© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. or or D.L. ( 9 Network, Toronto, Ontario, Canada. de Enfermedades Ismael Respiratorias Cosio Villegas, Mexico City, Mexico. University Health Network, Toronto, Ontario, Canada. (CRCHUM), Montreal, Québec, Canada. General Massachusetts Hospital, Harvard Medical School, Boston, USA. Massachusetts, 1 therapies. antifibrotic new for targets provide also will enhance further our understanding of the pathogenesis of fibrosis but not will and only ing of the migration activation regulation fibroblast progression IPF to contributes genes of set this that hypothesize to us leading course, clinical advancing rapidly more a and myofibroblast activation in the lungs of migration individuals with IPF with cell with associated genes of expression increased found phenotype myofibroblast effector an to tion activa excessive to their and/or injury of tissue to sites of fibroblasts recruitment excess the to attributable is proteins matrix of mulation scleroderma) (SSc, sclerosis systemic and (IPF) fibrosis pulmonary idiopathic ety including of human diseases, of distortion tissue architecture and ultimately organ failure in a vari accumulation of collagens and other matrix excess proteins by that results in characterized the is Fibrosis organs. most in fibrosis tissue of development the to lead can processes repair wound Dysregulated treatment of fibrotic diseases. tissue fibrogenesis and identify sEphrin-B2, its receptors EphB3 and EphB4 and ADAM10 as potential therapeutic targets in the fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of lavage and prevents lung fibrosis in mice. Consistent with the mouse data, signaling is ADAM10–sEphrin-B2 upregulated in induced inhibition activation. myofibroblast Pharmacological of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar is increased by transforming growth factor (TGF)- that a disintegrin and 10 metalloproteinase (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 expression EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and alveolar airspace after lung injury. Shedding of sEphrin-B2 promotes fibroblast chemotaxis and activation via EphB3 and/or functional and evidence translational that the ectodomain of ephrin-B2 membrane-bound is shed from fibroblasts into the Here we identify soluble ephrin-B2 (sEphrin-B2) as a new profibrotic mediator in lung and skin fibrosis. We provide molecular, architecture and organ function; however, the molecular mediators of activation myofibroblast have yet to be fully identified. matrix extracellular (ECM) deposition and tissue remodeling by activated leads myofibroblasts, to loss of proper tissue Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive Andrew M Tager Annie Pardo Katharine E Black Franklin Alicia David Lagares myofibroblast activation and organ fibrosis ADAM10-mediated ephrin-B2 shedding promotes nature medicine nature Received 18 November 2016; accepted 11 September 2017; published online 23 October 2017; These These authors contributed equally to this work. Division of Pulmonary and Critical Care Medicine, Fibrosis Research Center and Center for Immunology and Inflammatory Diseases, Department of Medicine, [email protected] 5 , , Moisés Selman 1

1 , 9 , , Daniela M Santos , advance online publication online advance 1 10 , 1 10 , , Parisa Ghassemi-Kakroodi , Rachel , Knipe Rachel & Mohit Kapoor ). 8 Departments Departments of Surgery and of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. 3 Division Division of Rheumatology, Jewish General Hospital, McGill University, Montreal, Québec, Canada. 6 , , Jiangping Wu 10 1 1 , , Meryem Blati These These authors jointly directed this work. should Correspondence be addressed to M.K. ( . This pathological accu pathological This . 1 , , Paula Grasberger 1– 5 Facultad Facultad de Ciencias, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico. 2 5 , . We have previously previously We have . 4 , 7 , 8 6 ,

10 b . If so, elucidat so, If . 1, 1, and sEphrin-B2 ADAM10-mediated generation is required for TGF-

2 , , Jean-Pierre Pelletier

2 , 9 2 , , Caroline Tremblay , , Murray Baron 7 Division Division of Genetics and Development, Krembil Research Institute, University Health 1 - - - - , , Neil Ahluwalia signaling) cells (reverse signaling) ligand-expressing and signaling) (forward tor-expressing the events recep between signaling initiates bidirectional interaction ligands that bind to Eph receptors at the of surface adjacent cells. This linked (ephrin-A1–6) and transmembrane (ephrin-B1– 3) cell surface exhibiting as promigratory activities described been previously has and ligands ephrin of family the to belongs which ephrin-B2, protein transmembrane the number sion found we set, data this in with IPF and from lated with SSc–ILD in individuals both fibroblasts controls as used of fibroblasts lung that healthy to (SSc–ILD) disease lung interstitial SSc-associated or IPF with the gene of from expression individuals isolated lung fibroblasts tions, we a analyzed data publicly microarray available set comparing To identify putative genes that regulate profibrotic fibroblast To func fibroblast profibrotic that putative genes regulate identify 2 Department Department of Medicine, University of Montreal Hospital Research Centre 3 , , Brian Wu d 2 o , , Johanne Martel-Pelletier i G : 2 1 , , Alba Santos 1 S 0 0 1 E . . Ephrin–Eph signaling controls tissue patterning patterning tissue controls signaling Ephrin–Eph . 1 , , Sydney B Montesi 1 0 7 3 2 8 4 / n 8, ; ; m 4 9 EFNB2 , , Hassan Fahmi . . Ephrins are glycosyl-phosphatidylinositol- . EFNB2 4 4 1 9 1 probe: 34334_at) probe: , , Clemens K Probst (Gene Expression Omnibus acces Omnibus Expression (Gene 7 . Among the genes upregu genes the Among . 1 , Barry S , Shea Barry 2 [email protected] , , Rajiv Gandhi 2 4 , The The Arthritis Program,

6 Instituto Instituto Nacional 7 s e l c i t r a . . EFNB2 1 ,

b

encodes encodes 1 1-

4

, , )  - - - - © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. ih nrtaha bemcn r B ( PBS or bleomycin intratracheal with from Ephrinb2 cells epithelial alveolar in preservation potential its compare from cells these in blotting mice, but we did not ephrin-B2 observe protein expression by western from cells epithelial alveolar isolated from those to compared mice in macrophages alveolar from extracts in protein B2 from those of fibroblasts lung from extracts in expression lower markedly demonstrated protein ephrin-B2 for as to referred herein are and controls as used were alone vehicle oil corn with treated Littermates as to referred herein mice, knockout ditional tion of the Col1a2 the for hemizygous and allele ‘floxed’ the for homozygous were that offspring of treatment ( 2) alpha I, type lagen, of promoter mouse the by driven recombinase Cre ible ( sites Wesuch as cells, fibroblasts. mice with crossed delete conditionally could we which development cardiovascular defective to owing at die midgestation ephrin-B2-deficient that are mice globally fibrosis of development the for required is ephrin-B2 last and protein analyses ( mRNAby demonstrated as subjects, control from tissue lung in that with compared IPF with individuals from isolated fibroblasts lung in higher is markedly of ligands, family of ephrin the members other not but ephrin-B2, of expression that confirmed studies initial Our Bleomycin-induced lung fibrosis requires ephrin-B2 in fibroblasts RESULTS fibrosis skin and activation fibroblast in role important an plays mechanism this that evidence translational and functional genetic, molecular, We provide activation. and ment recruit fibroblast promotes sEphrin-B2, as to referred herein main, ectodo ephrin-B2 the of shedding proteolytic ADAM10-mediated been investigated. Here we a describe new mechanism through which not has fibrosis tissue of development the to contribute may sig naling ephrin-B2 which through mechanisms molecular of and cellular spectrum full the disease, human and models mouse in fibrosis of development the to contributions vascular in implicated been has involved skin of individuals with SSc in muscle and vessels in smooth small in clinically increased vascular injury kidney after sis fibro and rarefaction capillary increased in resulting pericytes, by stabilization vascular is compromised have ephrin-B2 by attenuated, consequently signaling reverse which in ephrin-B2, of domain mice in permeability vascular increased and architecture microvessel disrupted in results of adhesion wall vessel and blood the of motility assembly during cell controls also receptors, EphB through signals which ligand, ephrin-B2 the activities, promigratory its with development embryonic during motility cell and A  observed fibrosis parenchymal lung the that revealed analysis cal

Ephrinb2 s e l c i t r Efnb2 Efnb2 -Cre in smooth muscle cells and pericytes during development development during pericytes and cells muscle smooth in -CKO mice. -CKO Efnb2 ERT loxP/loxP -CKO and and -CKO Ephrinb2 mice), as confirmed by as PCR ( mice), confirmed gene in fibroblasts and the generation of and gene the generation in fibroblasts in vivo in mice) -C mice. We observed preservation of Ephrin- of preservation Weobserved mice. -C 9 Fig. 1a . Mice lacking the intracellular PDZ signaling signaling PDZ intracellular the lacking Mice . 1 4 . in vitro in . In human disease, ephrin-B2 expression is is expression ephrin-B2 disease, human In . Col1a2 Ephrinb2 1 7 to mice that express a tamoxifen-induc a express that mice to Ephrinb2 , b ). ). We then investigated whether fibrob Col1a2 and in the development of the lung lung the of development the in and Ephrinb2 Ephrinb2 -Cre Ephrinb2 -C mice were then challenged challenged then were mice -C 1 ERT Ephrinb2 -C mice, and thus we could not not could we thus and mice, -C 5 . . Although ephrin-B2 signaling -Cre transgene ( transgene -Cre Efnb2 mice) ( mice) i. 1 Fig. -C mice ( mice -C -C mice. Western blotting blotting Western mice. -C 9, 13, 12 -CKO mice compared to to compared mice -CKO 1 , 6 in collagen-expressing collagen-expressing in Fig. Fig. 1 1 , we generated mice in in mice generated we , 3 -C -C and f . Genetic inactivation inactivation Genetic . Efnb2 Ephrinb2 . lne histologi Blinded ). Fig. 1 Fig. d 1 Fig. 1 Fig. 1 ), ), led to the dele . In accordance accordance In . flanked by loxP flanked Ephrinb2 Ephrinb2 c Efnb2 ). Tamoxifen ). Col1a2 -CKO mice. mice. -CKO e Efnb2 in vivo in ). We also also We ). loxP/loxP -CKO -CKO Efnb2 (col con . As . ------;

