articles

ADAM10-mediated ephrin-B2 shedding promotes

myofibroblast activation and organ fibrosis
David Lagares1,9,10, Parisa Ghassemi-Kakroodi2,9, Caroline Tremblay2, Alba Santos1, Clemens K Probst1,
Alicia Franklin1, Daniela M Santos1, Paula Grasberger1, Neil Ahluwalia1, Sydney B Montesi1, Barry S Shea1 ,
Katharine E Black1, Rachel Knipe1, Meryem Blati2, Murray Baron3, Brian Wu4, Hassan Fahmi2, Rajiv Gandhi4,
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

Annie Pardo5, Moisés Selman6, Jiangping Wu2, Jean-Pierre Pelletier2, Johanne Martel-Pelletier2,
Andrew M Tager1,10 & Mohit Kapoor2,4,7,8,10

Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive
extracellular matrix (ECM) deposition and tissue remodeling by activated myofibroblasts, leads to loss of proper tissue
architecture and organ function; however, the molecular mediators of myofibroblast activation have yet to be fully identified.
Here we identify soluble ephrin-B2 (sEphrin-B2) as a new profibrotic mediator in lung and skin fibrosis. We provide molecular,
functional and translational evidence that the ectodomain of membrane-bound ephrin-B2 is shed from fibroblasts into the
alveolar airspace after lung injury. Shedding of sEphrin-B2 promotes fibroblast chemotaxis and activation via EphB3 and/or
EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and
that a disintegrin and metalloproteinase 10 (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 expression
is increased by transforming growth factor (TGF)-b1, and ADAM10-mediated sEphrin-B2 generation is required for TGF-b1-
induced myofibroblast activation. Pharmacological inhibition of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar
lavage and prevents lung fibrosis in mice. Consistent with the mouse data, ADAM10–sEphrin-B2 signaling is upregulated in
fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of
tissue fibrogenesis and identify sEphrin-B2, its receptors EphB3 and EphB4 and ADAM10 as potential therapeutic targets in the
treatment of fibrotic diseases.

Dysregulated wound repair processes can lead to the development
of tissue fibrosis in most organs. Fibrosis is characterized by excess
accumulation of collagens and other matrix proteins that results in the
distortion of tissue architecture and ultimately organ failure in a vari-
ety of human diseases, including idiopathic pulmonary fibrosis (IPF)
and systemic sclerosis (SSc, scleroderma)1. This pathological accu-
mulation of matrix proteins is attributable to the excess recruitment
of fibroblasts to sites of tissue injury and/or to their excessive activa-
tion to an effector myofibroblast phenotype1–5. We have previously
found increased expression of genes associated with cell migration
and myofibroblast activation in the lungs of individuals with IPF with
a more rapidly advancing clinical course, leading us to hypothesize
that this set of genes contributes to IPF progression6. If so, elucidat-
ing the regulation of fibroblast migration and activation will not only
further enhance our understanding of the pathogenesis of fibrosis but
will also provide targets for new antifibrotic therapies.
To identify putative genes that regulate profibrotic fibroblast func-
tions, we analyzed a publicly available microarray data set comparing
the gene expression of lung fibroblasts isolated from individuals with
IPF or SSc-associated interstitial lung disease (SSc–ILD) to that of
healthy lung fibroblasts used as controls7. Among the genes upregu-
lated in fibroblasts from both individuals with IPF and with SSc–ILD
in this data set, we found EFNB2 (Gene Expression Omnibus acces-
sion number GSE1724; EFNB2 probe: 34334_at)7. EFNB2 encodes
the transmembrane protein ephrin-B2, which belongs to the family
of ephrin ligands and has previously been described as exhibiting
promigratory activities8,9. Ephrins are glycosyl-phosphatidylinositol-
linked (ephrin-A1–6) and transmembrane (ephrin-B1– 3) cell surface
ligands that bind to Eph receptors at the surface of adjacent cells. This
interaction initiates bidirectional signaling events between the recep-
tor-expressing (forward signaling) and ligand-expressing (reverse
signaling) cells10. Ephrin–Eph signaling controls tissue patterning

1Division of Pulmonary and Critical Care Medicine, Fibrosis Research Center and Center for Immunology and Inflammatory Diseases, Department of Medicine,
Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. 2Department of Medicine, University of Montreal Hospital Research Centre
(CRCHUM), Montreal, Québec, Canada. 3Division of Rheumatology, Jewish General Hospital, McGill University, Montreal, Québec, Canada. 4The Arthritis Program,
University Health Network, Toronto, Ontario, Canada. 5Facultad de Ciencias, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico. 6Instituto Nacional
de Enfermedades Respiratorias Ismael Cosio Villegas, Mexico City, Mexico. 7Division of Genetics and Development, Krembil Research Institute, University Health
Network, Toronto, Ontario, Canada. 8Departments of Surgery and of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
9These authors contributed equally to this work. 10These authors jointly directed this work. Correspondence should be addressed to M.K. (mkapoor@uhnresearch.ca)

or D.L. (dlagares@mgh.harvard.edu).

Received 18 November 2016; accepted 11 September 2017; published online 23 October 2017; corrected online 20 November 2017 (details online);
doi:10.1038/nm.4419

nature medicine VOLUME 23 | NUMBER 12 | DECEMBER 2017 1405

 Articles
and cell motility during embryonic development11. In accordance
with its promigratory activities, the ephrin-B2 ligand, which signals
through EphB receptors, also controls cell motility and adhesion
during assembly of the blood vessel wall9,12,13. Genetic inactivation
of Efnb2 in smooth muscle cells and pericytes during development
results in disrupted microvessel architecture and increased vascular
permeability in mice9. Mice lacking the intracellular PDZ signaling
domain of ephrin-B2, in which reverse signaling by ephrin-B2 is
consequently attenuated, have compromised vascular stabilization
by pericytes, resulting in increased capillary rarefaction and fibro-
sis after kidney injury14. In human disease, ephrin-B2 expression is
increased in small vessels and in vascular smooth muscle in clinically
involved skin of individuals with SSc15. Although ephrin-B2 signaling
has been implicated in vascular contributions to the development of
fibrosis in mouse models and human disease, the full spectrum of
cellular and molecular mechanisms through which ephrin-B2 sig-
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
naling may contribute to the development of tissue fibrosis has not
been investigated. Here we describe a new mechanism through which
ADAM10-mediated proteolytic shedding of the ephrin-B2 ectodo-
main, herein referred to as sEphrin-B2, promotes fibroblast recruit-
ment and activation. We provide molecular, genetic, functional and
translational evidence that this mechanism plays an important role
in fibroblast activation in vitro and in the development of the lung
and skin fibrosis in vivo.
RESULTS
Bleomycin-induced lung fibrosis requires ephrin-B2 in fibroblasts
Our initial studies confirmed that expression of ephrin-B2, but not
other members of the ephrin family of ligands, is markedly higher in
lung fibroblasts isolated from individuals with IPF compared with
that in lung tissue from control subjects, as demonstrated by mRNA
and protein analyses (Fig. 1a,b). We then investigated whether fibrob-
last ephrin-B2 is required for the development of fibrosis in vivo. As
mice that are globally ephrin-B2-deficient die at midgestation owing
to defective cardiovascular development13,16, we generated mice in
which we could conditionally delete Efnb2 in collagen-expressing
cells, such as fibroblasts. We crossed mice with Efnb2 flanked by loxP
sites (Efnb2loxP/loxP mice)17 to mice that express a tamoxifen-induc-
ible Cre recombinase driven by the mouse promoter of Col1a2 (col-
lagen, type I, alpha 2) (Col1a2-CreERT mice) (Fig. 1c). Tamoxifen
treatment of offspring that were homozygous for the ‘floxed’ Efnb2
allele and hemizygous for the Col1a2-Cre transgene (Efnb2loxP/loxP;
Col1a2-CreERT mice), as confirmed by PCR (Fig. 1d), led to the dele-
tion of the Efnb2 gene in fibroblasts and the generation of Efnb2 con-
ditional knockout mice, herein referred to as Ephrinb2-CKO mice.
Littermates treated with corn oil vehicle alone were used as controls
and are herein referred to as Ephrinb2-C mice. Western blotting
for ephrin-B2 protein demonstrated markedly lower expression in
extracts from lung fibroblasts of Ephrinb2-CKO mice compared to
those from Ephrinb2-C mice. We observed preservation of Ephrin-
B2 protein in extracts from alveolar macrophages in Ephrinb2-CKO
mice compared to those from Ephrinb2-C mice (Fig. 1e). We also
isolated alveolar epithelial cells from Ephrinb2-C and Ephrinb2-CKO
mice, but we did not observe ephrin-B2 protein expression by western
blotting in these cells from Ephrinb2-C mice, and thus we could not
compare its potential preservation in alveolar epithelial cells from
Ephrinb2-CKO mice.
Ephrinb2-CKO and Ephrinb2-C mice were then challenged
with intratracheal bleomycin or PBS (Fig. 1f). Blinded histologi-
cal analysis revealed that the lung parenchymal fibrosis observed
1406
in Ephrinb2-C at 14 d following bleomycin challenge was mark-
edly lower than that in Ephrinb2-CKO mice, which was associated
with substantially lower lung hydroxyproline (collagen) content
(Fig. 1g,h). Further, evaluation of fibrotic protein markers in total
lung homogenates showed markedly lower expression of both α-
smooth muscle actin (α-SMA, a marker of myofibroblast differen-
tiation) and type I collagen in bleomycin-challenged Ephrinb2-CKO
mice compared to controls (Fig. 1i).
Identification of soluble ephrin-B2 ectodomain in
bronchoalveolar lavage fluid
Considering the requirement for ephrin-B2 fibroblast expression
that we identified in bleomycin-induced lung fibrosis, we next inves-
tigated the regulation of ephrin-B2 expression in this model. Lung
homogenates taken from wild-type (WT) mice at 14 d following
bleomycin challenge did not demonstrate greater expression of the
full-length transmembrane ephrin-B2 (approximately 60 kDa, as pre-
viously described for fibroblasts, endothelial cells and cancer cells18)
compared to homogenates from WT mice following PBS challenge;
however, a lower-molecular-weight band (~50 kDa) appeared in the
homogenates following bleomycin challenge that was either absent or
expressed at low levels in lungs following PBS challenge (Fig. 2a).
Ephrin-B ligands, as well as EphB receptors, have been shown
to undergo ectodomain shedding to release active proteins19–22.
Proteolytic cleavage of mouse ephrin-B2 has recently been shown to
generate a 50-kDa band corresponding to the ectodomain of ephrin-
B2, consistent with our findings in Figure 2a23. These data led us to
hypothesize that ephrin-B2 is proteolytically cleaved following profi-
brotic lung injury and that the resulting sEphrin-B2 contributes to
the pathogenesis of fibrosis. To investigate this hypothesis, we first
evaluated the presence of sEphrin-B2 in bronchoalveolar lavage (BAL)
following bleomycin challenge. As demonstrated by western blotting
with an N-terminus-specific anti-ephrin-B2 antibody, sEphrin-B2
expression was observed at low basal levels in BAL from WT mice 14
d following PBS treatment but was markedly greater in BAL from WT
mice 14 d following bleomycin challenge (Fig. 2b,c). Further, using
an enzyme-linked immunosorbent assay (ELISA) with an ectodo-
main-specific anti-ephrin-B2 detection monoclonal antibody (mAb),
we observed greater sEphrin-B2 concentration in BAL from bleomy-
cin-challenged mice at 3, 7 and 14 d following bleomycin challenge
compared to PBS-challenged mice (Fig. 2d).
Oligomeric sEphrin-B2 directs fibroblast migration, invasion
and myofibroblast differentiation
To investigate whether generation of sEphrin-B2 was reduced in
Ephrinb2-CKO mice following bleomycin challenge, which could
contribute to the protection of these mice from lung fibrosis in this
model, we compared the concentrations of sEphrin-B2 in BAL of
Ephrinb2-CKO and Ephrinb2-C in mice 14 d following bleomycin
treatment. sEphrin-B2 concentration was lower, but not completely
eliminated, in BAL from Ephrinb2-CKO mice compared to Ephrinb2-
C mice 14 d following bleomycin treatment (Fig. 2e). We hypoth-
esized that this reduction was at least in part attributable to loss of
sEphrin-B2 generation from ephrin-B2-deficient fibroblasts: i.e., that
fibroblasts in WT mice are a source of the sEphrin-B2 generated in
response to lung injury. To test this hypothesis, we used ELISA to
assess sEphrin-B2 shedding by lung fibroblasts isolated from PBS- or
bleomycin-challenged WT mice. A low concentration of sEphrin-
B2 was present in medium conditioned by lung fibroblasts isolated
from PBS-challenged WT mice. In contrast, medium conditioned by
VOLUME 23 | NUMBER 12 | DECEMBER 2017 nature medicine
 articles

a c d

xP
re E ;
T
lo

R
P/
ol b2 lox
C
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2-
n
1a
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T
Healthy

