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ERS Annual Congress Vienna

15 September 2012

Postgraduate Course 11 Pulmonary fibrosis

Saturday, 1 September 2012 14:0017:30 Room: A3

Cellular plasticity in lung fibrosis: where are all fibroblasts coming from?
Dr. Robert M. Strieter 220 Massachusetts Ave Novartis Institutes for Biomedical Research Cambridge, MA 02139 United States of America Robert.strieter@novartis.com Aims
-To present potential cellular sources of fibroblasts in the lung during the pathogenesis of fibroproliferative disorders of the lung. -To present the contribution of these cellular sources to the pathogenesis of fibroproliferative disorders of the lung. -To show that fibrocytes have progenitor cell features. -To highlight the role of fibrocytes in mediating the fibroproliferative response in the pathogenesis of fibroproliferative disorders of the lung.

Summary
The pathogenesis of pulmonary fibrosis is complex, and our knowledge regarding the specific elements of this process continues to evolve. Idiopathic pulmonary fibrosis (IPF) is an example of a lung disorder in which the natural history remains to be fully elucidated. Historically, the pathogenesis of IPF was thought to be related to chronic inflammation of unclear origin. More recently, the cause of pulmonary fibrosis has been hypothesized to occur in the absence of preceding inflammation and is associated with epithelial injury of unknown mechanism, followed by impaired wound repair leading to fibrosis. Over more than a decade, several concepts have led to refinement of the pathogenesis of pulmonary fibrosis. These include the following: injury and loss of the integrity of the alveolarcapillary barrier basement membrane (BM) is both necessary and sufficient for contributing to impaired and aberrant repair of lung architecture; pathologic fibrosis is a point of no return due to loss of BM and the inability to re-establish normal re-epithelialization and re-endothelialization on a BM; transforming growth factor-beta is necessary but not entirely sufficient to promote lung fibrosis; persistent or recurrent injury of unknown cause occurs over time, which contributed to the histopathology of temporal heterogeneity (i.e. usual interstitial pneumonitis); and cellular sources other than local fibroblasts contribute to the pathogenesis of fibrosis, such as epithelial cells that undergo epithelial-to-mesenchymal transformation (EMT) and bone marrow-derived progenitor cells (i.e. fibrocytes) (reviewed in [1]). In this presentation, we will focus on the role of fibrocytes in the promotion of the pathogenesis of fibroproliferative disorders of the lung. Lung fibroblasts and myofibroblasts play an important role in extracellular matrix (ECM) deposition in pulmonary fibrosis. The origin of the expanded populations of these cells in the lung is of substantial interest and remains to be fully elucidated. The origin of fibroblasts/myofibroblasts during the pathogenesis of fibrosis has evolved over the last decade that includes the classic concept and two contemporary theories for the origin of these cells [1]. The classic concept is that tissue injury signals the activation of resident fibroblasts to undergo migration, proliferation, and expression of number of constituents of the ECM. One contemporary theory is that tissue injury in the presence of TGF-beta and other factors induces epithelial cells to undergo transition to a mesenchymal phenotype (i.e. EMT), the fibroblast/myofibroblast that subsequently contributes to fibroproliferation [1]. The second contemporary theory is that circulating fibrocytes are mesenchymal progenitor cells that are derived from the bone marrow (i.e. fibrocytes); enter the circulation, home, and traffic into sites of lung injury, differentiate into fibroblasts/myofibroblasts, and contribute to the generation of ECM during fibroproliferation. In regard to EMT, several studies [2, 3] have demonstrated that alveolar type II pneumocytes can serve as progenitor cells, and in response to TGF-beta, can undergo differentiation to fibroblast/myofibroblast-like cells and contribute to the generation of ECM. In addition, studies