expressed at low levels in lungs following PBS challenge ( challenge PBS following lungs in levels at low expressed homogenates following bleomycin challenge that was either absent or the in appeared kDa) (~50 band lower-molecular-weight a however, challenge; PBS following mice WT from homogenates to compared cells and cancer cells endothelial for fibroblasts, described viously transmembrane (approximatelyephrin-B2 full-length 60 kDa, as pre the of expression following greater d demonstrate not 14 did at challenge bleomycin mice (WT) wild-type from taken homogenates Lung model. this in expression ephrin-B2 of regulation the tigated expression we inves next lung fibrosis, fibroblast in bleomycin-induced that we identified ephrin-B2 for requirement the Considering bronchoalveolar lavage fluid Identification of soluble ephrin-B2 ectodomain in ( controls to compared mice bleomycin-challenged in I collagen type and tiation) ( actin both muscle of smooth expression lower markedly showed homogenates lung content (collagen) hydroxyproline ( lung lower substantially with in that than lower edly in from PBS-challenged WT mice. In contrast, medium conditioned by conditioned medium In mice. WT contrast, from PBS-challenged isolated fibroblasts lung by conditioned medium in present was sEphrin- B2 of concentration low A mice. WT bleomycin-challenged to from PBS- or by isolated lung shedding fibroblasts ELISA sEphrin-B2 assess used we hypothesis, this test To injury. lung to response in generated sEphrin-B2 the of source a are mice WT in fibroblasts i.e., fibroblasts: that of generation from sEphrin-B2 ephrin-B2-deficient loss to attributable part in least at was reduction this that esized ( treatment bleomycin following d 14 mice C eliminated, in BAL from completely not but lower, was concentration sEphrin-B2 treatment. of BAL in Ephrinb2 sEphrin-B2 of concentrations the compared we model, this in fibrosis lung from mice these of protection the to contribute in reduced was Ephrinb2 sEphrin-B2 of generation whether investigate To and myofibroblast differentiation Oligomeric sEphrin-B2 directs fibroblast migration, invasion ( mice PBS-challenged to compared challenge bleomycin following d 14 and 7 3, at mice cin-challenged in BAL concentration from bleomy sEphrin-B2 greater we observed monoclonal detection anti-ephrin-B2 antibody ectodo (mAb), main-specific an with (ELISA) assay immunosorbent enzyme-linked an ( challenge bleomycin following d 14 mice d following PBS treatment but was markedly greater in BAL from WT at in BAL levels low from basal 14 mice WT was observed expression sEphrin-B2 antibody, anti-ephrin-B2 N-terminus-specific an with blotting by As western demonstrated challenge. bleomycin following evaluated the presence of first sEphrin-B2 in we bronchoalveolar lavagehypothesis, (BAL) this Toinvestigate fibrosis. of pathogenesis the to contributes sEphrin-B2 resulting the that and injury lung brotic profi following cleaved is proteolytically that ephrin-B2 hypothesize in findings our with consistent B2, of ephrin- to ectodomain the corresponding band a generate 50-kDa proteins active to shown been recently has release ephrin-B2 of mouse cleavage Proteolytic to shedding ectodomain undergo to Fig. 1g Fig. Ephrin-B ligands, as well as EphB receptors, have been shown shown been have receptors, EphB as well as ligands, Ephrin-B Ephrinb2 , h -CKO and and -CKO -CKO mice following bleomycin challenge, which could could which challenge, bleomycin following mice -CKO ). Further, evaluation of fibrotic protein markers in total total in markers protein fibrotic of evaluation Further, ). C t 4 floig loyi calne a mark was challenge bleomycin following d 14 at -C advance online publication online advance Ephrinb2 α Ephrinb2 Ephrinb2 -SMA, a marker of myofibroblast differen myofibroblast of marker a -SMA, Fig. 1 Fig. -C in mice 14 d following bleomycin bleomycin following d 14 mice in -C -CKO mice, which was associated associated was which mice, -CKO -CKO mice compared to i ). Figure 2 Figure Fig. 2 Fig. d Fig. 2b Fig. ). a 2 3 Fig. 2 Fig. . These data led us to to us led data These . nature medicine nature , c ). Further, using using Further, ). e Ephrinb2 ). We hypoth We ). Fig. 2 Fig. Ephrinb2 -CKO 19– a ). 1 α 2 8 2 ------) .

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. conditioned by these fibroblasts transfected with nontargeting control medium to compared ephrin-B2 targeting RNA (siRNA) interfering or PBS- from bleomycin-challenged fibroblasts isolated that had been transfected with small fibroblasts lung by conditioned medium in Furthermore, the concentration of sEphrin-B2 was substantially lower ( sEphrin-B2 of concentration higher markedly a had lenge lung fibroblasts isolated from WT mice 7 d following bleomycin chal ** In values. maximum and minimum whiskers, percentiles; 75th t In shown. is replicates technical two of out representative One groups. all of homogenates treatment. PBS ( genotype. per group per ( challenge bleomycin or PBS after ( model fibrosis lung bleomycin-induced the shown. is replicates technical two of out representative One groups. to (normalized expression protein and Cre) (without mice WT for bp) (408 band Cre the and bp) (552 of generation ( shown. is replicates technical two of out representative One analysis. densitometry for control a loading as used was GAPDH ( detected. not ND, GAPDH. of level ( donors control healthy from fibroblasts lung human in ligands ephrin-B 1 Figure nature medicine nature ( siRNA -test. In In -test. P < 0.01, *** < 0.01, b a g 40 40 80 kD Bleomycin Saline mRNA expression a 10

0 2 4 6 8 Fig. 2 Fig. h Bleomycin-induced lung fibrosis is dependent on ephrin-B2 in fibroblasts. ( fibroblasts. in ephrin-B2 on dependent is fibrosis lung Bleomycin-induced EFNA1 2 1 , data are presented as mean mean as presented are , data Ephrin-B2 Healthy ND

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EFNB3 * g -actin) in lung fibroblasts and alveolar macrophages from from macrophages alveolar and fibroblasts lung in -actin) ). Scale bar, 100 Scale ). d b H ND ) Representative ( ) Representative ) Ephrin-B2 protein levels in human fibroblasts from healthy controls ( controls healthy from fibroblasts human in levels protein ) Ephrin-B2 i ) Representative western blot of of blot western ) Representative 2 ± -CKO s.d. and were analyzed with two-way ANOVA. Center lines, median values; +, the mean; box edges, 25th and and 25th edges, box mean; the +, values; median lines, Center ANOVA. two-way with analyzed were and s.d. -CKO mice at 14 d after bleomycin or PBS challenge. The color legend in in legend color The challenge. PBS or bleomycin d after 14 at mice -CKO f ) and Masson ) and c Efnb2 Ephrinb2 ( Ephrinb2 injectio Corn oil mous loxP/loxP µ h Efnb2 m. i.t., intratracheal; i.p., intraperitoneal. Representative images are presented from from presented are images Representative intraperitoneal. i.p., intratracheal; i.t., m. n

Hydroxyproline ( g/left lung) control = 30 mice per genotype) genotyping showing the WT band (450 bp), the ephrin-B2 floxed band band floxed ephrin-B2 the bp), (450 band WT the showing genotyping genotype) per mice = 30 µ e

10 15 20 25 n -C) ′ 0 5 s trichrome staining of lung sections from from sections lung of staining s trichrome loxP/loxP i PBS , data are presented as mean mean as presented are , data Supplementary Figure 3 Figure Supplementary ; Fig. 2 Fig. ** Col1a2 Efnb2 β * -actin was used as a loading control. ( control. a loading as used was -actin BLM ** α Col1a2 -SMA and type I collagen protein expression (normalized to to (normalized expression protein I collagen type and -SMA -Cre f n ). ). Ephrinb2 - = 4) and individuals with IPF ( IPF with individuals and = 4) loxP/loxP a injectio Tamoxife knockout mouse and and ( ER Ephrinb2 -Cre Ephrinb2 T studies have demonstrated that EphB receptor activation requires requires activation receptor EphB that demonstrated have studies ( domain the protein of Fc ephrin-B2 domains an full-length C-terminal to and fused transmembrane the ephrin-B2 replaces that of which ectodomain ephrin-B2-Fc, the mouse contains recombinant used we these experiments, In fibroblasts. of activities profibrotic directs injury lung ing follow BAL in present and fibroblasts from sEphrin-B2 as shed is Ephrinb2 Ephrinb2 ER n b We next determined whether the ephrin-B2 ectodomain that that ectodomain ephrin-B2 the whether determined next We ; ; conditional , data are presented as mean mean as presented are , data T n Col1a2 -CKO a -CKO -C -C and and -C ) Relative mRNA expression of genes encoding ephrin-A and and ephrin-A encoding genes of expression mRNA ) Relative ) . -Cre Ephrinb2 ERT ± Ephrinb2 s.d and were analyzed with two-way ANOVA. * ANOVA. two-way with analyzed were and s.d mice. ( mice. f e d i kDa α-SMA 42 40 80 Bleomycin –7 Ephrinb2 densitometry 10.0 -C and and -C ( T -CKO mice at 14 d after bleomycin (BLM) or (BLM) bleomycin d after 14 at mice -CKO ablation) 130 Ephrinb2 5.0 kD amoxifen i.t. e Lung fibroblasts 42 42 0 n ) Representative western blot of ephrin-B2 ephrin-B2 of blot western ) Representative a = 4). mRNA expression was normalized to the the to normalized was expression mRNA = 4). Ephrinb2 f n , g = 3) and individuals with IPF ( IPF with individuals and = 3) Ephrinb2 ) Schematic showing mice subjected to subjected mice showing ) Schematic –1 *** C -C mice and and mice -C ± PBS -C WT s.d. and were analyzed with Student’s Student’s with analyzed were and s.d. Fig. 3 Fig. i.t. injection * Bleomycin CKO

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5.0 -C 0 n β Ephrinb2 = 3 mice for all all for = 3 mice -CKO mice 14 d 14 mice -CKO s e l c i t r a Harvest -actin) in total lung lung total in -actin) i β Type Icollagen α + 14 . . -actin -SMA n *** = 6 mice for for = 6 mice -CKO β Ephrin-B2 Time (d) -actin * * n n = 8 mice = 8 mice P = 3). = 3). < 0.05, < 0.05,