C
IPF
8 Ephrin-B2 floxed (552 bp)
mRNA expression

WT (450 bp)
loxP/loxP ERT
6 Efnb2 Col1a2-Cre
Cre (408 bp)
4

KO

KO
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loxP/loxP ERT Ephrin-B2

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b Healthy IPF
40

kDa 1 2 3 4 5 6 42 β-actin
80 Corn oil Tamoxifen
Ephrin-B2 injection injection Lung fibroblasts Lung macrophages
40
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

Tamoxifen i.p.
40 GAPDH f Bleomycin
2.0 i.t.
densitometry

* Healthy
Ephrin-B2

IPF Tamoxifen
1.0 Ephrinb2 control Ephrinb2 conditional (Ephrinb2 Bleomycin
mouse knockout mouse ablation) i.t. injection Harvest
0 (Ephrinb2-C) (Ephrinb2-CKO)
Time (d)
–7 –1 0 + 14

g h
Ephrinb2-C Ephrinb2-CKO
** i kDa C
PBS
CKO C
BLM
CKO
*
25 ** 42 α-SMA
Saline

130 Type I collagen

Hydroxyproline (µg/left lung)

20 Ephrinb2-C
42 β-actin
Ephrinb2-CKO
15
*** ***
* *
* *
10
10.0 10.0

Type I collagen
Bleomycin

densitometry

densitometry
α-SMA

5
5.0 5.0

0
PBS BLM 0 0

Figure 1 Bleomycin-induced lung fibrosis is dependent on ephrin-B2 in fibroblasts. (a) Relative mRNA expression of genes encoding ephrin-A and
ephrin-B ligands in human lung fibroblasts from healthy control donors (n = 4) and individuals with IPF (n = 4). mRNA expression was normalized to the
level of GAPDH. ND, not detected. (b) Ephrin-B2 protein levels in human fibroblasts from healthy controls (n = 3) and individuals with IPF (n = 3).
GAPDH was used as a loading control for densitometry analysis. One representative out of two technical replicates is shown. (c) Schematic of the
generation of Ephrinb2-CKO mice. (d) Representative (n = 30 mice per genotype) genotyping showing the WT band (450 bp), the ephrin-B2 floxed band
(552 bp) and the Cre band (408 bp) for WT mice (without Cre) and Efnb2loxP/loxP; Col1a2-CreERT mice. (e) Representative western blot of ephrin-B2
protein expression (normalized to β-actin) in lung fibroblasts and alveolar macrophages from Ephrinb2-C and Ephrinb2-CKO mice. n = 3 mice for all
groups. One representative out of two technical replicates is shown. β-actin was used as a loading control. (f,g) Schematic showing mice subjected to
the bleomycin-induced lung fibrosis model (f) and Masson′s trichrome staining of lung sections from Ephrinb2-C mice and Ephrinb2-CKO mice 14 d
after PBS or bleomycin challenge (g). Scale bar, 100 µm. i.t., intratracheal; i.p., intraperitoneal. Representative images are presented from n = 8 mice
per group per genotype. (h) Hydroxyproline content measured in the lungs of Ephrinb2-C and Ephrinb2-CKO mice at 14 d after bleomycin (BLM) or
PBS treatment. n = 6 for all groups. (i) Representative western blot of α-SMA and type I collagen protein expression (normalized to β-actin) in total lung
homogenates of Ephrinb2-C and Ephrinb2-CKO mice at 14 d after bleomycin or PBS challenge. The color legend in h also applies to i. n = 6 mice for
all groups. One representative out of two technical replicates is shown. In a and b, data are presented as mean ± s.d. and were analyzed with Student’s
t-test. In h, data are presented as mean ± s.d. and were analyzed with two-way ANOVA. Center lines, median values; +, the mean; box edges, 25th and
75th percentiles; whiskers, minimum and maximum values. In i, data are presented as mean ± s.d and were analyzed with two-way ANOVA. *P < 0.05,
**P < 0.01, ***P < 0.001. Uncropped blot images are shown in Supplementary Figure 3.

lung fibroblasts isolated from WT mice 7 d following bleomycin chal- We next determined whether the ephrin-B2 ectodomain that
lenge had a markedly higher concentration of sEphrin-B2 (Fig. 2f). is shed as sEphrin-B2 from fibroblasts and present in BAL follow-
Furthermore, the concentration of sEphrin-B2 was substantially lower ing lung injury directs profibrotic activities of fibroblasts. In these
in medium conditioned by lung fibroblasts isolated from PBS- or experiments, we used recombinant mouse ephrin-B2-Fc, which
bleomycin-challenged fibroblasts that had been transfected with small contains the ectodomain of ephrin-B2 fused to an Fc domain
interfering RNA (siRNA) targeting ephrin-B2 compared to medium that replaces the transmembrane and C-terminal domains of the
conditioned by these fibroblasts transfected with nontargeting control full-length ephrin-B2 protein (Fig. 3a). Structural and functional
siRNA (Fig. 2f). studies have demonstrated that EphB receptor activation requires

nature medicine VOLUME 23 | NUMBER 12 | DECEMBER 2017 1407

 Articles

a Lung homogenates c platelet-derived growth factor (PDGF-BB) (Fig. 3d). These findings
PBS Bleomycin
kDa 1 2 3 4 1 2 3 4 *** PBS indicate that sEphrin-B2 contributes to fibroblast chemotaxis.
80
20 BLM The ephrin-B2 ligand signals through the EphB subfamily of recep-
Ephrin-B2 16
tor tyrosine kinases, which includes EphB1–4 and EphB6 (EphB5 is

densitometry
sEphrin-B2
12
present in chickens, but no mammalian homolog has been identi-
40 8 fied)25. Primary mouse lung fibroblasts expressed Ephb2, Ephb3 and
40
4 Ephb4 mRNA, but not Ephb1 or Ephb6 mRNA (Fig. 3e). Individual
GAPDH 0 siRNA knockdown of these receptors revealed that both fibroblast
chemotaxis and fibroblast invasion through Matrigel induced by
b Bronchoalveolar lavage d 1.50 PBS
*** BAL from bleomycin-treated mice required both EphB3 and EphB4
BLM (EphB3/4), but not EphB2, receptor signaling (Fig. 3f,g).
sEphrin-B2 in BAL (ng/ml)
PBS Bleomycin
1.25 ***
kDa 1 2 3 4 1 2 3 4 *** As EphB receptor activation requires higher-order ephrin-B2 oli-
1.00
80 gomers, as noted above24, we investigated the quaternary structure of
0.75
sEphrin-B2 in BAL by immunoblotting BAL proteins that had been
sEphrin-B2 0.50
40 separated by native PAGE on nonreducing gels. Our N-terminus-
0.25
specific anti-ephrin-B2 mAb recognized high-molecular-weight
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

0.00
Day 1 Day 3 Day 7 Day 14 species consistent with higher-order sEphrin-B2 oligomers in BAL
e f from bleomycin-challenged, but not control, mice (Fig. 3h). These
***
** Ephrinb2-C Nontargeting siRNA
higher-order species were not recognized in BAL samples pretreated
1.50 * Ephrinb2-CKO 0.6 ** siEfnb2 with SDS and 2-mercaptoethanol, further indicating that sEphrin-B2
*** is present in the form of higher-order oligomers in BAL recovered
sEphrin-B2 in BAL (ng/ml)

1.25 0.5
supernatants (ng/ml)

from mice following lung injury. Results from competition studies

sEphrin-B2 in

1.00 0.4

0.75 0.3 performed by preincubating our N-terminus-specific anti-ephrin-B2

0.50
0.25
0.00
PBS BLM
Figure 2 Ephrin-B2 ectodomain is shed by fibroblasts upon lung injury.
(a) Representative western blot showing ephrin-B2 expression levels in total
lung homogenates from C57BL/6N mice taken at 14 d
following PBS or bleomycin challenge. n = 4 mice for all groups. The
arrow indicates the appearance of the lower-molecular-weight band
(~50 kDa). One representative out of two technical replicates is shown.
(b,c) Representative western blot showing cleaved sEphrin-B2 levels in
BAL fluids from PBS- and bleomycin-challenged mice at day 14 after
treatment (b) and corresponding quantification of cleaved sEphrin-B2
protein (c). n = 4 mice for all groups. One representative out of three
technical replicates is shown. (d) Concentration of sEphrin-B2, as
determined by ephrin-B2 ELISA, in BAL fluid from C57BL/6N mice at
1, 3, 7 and 14 d following PBS or bleomycin challenge. n = 8 mice for
all groups. (e) Concentration sEphrin-B2 in BAL fluids recovered from
Ephrinb2-C and Ephrinb2-CKO mice at 14 d following PBS or bleomyc
in challenge. n = 5 mice for all groups. (f) Effect of Efnb2-targeting
siRNA versus a nontargeting siRNA control on sEphrin-B2 generation
by primary lung fibroblasts isolated from C57BL/6N mice 7 d following
PBS or bleomycin challenge. n = 4 for all groups. In c and d, data are
presented as mean ± s.d and were analyzed with Student’s t-test. In e
and f, data are presented as mean ± s.d and were analyzed with two-way
ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. In d and e, center lines,
median values; +, the mean; box edges, 25th and 75th percentiles;
whiskers, minimum and maximum values. Uncropped blot images are
shown in Supplementary Figure 4.
higher-order ephrin-B2 oligomers24; we consequently preclustered
ephrin-B2-Fc for these experiments by incubating mouse ephrin-
B2-Fc with IgG antibody in a 2:1 ratio immediately before use.
Treatment of mouse lung fibroblasts with preclustered ephrin-
B2-Fc, but not preclustered ephrin-B1-Fc or ephrin-B3-Fc, markedly
increased filopodia formation (Fig. 3b,c), suggesting that ephrin-
B2, but not ephrin-B1 or ephrin-B3, can regulate fibroblast motil-
ity. Chemotaxis assays demonstrated that preclustered ephrin-B2-Fc
induced fibroblast migration in a dose-dependent manner similar
to chemotactic agents, such as lysophosphatidic acid (LPA) or
0.2
mAb with recombinant ephrin-B2 before immunoblotting were con-
***
0.1
sistent with those expected from this antibody binding to sEphrin-B2
in the oligomers (Supplementary Fig. 1). Our results suggest that
0.0
PBS BLM sEphrin-B2 oligomers are present in BAL following lung injury and
are biologically active in that they are able to mediate profibrotic
functions of fibroblasts. In accordance with this hypothesis, depletion
of sEphrin-B2 in BAL from bleomycin-challenged mice with mag-
netic beads coated with the N-terminus-specific anti-ephrin-B2 mAb
markedly, though not completely, reduced the fibroblast chemotaxis
that was induced by these BAL samples (Fig. 3j).
We next determined whether ephrin-B2 signaling could also induce
fibroblast-to-myofibroblast differentiation. Treatment of primary
mouse lung fibroblasts with preclustered ephrin-B2-Fc markedly
increased Acta2 and Col1a1 mRNA and α-SMA and type I collagen
protein expression compared to IgG-Fc treatment (Fig. 3k,l).
Treatment with Ephrin-B2 ectodomain is sufficient to drive
tissue fibrosis in vivo
As ephrin-B2 ectodomain can direct profibrotic fibroblast functions
in vitro, we next investigated whether recombinant ephrin-B2-Fc can
induce tissue fibrosis in vivo. Mice were injected subcutaneously with
preclustered mouse ephrin-B2-Fc (100 µg per kg body weight per
mouse) or IgG-Fc as a control daily for 2 weeks. Blinded histological
analysis showed that ephrin-B2-Fc treatment produced robust dermal
fibrosis (Fig. 3m) associated with markedly increased dermal thick-
ness (Fig. 3n), hydroxyproline content (Fig. 3o) and α-SMA and type
I collagen expression (Fig. 3p) as compared to the results of IgG-Fc
treatment. Although these data demonstrate that ephrin-B2 forward
signaling by the ephrin-B2 ectodomain is sufficient to drive myofi-
broblast formation and tissue fibrosis in vivo, we have not investigated
potential contributions of this ectodomain on other cell types, such
as endothelial or immune cells.
In addition to lung fibrosis, we assessed the role of ephrin-B2 in
the development of skin fibrosis in vivo. We subjected Ephrinb2-CKO
mice and Ephrinb2-C mice to the bleomycin model of dermal fibrosis
(Fig. 3q). Blinded histological analysis showed markedly attenuated
skin fibrosis in bleomycin-treated Ephrinb2-CKO mice, which was