have suggested that EMT can be found in the lungs and contributes to fibrosis of IPF patients [4]. In contrast, recent studies have found little evidence for EMT in lungs undergoing fibrosis [5, 6]. However, further investigation is warranted to confirm or refute the presence and role of EMT in the promotion of pulmonary fibrosis. Fibrocytes were first identified in 1994 as circulating progenitor cells that migrated into wounds and contributed to wound repair [7]. Circulating fibrocytes appear to originate from the bone marrow, and these cells constitutively express markers of hematopoietic cells (CD45, major histocompatibility complex II, and CD34) and stromal cells (collagens I and III, and fibronectin) [7, 8, 9]. In addition, fibrocytes can undergo differentiation into fibroblast/myofibroblast-like cells with the gain in expression of alpha-smooth muscle actin in response to TGF-beta and endothelin [10]. In mouse models of pulmonary fibrosis, inhibition of the trafficking and extravasation of fibrocytes into the lungs is associated with a marked reduction in pulmonary fibrosis [8, 11, 12]. Patients with idiopathic fibrotic interstitial lung disease have been shown to have a marked increased number of circulating fibrocytes, as compared in healthy control subjects [13]. Moreover, lung tissue from IPF patients, as compared to normal lungs, have significantly increased fibrocytes that co-express markers of collagen production and alpha-smooth muscle actin, and the number of these cells directly correlates with the number of fibroblastic foci [14]. Furthermore, the measurement of elevated levels of circulating fibrocytes in patients with IPF directly correlates with exacerbations of their disease and serves as a biomarker for worse prognosis [15]. These findings underscore the importance of fibrocyte extravasation into the lungs during the pathogenesis of pulmonary fibrosis, and indicate that circulating fibrocytes may contribute to the expansion of the fibroblast/myofibroblast-like cells in patients with IPF and other fibroproliferative disorders of the lung. Fibrocytes appear to be critical cells in the regulation of fibrosis, and their biology has relevance for the future consideration of therapeutic targets to attenuate the progression of pulmonary fibrosis.

References
1. Strieter RM and Mehrad B. New mechanisms of pulmonary fibrosis. CHEST 2009; 136:13641370. 2. Willis BC, et al. Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis. Am J Pathol 2005; 166:13211332. 3. Kim KK, et. al. Epithelial cell alpha3beta1 integrin links beta-catenin and Smad signaling to promote myofibroblast formation and pulmonary fibrosis. J Clin Invest 2009; 119:213224. 4. Kim KK, et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A 2006; 103:1318013185. 5. Yamada M, et al. Dual immunohistochemistry provides little evidence for epithelialmesenchymal transition in pulmonary fibrosis. Histochem Cell Biol 2008; 129:453462. 6. Rock JR, et. al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc Natl Acad Sci U S A. 2011;108:E1475-483. 7. Bucala R, et al. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med 1994; 1:7181. 8. Phillips RJ, et al. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest 2004; 114:438446. 9. Quan TE, et al. Circulating fibrocytes: collagen-secreting cells of the peripheral blood. Int J Biochem Cell Biol 2004; 36:598606. 10. Hong KM, et al. Differentiation of human circulating fibrocyte sas mediated by transforming growth factor-beta and peroxisome proliferator activated receptor-gamma. J Biol Chem 2007; 282:2291022920. 11. Moore BB, et al. CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am J Pathol 2005; 166:675684. 12. Mehrad B, et. al. Fibrocyte CXCR4 regulation as a therapeutic target in pulmonary fibrosis. Int J Biochem Cell Biol. 2009;41:1708-1718.

13. Mehrad B, et al. Circulating peripheral blood fibrocytes in human fibrotic interstitial lung disease. Biochem Biophys Res Commun 2007; 353:104108 14. Andersson-Sjoland A, et al. Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis. Int J Biochem Cell Biol 2008; 40:2129-2140. 15. Moeller A, et al. Circulating fibrocytes are an indicator for poor prognosis in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2009; 179:588594.

