
VCAM1
6.27
0 103 104 105CD169
0
103
104
105
VCAM1
0.0225
0 103 104 105CD169
0
103
104
105
VCAM1
0.0417
0 103 104 105CD169
0
103
104
105
VCAM1
0.0801
0 103 104 105CD169
0
103
104
105
VCAM1
0.294
0 103 104 105CD169
0
103
104
105
VCAM1
0.983
a
g
0
2000
4000
6000 **
Iso VCAM1
Control
DTR
MFI
0 103 104 105CD169
0
103
104
105
VCAM1
80.8
0 103 104 105CD169
0
103
104
105
VCAM1
52.5
bBM macrophage Splenic RPM
c
WT!
WT
DTR!W
T
WT!
DTR
DTR!DTR
0
2000
4000
6000
8000 *
Sple
nic
VCAM
1 M
FI
CtrlDTR
Rat IgG
αVCAM1
0.0
0.5
1.0
1.5
2.0
2.5 n.s.
*
Spl
enic
ery
thro
blas
ts (x
105 )
Host Donor0.0
0.5
1.0
1.5
*
BM m
acro
phag
es (x
104 )
d
f e
Nature Medicine doi:10.1038/nm.3057
b
Supplementary Figure 11
e f g
i j k
l
c
0
5
10
15** ***
RB
C (x
109
per m
l)
16 d 25 d9 d0
2
4
6
8
WB
C (x
106
per m
l)
16 d 25 d9 d0
500
1000
1500* *
Pla
tele
ts (x
106
per m
l)
16 d 25 d9 d
Ctrl PV PV0.0
0.5
1.0
1.5
2.0
2.5***
PBS Clod
Pro
eryt
hrob
last
s (x
105 )
Ctrl PV PV0.0
0.5
1.0
1.5
2.0***
PBS Clod
Bas
ophi
lic E
B (x
106 )
Ctrl PV PV0.0
0.5
1.0
1.5 ***
PBS Clod
Pol
ychr
omat
ic E
B (x
105 )
Ctrl PV PV0.0
0.5
1.0
1.5
PBS Clod
n.s.
Orth
ochr
omat
ic E
B (x
106 )
Ctrl PV PV0
2
4
6 ***
PBS Clod
BM
mac
roph
ages
(x10
5 )
Ctrl PV PV0
2
4
6
8***
BM
ery
thro
blas
ts (x
106 )
PBS Clod
a
d
h
Ctrl PV PV0
1
2
3 ***
Spl
enic
RP
M (x
106 )
PBS ClodCtrl PV PV
0
1
2
3
4
5***
Spl
enic
ery
thro
blas
ts (x
106 )
PBS Clod
0 10 20 30 40 500
20
40
60
PV - PBS liposomesPV - Clodronate liposomes
* **
Ctrl
Time (d)
Hem
atoc
rit (%
)
Nature Medicine doi:10.1038/nm.3057
Supplementary Figure 12
a b
c d e
f
0 10 20 300
20
40
60
PBS liposomesClodronate liposomes
**
***
Deferoxamine
**
n.s.
Time (d)
Hem
atoc
rit (%
)
0 10 20 300
5
10
15
PBS liposomesClodronate liposomes
**** ** **
Time (d)
MC
H (p
g)
0 10 20 300
5
10
PBS liposomesClodronate liposomes
***
Time (d)
CH
r (pg
)
PBS PBS Clod Def0
1
2
3
4
5
**
n.s.
Ctrl PV
Spl
enic
ery
thro
blas
ts (x
106 )
PBS PBS Clod Def0
200
400
600
800
1000n.s.
*
PVCtrl
Ser
um ir
on (µ
g pe
r dl)
PBS PBS Clod Def0
20
40
60
80
100
PVCtrl
n.s.