1408 VOLUME 23 | NUMBER 12 | DECEMBER 2017 nature medicine

 articles

a b c d e f g
*

Ephrin-B2-Fc IgG-Fc
8 6.00 8 100 100

Chemotactic index
Number of filopodia

Chemotactic index
mRNA expression
Signal Cytoplasmic
Ectodomain TM 5.00 * **

Invasion index
(% reduction)
(% reduction)
peptide domain 6 6 75 75
4.00 * **
1–27 28–229 230–250 251–333 4 3.00 ** 4 50 50
Ephrin-B2 full-length ***
2
2.00 ** 2 25 25
Ephrin-B2-Fc Fc domain 1.00
27–227 ND ND
0 0.00 0 0 0
2 3 4
5 2 1 .5 .1 05 01 F A
0 0 0. 0. DG LP 1 2 3 4 6
hb hb hb hb hb NAhb hb hb 2 3 4
NAhb hb hb

Ephrin-B1 Fc
hr -B -Fc
-B Fc
Fc
Ep Ep Ep Ep Ep siR p p p siR p p p

in 2 -
3-
P g iE iE iE

EphrinlgG
in S S S g SiE SiE SiE
Ephrin-B2 (µg) t in
ge g et
ar ar

Ep
nt nt
No No

h Native PAGE
Control BLM
i Native PAGE
Control BLM
j k l
BAL BAL Ephrin-
BAL BAL kDa BAL BAL BAL
∆Ephrin-B2
BAL
∆Ephrin-B2 IgG-Fc B2-Fc
1 2 3 1 2 3 * * IgG-Fc
kDa
kDa 1,236 10.0 1.50 *** 4 42 α-SMA

mRNA expression
1,048 Ephrin-B2-Fc

Chemotactic index

sEphrin-B2 in BAL
1,236 720 sEphrin-B2 7.5 3 * 130 Type I collagen
1,048 42 β-actin
480 oligomer 1.00

(ng/ml)
720 sEphrin-B2 2
oligomer 242 5.0
480 Dimer

Type I collagen
0.50 ** **

densitometry
1

densitometry
242 2.5 10.0 10.0
Dimer Monomer

α-SMA
– + – + SDS + 2ME 0 5.0 5.0
0 0.00
a1 a2
l1 Act 0 0
Co
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

m n o IgG-Fc q Bleomycin
s.c. injection r
S.c. injection * Ephrin-B2-Fc ***
Dermal thickness (µm)

400 16 *
S.c. injection ephrin-B2-Fc or IgG-Fc * Tamoxifen i.p. 400
µg/mg of protein

ephrin-B2-Fc or IgG-Fc
Hydroxyproline

300

Thickness (µm)
12
300
200 8 Tamoxifen Bleomycin
Daily for 14 d (Ephrinb2 ablation) s.c. injection
Harvest 200
Time (d) 100 4
100
Control (IgG-Fc) Ephrin-B2-Fc Time (d)
0 0 –7 –1 0 +28 0
Daily for 28 d PBS BLM
p kDa Ephrinb2-C Ephrinb2-CKO
H&E

42
IgG-Fc Ephrin-B2-Fc
α-SMA
s
*
Saline

130 Type I collagen 40 * Ephrinb2-C

Trichrome staining

* Ephrinb2-CKO

µg/mg of tissue
Hydroxyproline
42 β-actin 30

20
Type I collagen

*** ***
densitometry
densitometry

10.0 10.0
Bleomycin

10
α-SMA

5.0 5.0
0
0 0 PBS BLM

Figure 3 The ephrin-B2 ectodomain directs fibroblast migration, invasion and myofibroblast differentiation in vitro and in vivo. (a) Domain structure
of the ephrin-B2 protein and the recombinant ephrin-B2 ectodomain fused to Fc. TM, transmembrane domain. (b) Representative phase contrast images
showing the effect of preclustered ephrin-B2-Fc or IgG-Fc (control) treatment for 24 h on filopodia formation in primary human lung fibroblasts.
White arrows indicate filopodia formation in fibroblasts. Scale bars, 50 µm. n = 25–50 cells per condition. (c) Blinded quantitation of the number
of filopodia per cell upon IgG-Fc, ephrin-B1-Fc, ephrin-B2-Fc or ephrin-B3-Fc treatment. n = 25–50 cells per condition. (d) Chemotactic indices
induced by ephrin-B2-Fc, LPA or PDGF-BB. Data are presented as fold increase over chemotaxis induced by IgG-Fc (dotted line). n = 4 for all groups.
(e) Relative mRNA expression of genes encoding EphB receptors in primary mouse lung fibroblasts. mRNA expression was normalized to the level of
GAPDH. n = 3 for all groups. (f,g) Effect of siRNA-mediated knockdown of EphB receptors on BAL-induced fibroblast chemotaxis (f) and invasion (g).
BAL from mice at day 7 following bleomycin challenge was used as a chemoattractant. n = 3 for all groups. (h) The oligomeric state of sEphrin-B2
from BAL samples from PBS- and bleomycin-challenged mice subjected to nondenaturing native gel electrophoresis. n = 3 for all groups. (i) Effect
of SDS and 2-β-mercaptoethanol (2ME) on the formation of high-molecular-weight sEphrin-B2 oligomers in BAL. (j) Effect of sEphrin-B2 depletion
by magnetic beads coated with anti-ephrin-B2 antibody on BAL-induced fibroblast chemotaxis of mouse lung fibroblasts. n = 5 for all groups.
(k) Effects of ephrin-B2-Fc or IgG-Fc on Col1a1 and Acta2 mRNA expression by mouse lung fibroblasts. Data are presented as fold increase over IgG-
Fc-treated fibroblasts. (l) Effects of ephrin-B2-Fc or IgG-Fc on α-SMA and type I collagen protein expression in mouse lung fibroblasts. Densitometry
data (normalized to β-actin) are presented. n = 3 for all groups. Blue and red represent data from lung fibroblasts that received IgG-Fc or ephrin-B2-Fc
treatment, respectively. (m) Top, schematic indicating that C57BL/6N mice were treated with daily subcutaneous (s.c.) injections of either preclustered
ephrin-B2-Fc (n = 6) or IgG-Fc (n = 6) as control for 14 d. Bottom, results of H&E and Masson’s trichrome staining for each treatment group. Scale
bars, 100 µm. (n–p) Effects of ephrin-B2-Fc or IgG-Fc treatment on skin fibrosis in mice as assessed by blinded analysis of dermal thickness of tissue
sections (n), quantification of hydroxyproline content in skin biopsies (o) and western blot showing α-SMA and type I collagen levels in skin (p). In n,
n = 5 for each treatment group; o, n = 6 for each treatment group; p, n = 4 for all groups. β-actin was used as a loading control for densitometry analysis.
One representative out of two technical replicates is shown. Densitometry data are presented in p. (q) Top, schematic showing the model through which
Ephrinb2-C and Ephrinb2-CKO mice were subjected to bleomycin-induced skin fibrosis. Mice received daily subcutaneous injections of either PBS or
bleomycin for 28 d. Bottom, representative images of Masson’s trichrome–stained skin of Ephrinb2-C and Ephrinb2-CKO mice 28 d following PBS or
bleomycin challenge. Scale bars, 100 µm. n = 6 for all groups. (r) Blinded analysis of dermal thickness of tissue sections from Ephrinb2-C mice and
Ephrinb2-CKO mice 28 d after PBS or bleomycin challenge. n = 6 for all groups. Blue and red represent data from Ephrinb2-C mice or Ephrinb2-CKO
mice, respectively. (s) Hydroxyproline content in 6-mm skin biopsies from Ephrinb2-C and Ephrinb2-CKO mice treated with PBS or bleomycin. n = 6 for
all groups. For c, d, j–l and n–p, data were analyzed with Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001 versus IgG-Fc treated fibroblasts. For
f and g, data were analyzed with Student’s t-test. *P < 0.05 versus nontargeting control siRNA. For r and s, data are presented as mean ± s.d and were
analyzed by two-way ANOVA. *P < 0.05, ***P < 0.001. In c, o and s, center lines, median values; +, the mean; box edges, 25th and 75th percentiles;
whiskers, minimum and maximum values. Data are presented as mean ± s.d. Uncropped blot images are shown in Supplementary Figure 5.

nature medicine VOLUME 23 | NUMBER 12 | DECEMBER 2017 1409

 Articles
associated with considerably lower dermal thickness and hydroxy-
proline content compared to control mice (Fig. 3r,s).
Identification of ADAM10 as the ephrin-B2 ectodomain
sheddase in fibroblasts
Previous studies have shown that ephrins can be cleaved by met-
alloproteinases, including matrix metalloproteinases (MMPs)
and ADAMs19–22. In order to investigate whether shedding of the
ephrin-B2 ectodomain is regulated by metalloproteinases in fibrob-
lasts, we tested the ability of BB-94, a broad-spectrum inhibitor of
both MMPs and ADAMs, to inhibit fibroblast generation of sEphrin-
B2. BB-94 treatment reduced sEphrin-B2 concentration in medium
conditioned by healthy human lung fibroblasts in a dose-dependent
manner, indicating that metalloproteinase(s) contribute to ephrin-
B2 cleavage (Fig. 4a). Of the MMPs and ADAMs, these fibroblasts
expressed MMP1, MMP2 and MMP14, as well as ADAM9, ADAM10,
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
ADAM12 and ADAM17 (Fig. 4b). In order to investigate whether
any of these proteases act as ephrin-B2 ectodomain sheddases in
human lung fibroblasts, we performed siRNA knockdowns of each
of these metalloproteinases individually. We first confirmed that
siRNA duplexes efficiently knocked down the MMPs and ADAMs
that we found to be expressed by cultured primary human lung fibrob-
lasts (Supplementary Fig. 2). We then found that siRNA-mediated
knockdown of ADAM10, but not the other genes encoding ADAMs
or MMPs expressed by fibroblasts, markedly lowered sEphrin-B2
concentration in fibroblast cell culture supernatant as assessed by
ELISA (Fig. 4c).
To further investigate whether ADAM10 is responsible for ephrin-
B2 ectodomain shedding in fibroblasts, we used phorbol 12-myristate
13-acetate (PMA), which activates limited proteolysis of membrane-
bound proteins, i.e., ectodomain shedding, by increasing the activ-
ity of ADAM10 and ADAM17 (ref. 26). Treatment of human lung
fibroblasts with PMA for 30 min resulted in a marked increase in the
sEphrin-B2 concentration in fibroblast cell culture supernatants com-
pared to that from fibroblasts treated with DMSO as control (Fig. 4c).
Knockdown of ADAM10 by siRNA also substantially reduced
sEphrin-B2 concentration in medium conditioned by PMA-stimulated
fibroblasts, whereas knockdown of ADAM17 or genes encod-
ing other ADAMs or MMPs had no pronounced effects compared
to human lung fibroblasts transfected with nontargeting siRNA.
Moreover, treatment of fibroblasts with GI254023X, an ADAM10-
specific inhibitor, reduced both constitutive and PMA-stimulated
sEphrin-B2 concentration in fibroblast supernatants in a dose-
dependent manner (Fig. 4d). In contrast, fibroblast treatment with
TAPI-0, an ADAM17-specific inhibitor, did not prevent sEphrin-B2
generation by fibroblasts (Fig. 4d), further implicating ADAM10 in
ephrin-B2 shedding.
ADAM10-mediated ephrin-B2 shedding drives TGF-b-induced
myofibroblast activation
Having identified ADAM10 as the major sheddase of the ephrin-B2
ectodomainin fibroblasts, we then investigated the regulation of this
shedding, and its contribution to the profibrotic activities of these
cells. TGF-β1 is a prototypical profibrotic cytokine that induces myofi-
broblast activation27,28. Whereas TGF-β1 treatment of human lung
fibroblasts did not induce expression of EFNB2 mRNA, it did result
in greater ADAM10 mRNA expression in these cells as compared to
untreated cells (Fig. 4e) and in a substantially greater sEphrin-B2
concentration in medium conditioned by these cells compared to that
conditioned by untreated cells (Fig. 4f).
1410
Considering that we found that recombinant ephrin-B2-Fc induced
fibroblast differentiation into activated myofibroblasts in vitro and
resulted in greater expression of the myofibroblast marker α-SMA
in mice in vivo, we investigated whether TGF-β-induced myofi-
broblast activation is dependent on ephrin-B2 shedding. Genetic
knockdown of ADAM10 or pharmacological inhibition with the
ADAM10-selective inhibitor GI254023X29 reduced TGF-β-induced
sEphrin-B2 production, confirming the role of ADAM10 in TGF-β-
induced ephrin-B2 shedding (Fig. 4f,g). TGF-β-induced expression
of the α-SMA protein by human lung fibroblasts was also markedly
reduced by siRNA knockdown of ADAM10 (Fig. 4h) or by pharmaco-
logical inhibition of ADAM10 with GI254023X, which reduced TGF-
β-induced ACTA2 mRNA expression dose-dependently (Fig. 4i).
GI254023X treatment also markedly and dose-dependently reduced
TGF-β-induced α-SMA protein expression by human lung fibrob-
lasts (Fig. 4j). Further, immunofluorescence staining of human lung
fibroblasts with an anti-α-SMA antibody and rhodamine–phalloidin
demonstrated that GI254023X treatment inhibited TGF-β-induced
formation of stress fibers and incorporation of α-SMA protein into
stress fibers, which are both phenotypic characteristics of activated
myofibroblasts (Fig. 4k). Taken together, these results indicate that
TGF-β-induced myofibroblast formation requires ADAM10-mediated
ephrin-B2 shedding.
Previous studies have shown that ADAM10 cleaves ephrin-B2
in the juxtamembrane region of its ectodomain20,30 (Fig. 4l). We
therefore tested whether this region is important for TGF-β-induced
ephrin-B2 shedding and myofibroblast activation. Forced overex-
pression of ephrin-B2∆Ecto, which lacks the entire ephrin-B2 ectodo-
main but preserves the transmembrane and intracellular domains,
markedly reduced the amount of sEphrin-B2 produced by these
cells in response to TGF-β compared to that produced by TGF-β
challenge of fibroblasts transfected with WT ephrin-B2 (Fig. 4l,m).
Likewise, overexpression of a mutant with a deletion of the juxtam-
embrane region, ephrin-B2∆Juxta, which lacks amino acids 168–218
of the ephrin-B2 ectodomain, abrogated TGF-β-induced ephrin-
B2 shedding (Fig. 4l,m). Further, deletions of specific sequences
in the juxtamembrane region (amino acids 182–194 or 197–218)
rendered ephrin-B2 resistant to TGF-β-induced ephrin-B2 shed-
ding (Fig. 4l,m).
To further demonstrate that the juxtamembrane region of ephrin-
B2 is required for ephrin-B2 shedding induced by TGF-β, we analyzed
ephrin-B2 protein levels in fibroblasts transfected with hemagglutinin
(HA)-tagged WT ephrin-B2 or HA-tagged, cleavage-resistant mutants.
As shown in Figure 4n, the amount of HA-tagged WT ephrin-B2 that
was detected by anti-HA antibodies in lysates of human lung fibrob-
lasts was substantially reduced by TGF-β treatment; this result is in
accordance with TGF-β-induced ephrin-B2 shedding. In contrast,
this TGF-β-induced reduction in HA-tagged ephrin-B2 expression
was largely prevented in human lung fibroblasts overexpressing HA-
tagged, cleavage-resistant ephrin-B2 mutants, further demonstrating
that the juxtamembrane region of ephrin-B2 is required for ectodo-
main cleavage (Fig. 4n).
We next investigated whether overexpression of cleavage-resistant
ephrin-B2 mutants prevents TGF-β-mediated myofibroblast activa-
tion, in addition to ephrin-B2 shedding. We found that human lung
fibroblasts overexpressing cleavage-resistant ephrin-B2 mutants had
reduced TGF-β-induced ACTA2 mRNA and α-SMA protein levels
compared to human lung fibroblasts transfected with WT ephrin-B2
(Fig. 4o,p), in accordance with a requirement for ephrin-B2 cleavage
for TGF-β-induced myofibroblast activation.
VOLUME 23 | NUMBER 12 | DECEMBER 2017 nature medicine
 articles