Evaluation
1. There are one classical and two contemporary theories as to the origin of fibroblasts/myofibroblasts in the lung during the pathogenesis of pulmonary fibrosis. True or False 2. Fibrocytes can be induced to express alpha-smooth muscle actin. True or False 3. A bone marrow-derived cell that has progenitor cell features and that can contribute to fibrosis is which cell? a. Epithelial cell b. Leukocyte c. Endothelial cell d. Fibrocyte 4. Elevated circulating fibrocytes have been associated with an improved prognosis in patients with IPF. True or False Please find all answers at the back of your handout materials

CELLULAR PLASTICITY IN LUNG FIBROSIS: WHERE ARE ALL FIBROBLASTS COMING FROM?
Robert M. Strieter, M.D., FCCP, MACP

Faculty disclosure
I am employed by Novartis Institutes for Biomedical Research I have no other conflicts of interest

10

Introduction
AIMS Aim 1-To present potential cellular sources of fibroblasts in the lung during the pathogenesis of fibroproliferative disorders of the lung. Aim 2-To present the contribution of these cellular sources to the pathogenesis of fibroproliferative disorders of the lung. Aim 3-To show that fibrocytes have progenitor cell features. Aim 4-To highlight the role of fibrocytes in mediating the fibroproliferative response in the pathogenesis of fibroproliferative disorders of the lung.

Isolation and Culture of Circulating Mesenchymal Progenitor Cells (Fibrocytes)

Peripheral Blood Mononuclear Cells

Non-adherent cells: Removed

DMEM, 20% FBS ~3 days

Isolated from human leukopheresis pack Centrifugation over Ficoll

Adherent cells: Additional 10-14 day incubation

Extraction with Accutase Crude fibrocyte preparation

Anti-CD2 Anti-CD14 Anti-CD19

Immunodepletion with magnetic beads

Resultant culture-enriched fibrocyte population >90% pure based on Col I/CXCR4/CD45RO staining (FACS)

Circulating Mesenchymal Progenitor Cell (CMPC) Surface Markers

ECM Markers
-smooth muscle actin Collagen I and III Fibronectin Vimentin Prolyl 4-hydroxylase

CD Markers
CD11a-LFA-1 CD11b-MAC 1 CD13-pan-myeloid-aminopeptidase-N CD18-2 integrin CD34-HSC and endothelial cells CD45RO-Leukocyte common antigen CD54-ICAM-1 CD58-LFA-3 CD71-Transferrin receptor CD80-B7.1-Co-stim binds CD28 CD80-B7.2-Co-stim binds CD28
Metz CN. Cell. Mol. Life Sci. 60:134250, 2003. Quan TE. Int, J. Biochem. Cell Biol. 36:598-606, 2004

MHC-Related Markers
MHC class II HLA-DP HLA-DQ HLA-DR

Chemokine Receptors
CXCR4 CCR3 CCR7

11

Circulating Fibrocytes Isolated from Peripheral Blood

Quan TE. Int, J. Biochem. Cell Biol. 36:598-606, 2004

Fibrocytes can Differentiate into Mesenchymal Phenotypes

Fibrocytes Can Differentiate to Osteoblasts and Chondrocytes

CD45 -tubulin CD45+Fibrocytes Time (days)
Osteogenic media

CD45 beads S

+ -

14

21

- +

- + -

Osteonectin Collagen I GAPDH Time (days) TGF-3 Aggrecan Collagen I GAPDH 0 CD45+Fibrocytes 9 18 28

- +

+ -

12

TGF-1 TRII
SMAD 2/3

Extracellular
TRI

Growth factors, Mitogens, Stress

Rho RAS DAXX TAK1 (+) TZD

TZD

TZD

Intracellular

TZD

PPAR

SMAD 7 SMAD 2/3

Activation of MAPK ERK p38 JNK (+)

TZD

Repressors

(-)

SMAD 4 SMAD 4 SMAD 2/3

RXR TZD PPAR

A

RXR

ATF2 (+)

cJUN (?)

