The Induction of PU.1 in IL-1-induced Myeloid Differentiation

Gena V. Topper

Passegué Lab

Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSFUniversity of California, San Francisco

Roundtable 8/6/13

Granulocyte/ Macrophage Progenitor (GMP)

Multipotent progenitor (MPP)

Common Lymphoid Progenitor (CLP)

Hematopoietic Stem Cell (HSC)

RBC

Meg

Platelets

Neu Eo Bas

Mon

Mac

T cell B cell NK cell

Common Myeloid Progenitor (CMP)

Megakaryocyte/Erythrocyte

Progenitor (MEP)

Hematopoiesis is the process of blood generation from HSCs

Spe

cial

izat

ion

Self-renew

al

• Quiescent

• Generates all blood lineages• High self-renewal capacity

• Compartment of amplification

• Very proliferative

• Progressively more defined lineage potential

• Terminally differentiated

• Most specialized and common, bulk of blood system

Current and Emerging Views of Myelopoiesis• Classical view: a process controlled by sequential activation of lineage-specific

transcription factors• This view ignores the potential of the microenvironment and/or systemic cytokine

environment to influence the outcome/direction of HSC differentiation

• Can cell-extrinsic factors alter HSCs fate choices to facilitate myeloid differentiation?

HSC

PU.1

PU.1

PU.1PU.1, NF-κB, C/EBPα

The Role of IL-1 in Myelopoiesis

• IL-1 is a protypical proinflammatory cytokine known to regulate myelopoiesis• IL-1 therapy given to sublethally irradiated mice resulted in faster recovery

of the myeloid compartment, but a smaller number of pre-B and lymphoid cells (Morrissey, et al. 1988)

• IL-1 known to drive expansion of myeloid cells in BM progenitors through autocrine GM-CSF signaling (Bot, et al. 1990)

• HSC and MPP proliferation in response to alum-induced inflammation has been shown to be IL-1R-dependent (Ueda, et al. 2009)

• IL-1 is an extrinsic signal that promotes myelopoiesis, but by what mechanism?

IL-1 Signaling Affects HSC Maturation in vitro

*** p<0.005

Methocult

IL-1β Accelerates HSC differentiation in vitro

% M

ac1+

/ Fc

Rγ +

Mac

1

Fc Rγ

-IL-1β

Days

-IL-1β

+IL-1β

% S

ca1+

/c-K

it+

0

25

50

75

100

0 2 4 6 8 10 12

**

Days

0

20

40

60

80

100

0 2 4 6 8 10 12

*

*

c-K

it

Sca1

+IL-1β

-IL-1β

+IL-1β

-IL-1β

+IL-1βDay 1 Day 4 Day 8

Day 1 Day 4 Day 8

12 0

87

1 0

396

1 5

9041 47

448

13 84

2129 65

32

HSCs

24hrs

Analysis

+/- IL-1β

1

8 19

667

5 47

3611

87 6

0769 7

24 0

1

28 2

69

28 1

170

1

PU.1 is upregulated by IL-1β in HSCsIs IL-1 altering expression of myeloid lineage determinants?

12h Liquid culture +/- IL-1β

0

0.5

1

1.5

2

2.5

Pu.1

Exp

ress

ion

(Log

2) v

s. -

IL-1β

C/ebpα

HSCs

24 hrsAnalysis

+/- IL-1β

eYFPPu.1

Pu.1-EYFP expression in HSCs

24 h

rs in

cul

ture

+IL

-1β -IL-1β

+IL-1β

FITC, Living Cells

Methocult

PU.1 is a Potent Transcription Factor

• Transcription factor that mediates myeloid and B cell differentiation

• Pu.1 expression increases as HSCs differentiate along the myeloid axis

• PU.1 drives a molecular program characterized by the induction of myeloid determinants such as CD18, CSFRs, Mac1, etc

• Pu.1 hypomorphs show impaired myeloid differentiation

Friedman AD (2007), Oncogene

IL-1β Upregulates PU.1 Target Genes in HSCs

0

2

4

6

8

Pu.1 Mcsfr Gmcsfr

Rel

ativ

e E

xpre

ssio

n V

alue

(Lo

g 2 v

s. -

IL-1

)β

Mcsf Gmcsf IL-60

2

4

6

8*

Rel

ativ

e E

xpre

ssio

n V

alue

(Lo

g 2 v

s. -

IL-1

)βMethocult

IL-1-driven PU.1 Expression is Sustained in HSCs

-IL-1β

+IL-1β

Days

MF

I (x1

02 )

MFI of PU.1 FITC

0

5

10

15

0 2 4 6

HSCs

24 hrs

Analysis

+/- IL-1β

eYFPPu.1

Num

ber

PU.1 FITC

Day 2Day 1 Day 4 Day 6

-IL-1β

+IL-1β

IL-1 Drives PU.1 Expression in HSCs but not in Populations that are Already Committed

Pu.1

Exp

ress

ion

(Log

2) V

S. -

IL-1β

HSC GMP

-1

0

1

2

3 Pu.1-EYFP expression in GMPs

24 h

rs in

cul

ture

+/-

IL-1β

FITC, Living Cells

HSCs

24 hrsAnalysis

+/- IL-1β

eYFPPu.1

Methocult

-IL-1β

+IL-1β

IL-1-driven PU.1 Expression is Minimal in GMPs

-IL-1β

+IL-1β

Days

MF

I (x1

03 )

MFI of PU.1 FITC

0

1

2

3

0 2 4 6

GMPs

24 hrs

Analysis

+/- IL-1β

eYFPPu.1

Num

ber

PU.1 FITC

-IL-1β

+IL-1β

Day 2Day 1 Day 4 Day 6

HSC

IL-1

IL-1RI

M-CSFR

Mac-1

FCgR

GM-CSFR

Pu.1 Pu.1 Division Division

GM-“primed” HSC

GM Progenitors GM Cells

Conclusions

• Extrinsic signals are capable of altering HSC fate• IL-1 directly activates a GM differentiation program in HSCs characterized

by increased expression of PU.1 and its downstream target genes• Restricted to only the most immature HSC compartments as GMPS are not

responsive to Il-1 in this way

HSC

IL-1

IL-1RI

M-CSFR

Mac-1

FCgR

GM-CSFR

Pu.1 Pu.1 Division Division

GM-“primed” HSCGM Progenitors GM

Cells

Future Questions

1. To what extent does IL-1 drive PU.1-dependent differentiation in intermediate progenitor compartments?

2. Does IL-1 drive myeloid differentiation through increased expression of cytokines such as M-CSF and GM-SCF in an autocrine loop?

Emmanuelle PasseguéEric PietrasMatt Warr

Stephanie Leong

Johanna FlachSietske Bakker

Latika Kohli

Silvia Alvarez

Cristina Mirantes-Barbeito

AcknowledgementsThank you all for a fantastic educational summer!!!

Have a wonderful 2013

8 19

667

5 47

3611