a b Sheddases c d
MMPs ADAMs DMSO PMA DMSO PMA
0.20 0.10 0.6 0.6
* * * * * * * * *

mRNA expression
0.08 0.5 0.5
0.15

sEphrin-B2
sEphrin-B2

sEphrin-B2
0.4 0.4
*
(ng/ml)

(ng/ml)
*

(ng/ml)
0.06
0.10 0.3
** 0.04
0.3
0.2 *
0.2
0.05 ** *
0.02 0.1 0.1 *
0.00 ND NDND ND ND ND 0.0 0.0
0
0
0.1
1
5
10
50

0
0.1
1
20
50

0
0.1
1
20
50

0
1
10
50
100
0
1
10
50
100
A 1 2 7 14 9 10 12 17 19
P1 P2 P3 P7 P8 P9 14 8 9 10 12 15 17 19 28 N P P P
M M M MP AM AM AM AM AM
M M M M M M P AMAM M M M M M M si
R
M M D
BB-94 (nM) M M M M M M MM AD AD DA DA DA DA DA DA
N
T si si siM siM siA iAD iAD iAD iAD GI254023X GI254023X TAPI-0 TAPI-0
A A A A A A s s s s (nM) (nM) (nM) (nM)
(Batimastat)

e *
DMSO
f DMSO g DMSO
h DMSO
i 24.00 DMSO
j

densitometry
2.5 10.0

mRNA expression
0.6 TGF-β 0.6 TGF-β
TGF-β TGF-β * * TGF-β
mRNA expression

densitometry
10.0 20.00 *

α-SMA
2.0 0.5 * * 0.5 * * ** 5.0

α-SMA
16.00

sEphrin-B2
sEphrin-B2

ACTA2
5.0
1.5 0.4 0.4 *
(ng/ml)
(ng/ml)

12.00 ND ND ND ND ND

1.0
0.3 0.3 0 8.00
* 0
kDa
0.2 0.2 kDa 42 α-SMA
42 α-SMA 4.00
0.5 0.1 0.1 37 GAPDH
37 GAPDH 0.00
0 0.0 0.0 TGF-β – – – – – + + + + +

0
0.1
1
20
50
0
0.1
1
20
50
TGF-β – + – +
10 B2
A 10 SO 23
X

0
0.1
1
20
50
0
0.1
1
20
50
N
iR AM

10
AM EFN s M
D 540 GI254023X GI254023X

AM
AD

R
D T l2 (nM) (nM)

si
A N si GI254023X GI254023X

AD
G

T
N

si
(nM) (nM)

k l m n
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

18
19

–2
TGF-β
*

2–
xt

97
*

FP
DMSO GI254023X TGF-β + GI254023X

8
u
kDa

∆1

∆1
Cytoplasmic

∆J
W
0.6

G
Ectodomain TM
domain DMSO
Phalloidin

28–229 230–250 251–333 0.5 * TGF-β 80

DAPI

sEphrin-B2
Ephrin-B2 WT 0.4 HA-tagged

(ng/ml)
∆Ecto 0.3 Ephrin-B2
∆Juxta 40
0.2
∆182–194
α-SMA
DAPI

0.1
∆197–218 37 GAPDH
0.0
T to ta 4 8 TGF-β – + – + – + – + – +
W c ux 19 21
2 ∆E ∆J 2– 7–
-B 8 9
hrin ∆1 ∆1
Ep
o DMSO
TGF-β
p q r DMSO s
60 * * TGF-β
* 60
mRNA expression

* *
densitometry

8
mRNA expression

10
*
mRNA expression

50 * *
* * 10

densitometry
50
α-SMA

40 6
*
ACTA2

* *

α-SMA
40
5 *
ACTA2

30
* * 4 30 *
5
20 ND ND ND ND
0 20
10 2 0
TGF-β –+ – + –+ – + 10 TGF-β – + – + – + – +
0.0
kDa 0 ND ND ND
0.0 kDa
T to ta 4 8 α-SMA
W c x 19 21 42 1 2 3 4 6
ed NA B3 B4 α-SMA
2 ∆E ∆Ju 2– 7– 42
B B B B B
H H H H H
ct H H
-B 8 9
∆1 ∆1
37 GAPDH EP EP EP EP EP fe siR P P 37 GAPDH
h rin ns NT siE siE
Ep tra
94
FP

18
T

T ed

si B3

4
W

n
–1

B
–2

N
G

U
N ect

H
R
82

97

EP

EP
si
f
∆1

ns
∆1

si
ra
nt
U

Figure 4 ADAM10-mediated sEphrin-B2 generation is required for TGF-β1-induced myofibroblast differentiation. (a) Effect of BB-94 (Batimastat) treatment
on the generation of sEphrin-B2 in primary human lung fibroblasts. *P < 0.05 versus control (DMSO). Blue indicates treatment with DMSO as control,
and red indicates treatment with BB-94. (b) Relative mRNA expression of genes encoding metalloproteinases (MMPs and ADAMs) in primary human lung
fibroblasts. mRNA expression was normalized to the level of GAPDH. n = 5 sets of human lung fibroblasts analyzed. (c) Effect of siRNA-mediated knockdown
of metalloproteinases on ephrin-B2 shedding by human lung fibroblasts. Concentration of sEphrin-B2 in the conditioned media was determined by ELISA.
Three sets of primary lung fibroblasts were used; n = 3 sets for all groups. *P < 0.05 versus control (DMSO). NT, nontargeting. (d) Effect of GI254023X
(ADAM10 inhibitor) and TAPI-0 (ADAM17 inhibitor) on constitutive and PMA-induced generation of sEphrin-B2 by primary human lung fibroblasts.
Three sets of primary lung fibroblasts were used; n = 3 sets for all groups. *P < 0.05 versus control (DMSO). (e) Relative mRNA expression of EFNB2 and
ADAM10 in primary human lung fibroblasts treated with or without TGF-β (10 ng/ml) for 48 h. GAPDH was used as a housekeeping gene. *P < 0.05 versus
TGF-β-treated fibroblasts. (f) Effect of siRNA-mediated knockdown of ADAM10 on TGF-β-induced generation of sEphrin-B2 in human lung fibroblasts.
Nontargeting siRNA was used as control. (g) Effect of ADAM10 inhibition with GI254023X (20 nM) on TGF-β-induced sEphrin-B2 generation in human
lung fibroblasts. DMSO was used as control. n = 5 for all groups. (h) Effect of siRNA-mediated knockdown of ADAM10 on TGF-β-induced α-SMA protein
expression and densitometry (GAPDH used as a loading control) in human lung fibroblasts. Nontargeting siRNA was used as control. n = 4 for all groups.
(i) Effect of ADAM10 inhibition with GI254023X (20 nM) on TGF-β-induced ACTA2 mRNA expression in human lung fibroblasts. GAPDH was used as a
housekeeping gene. n = 4 for all groups. *P < 0.05 versus TGF-β-treated fibroblasts. Data are presented as fold change relative to DMSO-treated cells.
(j) Effect of ADAM10 inhibition with GI254023X (20 nM) on TGF-β-induced α-SMA protein expression, with GAPDH used as a loading control, with GAPDH
used as a loading control; n = 4 for all groups. *P < 0.05 versus TGF-β-treated fibroblasts. (k) Representative immunofluorescence images (n > 6 images per
condition) of human lung fibroblasts treated with vehicle (DMSO) or the ADAM10 inhibitor GI254023X (20 nM) and stained for α-SMA (green) to
identify myofibroblasts, phalloidin (red) to visualize filamentous F-actin in fibroblasts and Hoechst 33342 (blue) to visualize nuclei. Scale bars,
50 µm. (l) Schematic of ephrin-B2 WT and HA-tagged ephrin-B2 mutants, including ephrin-B2 ∆Ecto-HA, ephrin-B2∆Juxta-HA, ephrin-B2∆182–194-HA and
ephrin-B2∆197–218-HA. (m) Effect of the ephrin-B2 mutants on TGF-β-induced sEphrin-B2 generation in human lung fibroblasts as assessed by ephrin-B2
ELISA. n = 4 for all groups. (n) Effect of ephrin-B2 mutants on TGF-β-induced ephrin-B2 shedding in human lung fibroblasts as assessed by western blot
analysis of HA-tagged ephrin-B2 WT and mutants. GAPDH was used as a loading control. (o) Effect of ephrin-B2 mutants on TGF-β-induced ACTA2 mRNA
expression in human lung fibroblasts. GAPDH was used as a housekeeping gene. n = 4 for all groups. Data are presented as fold change relative to DMSO-
treated cells. (p) Effect of ephrin-B2 mutants on TGF-β-induced α-SMA protein expression in human lung fibroblasts. GAPDH was used as a loading control.
n = 3 for all groups. (q) Relative mRNA expression of genes encoding EphB receptors in primary human lung fibroblasts. GAPDH was used as a housekeeping
gene. n = 3 for all groups. (r) Effect of siRNA-mediated knockdown of EphB receptors on TGF-β-induced ACTA2 mRNA expression in human lung fibroblasts.
GAPDH was used as housekeeping gene. n = 3 for all groups. Data are presented as fold change relative to DMSO-treated cells. (s) Effect of siRNA-mediated
knockdown of EphB receptors on TGF-β-induced α-SMA protein levels in human lung fibroblasts. GAPDH was used as a loading control. n = 3 for all groups.
All data are presented as mean ± s.d and were analyzed with Student’s t-test. *P < 0.05. Uncropped blot images are shown in Supplementary Figure 6.