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. induced fibroblast migration in a dose-dependent manner similar similar manner dose-dependent a in migration fibroblast induced ephrin-B2-Fc preclustered that demonstrated assays motil ity. Chemotaxis fibroblast regulate can ephrin-B3, or ephrin-B1 not but B2, ( formation filopodia increased ephrin- preclustered with use. markedly fibroblasts or B2-Fc, but ephrin-B3-Fc, not ephrin-B1-Fc preclustered before lung immediately mouse ratio of 2:1 a Treatment in antibody IgG with ephrin- mouse B2-Fc incubating by experiments these for ephrin-B2-Fc oligomers ephrin-B2 higher-order shown in blot are images and Uncropped minimum values. maximum whiskers, +, values; median the mean; box 25th edges, and 75th percentiles; ANOVA. * and as presented mean PBS or challenge. bleomycin mice from 7 C57BL/6N isolated by lung d primary fibroblasts following generation on siRNA control siRNA a versus sEphrin-B2 nontargeting in challenge. Ephrinb2 all ( groups. 1, 3, 7 and 14 d PBS or following challenge. bleomycin by in ELISA, ephrin-B2 BAL determined fluid mice from at C57BL/6N is ( shown. replicates technical ( protein ( treatment mice at BAL day from fluids 14 PBS- and after bleomycin-challenged ( out is of (~50 kDa). One shown. replicates two representative technical of the the arrow appearance indicates lower-molecular-weight band PBS or following challenge. bleomycin mice from taken C57BL/6N at lung homogenates 14 d ( 2 Figure A  or (LPA) acid lysophosphatidic as such agents, chemotactic to b a kDa e

b a ) Representative western blot showing ephrin-B2 expression levels in levels total expression blot ephrin-B2 western showing ) Representative , kDa 40 80 40 40 80 c

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c -C -C and Ephrin-B2 ectodomain is shed upon ectodomain by lung Ephrin-B2 injury.fibroblasts Supplementary Figure 4 Figure Supplementary ). ). P 2 2 Bronchoalveolar lavage PBS PBS < 0.05, ** PBS b n e Lung homogenates ) and corresponding quantification of cleaved sEphrin-B2 of sEphrin-B2 cleaved ) quantification and corresponding ) Concentration sEphrin-B2 in from BAL sEphrin-B2 recovered fluids ) Concentration = 4 mice out for of all One three groups. representative n ** 3 3 = 5 mice for all ( groups. * Ephrinb2 ** BLM 4 4 * ± 1 s.d and with were Student’sanalyzed 1 P Bleomycin Bleomycin < 0.01, *** 2 Ephrinb Ephrinb 2 -CKO -CKO mice at 14 d PBS or following bleomyc 3 3 n 2 2 4 = 4 for all In groups. -CKO -C d 4 ) Concentration of sEphrin-B2, as of ) sEphrin-B2, Concentration sEphrin-B2 . GAPD Ephrin-B2 ± s.d and with were two-way analyzed P Fig. 3b Fig. < In 0.001. 2 H n f 4 f = 4 mice for all The groups.

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performed by preincubating our N-terminus-specific anti-ephrin-B2 anti-ephrin-B2 by performed our preincubating N-terminus-specific studies competition from Results injury. lung following mice from recovered BAL in oligomers higher-order of form the in present is that indicating sEphrin-B2 with SDS further and 2-mercaptoethanol, pretreated in BAL samples were not recognized species higher-order ( mice control, not but bleomycin-challenged, from BAL in oligomers sEphrin-B2 high-molecular-weight higher-order with consistent recognized species mAb anti-ephrin-B2 specific N-terminus- Our gels. nonreducing on PAGE native by separated been had that proteins BAL immunoblotting by BAL in sEphrin-B2 gomers, as noted above ( signaling receptor EphB2, not but (EphB3/4), by EphB4 and EphB3 both induced required mice Matrigel bleomycin-treated from BAL through invasion fibroblast and chemotaxis fibroblast both that revealed receptors these of knockdown siRNA Ephb4 identi fied) been has homolog mammalian no but chickens, in present is (EphB5 EphB6 and EphB1–4 includes which kinases, tyrosine tor chemotaxis. fibroblast to contributes sEphrin-B2 that indicate ( (PDGF-BB) factor growth platelet-derived skin fibrosis in bleomycin-treated bleomycin-treated in fibrosis skin ( mice and the development of skin fibrosis cells. immune or endothelial as such types, cell other on ectodomain this of contributions potential broblast formation and tissue fibrosis myofi drive to sufficient is ectodomain ephrin-B2 the by signaling forward ephrin-B2 that demonstrate data these Although treatment. ( expression collagen I ness ( ( fibrosis analysis showed that ephrin-B2-Fc treatment produced robust dermal histological Blinded for 2 weeks. daily a as or control IgG-Fc mouse) (100 ephrin-B2-Fc mouse preclustered induce tissue fibrosis in vitro functions fibroblast profibrotic direct can ectodomain As ephrin-B2 tissue fibrosis Treatment with Ephrin-B2 ectodomain is sufficient to drive ( treatment IgG-Fc to compared expression protein markedly ephrin-B2-Fc increased preclustered with fibroblasts lung primary mouse of Treatment differentiation. fibroblast-to-myofibroblast ( samples BAL by these induced was that chemotaxis fibroblast the completely, reduced not though markedly, mAb anti-ephrin-B2 netic coated beads with the N-terminus-specific mag with mice bleomycin-challenged from BAL in sEphrin-B2 of profibrotic mediate to able functions of fibroblasts. In are accordance with this hypothesis, depletion they that in active biologically are and injury lung following BAL in present are oligomers sEphrin-B2 ( oligomers the in from sistent this binding with antibody expected to those sEphrin-B2 were con before immunoblotting mAb ephrin-B2 recombinant with Fig. 3 Fig. As EphB receptor activation requires higher-order ephrin-B2 oli ephrin-B2 higher-order requires activation receptor EphB As The ephrin-B2 ligand signals through the EphB subfamily of recep In addition to lung fibrosis, we assessed the role of ephrin-B2 in in ephrin-B2 of role the assessed we fibrosis, lung to addition In We next determined whether ephrin-B2 signaling could also induce 2 Fig. Fig. 3 5 mRNA, but not not but mRNA, . Primary mouse lung fibroblasts expressed expressed fibroblasts lung mouse . Primary q , we next investigated whether recombinant ephrin-B2-Fc can , recombinant ephrin-B2-Fc we whether next investigated ). Blinded histological analysis showed markedly attenuated attenuated markedly showed analysis histological Blinded ). Fig. 3 Fig. Ephrinb2 Acta2 n ), ), hydroxyproline content ( m in vivo advance online publication online advance and and ) associated with markedly increased dermal thick dermal increased markedly with associated ) Supplementary Fig. 1 Fig. Supplementary -C -C mice to the bleomycin model of fibrosis dermal Col1a1 in in vivo. Fig. 3 Fig. 2 Ephb1 4 , we investigated the quaternary structure of, structure we the investigated quaternary mRNA and and mRNA Mice with were subcutaneously injected p or or ) as compared to the results of IgG-Fc IgG-Fc of results the to compared as ) in vivo Ephb6 Ephrinb2 in vivo . . We subjected Fig. Fig. 3 mRNA ( mRNA µ Fig. 3 Fig. α ). Our results suggest that that suggest results Our ). g per kg body weight per per weight body kg per g -SMA and type I collagen collagen I type and -SMA , , we have not investigated Fig. 3 Fig. -CKO mice, which was was which mice, -CKO o ) ) and j Fig. 3f Fig.

). Fig. 3 Fig. nature medicine nature d Ephb2 Fig. 3k Fig. ). These findings findings These ). α Fig. 3 Fig. Ephrinb2 -SMA -SMA and type , g e ). ). Individual Individual ). , , Ephb3 , h l ). ). These These ). -CKO and ------© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. bleomycin challenge. Scale bars, 100 100 bars, Scale challenge. bleomycin of skin trichrome–stained Masson’s of images representative Bottom, d. 28 for bleomycin Ephrinb2 in presented are data Densitometry shown. is replicates technical two of out representative One n ( sections 100 bars, ( ephrin-B2-Fc ( respectively. treatment, to (normalized data ( fibroblasts. Fc-treated ( fibroblasts. lung mouse of chemotaxis fibroblast BAL-induced on antibody anti-ephrin-B2 with coated beads magnetic by 2- and SDS of electrophoresis. gel native nondenaturing to subjected mice bleomycin-challenged and PBS- from samples BAL from a as chemoattractant. used was challenge bleomycin 7 following day at mice from BAL GAPDH. ( line). (dotted IgG-Fc by induced chemotaxis over increase fold as presented are Data LPA PDGF-BB. or ephrin-B2-Fc, by induced treatment. ephrin-B3-Fc or ephrin-B2-Fc ephrin-B1-Fc, IgG-Fc, upon cell per filopodia of 50 bars, Scale fibroblasts. in formation filopodia indicate arrows White fibroblasts. lung human primary in formation h 24 filopodia on for treatment (control) IgG-Fc or ephrin-B2-Fc preclustered of effect the showing ( domain. transmembrane TM, Fc. to fused ectodomain ephrin-B2 recombinant the and protein ephrin-B2 the of 3 Figure nature medicine nature whiskers, minimum and maximum values. Data are presented as mean mean as presented are Data values. maximum and minimum whiskers, * ANOVA. two-way by analyzed f For groups. all ( respectively. mice, Ephrinb2 k e and and = 5 for each treatment group; group; treatment = each 5 for ) Effects of ephrin-B2-Fc or IgG-Fc on on IgG-Fc or ephrin-B2-Fc of ) Effects of level the to normalized was expression mRNA fibroblasts. lung mouse primary in receptors EphB encoding genes of expression mRNA ) Relative 1,04 1,23 Ephrin-B2 full-length h a m kD Trichrome staining H&E 24 48 72 g a 2 0 0 8 6 , data were analyzed with Student’s Student’s with analyzed were , data Control (IgG-Fc) Ephrin-B2-F