Elk-1

Nucleus
(+)

Activators

(-) TZD PPAR PPRE RXR Adipocytes

SMAD 4 SMAD 2/3 Myofibroblasts SRE

Fibrocyte Constitutive Gene Expression

Name of Gene
Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Chemokine receptor (CCR2) Chemokine receptor (CCR5) Chemokine receptor (CCR7) Chemokine receptor (CXCR4) Collagen, type I, alpha 1 Collagen, type I, alpha 2 Collagen, type III, alpha 1 Collagen, type IV, alpha 2

Relative Expression
204078 91 353 385 7086 223848 30141 122958 120161

Hierarchy of Chemokine Receptor Expression on Fibrocytes in Peripheral Blood of Normal Subjects

2.5
Isotype control Isotype control Isotype control

Cells(105)/ml)

2.0

Anti-CXCR4

Anti-CCR2

Anti-CCR7

1.5

1.0

0.5

0
CD45+ COL1+ CD45+ COL1+ CXCR4+ CD45+ COL1+ CCR2+ CD45+ COL1+ CCR7+ CD45+ COL1+ CCR2+ CCR7+ CD45+ COL1+ CCR2+ CXCR4+ CD45+ COL1+ CCR7+ CXCR4+

13

Fibrocytes Produce Significant Collagen In Vitro

Soluble Collagen (Sircol; ug/ml)
250 200 150 100 50 0 -50 Monocyte conditioned media Fibrocyte + TGF Conditioned Media Fibroblast Conditioned Media Fibrocyte Conditioned Media Fibroblast + TGF Conditioned Media

Week 1

Week 2

Week 3

Note: TGF (10ng/ml)

Fibrocytes have the Capacity to Differentiate to SMA+ cells in Response to TGF-

SMA Expression Fold Change 2-CT

TGF- Promotes Differentiation of Fibrocytes to Myofibroblast-like cells Expressing SMA

9 8 7 6 5 4 3 2 1 0

* * *

TGF- ng/ml

0.3

10

30

100

SMA Expression Fold Change 2-CT

9 8 7 6 5 4 3 2 1 0

TGF- 10ng/ml *

* *

Time(hrs)

0
SMA GAPDH Time (hrs)

* 16 *

24

48

24

48

72

14

Fibrocytes Gain -Smooth muscle Actin and Lose CD34 and CD45 in the Presence of TGF-
90 80 70

Week 1 Post-Purification TGF- (1ng/ml) No TGF-

90 80 70

Week 2 Post-Purification TGF- (1ng/ml) No TGF-

Percent Positive Cells

60 50 40 30 20 10 0 SMA CD34 CD45 CXCR4 Col I

Percent Positive Cells

60 50 40 30 20 10 0 SMA CD34 CD45 CXCR4 Col I

Circulating Fibrocytes Contribute to Pulmonary Fibrosis

Presence of CD45+Col1+GFP+ Cells in the Bone Marrow and Lungs of GFP BM Chimeric Mice During Bleomycin-Induced Pulmonary Fibrosis
4.5

Bone Marrow

4.5

Lung

Bleo

Bleo

Cells/ml (x105)

Saline

Cells/lung (x106)

Saline
3.0

3.0

1.5

1.5

GFP+ GFPCD45+ Col1+

GFP+ GFPCD45+ Col1+

15

Presence of CD45+Type I and III Pro-Collagen+ Cells in the Lungs During Bleomycin-Induced Pulmonary 2.0 Fibrosis Bleomycin

Cells(106)/Lung

Saline CTRL

1.5

0.5

CD45+Type I Pro-collagen C-terminus

CD45+Type I Pro-collagen N-terminus

CD45+Type III Pro-collagen C-terminus

Presence of CD45+Col1+SMA+ Cells in the Bone Marrow and Lungs of GFP BM Chimeric Mice During BleomycinInduced Pulmonary Fibrosis