nature medicine VOLUME 23 | NUMBER 12 | DECEMBER 2017 1411

 Articles

a c e ***
BLM
*** ***
1.0 * * 300 * Vehicle
1.50
Vehicle

sEphrin-B2 in BAL (ng/ml)

Densitometry
kDa Day 0 Day 1 Day 3 Day 7 Day 14
* * * GI254023X 1.25 GI254023X
80
ADAM10 0.5 225

Hydroxyproline
* 1.00

(µg/left lung)
42 β-actin
0 150 0.75
0 1 3 7 14
b
0.50
Time following
Vehicle GI254023X 75
BLM (d) 0.25

0 0.00
PBS BLM PBS BLM

d f
Saline

BLM +
kDa PBS BLM GI25 100
BLM + vehicle

Percentage survival
42 α-SMA BLM + GI254023X
75

42 β-actin
50

** ***
1.0 Vehicle 25
Bleomycin

densitometry
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

GI254023X

α-SMA
0
0.5
0 5 10 15
Time after
0
PBS BLM challenge (d)

Figure 5 ADAM10 inhibition prevents ephrin-B2 shedding, myofibroblast formation and lung fibrosis in mice. (a) Western blot (left) and densitometry
(right) showing ADAM10 levels in lung homogenates after bleomycin challenge. β-actin was used as a loading control. n = 4 mice for all groups.
(b) Representative images (4 images from n = 10 mice per group) of Masson’s trichrome staining of lung sections from mice at 14 d after PBS or
bleomycin challenge that were treated with ADAM10 inhibitor GI254023X or vehicle control. Scale bar, 100 µm. (c) Hydroxyproline content measured
in the lungs of mice 14 d after bleomycin or PBS challenge treated with ADAM10 inhibitor GI254023X or vehicle control. n = 8 for all groups.
(d) α-SMA protein expression assessed by western blot (top) and densitometry (bottom; normalized to β-actin) in total lung homogenates of
mice 14 d after bleomycin or PBS challenge treated with ADAM10 inhibitor GI254023X (GI25) or vehicle control. This representative blot shows
3 samples per group (n = 6 mice for all groups). (e) Concentration of sEphrin-B2 in BAL fluids recovered from mice at 14 d following PBS or
bleomycin challenge and treated with ADAM10 inhibitor GI254023X or vehicle control. n = 8 mice for all groups. (f) Survival curves for mice
challenged with bleomycin and treated with ADAM10 inhibitor GI254023X or vehicle control. n = 10 mice per group. In a, data are presented
as mean ± s.d and were analyzed with Student’s t-test. *P < 0.05 versus Day 0. In c–e, data are presented as mean ± s.d and were analyzed with
two-way ANOVA. **P < 0.01, ***P < 0.001. In f, data were analyzed with the log-rank test. ***P < 0.001. In c and e, center lines, median
values; +, the mean; box edges, 25th and 75th percentiles; whiskers, minimum and maximum values. Uncropped blot images are shown in
Supplementary Figure 7.

As expression of the recombinant ephrin-B2 ectodomain was suf-
ficient to drive myofibroblast activation and TGF-β-induced myofi-
broblast activation required ADAM10-mediated ephrin-B2 shedding,
we hypothesized that TGF-β-induced myofibroblast activation is con-
trolled by autocrine sEphrin-B2–EphB receptor signaling. Of the five
identified EphB receptors, primary human lung fibroblasts expressed
proteins encoded by EPHB3 and EPHB4, but not EPHB1, EPHB2
or EPHB6 (Fig. 4q). We found that siRNA-mediated knockdown of
EPHB3 or EPHB4 markedly reduced the TGF-β-induced increase in
both ACTA2 mRNA and α-SMA protein levels compared to human
lung fibroblasts transfected with nontargeting siRNA (Fig. 4r,s),
demonstrating that both receptors mediate profibrotic activities of
sEphrin-B2 in lung fibroblasts.
Pharmacological inhibition of ADAM10 prevents bleomycin-
induced lung fibrosis in mice
On the basis of our findings above, we hypothesized that therapeutic
targeting of ADAM10 with the ADAM10-selective pharmacologi-
cal inhibitor GI254023X29 could prevent lung fibrosis by inhibiting
ephrin-B2 shedding and myofibroblast activation in vivo. Previous
studies show that bleomycin challenge rapidly increases levels of
activated TGF-β within areas of lung injury31,32, which led us to
hypothesize that ADAM10 levels would be similarly increased upon
lung injury. Indeed, ADAM10 expression levels were markedly higher
in vivo after bleomycin-induced lung injury compared to those in
PBS-challenged mice (Fig. 5a). To examine the ability of ADAM10
inhibition to mitigate lung fibrosis in vivo, mice were subjected to
the bleomycin-induced lung fibrosis model and treated daily with
GI254023X (200 mg per kg body weight per day) or vehicle control by
intraperitoneal injection. Blinded histological analysis revealed that
the lung parenchymal fibrosis produced 14 d following bleomycin
challenge in vehicle-treated mice was mitigated in mice treated with
GI254023X (Fig. 5b), and this was associated with marked reduc-
tion in lung hydroxyproline levels (Fig. 5c). ADAM10 inhibition also
resulted in a considerably lower level of α-SMA expression in bleomy-
cin-challenged mice compared to PBS-challenged mice (Fig. 5d).
To gain insight into the mechanism of action of GI254023X in this
lung fibrosis model, we assessed sEphrin-B2 concentration in BAL
samples. In accordance with our in vitro studies demonstrating that
ADAM10 is responsible for ephrin-B2 cleavage to generate sEphrin-
B2, GI254023X treatment reduced sEphrin-B2 concentration in the
BAL of bleomycin-challenged mice (Fig. 5e), suggesting that the
ADAM10–sEphrin-B2 axis drives myofibroblast activation in lung
fibrosis in vivo. Further, ADAM10 inhibition with GI254023X mark-
edly reduced mortality caused by bleomycin-induced lung injury and
fibrosis in mice (Fig. 5f).
ADAM10-sEphrin-B2 signaling is upregulated in idiopathic
pulmonary fibrosi
To determine the relevance of our studies to human disease, we inves-
tigated the role of ADAM10–sEphrin-B2 signaling in fibroblasts iso-
lated from the lungs of individuals with IPF and healthy controls. IPF

1412 VOLUME 23 | NUMBER 12 | DECEMBER 2017 nature medicine

 articles

a b c d * e
0.6 3.0 0.6 * DMSO 0.6 * Nontargeting siRNA 3.0 * DMSO
* * *
*
supernatants (ng/ml)

supernatants (ng/ml)
supernatants (ng/ml)
0.5 2.5 0.5 GI254023X 0.5 siADAM10 GI254023X

mRNA expression

COL1A1 mRNA
sEphrin-B2 in

sEphrin-B2 in
sEphrin-B2 in
0.4 2.0 0.4 0.4 * 2.0

expression
ADAM10
*
0.3 1.5 0.3 0.3 *
0.2 1.0 0.2 0.2 1.0
0.1 0.5 0.1 0.1
0.0 0.0 0.0 0.0 0.0
y

y
F

F
F

F
lth

lth

lth

lth

lth
IP

IP

IP
IP

IP
ea

ea

ea

ea

ea
H

H
f *
g Nontargeting
h Nontargeting
i SDS-PAGE
3.0
* DMSO
3.0 * 3.0
*
* siRNA
* siRNA Control BAL IPF BAL
GI254023X siADAM10 siADAM10 kDa 1 2 3 4 5 6 1 2 3 4 5 6
COL1A1 mRNA

ACTA2 mRNA
ACTA2 mRNA

2.0 2.0
expression

2.0

expression
expression

60
* sEphrin-B2
*
1.0 1.0 1.0
37
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

0.0 0.0 0.0

y

y
y

F
lth

lth
lth

IP

IP

IP
ea

ea
ea

H
H

j Cohort 1 Cohort 2 k l Homeostasis Fibrosis

** ** Autocrine
6.0 n = 11 6.0 9.0 signaling sEphrin-B2
**
sEphrin-B2 in plasma (ng/ml)

n=5
sEphrin-B2 in BAL (ng/ml)
sEphrin-B2 in BAL (ng/ml)

ADAM10 Ephrin-B2 ADAM10

5.0 5.0 7.5 n = 30 EphB3 EphB4
Paracrine
TGF-β signaling

4.0 4.0 6.0

Collagen
+
α-SMA α-SMA EphB4 EphB3
3.0 3.0 4.5 n = 30

2.0 n=4 2.0 3.0 Collagen

α-SMA
n=4
1.0 1.0 1.5

0.0 0.0 0.0 Quiescent Activated Therapeutic targets

Ephrin-B2
fibroblast α-SMA + myofibroblast
y

y
F
F

F
lth

lth

lth

ADAM10
IP
IP

IP
ea

ea

ea

EphB3/4
H

Figure 6 ADAM10–sEphrin-B2 signaling is upregulated in IPF. (a) Concentration of sEphrin-B2 as determined by ELISA in conditioned media from primary
lung fibroblasts from control (healthy) donors (n = 5) and individuals with IPF (n = 5). (b) Relative mRNA expression of ADAM10 in primary lung fibroblasts
from control donors (n = 5) and individuals with IPF (n = 5). mRNA expression was normalized to the level of GAPDH. (c) Effect of pharmacological
inhibition of ADAM10 with GI254023X (20 nM) on sEphrin-B2 concentration in conditioned media. DMSO was used as vehicle control. n = 3 for all groups.
(d) Effect of siRNA-mediated knockdown of ADAM10 on sEphrin-B2 concentration in conditioned media. Nontargeting siRNA was used as a control.
n = 3 for all groups. (e,f) Effect of pharmacological inhibition of ADAM10 with GI254023X (20 nM) on TGF-β-induced COL1A1 (e) and ACTA2 (f) mRNA
expression in human lung fibroblasts versus treatment with vehicle (DMSO) control. mRNA expression was normalized to the level of GAPDH. n = 4 for
all groups. Data are presented as fold change relative to DMSO-treated cells. (g,h) Effect of siRNA-mediated knockdown of ADAM10 on TGF-β-induced
COL1A1 (g) and ACTA2 (h) mRNA expression in human lung fibroblasts. Nontargeting siRNA was used as control. mRNA expression was normalized to the
level of GAPDH. n = 4 for all groups. Data are presented as fold change relative to untransfected cells. (i) Concentration of sEphrin-B2 in BAL fluid from
control donors (n = 6) and individuals with IPF (n = 6) assessed by western blotting. (j) Concentration of sEphrin-B2 in BAL fluids from control donors (n
= 8) and individuals with IPF (n = 16) assessed by ELISA. Data are stratified into two cohorts obtained from separate institutions. (k) Concentration of
sEphrin-B2 in plasma from control donors (n = 30) and individuals with IPF (n = 30) assessed by ELISA. All data are presented as mean ± s.d and were
analyzed with a Student’s t-test. *P < 0.05, **P < 0.01. In j and k, center lines, median values; +, the mean; box edges, 25th and 75th percentiles;
whiskers, minimum and maximum values. Uncropped blot images are shown in Supplementary Figure 8. (l) Schematic of the working model of ADAM10-
mediated ephrin-B2 shedding in fibroblasts. Our results suggest that in fibroblasts during the development of fibrosis, sEphrin-B2 is produced by ADAM10
upregulation in TGF-β-stimulated fibroblasts. sEphrin-B2 then acts through EphB3/4 receptors to further drive and/or amplify profibrotic fibroblast effector
functions, including migration, invasion, myofibroblast differentiation and extracellular matrix production in an autocrine and/or paracrine fashion.