2 1 n ephrin-B2-Fc orIgG-Fc -CKO mice 28 d after PBS or bleomycin challenge. challenge. bleomycin or PBS d 28 after mice -CKO and -C Control The ephrin-B2 ectodomain directs fibroblast migration, invasion and myofibroblast differentiation differentiation myofibroblast and invasion migration, fibroblast directs ectodomain ephrin-B2 The n Native P = 3 for all groups. ( groups. = all 3 for µ BAL ), quantification of hydroxyproline content in skin biopsies ( biopsies skin in content hydroxyproline of quantification ), m. ( m. S.c. injectio Daily for14 1 3 β n

c -mercaptoethanol (2ME) on the formation of high-molecular-weight sEphrin-B2 oligomers in BAL. ( BAL. in oligomers sEphrin-B2 high-molecular-weight of formation the on (2ME) -mercaptoethanol n = 6) or IgG-Fc ( = IgG-Fc or 6) AGE , , Ephrinb2 c – 3 2 BLM BAL peptide d Signal p 1–2 , , n ) Effects of ephrin-B2-Fc or IgG-Fc treatment on skin fibrosis in mice as assessed by blinded analysis of dermal thickness of tissue tissue of thickness dermal of analysis blinded by assessed as mice in fibrosis skin on treatment IgG-Fc or ephrin-B2-Fc of ) Effects d s β j

– ) Hydroxyproline content in 6-mm skin biopsies from from biopsies skin 6-mm in content ) Hydroxyproline -actin) are presented. presented. are -actin) 7 Time (d) l and and advance online publication online advance sEphrin-B2 Dimer l oligomer Ectodomai ) Effects of ephrin-B2-Fc or IgG-Fc on on IgG-Fc or ephrin-B2-Fc of ) Effects 28–229 m ephrin-B2-Fc orIgG-Fc Ephrin-B2-Fc 27–227 -CKO mice were subjected to bleomycin-induced skin fibrosis. Mice received daily subcutaneous injections of either PBS or PBS either of injections subcutaneous daily received Mice fibrosis. skin bleomycin-induced to subjected were mice -CKO ) Top, schematic indicating that C57BL/6N mice were treated with daily subcutaneous (s.c.) injections of either preclustered preclustered either of injections (s.c.) subcutaneous daily with treated were mice C57BL/6N that ) indicating Top, schematic n S.c. injectio – p f P , , data were analyzed with Student’s Student’s with analyzed were , data 230–25 n g < 0.05, *** < 0.05, o n 1,048 1,236 i ) Effect of siRNA-mediated knockdown of EphB receptors on BAL-induced fibroblast chemotaxis ( chemotaxis fibroblast BAL-induced on receptors EphB of knockdown siRNA-mediated of ) Effect , , TM = 6) as control for 14 d. Bottom, results of H&E and Masson’s trichrome staining for each treatment group. Scale Scale group. treatment each for staining trichrome Masson’s and H&E of results Bottom, d. 14 for = control as 6) kD 24 48 72 n Fc domain n a Native P 2 0 0 = 6 for each treatment group; group; treatment = each 6 for Control 0 BAL Cytoplasmi – – µ 251–333 domain + + m. m. Col1a1 AGE BL BAL t n p n n -test. * -test. M SDS +2ME = 6 for all groups. ( groups. = all 6 for

P = 3 for all groups. Blue and red represent data from lung fibroblasts that received IgG-Fc or ephrin-B2-Fc ephrin-B2-Fc or IgG-Fc received that fibroblasts lung from data represent red and Blue groups. = all 3 for sEphrin-B2 130 α-SMA Dermal thickness ( m) Monomer Dimer µ c oligomer < 0.001. In In < 0.001. kD 42 42 densitometry 100 200 300 400 and and 10.0 a 0 b 5.0 0

P Ephrin-B2-Fc IgG-Fc IgG-Fc < 0.05 versus nontargeting control siRNA. For For siRNA. control nontargeting versus < 0.05 * Acta2 *** j Chemotactic index o

10.0 Ephrin-B2-Fc mRNA expression by mouse lung fibroblasts. Data are presented as fold increase over IgG- over increase fold as presented are Data fibroblasts. lung mouse by expression mRNA 2.5 5.0 7.5 c Hydroxyproline BAL BAL , , Type I collagen 0

n µg/mg of protein o

= 6 for all groups. Blue and red represent data from from data represent red and Blue groups. = all 6 for densitometry 12 16 c and and ∆ α 10.0 0 4 8 Ephrin-B2 5.0 r -SMA and type I collagen protein expression in mouse lung fibroblasts. Densitometry Densitometry fibroblasts. lung mouse in expression protein I collagen type and -SMA Number of filopodia *

) Blinded analysis of dermal thickness of tissue sections from from sections tissue of thickness dermal of analysis ) Blinded Ephrin-B1-Fc 0 lgG-Fc 0 2 4 6 8 Ephrin-B2-Fc p t * β T α s *** , , -test. * -test. Ephrin-B3-Fc -acti ype Icollagen -SMA , center lines, median values; +, the mean; box edges, 25th and 75th percentiles; percentiles; 75th and 25th edges, box mean; the +, values; median lines, , center Ephrin-B2-F IgG-F * n o = 4 for all groups. groups. = all 4 for ± n µ ) and western blot showing showing blot western ) and

s.d. Uncropped blot images are shown in in shown are images blot Uncropped s.d. sEphrin-B2 in BAL

m. m. c

Ephrinb2 (ng/ml) P 0.00 0.50 1.00 1.50 n d < 0.05, ** < 0.05, = 25–50 cells per condition. ( condition. per cells = 25–50 c Chemotactic index BAL BAL 0.00 1.00 2.00 3.00 4.00 5.00 6.00 *** q

Bleomycin Saline ∆ Ephrin-B2 -C and and -C Ephrin-B2

5 *

2 * ( Ephrinb n Ephrinb2 1 * P 0.5 = 3 for all groups. ( groups. = all 3 for * –7 < 0.01, *** < 0.01, Tamoxife Ephrinb2 (

β 0.1 n * µ 0.05 Ephrinb2 g) -actin was used as a loading control for densitometry analysis. analysis. densitometry for control a as loading used was -actin = 25–50 cells per condition. ( condition. per cells = 25–50 0.01 * k 2 ablation) mRNA expression PDGF * -C

–1 Col1a1 0 1 2 3 4 * n LPA p * Daily for28 s.c. injectio 0 . . ( Bleomyci Acta2 * α -C and and -C q r e -CKO mice treated with PBS or bleomycin. bleomycin. or PBS with treated mice -CKO and and s.c. injectio ) Top, schematic showing the model through which which through model the ) showing Top, schematic -SMA and type I collagen levels in skin ( skin in levels I collagen type and -SMA * Bleomyci

P mRNA expression n < 0.001 versus IgG-Fc treated fibroblasts. For For fibroblasts. treated IgG-Fc versus < 0.001 Ephrinb 0 2 4 6 8 n Ephb1 Tamoxifen i.p. d ND s

Ephrin-B2-F IgG-Fc Ephb2 Ephrinb2 , data are presented as mean mean as presented are , data n

n Ephb3 in vitro in c

h Ephb4 Harvest 2 ) Blinded quantitation of the number number the of quantitation ) Blinded +2 -CK ) The oligomeric state of sEphrin-B2 sEphrin-B2 of state oligomeric ) The Ephb6 8 ND b O Time (d) ) Representative phase contrast images images contrast phase ) Representative j Supplementary Figure 5 Figure Supplementary c and and ) Effect of sEphrin-B2 depletion depletion sEphrin-B2 of ) Effect Ephrinb2 -CKO mice 28 d following PBS or PBS d 28 following mice -CKO f n

l Nontargeting siRNA kD = 3 for all groups. ( groups. = all 3 for 130 Chemotactic index

-SMA 42 42

in vivo in α s a (% reduction) 100 r densitometry n d 25 50 75

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µ 100 200 300 400 -C mice or or mice -C 0 0 0 Ephb2 10 20 30 40 Si . . ( 0 0 Ephb3 Ephrinb2

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Ephrinb2 densitometry Nontargeting siRNA 10. ± Invasion index -C mice and and mice -C β Ty α 5. s.d and were were and s.d -acti -SMA (% reduction) 0 0 0 pe Icollagen . i 100 ) Effect ) Effect 25 50 75 Ephrinb Ephrinb 0 n p **

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 © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. concentration concentration in medium conditioned by these cells compared to that ( cells untreated greater in of expression induce not did fibroblasts activation broblast these cells. TGF- of activities profibrotic the to contribution its and shedding, of this regulation the investigated we then fibroblasts, ectodomainin ephrin-B2 the of sheddase major the as ADAM10 identified Having myofibroblast activation ADAM10-mediated ephrin-B2 shedding drives TGF- shedding. ephrin-B2 ( fibroblasts by generation sEphrin-B2 prevent not did inhibitor, dose- ADAM17-specific TAPI-0, an a in supernatants ( manner dependent fibroblast in concentration PMA-stimulated sEphrin-B2 and constitutive both reduced inhibitor, specific ADAM10- an siRNA. GI254023X, with nontargeting fibroblasts of with treatment Moreover, transfected fibroblasts lung human to compared effects pronounced no had MMPs or ADAMs of other ing knockdown whereas fibroblasts, sEphrin-B2 concentration in medium conditioned by PMA-stimulated of Knockdown pared to that from fibroblasts treated with DMSO as control ( sEphrin-B2 concentration in fibroblast cell culture supernatants com in the in a increase for PMA with marked 30 resulted min fibroblasts (ref. ADAM17 and ADAM10 of ity activ the increasing by shedding, ectodomain i.e., proteins, bound of membrane- proteolysis limited activates which (PMA), 13-acetate B2 ectodomain shedding in fibroblasts, we used phorbol 12-myristate by ( assessed ELISA as supernatant culture cell fibroblast in sEphrin-B2 concentration lowered markedly fibroblasts, by expressed MMPs or of knockdown ( lasts that we found ADAMs to be expressedand by MMPs cultured primary the human lungdown fibrob that knocked confirmed efficiently duplexes first siRNA We individually. metalloproteinases these of each of knockdowns in siRNA performed sheddases we fibroblasts, ectodomain lung human ephrin-B2 as act proteases these of any ( ADAM17 and ADAM12 expressed MMP1, MMP2 and MMP14, as well as ADAM9, ADAM10, ( cleavage B2 ephrin- to contribute metalloproteinase(s) that indicating manner, dose-dependent a in fibroblasts lung human healthy by conditioned medium in concentration sEphrin-B2 reduced treatment BB-94 B2. of MMPs both and inhibitor ADAMs, of to generation sEphrin- fibroblast inhibit broad-spectrum a BB-94, of ability the tested we lasts, fibrob in (MMPs) metalloproteinases by regulated is ectodomain ephrin-B2 metalloproteinases matrix ADAMs and including met by cleaved be alloproteinases, can ephrins that shown have studies Previous sheddase in fibroblasts Identification of ADAM10 as the ephrin-B2 ectodomain ( mice control to compared content proline hydroxy and thickness dermal lower considerably with associated A  ( cells by untreated conditioned