Tra
nsfe
rrin
sat
urat
ion
(%)
g
Ctrl PV PV0
1
2
**
End
ogen
ous
CF
U-E
(x10
4 )
PBS Clod
Nature Medicine doi:10.1038/nm.3057
Supplementary Figure 1: Macrophage depletion reduces erythroblast islands. a) FACS plots of multiplets gated as shown on DAPI- BM from WT and CD169-DTR mice after DT administration. b) Ter119+F4/80+ multiplets were sorted and cytospun onto slides. Representative confocal image is shown. Red=Ter119 and Green=F4/80. Supplementary Figure 2: Macrophage depletion reduces all committed erythroid progenitors. a) FACS plot of complementary gating strategy with FSC and CD44. b) Quantitation of erythroblasts with complementary gating strategy at various time points of depletion (n=3-8). Absolute numbers of erythroblasts per femur were normalized such that average values of Ctrl mice were set to 100% at each time point. c-d) Quantitation of proerythroblasts (Gate I), basophilic (Gate II), polychromatic (Gate III), and orthochromatic (Gate IV) erythroblasts per femur of Ctrl and DTR mice after c) 24 hours and d) 4 weeks of depletion (n=5, representative of two independent experiments). e) Quantification of erythroblasts in successive stages of erythroid maturation with complementary gating strategy after 4 weeks of depletion (n=4, representative of two independent experiments). f) Quantitation of CD11b-CD45-Ter119+CD71+ sorted erythroblasts after culture for 24 or 48 hours. g) Quantitation of viable erythroblasts (DAPI-Annexin-) after culture for 24 or 48 hours. h) Quantitation of CFU-E per femur of Ctrl or DTR mice after 4 weeks of depletion (n=4-5). i) Quantitation of BFU-E per femur of Ctrl or DTR mice after 4 weeks of depletion (n=4-5). Supplementary Figure 3: Macrophage depletion does not affect erythroblast proliferation or viability in the steady state. a) FACS plots of representative BrdU incorporation rates of erythroblasts (CD45- CD11b- Ter119+) in Ctrl or DTR mice 8hr after DT administration. b-e) Quantification of BrdU incorporation rates of erythroblasts (CD45- CD11b- Ter119+) in mice not injected with DT (-) or at various time points after DT administration (n=3-9). Gates were set based on cells from mice that were not administered BrdU. f) FACS plot and g-j) quantitation of viable (DAPI- Annexin-) erythroblasts in mice not injected with DT (-) or at various time points after DT administration (n=3-9). Gates were set based on cells unstained with DAPI and Annexin V. Supplementary Figure 4: Macrophage depletion mobilizes erythroblasts into the peripheral circulation. a) FACS plots of CD45- CD11b- single peripheral blood cells in Ctrl or DTR mice 24hr after DT administration. b) Quantitation of PB erythroblasts (CD45- CD11b- Hoechst 33342+ Ter119+) 24hr after a single injection of 10µg/kg of DT (n=5, representative of two independent experiments). c) Quantitation of percentage of BM or peripheral blood erythroblasts that were Annexin+ 24hr after DT administration (n=5, representative of two independent experiments). d) Quantitation of percentage of BM or peripheral blood erythroblasts that incorporated BrdU 24hr after DT administration (n=5, representative of two independent experiments). e) Quantitation of Hoechst 33342+ Ter119+ cells in peripheral blood of mice treated with DT for 1 day or 4 weeks (n=4-5). f-i) Quantitation of f) proerythroblasts, g) basophilic EB, h) polychromatic EB, and i) orthochromatic EB per ml of blood 1 day after administration of DT (n=4-5). Supplementary Figure 5: Macrophage depletion in steady-state mice augments splenic erythroblasts, but splenectomy does not result in anemia. a) Serum erythropoietin (EPO) levels in Ctrl mice or DTR mice treated with DT for 1 day or 4 weeks (n=4-5). b) FACS plots of DAPI- CD11b- CD45- single cells and c) quantitation of erythroblasts from liver of Ctrl or DTR mice after 4 weeks of depletion (n=10, pooled from two independent experiments). d) FACS plots of DAPI- CD11b- CD45- single cells from spleen of Ctrl
