Mul;plets& 4.14%& 4.75%& Singlets& SSC2A& Ctrl& DTR… · DTR Gate i x10 6 Ctrl DTR 0.00 0.05 0.10...

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


– 24 hr0

20

40

60

80

100

Time after DT

Viab

le (%

)

– 36 hr 48 hr0

20

40

60

80

100

Time after DT

Viab

le (%

)

– 12 hr0

20

40

60

80

100

Viab

le (%

)

Time after DT – 4 hr 8 hr

0

20

40

60

80

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


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+

(%)



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)


![#$]!& = [(1[*ø+][#$,] + .(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


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 )



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 )



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 )



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


b


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 (%

)



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


**** ** **

Time (d)

MC

H (p

g)

0 10 20 300

5

10


***

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


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


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).


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.


References!1.! Chasis,!J.A.!&!Mohandas,!N.!Erythroblastic!islands:!niches!for!erythropoiesis.!Blood!112,!470@478!

(2008).!2.! Manwani,!D.!&!Bieker,!J.J.!The!erythroblastic!island.!Current+topics+in+developmental+biology!82,!

23@53!(2008).!!!


Mul;plets& 4.14%& 4.75%& Singlets& SSC2A& Ctrl& DTR… · DTR Gate i x10 6 Ctrl DTR 0.00 0.05 0.10...

Documents

Transcript of Mul;plets& 4.14%& 4.75%& Singlets& SSC2A& Ctrl& DTR… · DTR Gate i x10 6 Ctrl DTR 0.00 0.05 0.10...