To further investigate whether To ADAM10 for is whether investigate responsible ephrin- further s e l c i t r Supplementary Fig. 2 Fig. Supplementary Fig. 4 Fig. ADAM10 β 1 is a prototypical profibrotic cytokine that induces myofi 19– Fig. 4 Fig. c ). ADAM10 2 Fig. 4 Fig. 2 ADAM10 . In order to investigate whether shedding of the the of shedding whether investigate to order In . 27, a mRNA expression in these cells as compared to to compared as cells these in expression mRNA ). Of the MMPs and ADAMs, these fibroblasts fibroblasts these ADAMs, and MMPs the Of ). Fig. 4 Fig. 2 8 e . Whereas TGF- Whereas . ) and in a substantially greater sEphrin-B2 sEphrin-B2 greater substantially a in and ) , but not the other genes encoding ADAMs ADAMs encoding genes other the not but , Fig. 4 Fig. Fig. 4 Fig. d by siRNA also substantially reduced reduced substantially also siRNA by ). In contrast, fibroblast treatment with with treatment fibroblast contrast, In ). ). We then found that siRNA-mediated siRNA-mediated that found We then ). Fig. 4 Fig. d b ), further implicating ADAM10 in in ADAM10 implicating further ), ). In order to investigate whether whether investigate to order In ). 2 f 6 ). ADAM17 β ). Treatment of human lung lung human of Treatment ). EFNB2 1 treatment of human lung lung human of treatment 1 Fig. 3r Fig. mRNA, it did result result did it mRNA, r ee encod genes or , s ). b -induced Fig. Fig. 4 c ). ). ------

ephrin-B2 shedding. ephrin-B2 TGF- ( myofibroblasts activated of characteristics phenotypic both are which fibers, stress of incorporation and fibers stress of formation TGF- inhibited treatment GI254023X that demonstrated with an fibroblasts anti- ( lasts TGF- reduced dose-dependently and markedly also treatment GI254023X β inhibition of logical ADAM10 which with reduced TGF-GI254023X, reduced by siRNA knockdown of the of ( shedding ephrin-B2 induced TGF- in ADAM10 of role the confirming production, sEphrin-B2 GI254023X inhibitor ADAM10-selective Genetic of shedding. knockdown ephrin-B2 on dependent is activation broblast mice in marker myofibroblast the of expression greater in resulted myofibroblasts activated into differentiation fibroblast therefore tested whether this region is important for is TGF- region important this whether tested therefore ectodomain its of region juxtamembrane the in for TGF- for ( ephrin-B2 WT with transfected to compared human lung fibroblasts TGF- reduced mutants had ephrin-B2 cleavage-resistant overexpressing fibroblasts lung human We that found shedding. ephrin-B2 to addition in tion, TGF- prevents mutants ephrin-B2 ( cleavage main ectodo for required is ephrin-B2 of region juxtamembrane the that demonstrating mutants, further ephrin-B2 cleavage-resistant tagged, HA- overexpressing fibroblasts lung human in prevented largely was TGF- this TGF- with accordance TGF- by reduced substantially was lasts fibrob lung of human lysates in by antibodies anti-HA detected was As shown in (HA)-tagged WT ephrin-B2 or HA-tagged, cleavage-resistant mutants. ephrin-B2 protein levels in fibroblasts transfected with hemagglutinin B2 is required for ephrin-B2 shedding induced by TGF- ( ding TGF- to resistant 197–218) or ephrin-B2 182–194 rendered acids (amino region juxtamembrane the in ( shedding B2 TGF- abrogated ectodomain, ephrin-B2 the of ephrin-B2 region, embrane juxtam the of a deletion with a of mutant overexpression Likewise, ( ephrin-B2 WT with transfected of fibroblasts challenge these TGF- by to response produced in cells sEphrin-B2 of amount the reduced markedly domains, intracellular and transmembrane the preserves but main overex of pression ephrin-B2 Forced activation. myofibroblast and shedding ephrin-B2 Fig. Fig. 4o -induced -induced Considering Considering that we found that recombinant ephrin-B2-Fc induced Previous studies have shown that ADAM10 cleaves ephrin-B2 ephrin-B2 cleaves ADAM10 that shown have studies Previous We next investigated whether overexpression of cleavage-resistant of cleavage-resistant overexpression We whether investigated next of ephrin- region juxtamembrane the that Todemonstrate further β β Fig. 4 Fig. Fig. 4l Fig. α -induced myofibroblast formation requires ADAM10-mediated -induced -induced , -SMA protein by human lung fibroblasts was also markedly markedly also was fibroblasts lung human by protein -SMA p β n vivo in ), in accordance with a requirement for ephrin-B2 cleavage cleavage a ), with for in requirement ephrin-B2 accordance β -induced myofibroblast activation. myofibroblast -induced -induced reduction in HA-tagged ephrin-B2 expression expression ephrin-B2 HA-tagged in reduction -induced ACTA2 j ). Further, immunofluorescence staining of human lung of human staining immunofluorescence Further, ). Figure Figure 4 , m β Fig. 4l Fig. ). Fig. 4 Fig. advance online publication online advance -induced -induced ADAM10 w ivsiae wehr TGF- whether investigated we , Fig. 4 Fig. α -SMA protein expression by human lung fibrob lung human by expression protein -SMA RA xrsin oedpnety ( dose-dependently expression mRNA n n , ∆ m , , the amount of HA-tagged that WT ephrin-B2 k ). Ecto β α ). Taken together, these results indicate that that indicate results these together, Taken ). ). Further, deletions of specific sequences sequences specific of deletions Further, ). -induced ephrin-B2 shedding. In contrast, contrast, In shedding. ephrin-B2 -induced -SMA antibody and rhodamine–phalloidin and -SMA antibody rhodamine–phalloidin ACTA2 or pharmacological inhibition with the the with inhibition pharmacological or , which lacks the entire ephrin-B2 ectodo ephrin-B2 entire the lacks , which β ∆ compared to that produced by TGF- by produced that to compared Juxta Fig. 4f Fig. ADAM10 , which lacks amino acids 168–218 168–218 acids amino lacks which , mRNA and and mRNA β -mediated myofibroblast activa myofibroblast -mediated , g ). TGF- ). β β -induced ephrin-B2 shed ephrin-B2 -induced 2 treatment; this result is in in is result this treatment; 9 ( reduced TGF- reduced Fig. 4 α β

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. All data are presented as mean knockdown of EphB receptors on TGF- GAPDH was used as housekeeping gene. gene. n treated cells. ( expression in human lung fibroblasts. GAPDH was used as a housekeeping gene. analysis of HA-tagged ephrin-B2 WT and mutants. GAPDH was used as a loading control. ( ELISA. ephrin-B2 50 identify myofibroblasts, phalloidin (red) to visualize filamentous F-actin in fibroblasts and Hoechst 33342 (blue) to visualize nuclei. Scale bars, condition) of human lung fibroblasts treated with vehicle (DMSO) or the ADAM10 inhibitor GI254023X (20 nM) and stained for used as a loading control; ( housekeeping gene. ( expression and densitometry (GAPDH used as a loading control) in human lung fibroblasts. Nontargeting siRNA was used as control. lung fibroblasts. DMSO was used as control. Nontargeting siRNA was used as control. ( TGF- ADAM10 Three sets of primary lung fibroblasts were used; (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; of metalloproteinases on ephrin-B2 shedding by human lung fibroblasts. Concentration of sEphrin-B2 in the conditioned media was determined by ELISA. fibroblasts. mRNA expression was normalized to the level of GAPDH. and red indicates treatment with BB-94. ( on the generation of sEphrin-B2 in primary human lung fibroblasts. * Figure 4 nature medicine nature j i = 3 for all groups. ( ) Effect of ADAM10 inhibition with GI254023X (20 nM) on TGF- ) Effect of ADAM10 inhibition with GI254023X (20 nM) on TGF- µ m. ( β o k e a n -treated fibroblasts. (

= 3 for all groups. ( ACTA2 n

Ephrin-B2 WT -SMA Phalloidin l = 4 for all groups. ( α mRNA expression sEphrin-B2 in primary human lung fibroblasts treated with or without TGF- ) Schematic of ephrin-B2 WT and HA-tagged ephrin-B2 mutants, including ephrin-B2 ADAM10-mediated sEphrin-B2 generation is required for TGF- mRNA expression ∆ DAPI DAPI 0. 1. 1. 2. 2. 0. (ng/ml) 10 20 30 40 50 60 197–218-HA 0 5 0 5 0 5 0

ADAM10 0.00 0.05 0.10 0.15 0.20 DMS p * * ) Effect of ephrin-B2 mutants on TGF- O

∆ TGF- DMS (Batimastat)

EFNB2 Ecto BB-94 (nM)

∆ GI254023X 0 ∆ Juxta

TGF- DMS 0.1 O

182–194 β n *

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) Relative mRNA expression of genes encoding EphB receptors in primary human lung fibroblasts. GAPDH was used as a housekeeping 5 * 197–218 * advance online publication online advance m O β *

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- sEphrin-B2 ) Effect of ephrin-B2 mutants on TGF- β b ± + GI254023X (ng/ml) mRNA expression 0. 0. 0. 0. 0. 0. 0. s.d and were analyzed with Student’s NT siRNA TGF- 0.06 0.08 -SMA 0.02 0.04 0.10 α TGF p 0 1 2 3 4 5 6 kDa 0

37 42 densitometry si MMP1 10 β - ADAM10 * 0 5 β MMP2 β GFP TGF- DMS MMP3 -induced * ND – – MMP7 n + + b P MMPs g O β ND = 3 for all groups. Data are presented as fold change relative to DMSO-treated cells. ( MMP8 ) Effect of ADAM10 inhibition with GI254023X (20 nM) on TGF- ) Relative mRNA expression of genes encoding metalloproteinases (MMPs and ADAMs) in primary human lung ND < 0.05 versus TGF- ∆ W l

Ephrin-B2 WT MMP9 182–194T ND n MMP14 = 5 for all groups. ( ND Sheddase ND ∆ ∆ – – ∆ * 197–21 182–19 P 197–218 sEphrin-B2 ADAM8 + + * ∆ α n n < 0.05 versus TGF-

∆ ADAM9

Juxt (ng/ml) ND = 3 sets for all groups. * = 3 sets for all groups. * ND -SMA protein levels in human lung fibroblasts. GAPDH was used as a loading control. Ect

0. 0. 0. 0. 0. 0. 0. ADAM10 0 1 2 3 4 5 6 a o 8 4 DMSO ADAM12 ADAM s

β Gl254023X GAPDH α ADAM15 Ectodomain -induced -SMA * ADAM17 28–229 TGF- DMS ADAM19 ND * s

ADAM28 O β 230–25 ND TM β r q h

α -treated fibroblasts. Data are presented as fold change relative to DMSO-treated cells. 0

mRNA expression Cytoplasmi h -SMA protein expression in human lung fibroblasts. GAPDH was used as a loading control.