Nature Medicine doi:10.1038/nm.3057
or DTR mice after 4 weeks of depletion. e) Quantitation of splenic erythroblasts in Ctrl and DTR mice at various time points after macrophage depletion (n=4-5, representative of three independent experiments). f) Hematocrit assessment of sham-operated and splenectomized Ctrl and DTR mice (n=4-6). g) Bone marrow, h) splenic red pulp, and i) liver Kupffer macrophages are reduced in DTR animals after 4 weeks of DT administration (n=5). Bone marrow macrophages were identified as Gr1loCD115intF4/80+B220-SSClo. Splenic red pulp and liver kupffer macrophages were identified as CD45+B220-F4/80+CD11blo. Supplementary Figure 6: Mathematical model demonstrating lack of effect of macrophage content on RBC homeostasis in the steady state. Supplementary Figure 7: Macrophage depletion with clodronate liposomes impairs BM and splenic recovery from hemolytic anemia. a) Quantitation of splenic red pulp macrophages on day 4 of experiment after a single dose of liposomes (liposomes on day -5) or after the last of three administrations of DT into WT (Ctrl) or DTR (DTR) mice (DT on days -5, -2, 0) (n=4-5). b-e) Quantitation of macrophages (b,d) and erythroblasts (c,e) on day 4 of experiment in the BM (b,c) and spleen (d,e) in liposome pre-treated and PHZ-challenged mice (liposomes on day -5 and PHZ on days 0,1) (n=3-4). f) Representative images of F4/80, BMP4, and DAPI staining from spleens of animals pre-treated with liposomal PBS (Lipo PBS) and Clodronate (Lip Clod) and challenged with PHZ (liposomes on day -5, PHZ on day 0, harvest day 1). Scale bar = 100µm. g) Quantitation of stress BFU-E in spleens of PBS and Clodronate liposome pre-treated animals on day 4 of experiment (liposomes on day -5 and PHZ on days 0,1) (n=3-4). Supplementary Figure 8: Macrophage depletion delays reticulocytosis and hematocrit recovery following 5-fluorouracil challenge. a) Immunofluorescence image of sternal BM of Ctrl (left) and DTR (right) animals 16 days after challenge with 250mg/kg 5FU. Red=Ter119; Blue=Hoechst 33342; Green=CD68. Scale bar=100µm. b-e) Macrophage (b,d) and erythroblast (c,e) counts in BM (b,c) and spleens (d,e) of Ctrl (blue) and DTR (red) animals 16 days after challenge with 250mg/kg 5FU. 5FU non-challenged (black) are displayed for comparison (n=6-9, pooled from two independent experiments). f) Reticulocyte and g) hematocrit assessment in Ctrl (blue) and DTR (red) animals following challenge with 250mg/kg 5FU (n=14-15, representative of two independent experiments). h) Quantitation of biotinylated RBC in Ctrl and DTR animals following BMT (n=5). Supplementary Figure 9: Iron perturbations after macrophage depletion. a-b) Quantification of a) serum iron and b) transferrin saturation in Ctrl, 1 day- and 4 week- depleted animals (n=5). c) Mean corpuscular hemoglobin (MCH) in Ctrl and DTR animals at various times after depletion (n=14, pooled from three independent experiments). d) Reticulocyte hemoglobin content (CHr) in Ctrl and DTR animals at various times after depletion (n=17-23, pooled from four independent experiments). Data analysed with two-way ANOVA with Bonferroni post-test. e-f) Quantification of e) serum iron and f) transferrin saturation in untransplanted (black) and Ctrl (blue) and DTR (red) mice 7 days after BMT (n=4-5). g-h) MCH and CHr in Ctrl and DTR animals at various times after depletion (n=26-28, pooled from five independent experiments). Data analysed with two-way ANOVA with Bonferroni post-test. i-j) Quantitation of erythroblasts in the i) BM and j) spleen 7 days after BMT in Ctrl, DTR, and DTR mice infused with iron (n=3-4). k) Quantitation of host versus donor splenic macrophages 7 days after BMT (n=6). l) Quantitation of splenic red pulp macrophages (RPM) in reciprocally-transplanted and DT-treated mice 7 days after BMT (n=5).