Bone Marrow
2.0 Bleo Saline 1.5 1.0 0.5 0

3.0

Lung
Bleo Saline

Cells/Lung (x105)

Cells/ml (x105)

2.0

1.0

GFP+ GFPCD45+ Col1+ SMA+

GFP+ GFPCD45+ Col1+ SMA+

Depletion of CXCL12 Reduces the Presence of Fibrocytes but not other Leukocyte Subpopulations in the Lung During Bleomycin-Induced Fibrosis in GFP BM Chimeric Mice * 9
Bleomycin CTRL Ab Bleomycin Anti-CXCL12 Ab

Cells (106)/Lung

7 5 3 1 0 GFP+Col1+ GFP-Col1+

16

Depletion of CXCL12 Reduces the Presence of Fibrocytes but not other Leukocyte Subpopulations in the Lung During Bleomycin-Induced Fibrosis in GFP BM Chimeric Mice

3.5 3 2.5 2 1.5 1 0.5 0 Bleomycin-Control Ab

Cells/Lungs (x107)

Bleomycin-Anti-CXCL12 Ab

CD3

CD4

CD8

NK1.1

Ly6

Mac

Depletion of CXCL12 Attenuates Bleomycin-Induced Pulmonary Fibrosis

A
Bleo + Control Ab

B
20,000

Picrosirius Red Staining (Square pixels; HPF 400x)

* P < 0.001

16,000 12,000 8,000 4,000 0

Bleo + Anti-CXCL12

*
Control Ab Anti-CXCL12

-SMA immunostaining

Bleomycin

Fibrocytes are Markedly Elevated in Patients with Pulmonary Fibrosis

17

CXCL12 is Elevated in the Lungs and Plasma of Patients with Pulmonary Fibrosis

Levels of Fibrocytes are Increased in the Circulation of Patients with Pulmonary Fibrosis (Idiopathic)
10

*
CD45+Col1+ CXCR4+ Cells 105/ml

10

10

CD45+Col1+ CXCR4- Cells 105/ml

CD45+Col1+ Cells 105/ml

*
6

Normal UIP/NSIP

Normal UIP/NSIP

Normal UIP/NSIP

Levels of Fibrocytes Expressing Alpha-Smooth Muscle Actin are Increased in the Circulation of Patients with Pulmonary Fibrosis (Idiopathic)
6 6 6

CD45+Col1+ SMA+ CXCR4+ Cells 104/ml

CD45+Col1+ SMA+ CXCR4- Cells 104/ml

CD45+Col1+ SMA+ Cells 104/ml

Normal

UIP/NSIP

Normal

UIP/NSIP

Normal UIP/NSIP

18

Circulating Fibrocytes are an Indicator for Poor Prognosis in Idiopathic Pulmonary Fibrosis

Moeller A, et. al. Am J Respir Crit Care Med.179:588-94, 2009

CD45+ Col1+ cells 105 /ml

Levels of Fibrocytes are Increased in the Circulation of Patients with 25 Hermansky-Pudlak Syndrome 1
20

15

10

Normal

HPS-1 without Pulmonary Fibrosis

HPS-1 with Pulmonary Fibrosis

The Majority of Fibrocytes in the Circulation of Patients with Hermansky-Pudlak Syndrome 1 and Pulmonary Fibrosis are CXCR4+
12
CD45+ COL1+ CD45+ COL1+ CXCR4+ CD45+ COL1+ CCR2+ CD45+ COL1+ CCR7+

Cells (105)/ml

10 8 6 4 2 0

Normal

HPS 1 with PF

19

Airway Remodeling in Patients with Asthma is Associated with Elevated Fibrocytes

Circulating Fibrocytes are Markedly Elevated in Patients with Severe Asthma Number of Cells in Peripheral Blood (per ml X 103)
4000 3000

CD45+ Col1+ CD45+ CD34+ Col1+ *p<0.05 N=9

2000

N=9
1000 0

N=6

*
N=6

Circulating Fibrocytes in Severe Asthma Correlate with GINA Treatment Step and BMI
3000