β β α-SMA 251–33 0 4 2 6 8 domain ) Effect of siRNA-mediated knockdown of β -induced -induced EPHB1 TGF β densitometry -induced sEphrin-B2 generation in human lung fibroblasts as assessed by ephrin-B2 kDa P ND β 10.0

n EPHB2 -induced ephrin-B2 shedding in human lung fibroblasts as assessed by western blot ADAM10 c 5. < 0.05 versus control (DMSO). Blue indicates treatment with DMSO as control, 42 37 -treated fibroblasts. ( 3 = 5 sets of human lung fibroblasts analyzed. (

EPHB3 ND t NT siRNA - c 0 0 β

-test. * sEphrin-B2

β EPHB4 β

1-induced myofibroblast differentiation. ( EPHB6 (10 ng/ml) for 48 h. GAPDH was used as a housekeeping gene. * (ng/ml) – – ND 0. 0. 0. 0. 0. 0. 0. P P si * 0 1 2 3 4 5 6 ADAM10 + + NT siRNA < 0.05 versus control (DMSO). ( < 0.05 versus control (DMSO). NT, nontargeting. ( α ACTA2 m * -SMA protein expression, with GAPDH used as a loading control, with GAPDH P on TGF- sEphrin-B2 * Ephrin-B2 WT si < 0.05. Uncropped blot images are shown in MMP1 * *

n (ng/ml) 0. 0. 0. 0. 0. 0. 0. GAPD α = 4 for all groups. Data are presented as fold change relative to DMSO- si TGF- DMSO

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MMP7 * * H

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β ∆

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) Effect of ephrin-B2 mutants on TGF- 182–194 k NT siRNA si ) Representative immunofluorescence images (

* mRNA expression ∆ * ADAM10 12.00 16.00 20.00 24.00 β 197–218 0.00 4.00 8.00 *

∆ si -induced si * EPHB3 ADAM12 DMSO Ecto-HA

si GI254023 si * EPHB4 TGF- DMSO ADAM17 (nM * * * si

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1 PMA

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e -SMA -induced sEphrin-B2 generation in human α * n TGF- 1 ) Relative mRNA expression of

densitometry 20 * mRNA expression in human lung fibroblasts. d kD TGF- Untransfected kD 50 37 40 80 X

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. H

 © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. in vivo in lung injury. Indeed, ADAM10 expression levels were markedly higher upon increased similarly be would levels ADAM10 that hypothesize of levels TGF- increases activated rapidly challenge bleomycin that show studies activation myofibroblast and shedding ephrin-B2 GI254023X inhibitor cal pharmacologi ADAM10-selective the with ADAM10 of targeting that therapeutic we above, hypothesized of our On basis findings the induced lung fibrosis in mice Pharmacological inhibition of ADAM10 prevents bleomycin- fibroblasts. lung in sEphrin-B2 of activities profibrotic mediate receptors ( both that siRNA demonstrating nontargeting with transfected fibroblasts lung both EPHB3 or by encoded proteins EphB human receptors, primary lung identified expressed fibroblasts Of the five receptor signaling. by sEphrin-B2–EphB trolled autocrine we hypothesized that TGF- broblast activation required ADAM10-mediated ephrin-B2 shedding, TGF- and activation myofibroblast drive to ficient A  ( mice PBS-challenged bleomycin challenge and treated with ADAM10 inhibitor GI254023X or vehicle control. control. vehicle or GI254023X inhibitor ADAM10 with treated and challenge bleomycin ( group per shows blot samples 3 representative This control. vehicle or (GI25) GI254023X inhibitor ADAM10 with treated challenge PBS or bleomycin after d 14 mice ( control. vehicle or GI254023X inhibitor ADAM10 with treated challenge PBS or bleomycin after d 14 mice of lungs the in bar, 100 Scale control. vehicle or GI254023X inhibitor ADAM10 with treated were that challenge bleomycin ( challenge. bleomycin after homogenates lung in levels ADAM10 showing (right) 5 Figure Supplementary Figure 7 Figure Supplementary in shown are images blot Uncropped values. maximum and minimum whiskers, percentiles; 75th and 25th edges, box mean; the +, values; ** ANOVA. two-way mean as control. vehicle or GI254023X inhibitor ADAM10 with treated and bleomycin with challenged d b

) ) from images (4 images Representative ) kDa As expression of the recombinant ephrin-B2 ectodomain was suf was ectodomain ephrin-B2 recombinant the of expression As b a 42 80 s e l c i t r EPHB6 α

-SMA protein expression assessed by western blot (top) and densitometry (bottom; normalized to to normalized (bottom; densitometry and (top) blot western by assessed expression protein -SMA Bleomycin Saline ACTA2 after bleomycin-induced lung injury compared to those in in those to compared injury lung bleomycin-induced after or

± ADAM10 inhibition prevents ephrin-B2 shedding, myofibroblast formation and lung fibrosis in mice. ( mice. in fibrosis lung and formation myofibroblast shedding, ephrin-B2 prevents inhibition ADAM10 Day s.d and were analyzed with Student’s Student’s with analyzed were and s.d ( EPHB4 Fig. 4 Fig. 0 mRNA and and mRNA Vehicle β Day 1 ihn ra o ln injury lung of areas within q P markedly reduced the TGF- the reduced markedly ). We found that siRNA-mediated knockdown of of knockdown siRNA-mediated that We ). found < 0.01, *** 0.01, < n = 6 mice for all groups). ( groups). all for mice 6 = EPHB3 . Day 3 Fig. 5 Fig. BLM 2 α 9 β could prevent lung fibrosis by inhibiting inhibiting by fibrosis lung prevent could

-SMA protein levels compared to human human to compared levels protein -SMA -induced -induced myofibroblast activation is con a and and Day 7 ). To examine the ability of ADAM10 ADAM10 of ability the Toexamine ). P < 0.001. In In 0.001. < EPHB4 Day 14 GI254023X n = 10 mice per group) of Masson’s trichrome staining of lung sections from mice at 14 d after PBS or or PBS after d 14 at mice from sections lung of staining trichrome Masson’s of group) per mice 10 = , but not not but , f β ADAM10 , data were analyzed with the log-rank test. *** test. log-rank the with analyzed were data , t -test. * -test. e -actin 31, β ) Concentration of sEphrin-B2 in BAL fluids recovered from mice at 14 d following PBS or or PBS following d 14 at mice from recovered fluids BAL in sEphrin-B2 of Concentration ) -induced increase in increase -induced 3 2 , which led us to to us led which , β in vivo in P -induced myofi -induced EPHB1 < 0.05 versus Day 0. In In 0. Day versus 0.05 < Densitometry 0.5 1.0 42 kDa d 42 i. 4r Fig. 0 . Previous Previous . , , Time following EPHB2 7 3 1 0 BLM (d) α-SMA * PBS * , densitometry s * ), ), 0.5 1.0 - - - - 14 0

* PBS latedfrom lungs the of individuals with IPF and healthy controls. IPF iso fibroblasts in signaling ADAM10–sEphrin-B2 of role the tigated To determine the relevance of our studies to human disease, we inves pulmonary fibrosi ADAM10-sEphrin-B2 signaling is upregulated in idiopathic ( mice in fibrosis caused by edly reduced lung mortality bleomycin-induced and injury fibrosis lung in activation myofibroblast drives axis ADAM10–sEphrin-B2 ( mice bleomycin-challenged of BAL the in concentration sEphrin-B2 reduced treatment GI254023X B2, sEphrin- to generate cleavage for ephrin-B2 responsible is ADAM10 our with accordance In samples. BAL in concentration sEphrin-B2 assessed we model, fibrosis lung ( mice PBS-challenged to compared mice cin-challenged resulted in a considerably lower level of tion in lung hydroxyproline ( levels ( GI254023X with treated mice in mitigated was mice vehicle-treated in challenge bleomycin following d 14 produced fibrosis parenchymal lung the that revealed analysis histological Blinded injection. intraperitoneal with GI254023X (200 mg daily per kg body weight per day) treated or vehicle controland by model fibrosis lung bleomycin-induced the fibrosis lung mitigate to inhibition BLM c β To gain insight into the mechanism of action of GI254023X in this of To of GI254023X action into mechanism the insight gain -actin was used as a loading control. control. loading a as used was -actin

BLM Hydroxyproline ** c

– (µg/left lung) e 150 225 300 in in vivo , data are presented as mean mean as presented are data , 75 BLM GI25 0 n = 8 mice for all groups. ( groups. all for mice 8 = + PBS GI254023X Vehicle Fig. 5 Fig. . Further, ADAM10 inhibition with GI254023X mark . Further, GI254023X with ADAM10 inhibition β α n * advance online publication online advance -actin -SMA P = 10 mice per group. In In group. per mice 10 = Fig. 5 Fig. * < 0.001. In In 0.001. < BLM b * ), and this was associated with marked reduc marked with associated was this and ), β f -actin) in total lung homogenates of of homogenates lung total in -actin) ). GI254023X Vehicle

Percentage survival f µ c 100 m. ( m. 25 50 75 and and a 0 ) Western blot (left) and densitometry densitometry and (left) blot Western ) in vitro in 0 c challenge (d) f Fig. Fig. 5 ) Hydroxyproline content measured measured content Hydroxyproline ) ) Survival curves for mice mice for curves Survival ) ± Time after e s.d and were analyzed with with analyzed were and s.d in vivo in , center lines, median median lines, center , e 5 n sEphrin-B2 in BAL (ng/ml) i. 5 Fig. α = 4 mice for all groups. groups. all for mice 4 = a 10 0.00 0.25 0.50 0.75 1.00 1.25 1.50 -SMA expression in bleomy c studies demonstrating that that demonstrating studies , data are presented presented are data , n ). ). ADAM10 also inhibition *** = 8 for all groups. groups. all for 8 = 15 , mice were subjected to to subjected were mice , e PBS ), suggesting that the the that suggesting ), *** nature medicine nature BLM +GI254023X BLM +vehicle *** BLM *** Fig. 5 Fig.