Nature Medicine doi:10.1038/nm.3057
Supplementary Figure 10: VCAM1 on bone marrow macrophages is critical for erythropoiesis. a) Gene expression of Vcam1 in Gr1hi monocytes, Gr1lo monocytes, and macrophages sorted form the BM, as assessed by microarray. b) FACS plots showing double staining of VCAM1 and CD169 in BM and splenic RPM. Gating was set based on isotype and negative staining controls for VCAM1 and CD169, respectively. c) Quantitation of mean fluorescence of VCAM1 on BM DAPI- single cells in Ctrl (blue) and DTR (red) animals after 4 weeks of CD169+ macrophage depletion (n=5-10, representative of three independent experiments). Isotype control fluorescence (Iso) is indicated by the black bar. d) Quantitation of host versus donor BM macrophages 7 days after BMT (n=6). e) Quantitation of splenic VCAM1 mean fluorescent intensity (MFI) in reciprocally-transplanted and DT-treated mice 7 days after BMT (n=5). f) Quantitation of splenic erythroblasts 7 days after BMT of Ctrl (blue), DTR (full red), rat IgG-treated (striped blue) or anti-VCAM1-treated (striped red) animals (n=3-5). g) FACS plots of CD15- CD163+ population depicted in Fig. 5a without the CD169 or VCAM1 antibody (Ab), respectively. Supplementary Figure 11: Long-term macrophage depletion reduces bone marrow and splenic erythroblasts in JAK2V617-induced polycythemia vera. a-c) Quantitation of recovery of a) red blood cells (RBC), b) white blood cells (WBC) and c) platelets at 9, 16 and 25 days post transplant of WT (white, Ctrl) or JAK2V617 (black, PV) transgenic BM (n=10, pooled from two independent experiments). d-g) Quantitation of splenic d) proerythroblasts, e) basophilic EB, f) polychromatic EB, and g) orthochromatic EB 7 days after the last of four weekly liposome administrations into PV animals (day 28 of experiment, 12 weeks post BMT) (n=4-6). h-k) Macrophage (h,j) and erythroblast (i,k) counts in BM (h,i) and spleen (j,k) 7 days after a single liposome administration into PV animals (day 7 of experiment, 6 weeks post BMT) (n=4). Ctrl animals are shown in black. l) Hematocrit levels of Ctrl (black) and PV mice that were treated with a single dose of PBS (blue) or clodronate (red) liposomes (n=5). Data analysed with two-way ANOVA with Bonferroni post-test. Day 0 corresponds to first day of liposome injection and 5 weeks after BMT. Supplementary Figure 12: Macrophage depletion reduces erythrocyte iron content, but serum iron reduction is not sufficient to normalize the erythron in polycythemia vera. a) Mean corpuscular hemoglobin (MCH) and b) reticulocyte hemoglobin content (CHr) in mice that were transplanted with JAK2V617F BM and subsequently treated with PBS liposomes (blue), clodronate liposomes (red) (n=4-8). Data analysed with two-way ANOVA with Bonferroni post-test. c-e) Quantitation of c) serum iron, d) transferrin saturation, and e) splenic erythroblasts in Ctrl (black) and PV mice that were treated with PBS liposomes (blue), clodronate liposomes (red), or deferoxamine (orange) (n=4-8). f) Hematocrit measurement in PV mice that were treated with PBS liposomes (blue), clodronate liposomes (red), or deferoxamine (orange) (n=4-8). Data analysed with two-way ANOVA with Bonferroni post-test. Day 0 corresponds to first day of liposome or deferoxamine injection and 8 weeks after BMT. g) Quantitation of endogenous CFU-E in spleens of Ctrl (black) and PV mice treated with PBS (blue) or clodronate (red) liposomes (n=4-6) and harvested on day 30 of experiment.
Nature Medicine doi:10.1038/nm.3057
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