lung fibroblasts had a substantially higher concentration of sEphrin- markedly inhibited the increased COL1A1 and ACTA2 expression of
B2 in culture medium and expressed markedly increased ADAM10 IPF fibroblasts compared to controls (Fig. 6e–h).
mRNA compared to normal lung fibroblasts in vitro (Fig. 6a,b). Considering the relevance of the ADAM10–sEphrin-B2 axis in
Pharmacological inhibition of ADAM10 with GI254023X or siRNA IPF lung fibroblasts, we next examined concentration of sEphrin-B2
knockdown of ADAM10 markedly reduced sEphrin-B2 production in both BAL fluid and plasma samples from individuals with IPF
by both IPF and control lung fibroblasts (Fig. 6c,d), confirming that and healthy volunteers. Quantification of sEphrin-B2 in these sam-
ADAM10 is responsible for ephrin-B2 shedding in IPF and control ples by ELISA and by western blotting of a subset of these samples
lung fibroblasts. IPF lung fibroblasts are characterized by increased showed a markedly increased concentration of sEphrin-B2 in the BAL
type I collagen and α-SMA expression33. We examined whether fluid of 16 individuals with IPF compared to samples from 8 healthy
ADAM10–sEphrin-B2 signaling contributes to this increased expres- volunteers (Fig. 6i,j). We then compared plasma sEphrin-B2 con-
sion of COL1A1 and ACTA2 through inhibition of ADAM10 in IPF lung centration in 30 individuals with IPF and 30 gender-matched, non-
fibroblasts. Treatment with GI254023X or a siRNA targeting ADAM10 smoking controls (subject demographics in Supplementary Table 1)

nature medicine VOLUME 23 | NUMBER 12 | DECEMBER 2017 1413

 Articles
and observed a markedly higher concentration of plasma sEphrin-
B2 in the individuals with IPF compared to the samples from the
control group (Fig. 6k).
DISCUSSION
Using a combination of mouse models and clinical samples obtained
from individuals with IPF, we demonstrate that a soluble form of
ephrin-B2, composed of its cleaved ectodomain, is a potent media-
tor of tissue fibrosis. This soluble form, sEphrin-B2, was markedly
elevated in the lung tissue and the BAL of mice in our pulmonary
fibrosis model and in the BAL of individuals with IPF, suggesting
ephrin-B2 shedding is increased in response to tissue injury. We dem-
onstrate that treatment with recombinant Ephrin-B2 ectodomain was
sufficient to induce dermal fibrosis in mice when injected subcutane-
ously by promoting collagen and α-SMA expression consistent with
that of myofibroblast activation. Conversely, loss of ephrin-B2 in the
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
fibroblasts of mice resulted in decreased sEphrin-B2 generation and
protection from lung and dermal fibrosis in vivo, identifying ephrin-
B2 as a critically important profibrotic mediator. Although loss of
ephrin-B2 in fibroblasts provided substantial protection from bleomy-
cin-induced skin and lung fibrosis, we did not observe full protection
in both models, suggesting additional cell types, such as inflammatory
cells, may also be sources of sEphrin-B2 during the development of
tissue fibrosis, which will be the focus of future investigations.
It has previously been demonstrated that both ephrin-B ligands and
EphB receptors undergo ectodomain shedding to release active pro-
teins19–22. In cancer cells, shedding of membrane-bound ephrin-B lig-
ands regulates invasion34. To the best of our knowledge, however, our
study is the first to describe ephrin-B2 shedding in tissue fibrosis and
to implicate sEphrin-B2 in human fibrotic disease. Our results suggest
a new regulatory mechanism, through which proteolytic shedding of
the ectodomain of ephrin-B2 amplifies myofibroblast activation and
profibrotic functions, occurs during tissue fibrogenesis. Our studies
demonstrate that the generation of sEphrin-B2 by fibroblasts induced
ephrin-B2 forward signaling through EphB3/4, which is involved in
multiple fibroblast functions, including migration, invasion, myofi-
broblast differentiation and collagen production.
Our study also identified ADAM10 as the major metallopro-
tease responsible for the generation of sEphrin-B2 by human lung
fibroblasts. Ephrin-B2 cleavage by ADAM10 has recently been dem-
onstrated during Xenopus development20, suggesting that there is
an evolutionarily conserved mechanism for ephrin-B2 cleavage by
ADAM10 in both development and tissue fibrogenesis. The aberrant
reactivation of multiple developmental pathways has been impli-
cated in the pathogenesis and progression of lung fibrosis35,36, and
our results indicate that ADAM10–ephrin-B2 may be another such
pathway. Consistently, our results suggest that the ADAM10-mediated
ephrin-B2 shedding pathway is reactivated by tissue injury, and its
sustained activation promotes organ fibrosis by sustaining and ampli-
fying myofibroblast activation.
We also identified EphB3/4 as the receptors through which the
ADAM10–sEphrin-B2 pathway directs myofibroblast activation.
Importantly, our results demonstrate that sEphrin-B2 produced in the
alveolar space upon lung injury is the form of active high-molecular
-weight oligomers. Activation of forward EphB receptor signaling
requires Eph receptor clustering induced by binding of oligomeric
ephrins. Our results demonstrate that this oligomeric state of sEphrin-
B2 in fibrotic tissues is biologically active and regulates myofibroblast
functions. Consequently, strategies to interrupt the elaboration of
sEphrin-B2 by fibroblasts by targeting ADAM10 have the potential
1414
to serve as new therapeutic strategies for fibrotic diseases. As we
found, therapeutic targeting of ADAM10 with GI254023X was able
to substantially reduce lung fibrosis in mice by inhibiting ephrin-B2
shedding and myofibroblast activation in vivo, providing preclinical
evidence that inhibition of ADAM10 could mitigate fibrosis in vivo.
During our investigations into the molecular mechanism(s)
through which ADAM10-mediated ephrin-B2 shedding is acti-
vated in fibroblasts, we determined that this pathway was activated
by TGF-β, the prototypical cytokine that drives myofibroblast
activation. We found that fibroblast ADAM10 expression, but not
expression of ephrin-B2 itself, is induced by TGF-β signaling. Further,
we found that genetic or pharmacological inhibition of ADAM10-
mediated ephrin-B2 shedding blocks myofibroblast activation by
TGF-β, indicating that ephrin-B2 cleavage is required for the genera-
tion of myofibroblasts, which are central effector cells in the develop-
ment of fibrosis. Currently, it is not fully understood how ADAM10
mediates ephrin-B2 cleavage in fibroblasts. Previous studies have
shown that ADAM10 cleaves the juxtamembrane regions of ephrin-
A2 (ref. 22) and ephrin-B2 (ref. 20) to release active ectodomains.
Consistent with these data, our genetic studies overexpressing cleav-
age-resistant ephrin-B2 mutants confirmed the requirement for the
juxtamembrane region of ephrin-B2 in ADAM10-mediated ephrin-
B2 shedding and myofibroblast activation by TGF-β. The molecular
requirement for ADAM10-mediated ephrin-B2 shedding in myofi-
broblast activation by TGF-β warrants further investigation. Previous
studies have shown that ephrin-B2 and EphB4 receptor interact on
the surface of the same cell (i.e., ‘in cis’), and this interaction attenu-
ates the ability of ephrin-B2 expressed in other cells to activate for-
ward ephrin-B2–EphB4 receptor signaling ‘in trans’37. Our studies on
human lung fibroblasts may suggest a molecular mechanism through
which high expression of membrane-bound ephrin-B2 in quiescent
fibroblasts prevents activation of forward EphB3/4 receptor signal-
ing via cis negative interaction. Upon tissue injury, TGF-β-mediated
ADAM10 upregulation would lead to increased ephrin-B2 shed-
ding in activated myofibroblasts. Consequently, ADAM10-mediated
ephrin-B2 shedding could promote (a) de-repression of the negative
cis inhibitory ephrin-B2–EphB3/4 receptor interaction and/or (b)
activation of profibrotic forward EphB3/4 receptor signaling activated
by autocrine sEphrin-B2, although ephrin-B2 expressed in other cells
implicated in pathological fibrosis could be involved in activation
of EphB3/4 receptors in trans. Further research focused on gather-
ing mechanistic understanding of ephrin-B2 signaling in activated
myofibroblasts is needed.
Although our study does not address whether ephrin-B2 reverse
signaling may also regulate myofibroblast activation or function,
ephrin-B2 ectodomain shedding would concomitantly terminate
ephrin-B2 reverse signaling through the cytoplasmic tail of ephrin-
B2 in fibroblasts previously bearing full-length ephrin-B2. Reverse
ephrin-B2 ligand signaling has recently been shown to play an anti-
fibrotic role in mouse models of kidney fibrosis protecting against
peritubular capillary rarefaction through promotion of angiogenesis
and vascular stability during kidney injury and, most relevant to our
study, through inhibition of pericyte-to-myofibroblast transition and
myofibroblast activation14. Thus ephrin-B2 ectodomain shedding
may not only promote myofibroblast activation through promotion
of profibrotic ephrin-B2 forward signaling but also by terminating
antifibrotic ephrin-B2 reverse signaling.
Our studies also show that ADAM10–sEphrin-B2 signaling is
upregulated in fibroblasts from individuals with IPF and an elevated
concentration of sEphrin-B2 in plasma and in BAL from individuals
VOLUME 23 | NUMBER 12 | DECEMBER 2017 nature medicine