CD45+Col1+ Cells in Peripheral Blood (per ml x 103)

2500

CD45+Col1+ Cells in Peripheral Blood (per ml x 103)

R2 = 0.59 p<0.001

3000

2500

R2 = 0.49 p<0.05

2000

2000

1500

1500

1000

1000

500

500

0 1 2 3 4 5

20

25

30

35

40

GINA Treatment Step

Body Mass Index

20

Number of Cells in Peripheral Blood (per ml X 103)

2000

p<0.05 N=9

CD45+Col1+SMA+ Cells (per ml x 103)

Circulating Fibrocytes in Severe Asthma Exhibit an Activated/Differentiated Phenotype 4000 CD45+ Col1+ 2000 Asthmatic Patients CD45+ Col1+ CXCR4+ CD45+ Col1+ SMA+ R2 = 0.52 + + + + 3000 1500 CD45 Col1 SMA CXCR4 p<0.05 * * 1000 * *
500

1000

Control

Asthma

CD45+Col1+p-SMAD2/3+ Cells (per ml x 103) Asthmatic Patients R2 = 0.99 p<0.0001

500

1000

1500

2000

CD45+Col1+SMA+ Cells (per ml x 103)

2000

1500

Asthmatic Patients R2 = 0.56 p<0.05

CD45+Col1+p-SMAD2/3+ Cells (per ml x 103)

2500 2000 1500 1000 500 0 0

1000

500

00

CD45+Col1+p-AKT+ Cells (per ml x 103)

500

1000

1500

2000

CD45+Col1+p-AKT+ Cells (per ml x 103)

500

1000

1500

2000

Bronchiolitis Obliterans Associated with Lung Transplantation

Tracheal Transplant Model of BOS .

BALB/c mouse trachea donor

C57BL/6 mouse recipient

21

Time Course of BO in the Murine Model

Syngeneic

Allogeneic

14

21

28

Days Post Transplantation

Collagen Deposition During Development of BO

Allo
175

Syn
* *

Hydroxyproline (ug/ml)

150 125 100 75 50 25 0 0 7 14

21

28

Days Post Transplantation

CXCL12 and CXCR4 are Markedly Expressed During BO

CXCL12 mRNA fold increase Allo:Syn
10

* * *

Syn Naive *

* *

CXCR4 mRNA fold increase Allo:Syn

Allo

10

* *

30

CXCL12 (ng/ml)

20

10

* * * *

0 0 7 14 21 28

DAYS

DAYS

14 21 28

DAYS

14 21 28

22

Fibrocyte Extravasation is Markedly Increased During BO

8 CD45+ Col1+ CXCR4+ cells (x105) 7 6 5 4 3 2 1 0
7 14 21 Days Post-Transplant

Isograft Allograft 4

21 Days PostTransplantation

Cells (x105)

Iso

Allo

Iso

Allo

CD45+Col1+CXCR4+ CD45+Col1+CCR2+

Fibrocytes are Elevated in Patients with Bronchiolitis Obliterans Syndrome Post-Lung Transplantation

Damien J. LaPar, D.J. et. al. Ann Thorac Surg 92:470 7, 2011

Fibrocytes are Elevated in Patients with Bronchiolitis Obliterans Syndrome Post-Lung Transplantation

Damien J. LaPar, D.J. et. al. Ann Thorac Surg 92:470 7, 2011

23

Conclusions
Fibrocytes are bone marrow-derived progenitor cells that have the capacity to differentiate along mesenchymal lineage. Fibrocytes contribute to animal models of lung fibrosis relevant to fibrosis of the lung parenchyma and remodeling of the airway. Fibrocytes may represent a biomarker of the presence and progression of lung fibrosis in humans. Fibrocytes may serve as a biomarker for prognosis related to fibrotic disorders of the lung of humans.

24