GI254023X Vehicle

d ). - - - - - © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. sion of expresADAM10–sEphrin-B2 contributesincreased this signaling to and collagen I type increased by characterized are fibroblasts lung IPF fibroblasts. lung control and IPF in shedding ephrin-B2 for responsible is ADAM10 ( fibroblasts lungcontrol and IPF both by of knockdown siRNA or GI254023X with ADAM10 of inhibition Pharmacological fibroblasts lung normal to compared mRNA increased markedly expressed and medium culture in B2 sEphrin- of concentration higher substantially a had fibroblasts lung functions, including migration, invasion, myofibroblast differentiation and extracellular matrix production in an autocrine and/or paracrine fashion. upregulation in TGF- mediated ephrin-B2 shedding in fibroblasts. Our results suggest that in fibroblasts during the development of fibrosis, sEphrin-B2 is produced by ADAM10 whiskers, minimum and maximum values. Uncropped blot images are shown in analyzed with a Student’s sEphrin-B2 in plasma from control donors ( = 8) and individuals with IPF ( control donors ( level of GAPDH. COL1A1 all groups. Data are presented as fold change relative to DMSO-treated cells. ( expression in human lung fibroblasts versus treatment with vehicle (DMSO) control. mRNA expression was normalized to the level of GAPDH. n ( inhibition of ADAM10 with GI254023X (20 nM) on sEphrin-B2 concentration in conditioned media. DMSO was used as vehicle control. from control donors ( lung fibroblasts from control (healthy) donors ( Figure 6 nature medicine nature fibroblasts. Treatment with GI254023X or a siRNA targeting j f a d = 3 for all groups. ( ) Effect of siRNA-mediated knockdown of ACTA2 mRNA sEphrin-B2 in sEphrin-B2 in BAL (ng/ml) expression supernatants (ng/ml) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0.0 1.0 2.0 3.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 COL1A1

( Healthy Healthy ADAM10–sEphrin-B2 signaling is upregulated in IPF. (

g Healthy n ) and Cohort = * *

4 IPF ** n * n

IPF =11 n ACTA2 = 6) and individuals with IPF ( and ADAM10 1 = 4 for all groups. Data are presented as fold change relative to untransfected cells. ( IPF * c b β n

e

-stimulated fibroblasts. sEphrin-B2 then acts through EphB3/4 receptors to further drive and/or amplify profibrotic fibroblast effector ADAM10 ACTA2 = 5) and individuals with IPF ( , f advance online publication online advance ( ) Effect of pharmacological inhibition of ADAM10 with GI254023X (20 nM) on TGF- mRNA expression GI254023X DMSO α h 0.0 0.5 1.0 1.5 2.0 2.5 3.0 t SA expression -SMA ) mRNA expression in human lung fibroblasts. Nontargeting siRNA was used as control. mRNA expression was normalized to the -test. * Healthy markedly reduced sEphrin-B2 production production sEphrin-B2 reduced markedly sEphrin-B2 in BAL (ng/ml) through inhibition of ADAM10 in IPF lung 0.0 1.0 2.0 3.0 4.0 5.0 6.0 n

= 16) assessed by ELISA. Data are stratified into two cohorts obtained from separate institutions. ( Healthy IPF * P n Cohort < 0.05, ** = g 4 **

IPF n COL1A1 mRNA =

2 expression sEphrin-B2 in 5 0.0 1.0 2.0 3.0

n supernatants (ng/ml) ADAM10 = 30) and individuals with IPF ( 0.6 0.0 0.1 0.2 0.3 0.4 0.5 3

n Healthy P Fig. 6c Fig. 3 = 5) and individuals with IPF ( < 0.01. In n . We examined whether whether examined We . Healthy = 6) assessed by western blotting. ( * * * on sEphrin-B2 concentration in conditioned media. Nontargeting siRNA was used as a control. n k n vitro in * , = 5). mRNA expression was normalized to the level of GAPDH. ( IPF d sEphrin-B2 in plasma (ng/ml) * ), confirming that confirming ), 0.0 1.5 3.0 4.5 6.0 7.5 9.0 IPF *

j Healthy and si siRNA Nontargeting n ADAM10 =30 ( GI254023X DMSO a i. 6a Fig. k ) Concentration of sEphrin-B2 as determined by ELISA in conditioned media from primary n ** ADAM10 ADAM10 =30 , center lines, median values; +, the mean; box edges, 25th and 75th percentiles; IPF , b l ). ). d - h

ACTA2 mRNA

n sEphrin-B2 in g = 30) assessed by ELISA. All data are presented as mean n , Homeostasis expression supernatants (ng/ml) h Supplementary Figure 8 smoking controls (subject demographics in demographics controls smoking (subject non gender-matched, 30 and IPF with individuals 30 in centration ( volunteers 8 healthy from samples to compared IPF with individuals of 16 fluid showed a samples markedly increased concentration these of sEphrin-B2 of in the BAL subset a of blotting western by and ELISA by ples sam these in IPF sEphrin-B2 of with Quantification volunteers. individuals healthy and from samples plasma and fluid BAL both in of sEphrin-B2 concentration examined we next fibroblasts, lung IPF IPF fibroblasts compared to controls ( increased the inhibited markedly = 5). ( 0.0 1.0 2.0 3.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 ) Effect of siRNA-mediated knockdown of Considering the relevance of the ADAM10–sEphrin-B2 axis in in axis ADAM10–sEphrin-B2 the of relevance the Considering Healthy Healthy Quiescent fibroblast j ) Concentration of sEphrin-B2 in BAL fluids from control donors ( b * ) Relative mRNA expression of ADAM1 * *

IPF IPF 0 * Fig. 6i Fig. * Ephrin-B2 EphB si siRNA Nontargeting si Nontargeting siRNA ADAM10 ADAM10 3 , j EphB ). We then compared plasma sEphrin-B2 con sEphrin-B2 plasma compared then We ). i . ( ) Concentration of sEphrin-B2 in BAL fluid from 4 l ) Schematic of the working model of ADAM10- β -induced kDa α i 37 60 Fibrosis -SMA +myofibroblast COL1A1 TGF- 1 Activated c COL1A1 ADAM10 e Control BAL ) Effect of pharmacological β 2

Fig. 6e COL1A1 mRNA α 3 -SM ADAM1 ADAM10 expression A 0.0 1.0 2.0 3.0 4 + and 0 SDS-PAGE 5 ( in primary lung fibroblasts Supplementary Table Supplementary 1 – e

Autocrine Healthy signaling 6 ) and h k ). on TGF- ) Concentration of * 1 ACTA2 Collagen α * -SMA 2 Therapeutic target s e l c i t r a IPF BAL ACTA2 n ±

3 IPF = 3 for all groups. s.d and were EphB Ephrin-B2 * ADAM1 EphB3/ sEphrin-B2 4 β expression of expression 4 n 5 -induced Paracrine signaling Collagen EphB ( 4 α = 4 for 0 -SMA GI254023X DMSO 6 f ) mRNA 3 s sEphrin-B2

n

 - - - )

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. functions. Consequently, strategies to interrupt the elaboration of of elaboration the interrupt to strategies Consequently, functions. B2 in fibrotic tissues is active and biologically regulates myofibroblast ephrins. Our results demonstrate oligomeric that this of oligomeric state binding of sEphrin- by induced clustering receptor Eph signaling requires receptor EphB forward of Activation oligomers. -weight high-molecular active of form the is injury lung upon space alveolar activation. Importantly, myofibroblast our results demonstrate that sEphrin-B2 produced in directs the pathway ADAM10–sEphrin-B2 activation. myofibroblast fying sustained activation promotes organ fibrosis by its sustaining and and ampli injury, tissue by reactivated is pathway shedding ephrin-B2 pathway. Consistently, our results suggest such that the another ADAM10-mediatedbe may ADAM10–ephrin-B2 that indicate results our fibrosis lung of progression and impli pathogenesis been the in has cated pathways developmental multiple of reactivation The aberrant fibrogenesis. and tissue ADAM10 in development both by cleavage ephrin-B2 for mechanism conserved evolutionarily an during onstrated dem been recently has by ADAM10 cleavage Ephrin-B2 lung fibroblasts. human by sEphrin-B2 of generation the for responsible tease production. collagen and differentiation broblast myofi invasion, migration, including functions, fibroblast multiple in involved is which EphB3/4, through signaling forward ephrin-B2 demonstrate that the generation of sEphrin-B2 by fibroblasts induced studies Our fibrogenesis. tissue during occurs functions, profibrotic and activation myofibroblast amplifies of ephrin-B2 ectodomain the of shedding proteolytic which through mechanism, a regulatory new to implicate sEphrin-B2 in human fibrotic disease. Our results suggest in and shedding study fibrosis tissue is ephrin-B2 the to first describe ands regulates invasion teins pro active release to shedding ectodomain undergo receptors EphB investigations. future of focus the be will which fibrosis, tissue of development the during sEphrin-B2 of sources be also may cells, in both models, suggesting additional cell types, such as inflammatory skin and cin-induced lung we fibrosis, did protection full not observe of ephrin-B2 loss in fibroblasts providedAlthough substantial mediator. protection from bleomyprofibrotic important critically a as B2 fibrosis from lung and dermal protection and generation sEphrin-B2 decreased in resulted mice of fibroblasts in the of Conversely, ephrin-B2 loss activation. of that myofibroblast and collagen promoting by ously to sufficient induce fibrosis in dermal mice subcutane when injected onstrate that treatment with recombinant Ephrin-B2 was ectodomain suggesting IPF, ephrin-B2 shedding with is increased in response to tissue injury. individuals We of dem BAL the in and model pulmonary our fibrosis in mice of BAL the and tissue lung the in elevated markedly was sEphrin-B2, form, soluble This fibrosis. tissue of tor media potent a is ectodomain, of cleaved its of form composed soluble ephrin-B2, a that demonstrate we IPF, with individuals from obtained samples and clinical Using of models a mouse combination DISCUSSION the from samples ( group the control to compared IPF with individuals the in B2 sEphrin- plasma of concentration higher markedly a observed and A 1 potential the have ADAM10 targeting by fibroblasts by sEphrin-B2 0 We also identified EphB3/4 as the receptors through which the the which through receptors the as EphB3/4 identified also We metallopro major the as ADAM10 identified also study Our It has previously been demonstrated that both ephrin-B ligands and

s e l c i t r 19– 2 2 . . In cancer cells, shedding of membrane-bound ephrin-B lig Fig. 6 Fig. Xenopus k ). 3 4 . . To the best of our however,knowledge, our development α -SMA expression consistent with with consistent expression -SMA 2 in in vivo 0 , suggesting that there is is there that suggesting , , identifying ephrin- , identifying 35, 3 6 , and and , ------