with IPF. These data further emphasize on the relevance of ADAM10–
sEphrin-B2 signaling in human disease.
In summary, we have identified a new pathway that promotes
myofibroblast activation and effector functions through cleavage of
ephrin-B2 by ADAM10 to generate sEphrin-B2 that signals through
EphB3/4. This pathway is required for TGF-β-induced myofibroblast
activation, suggesting it plays a central role in the pathogenesis of
fibrotic diseases. Our study also identifies sEphrin-B2, its receptors
EphB3/4 and its sheddase ADAM10 as potential therapeutic targets
for this class of diseases (Fig. 6l).
Methods
Methods, including statements of data availability and any associated
accession codes and references, are available in the online version of
the paper.
Note: Any Supplementary Information and Source Data files are available in the
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
online version of the paper.
Acknowledgments
Authors would like to thank P. Datta, S. Nakamura, H. Endisha and J. Rockel (all
from the University Health Network) for their technical assistance with mouse
breeding and genotyping. The authors gratefully acknowledge funding support
by University of Montreal Hospital Research Centre and University of Montreal
(M.K.); Campaign to Cure Arthritis via the Toronto General and Western
Foundation, University Health Network, Toronto (M.K.); an American Thoracic
Society Foundation and Pulmonary Fibrosis Foundation Research Grant and the
Marie A. Coyle Research Grant from the Scleroderma Foundation (D.L.), and by
the National Institutes of Health, HL108975 and a grant from the Scleroderma
Research Foundation (A.M.T).
AUTHOR CONTRIBUTIONS
D.L. designed most of the experiments, performed in vitro and in vivo mouse
experiments, analyzed the data and generated the figures. P.G.-K. and M.B. were
involved in the generation of Ephrinb2-CKO mice. A.S., P.G., N.A. and D.M.S.
performed and analyzed in vitro experiments related to the ADAM10–ephrin-B2
–EphB3/4 pathway in fibroblasts. C.K.P. and A.F. performed in vivo studies with
ADAM10 inhibitor. C.T. was involved in histological characterization of mouse
experiments in skin fibrosis model. M.S., A.P., S.B.M., R.K., K.E.B. and B.S.S.
provided human lung fibroblasts, plasma and bronchoalveolar lavage fluid from
individuals with IPF and healthy controls. M.B. and R.G. provided intellectual
input on project design and troubleshooting. B.W. performed protein expression
studies in mouse samples. J.W., H.F., J.-P.P. and J.M.-P. were involved in the
characterization of mouse phenotype and troubleshooting with experiments
related to ephrin biology. M.K. designed the original concept and led the entire
team during the course of this study. D.L., A.M.T. and M.K. designed the study
experiments, supervised the project and took overall responsibility for writing the
manuscript with the help of all the authors.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html. Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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1415
 ONLINE METHODS
Plasmids, antibodies and reagents. Ephrin-B2 plasmids, including HA-tagged
full-length WT EphrinB2 and the HA-tagged mutants EphrinB2∆Ecto-HA ,
EphrinB2∆Juxta-HA, EphrinB2∆182–194-HA and EphrinB2∆197–218-HA were kindly
shared by I.O. Daar (National Cancer Institute, National Institutes of Health,
USA). Mouse and human recombinant PDGF-BB, ephrin-A1, ephrin-A2,
ephrin-A3, ephrin-A4, ephrin-A5, ephrin-B1, ephrin-B2 and ephrin-B3
were purchased from R&D Systems, and 18:1 LPA was purchased from
Avanti Polar Lipids. Antibodies used for western blotting include: mouse
polyclonal anti-α-SMA (clone 1A4; Sigma-Aldrich), rabbit polyclonal col-
lagen type I (ab34710, Abcam), rabbit monoclonal GAPDH (clone D16H11,
Cell Signaling), mouse monoclonal β-actin (AC-15, Sigma), ephrin-B2 (P-20
Santa Cruz and HPA008999 Sigma), ADAM10 (#14194, Cell Signaling) and
rabbit monoclonal HA-tag (C29F4, Cell Signaling). Pharmacological inhibi-
tors, including BB-94 (Batimastat), GI254023X and TAPI-0, as well as phor-
bol 12-myristate 13-acetate (PMA), 4-hydroxytamoxifen and fibronectin were
purchased from Sigma-Aldrich. Recombinant human and mouse TGF-β-1
protein was purchased from R&D.
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
Cell lines. Human lung fibroblasts (IMR-90) were purchased from ATCC and
cultured according to the vendor’s protocol. Cell cultures were tested for myco-
plasma infection.
Animals. Pathogen-free female C57BL/6N (6- to 8-week-old) mice pur-
chased from the National Cancer Institute (NCI) Frederick Mouse Repository
(Frederick, MD, USA) were used throughout this study. All experiments were
performed in accordance with National Institute of Health guidelines and pro-
tocols were approved by the Massachusetts General Hospital Subcommittee on
Research Animal Care, and all mice were maintained in a specific pathogen–free
(SPF) environment certified by the American Association for Accreditation of
Laboratory Animal Care (AAALAC).
Generation of ephrin-B2 conditional-knockout mice. To generate mice in which
ephrin-B2 could be conditionally deleted specifically in collagen-expressing
cells such as fibroblasts (Ephrinb2-CKO mice), mice carrying a tamoxifen-
inducible Cre-recombinase (Col1a2-CreERT) (Jackson Laboratory) were
crossed with Efnb2loxP/loxP mice17. Mice homozygous for the floxed Efnb2 allele
and hemizygous for the Col1a2-CreERT allele (Efnb2loxP/loxP; Col1a2-CreERT)
were generated. Genotyping was performed using specific primers for cre and
Ephrinb2 (Supplementary Table 2). Three-week-old Efnb2loxP/loxP; Col1a2-
CreERT mice were treated with intraperitoneal injections of the tamoxifen sus-
pension (0.1 ml of 10 mg/ml) to delete Ephrinb2 or with corn oil (as control) for
7 d. Successful loss of ephrin-B2 expression upon tamoxifen treatment in lung
fibroblasts was confirmed by western blotting using anti-ephrin-B2 antibody.
All animal procedures and protocols were approved by the Comité Institutionnel
de protection des animaux (Institutional Animal Protection Committee) of the
University of Montreal Hospital Research Centre (CRCHUM), animal care
committee of the Krembil Research Institute (University Health Network) and
Massachusetts General Hospital Subcommittee on Research Animal Care.
Mouse model of pulmonary fibrosis. Six-week-old mice were anesthetized
with ketamine and xylazine before exposure of the trachea. Lung fibrosis was
induced by intratracheal administration of bleomycin (50 µl at 1.2 U per kg body
weight) or PBS as control as previously described38,39. Mice were euthanized at
the indicated time points. Lungs and BAL were harvested for histological studies,
collagen determination and biochemical analyses.
For in vivo drug studies with the ADAM10 inhibitor, GI254023X was diluted
in 0.1 M carbonate buffer and mice were administered 200 mg per kg body
weight once daily by intraperitoneal injection with a total volume of 100 µl.
Drug dosing began 3 d before bleomycin or PBS treatment and was maintained
daily throughout the course of the study.
Mouse model of skin fibrosis. Skin fibrosis in 6- to 8-week-old mice was
induced by daily subcutaneous injection of bleomycin (100 µl from 10 µg/ml
stock) for 28 d as previously described27. Sterile saline was used as control. Mice
were then euthanized, and full-thickness 6-mm punch biopsies were obtained to
conduct histological, immunohistochemical and hydroxyproline analysis.
nature medicine
Generation of ephrin-B2 immune complexes. To generate ephrin-B2 immune
complexes, recombinant mouse or human ephrin-B2-Fc (R&D systems) was
incubated at a 2:1 ratio (wt/wt) with a goat antibody against human IgG (Jackson
ImmunoResearch) for 90 min at 4 °C before immediate use.
In vivo ephrin-B2-Fc injection. Subcutaneous injections using mouse recom-
binant ephrin-B2-Fc were performed using the methodology previously reported
for bleomycin-induced model of skin fibrosis40. 6- to 8-week-old mice received
100 µl subcutaneous injections of preclustered ephrin-B2-Fc (100 µg per kg
body weight per mouse) into a single location on the shaved back of mice once
daily for 2 weeks. Control mice received subcutaneous injections of IgG-Fc for
2 weeks. Following two-week treatment with either IgG-Fc or ephrin-B2-Fc,
mice were further housed for 2 weeks and euthanized by CO2 euthanasia, and
skin samples were collected for histological studies, collagen determination
and biochemical analyses. All animal procedures and protocols were approved
by the Comité Institutionnel de protection des animaux (Institutional Animal
Protection Committee) of the University of Montreal Hospital Research
Centre (CRCHUM), animal care committee of the Krembil Research Institute
(University Health Network) and Massachusetts General Hospital Subcommittee
on Research Animal Care.
Histological analysis. Excised skin and lung biopsies were fixed in 10% buffered
formalin and multiple paraffin-embedded 5-µm sections of the entire mouse
lung and skin were stained with H&E or Masson’s trichrome according to the
standard protocols of our laboratory as previously described27,39.
Dermal thickness measurement. Dermal thickness was determined with the
use of photomicrographs (100× magnification) of H&E-stained sections, meas-
uring the distance between the epidermal–dermal junction and the dermal–fat
junction at five randomly selected sites per high-power field in ten high-power
fields per section as previously shown27. All investigations were performed in
a blinded fashion.
Hydroxyproline assay. Collagen content in mouse lungs and skin was measured
using hydroxyproline assay as previously described27,38,39.
Mouse BAL recovery. To obtain BAL samples for chemoattractant activity analy-
sis, ephrin-B2 ELISA and total protein concentration determination, lungs were
lavaged with six 0.5-ml aliquots of PBS. BAL samples were centrifuged at 3,000g
for 20 min at 4 °C and transferred the supernatants to siliconized low-binding
Eppendorf tubes (PGC Scientifics) for subsequent analysis.
BAL total protein. Total protein concentration in BAL samples was determined
using a commercially available bicinchoninic acid (BCA) Protein Assay Kit
(Pierce) per manufacturer’s protocol.
Determination of ephrin-B2 levels in BAL and plasma. sEphrin-B2 levels in human
and mouse BAL fluids and in human plasma were determined by ELISA (Uscn Life
Science Inc., SEE112Hu, SEE112Mu) according to the manufacturer’s protocol.
Preparation of ephrin-B2 antibody-coated beads. Ephrin-B2 antibody
(1–2 µg) was chemically cross-linked to 2.8 µm superparamagnetic Dynabeads
M-270 Epoxy beads (Life Technologies) according to the manufacturer’s proto-
col. Beads were washed with PBS and then blocked for 1 h in 2% (w/v) BSA and
PBS before resuspension in PBS. Ephrin-B2 was immunoprecipitated by incu-
bation of BAL fluids with ephrin-B2 antibody–coated beads overnight at 4 °C.
Ephrin-B2-depleted BALs were used as chemoattractants in migration and inva-
sion assays.
Isolation of primary mouse lung fibroblasts. Primary lung fibroblasts were
isolated as previously described38.
Fibroblast chemotaxis and invasion assay. Fibroblast chemotaxis was assayed
using a 96-Multiwell FluoroBlok Inserting system (Fisher Scientific; pore size: 8 µm)
precoated with fibronectin at 10 µg/ml (Sigma). Briefly, cells were labeled
with DiIC12(3) fluorescent dye for 1 h before chemotaxis. 50,000 cells in 50 µl
serum-free DMEM were then added to the apical chambers and exposed to BAL
doi:10.1038/nm.4419
 or BAL fractions, ephrin-B2-Fc, 18:1 LPA (Avanti Polar Lipids) or PDGF-BB
(R&D Systems) as chemoattractants. The plate was then incubated for 4 h at
37 °C in a 5% CO2 atmosphere. Fluorescence of migrated cells was recorded
at 544/590 nm (Ex/Em) on a bottom-reading fluorescent plate reader using
Fluoroskan Ascent FL (Thermo). The data were expressed as the ratio of migrated
cells toward any chemoattractant to cells migrated toward serum-free DMEM.
Experiments were performed in triplicate. For invasion assays, a 96-well BioCoat
Tumor Invasion System (Fisher Scientific) was used, and the plate was read at
48 h. Cells at passages 3 to 5 were used for chemotaxis and invasion assays. To
test the effect of receptor inhibition, fibroblasts were transfected with siRNA to
EPHB2, EPHB3 or EPHB4 before addition of BAL as a chemoattractant.
siRNA and plasmid transfection. The siRNA duplexes targeting human ephrin-
B2, human MMP1, human MMP2, human MMP7, human MMP14, human
ADAM9, human ADAM10, human ADAM12, human ADAM17, human
ADAM19, human EPHB3, human EPHB4, mouse ephrin-B2, mouse Ephb2,
mouse Ephb3 and mouse Ephb4 mRNA were On-Target Plus Smart Pools and
were obtained from Dharmacon Inc. (Thermo Scientific). The siRNAs (20 nM)
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
were transiently transfected into human and mouse primary fibroblasts using
HiPerFect Reagent (Qiagen) at a siRNA/HiPerFect ratio of 1:4 (µg/µl). siRNAs
from the On-Target Plus nontargeting siRNA pool were used as a nonspecific
control. Cells were harvested and mRNA levels were assessed 48 h after trans-
fection. Transient transfection experiments with ephrin-B2 mutants were per-
formed on primary human and mouse lung fibroblasts seeded on 6-well plates
(60–70% confluency) using Lipofectamine 2000 (Thermo Fisher Scientific).
Filopodia formation. Filopodial protrusions at the cell front were analyzed from
20 microscopic fields on 25–50 cells per conditions.
RT-PCR and qRT-PCR. Total RNA was extracted using Qiagen extraction
kits according to the manufacturer’s protocol, and cDNAs were generated by
reverse transcription using iScript cDNA Synthesis Kit (BioRad) as previously
reported39. qPCR was performed using fluorogenic SYBR Green and Mx4000
Multiplex Quantitative PCR System (Stratagene) as previously described39.
GAPDH was used as reference gene in all qRT-PCR reactions. PCR was per-
formed using the primers listed in Supplementary Table 2 at a final concen-
tration of 100 nM. Relative transcript abundance of a gene is expressed in ∆Ct
values (∆Ct = Ctreference – Cttarget). Relative changes in transcript levels compared
with controls are expressed as ∆∆Ct values (∆∆Ct = ∆Cttreated – ∆Ctcontrol) as
previously described39.
Western blot analysis. Cells and tissues were harvested, lysed in RIPA buffer
(Thermo Scientific) and supplemented with Halt Protease and Phosphatase
Inhibitor Cocktail (Thermo Scientific). Protein extracts were subjected to cen-
trifugation (6,000g) at 4 °C, and protein concentrations were determined using
BCA assay (Pierce). Protein was separated on either NativePAGE Bis-Tris Gel
System or 4–12% SDS-Tris-Glycine Protein Gel. Separated proteins were trans-
ferred onto polyvinylidene difluoride (PDVF) membranes (Invitrogen), and
membranes were blocked with 5% nonfat dry milk in TBS and incubated with
the indicated primary antibodies. After washing, membranes were incubated
with appropriate secondary, HRP-linked antibodies (Pierce). Proteins were visu-
alized by enhanced chemiluminescence and autoradiography (ECL; Amersham
Biosciences, GE Healthcare).
Immunofluorescence analysis. Cells were plated on glass coverslips, treated
with GI254023X (20 nM) or TGF-β as described and fixed with 4% PFA for
10–15 min. The cells were then washed three times with PBS, blocked for one
hour in blocking solution (10% goat serum; 0.1% triton-X in PBS) and incubated
overnight at 4 °C with a primary antibody against α-SMA (clone 1A4, Sigma-
Aldrich; 1:100 in blocking solution). The next day, samples were washed three
times with PBS and incubated for one hour with goat anti–mouse Alexa Fluor
488 and rhodamine phalloidin antibodies (Thermo Fisher Scientific) diluted
to 1:400 and 1:200, respectively, in blocking solution. Samples were washed
doi:10.1038/nm.4419
three times with PBS and mounted with VECTASHIELD Antifade Mounting
Medium with DAPI (Vectorlabs). Images were acquired with a Zeiss LSM780
confocal microscope (Zeiss).
Human plasma samples. Subjects with IPF were identified from those receiving
care at the Massachusetts General Hospital. For study inclusion, IPF subjects
had to satisfy IPF diagnostic criteria based on the 2011 recent joint consen-
sus statement of the American Thoracic Society (ATS), European Respiratory
Society (ERS), Japanese Respiratory Society41 and Latin American Thoracic
Association41 as determined by two investigators41 (Supplementary Table 1).
Partners Healthcare System Research Study Volunteer Program (RSVP) was used
to recruit controls. Controls were at least 50 years of age and were nonsmokers
without a history of chronic lung disease. Approval for plasma collection was
obtained through the Partners Institutional Review Board. All subjects provided
informed consent. Blood was obtained via venipuncture into tubes containing
CTAD (citrate-theophylline-adenosine-dipyridamole). Whole blood was centri-
fuged at 1,500g for 15 min to obtain plasma, which was then placed in aliquots
and immediately frozen and stored at −80 °C until analysis.
Human BAL samples. BAL samples from individuals with IPF and healthy
volunteer donors were recovered after instillation of sterile 0.9% saline by flexible
fiber-optic bronchoscopy. Bronchoscopies were performed at both the Instituto
Nacional de Enfermedades Respiratorias (INER), Mexico and the Massachusetts
General Hospital (MGH), as previously described38. These studies were
approved by the INER Ethics Committee and the MGH Institutional Review
Board, and informed consent was obtained from all participants. BAL super-
natants that were collected at MGH were immediately transferred to siliconized
low-binding Eppendorf tubes, and supernatants from both institutions were kept
at −70 °C until use.
Statistical analysis. Sample sizes were determined by power analysis on the
basis of our previous studies. In each experiment evaluating the effects of genetic
depletion of ephrin-B2 in fibroblasts or ADAM10 inhibition on the extent of
dermal and lung fibrosis produced in mice, n ≥ 8 mice per group were used to
account for the inherent variability in the fibrotic response of mice. Sample
sizes were estimated based on an 80% power to detect a 50% reduction in the
amount of fibrosis present in Ephrinb2-CKO mice or mice treated with ADAM10
inhibitor compared with WT control mice, accepting a type I error rate of 0.05.
No animals were excluded from analysis. Animals were distributed into groups
of equal body weight and genotype before treatments. Experimental data were
analyzed by unpaired Student′s t-test for differences between each of the experi-
mental conditions, two-way ANOVA for overall condition effects or log-rank
test for differences in survival times using GraphPad Prism Software 5.0. The
P values obtained are indicated in the figures and figure legends when statisti-
cally significant. *P < 0.05, **P < 0.01, ***P < 0.001 was considered significantly
different between groups. All data displayed a normal distribution. Data are
reported as mean ± s.d.
Data availability. Data are available from the corresponding authors upon rea-
sonable request. A Life Sciences Reporting Summary for is available.
38. Tager, A.M. et al. The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis
to lung injury by mediating fibroblast recruitment and vascular leak. Nat. Med. 14,
45–54 (2008).
39. Lagares, D. et al. Inhibition of focal adhesion kinase prevents experimental lung
fibrosis and myofibroblast formation. Arthritis Rheum. 64, 1653–1664 (2012).
40. Kapoor, M. et al. Loss of peroxisome proliferator-activated receptor gamma in mouse
fibroblasts results in increased susceptibility to bleomycin-induced skin fibrosis.
Arthritis Rheum. 60, 2822–2829 (2009).
41. Raghu, G. et al. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary
fibrosis: evidence-based guidelines for diagnosis and management. Am. J. Respir.
Crit. Care Med. 183, 788–824 (2011).
nature medicine
 cO r r i G e n da

Corrigendum: Persisting positron emission tomography lesion activity and

Mycobacterium tuberculosis mRNA after tuberculosis cure
Stephanus T Malherbe, Shubhada Shenai, Katharina Ronacher, Andre G Loxton, Gregory Dolganov, Magdalena Kriel, Tran Van,
Ray Y Chen, James Warwick, Laura E Via, Taeksun Song, Myungsun Lee, Gary Schoolnik, Gerard Tromp, David Alland,
Clifton E Barry III, Jill Winter, Gerhard Walzl, the Catalysis TB–Biomarker Consortium
Nat. Med. 22, 1094–1100 (2016); published online 5 September 2016; corrected after print 19 October 2016; corrected after print 10
November 2017

In the version of this article initially published, two authors in the Catalysis TB–Biomarker Consortium were incorrectly identified as Lani Theart
and Coenie Kogelenberg. The authors’ names are Lani Thiart and Coenie Koegelenberg. Also, in Figure 3c, the three patients who initiated TB
retreatment after EOT + 1y had ‘mixed’ response patterns at EOT + 1y, as is written in the text, and not ‘resolved’ response patterns, as is shown
in the original version of the figure. The errors have been corrected in the HTML and PDF versions of the article.

Corrigendum: ADAM10-mediated ephrin-B2 shedding promotes

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

myofibroblast activation and organ fibrosis

David Lagares, Parisa Ghassemi-Kakroodi, Caroline Tremblay, Alba Santos, Clemens K Probst, Alicia Franklin, Daniela M Santos,
Paula Grasberger, Neil Ahluwalia, Sydney B Montesi, Barry S Shea, Katharine E Black, Rachel Knipe, Meryem Blati, Murray Baron,
Brian Wu, Hassan Fahmi, Rajiv Gandhi, Annie Pardo, Moisés Selman, Jiangping Wu, Jean-Pierre Pelletier, Johanne Martel-Pelletier,
Andrew M Tager & Mohit Kapoor
Nat. Med.; doi:10.1038/nm.4419; corrected online 20 November 2017

In the version of this article initially published online, the positions of the colored boxes in the key of Figure 5f were inverted. The treatment
group is represented by the red line of the graph and the control group by the blue line. The error has been corrected in the print, PDF and HTML
versions of this article.

nature medicine VOLUME 23 | NUMBER 12 | DECEMBER 2017 1499

 nature research | life sciences reporting summary
Corresponding author(s): Dr. David Lagares and Dr. Mohit Kapoor
Initial submission Revised version Final submission

Life Sciences Reporting Summary

Nature Research wishes to improve the reproducibility of the work that we publish. This form is intended for publication with all accepted life
science papers and provides structure for consistency and transparency in reporting. Every life science submission will use this form; some list
items might not apply to an individual manuscript, but all fields must be completed for clarity.
For further information on the points included in this form, see Reporting Life Sciences Research. For further information on Nature Research
policies, including our data availability policy, see Authors & Referees and the Editorial Policy Checklist.

Experimental design
1. Sample size
Describe how sample size was determined. In each experiment evaluating the effects of genetic depletion of ephrin-B2 in
fibroblasts on the extent of dermal and lung fibrosis produced in mice, we use > or
= 8 mice per group to achieve statistical significance and account for the inherent
variability in the fibrotic response of mice. Assuming a 50% reduction in the
amount of fibrosis present in ephrin-b2 CKO mice compared with wild-type control
mice, then at least 8 mice per group were needed to achieve a power of 80%,
accepting a Type I error rate of 0.05. Histological analyses were done using n=8
mice per group. Collagen determinations by hydroxyproline levels were performed
using n=6 mice per group. Western blot analyses from our in vivo mouse model of
lung fibrosis includes 6 samples per condition. Representative blot shows at least
n=4 samples/group. Protein densitometry is also included from n=6 samples per
group. Similarly, studies involving injection of recombinant ephrin-B2-Fc in mouse
skin include n=6 mice/group. Western blots from 4 individual mice per group are
presented.
In each experiment evaluating the effects of a ADAM10 selective inhibitor
GI254023X on the extent of lung fibrosis produced in mice, we use > or = 10 mice
per group to achieve statistical significance and account for the inherent variability
in the fibrotic response of mice. Assuming a 50% reduction in the amount of
fibrosis present in mice treated with active drug compared with vehicle control,
then 10 mice per group were needed to achieve a power of 80%, accepting a Type
I error rate of 0.05. Collagen determinations by hydroxyproline levels were
performed using n=8 mice per group. Western blot analyses from our in vivo
mouse model of lung fibrosis includes 6 samples per condition. Representative blot
shows at least n=3 samples/group. Protein densitometry is also included from n=6
samples per group. Determination of soluble ephrin-B2 levels in BAL fluid was
performed using n = 8 mice for all groups.
2. Data exclusions
Describe any data exclusions. No animals were excluded from the analysis.
3. Replication
Describe whether the experimental findings were All attempts of replication were successful.
reliably reproduced.
4. Randomization
Describe how samples/organisms/participants were For experiments with control and conditional KO mice, animals were first
allocated into experimental groups. genotyped and then randomly assigned to receive either bleomycin or PBS. For
experiments with IgG and Ephrin B2Fc, wild type C57BL/6N mice were randomly
divided in cages of 5 mice/cage who received either IgG or Ephrin B2 Fc.
June 2017

For experiments with ADAM10 inhibitor, all mice used were wild type (C57Bl/6N)
animals, and were purchased commercially and randomized to experimental
groups by the cages our animal facility put them in upon their arrival from the
vendor.
5. Blinding
Describe whether the investigators were blinded to Blinded analysis was performed for histopathological analysis of skin and lung

1
Nature Medicine: doi:10.1038/nm.4419
 group allocation during data collection and/or analysis. fibrosis, filopodia quantitation and immunofluorescence studies by two blinded
observers.

nature research | life sciences reporting summary

Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used.

6. Statistical parameters
For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or in the
Methods section if additional space is needed).

n/a Confirmed

The exact sample size ( ) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.)
A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same
sample was measured repeatedly
A statement indicating how many times each experiment was replicated
The statistical test(s) used and whether they are one- or two-sided (note: only common tests should be described solely by name; more
complex techniques should be described in the Methods section)
A description of any assumptions or corrections, such as an adjustment for multiple comparisons
The test results (e.g. values) given as exact values whenever possible and with confidence intervals noted
A clear description of statistics including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range)
Clearly defined error bars

Software
Policy information about availability of computer code
7. Software
Describe the software used to analyze the data in this GraphPad Prism Software 5.0 was used for unpaired Student's t, Two way ANOVA
study. and log-rank test to determine significance.
For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be made
available to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). guidance for
providing algorithms and software for publication provides further information on this topic.

Materials and reagents

Policy information about availability of materials
8. Materials availability
Indicate whether there are restrictions on availability of No unique materials were used.
unique materials or if these materials are only available
for distribution by a for-profit company.
9. Antibodies
Describe the antibodies used and how they were validated Antibodies used for Western blotting include: mouse polyclonal anti– -SMA (clone
for use in the system under study (i.e. assay and species). 1A4; Sigma-Aldrich), rabbit polycloncal collagen type I (ab34710, Abcam), mouse
monoclonal anti-collagen type I (abcam), rabbit monoclonal GAPDH (clone
D16H11, Cell Signaling), mouse monoclonal -actin (AC-15, Sigma), ephrin-B2 (P-20
Santa Cruz and HPA008999 Sigma), ADAM10 (#14194,Cell Signaling), rabbit
monoclonal HA-Tag (C29F4, Cell Signaling), goat-anti-rabbit (31462, ThermoFisher
Scientific) and goat-anti-mouse (31432, ThermoFisher Scientific). The same anti– -
SMA antibody was used for immunofluorescence. The same ephrin-B2 antibodies
and goat antibody against IgG (Jackson ImmunoResearch) were used for
immunoprecipitation.
June 2017

2
Nature Medicine: doi:10.1038/nm.4419
 10. Eukaryotic cell lines
a. State the source of each eukaryotic cell line used.
b. Describe the method of cell line authentication used.
c. Report whether the cell lines were tested for
mycoplasma contamination.
d. If any of the cell lines used are listed in the database
of commonly misidentified cell lines maintained by
ICLAC, provide a scientific rationale for their use.
Animals and human research participants
Policy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines
11. Description of research animals
Provide details on animals and/or animal-derived
materials used in the study.
Policy information about studies involving human research participants
12. Description of human research participants
Describe the covariate-relevant population
characteristics of the human research participants.
Nature Medicine: doi:10.1038/nm.4419
nature research | life sciences reporting summary
Human lung fibroblasts (IMR-90) were purchased from ATCC and cultured
according to vendor’s protocol.
Cell lines were authenticated by the ATCC.
Cell lines were not tested for mycoplasma contamination.
No commonly misidentified cell lines were used.
Adult sex- and age-matched C57BL/6 mice at 6-8 weeks of age were purchased
from the National Cancer Institute (NCI)-Frederick Mouse Repository (Frederick,
MD, USA) and used throughout this study. C57BL/6J mice carrying a tamoxifen-
inducible Cre-recombinase [CreER(T)] under the control of a fibroblast-specific
promoter from the pro 2(I) collagen gene [C57BL/6J-Tg(COL1a2-CreER(T); Jackson
laboratory] were crossed with ephrin B2F/F mice (Obtained from Dr Jianping Wu),
co-author on this study.
Human BAL samples. We recovered BAL samples from individuals with IPF and
normal controls after instillation of sterile 0.9% saline by flexible fiber-optic
bronchoscopy. We performed these bronchoscopies both at the Instituto Nacional
de Enfermedades Respiratorias (INER), Mexico and the Massachusetts General
Hospital (MGH). These studies were approved by the INER Ethics Committee and
the MGH Institutional Review Board, and informed consent was obtained from all
participants. We immediately transferred BAL
Human plasma samples. Subjects with IPF were identified from those receiving
care at the Massachusetts General Hospital. For study inclusion, IPF subjects had to
satisfy IPF diagnostic criteria based on the 2011 recent joint consensus statement
of the American Thoracic Society (ATS), European Respiratory Society (ERS),
Japanese Respiratory Society (JRS), and Latin American Thoracic Association (ALAT)
as determined by two investigators. Partners Healthcare System Research Study
Volunteer Program (RSVP) was used to recruit controls. Controls were at least 50
years of age, non-smokers, and without a history of chronic lung disease. Approval
for plasma collection was obtained through the Partners Institutional Review
Board. All subjects provided informed consent.
June 2017
3