B2 shedding and myofibroblast activation by TGF- by activation myofibroblast and shedding B2 ephrin- ADAM10-mediated in ephrin-B2 of region juxtamembrane the for requirement the confirmed mutants ephrin-B2 age-resistant cleav overexpressing studies our genetic data, these with Consistent (ref. A2 have ephrin- of regions juxtamembrane studies the cleaves ADAM10 that shown Previous fibroblasts. in cleavage ephrin-B2 mediates ADAM10 how understood fully not is it Currently, fibrosis. of ment tion of are which myofibroblasts, cells in effector the central develop by activation TGF- myofibroblast blocks shedding ephrin-B2 mediated ADAM10- of inhibition pharmacological or genetic that found we expression not of ephrin-B2 but itself, is induced by TGF-expression, ADAM10 fibroblast that found We activation. TGF- by acti activated is was pathway this that shedding determined we fibroblasts, in vated ephrin-B2 ADAM10-mediated which through fibrosis mitigate could ADAM10 of inhibition that evidence activation myofibroblast and shedding ephrin-B2 inhibiting by mice in fibrosis lung reduce substantially to we able was As GI254023X with ADAM10 diseases. of targeting fibrotic therapeutic for found, strategies therapeutic new as serve to concentration of sEphrin-B2 in plasma and in BAL from individuals individuals BAL in from and plasma in of sEphrin-B2 concentration an and elevated IPF with individuals from fibroblasts in upregulated signaling. reverse ephrin-B2 antifibrotic terminating by also but signaling forward ephrin-B2 profibrotic of promotion through activation myofibroblast promote only not may activation myofibroblast and transition study, of inhibition through pericyte-to-myofibroblast to our relevant most and, injury kidney during stability and vascular of angiogenesis promotion through rarefaction capillary peritubular against protecting fibrosis kidney of models mouse in role fibrotic anti an play to shown been recently has signaling ligand ephrin-B2 Reverse ephrin-B2. full-length bearing previously fibroblasts in B2 ephrin- of tail cytoplasmic the terminate through signaling reverse concomitantly ephrin-B2 would shedding function, ectodomain or ephrin-B2 activation myofibroblast regulate also may signaling needed. is myofibroblasts activated in signaling ephrin-B2 of understanding mechanistic ing receptors activation EphB3/4 of in involved be could fibrosis pathological in implicated by expressed in autocrine although ephrin-B2 sEphrin-B2, other cells activation of profibrotic forward EphB3/4 receptor signaling activated cis negative of the de-repression (a) promote could shedding ephrin-B2 shed ephrin-B2 ADAM10-mediated Consequently, myofibroblasts. in ding activated increased to lead would upregulation ADAM10 via ing signal receptor EphB3/4 forward of activation prevents fibroblasts quiescent in ephrin-B2 membrane-bound of expression high which human lung may fibroblasts through mechanism a suggest molecular ward receptor ephrin-B2–EphB4 ‘ signaling for activate to cells other in expressed ephrin-B2 of ability the ates ‘ (i.e., cell same the of surface the on interact receptor EphB4 and ephrin-B2 that shown have studies broblast activation by TGF- myofi in shedding ephrin-B2 ADAM10-mediated for requirement During our investigations into the molecular mechanism(s) mechanism(s) molecular the into investigations our During Our studies also show that ADAM10–sEphrin-B2 signaling is is signaling ADAM10–sEphrin-B2 that show also studies Our reverse ephrin-B2 whether address not does study our Although inhibitory ephrin-B2–EphB3/4 receptor interaction and/or (b) (b) and/or interaction receptor ephrin-B2–EphB3/4 inhibitory β , indicating that ephrin-B2 cleavage , is that for required indicating ephrin-B2 the genera cis 2 β negative interaction. Upon tissue injury, TGF- Upon tissue interaction. negative 2 , the prototypical cytokine that drives myofibroblast myofibroblast drives that cytokine prototypical the , ) and ephrin-B2 (ref. (ref. ephrin-B2 and ) advance online publication online advance in trans in 1 4 . Thus ephrin-B2 ectodomain shedding shedding ectodomain ephrin-B2 Thus . β warrants investigation. further Previous . Further research focused on gather on focused research Further . in cis’in 2 0 ) to release active ectodomains. ectodomains. active release to ) ), and this interaction attenu interaction this and ), in vivo in in trans , providing preclinical preclinical providing ,

β nature medicine nature signaling. Further, ’ 3 β 7 . The molecular molecular The . . . Our studies on β -mediated -mediated in vivo in ------.

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 7. 6. 5. 4. 3. 2. 1. claims jurisdictional in published maps and affiliations. institutional r Reprints and permissions information is available online at at online available is information permissions and Reprints The authors declare no competing interests.financial manuscript with the help of all the authors. the project experiments, supervised and took overall responsibility for writing the team during the course of this study. D.L., A.M.T. and M.K. the designed study related to ephrin biology. M.K. the designed original concept and led the entire characterization of mouse phenotype and troubleshooting with experiments studies in mouse samples. J.W., H.F., J.-P.P. and J.M.-P. were involved in the input on project design and troubleshooting. B.W. performed protein expression individuals with IPF and healthy controls. M.B. and providedR.G. intellectual provided human lung fibroblasts, plasma and bronchoalveolar lavage fluid from experiments in skin fibrosis model. M.S., A.P., S.B.M., K.E.B.R.K., and B.S.S. ADAM10 inhibitor. C.T. was involved in histological characterization of mouse –EphB3/4 pathway in fibroblasts. C.K.P. and A.F. performed performed and analyzed involved in the generation of experiments, analyzed the data and generated the figures. P.G.-K. and M.B. were D.L. most designed of the experiments, performed Research Foundation (A.M.T). the National Institutes of Health, HL108975 and a grant from the Scleroderma Marie A. Coyle Research Grant from the FoundationScleroderma (D.L.), and by FoundationSociety and FibrosisPulmonary Foundation Research Grant and the Foundation, University Health Network, Toronto (M.K.); an American Thoracic (M.K.); Campaign to Cure via the Arthritis Toronto andGeneral Western by University of Montreal Hospital Research Centre and University of Montreal breeding and genotyping. The authors gratefully acknowledge funding support from the University Health Network) for their technical assistance with mouse Authors would like to thank P. Datta, S. Nakamura, H. Endisha and J. Rockel (all o Note: Any Supplementary Information and Source Data files are available in the t the in available are references, and codes accession statements of including Methods, and data availability any associated M ( diseases of class this for targets therapeutic potential as ADAM10 sheddase its and EphB3/4 receptors its sEphrin-B2, identifies also study of Our diseases. pathogenesis fibrotic the in role central a plays it suggesting activation, EphB3/4. This pathway is required for TGF- through signals that sEphrin-B2 generate to by ADAM10 ephrin-B2 of cleavage through functions effector and activation myofibroblast disease. human in signaling sEphrin-B2 with IPF. These data further emphasize on the relevance of ADAM10– nature medicine nature C AUTH A e h n ckn O p

l ethods e In summary, we have identified a new pathway that promotes promotes that pathway new a identified have we summary, In i r MPETING FINANCIAL INTERESTS FINANCIAL MPETING n Renzoni, E.A. Renzoni, M. Selman, fibrosis. kidney in mechanisms molecular and Cellular J.S. Duffield, and patterns fibrosis: Mercer,P.F. Pulmonary injury,& repair,and epithelial R.C. alveolar Chambers, of Mechanisms D. Jiang, & C.E. Barkauskas, P.W., Noble, for translation therapeutic fibrosis: of T.R.Mechanisms Ramalingam, T.A. Wynn,& of component lethal Fibrosis—a M. Kapoor, & A.M. Tager, D., Y.Y., Lagares, Ho, lung fibroblasts. lung pattern. expression gene and behavior 124 fibrosis. perpetrators. disease. fibrotic sclerosis. systemic

i p n e t

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Nat. Med. Nat. Respir. Res. Respir. p a Accelerated variant of idiopathic pulmonary fibrosis: clinical fibrosis: pulmonary idiopathic of variant Accelerated Nat. Rev. Rheumatol. Rev. Nat. p

Gene expression profiling reveals novel TGF novel reveals profiling expression Gene e r in vitro advance online publication online advance . O NS Ephrinb2

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. were then euthanized, and full-thickness 6-mm punch biopsies were obtained to stock) for 28 d as previously described (100 bleomycin of injection subcutaneous daily by induced fibrosis. skin of model Mouse daily throughout the course of the study. Drug dosing began 3 d before bleomycin or 100 PBS of treatment andvolume was maintainedtotal a with injection intraperitoneal by daily once weight body kg per mg 200 administered were mice and buffer carbonate M 0.1 in collagen determination and biochemical analyses. the indicated time points. Lungs and BAL were harvested for histological studies,weight) or PBS as control as previously described induced by intratracheal administration of bleomycin (50 was fibrosis Lung trachea. the of exposure before xylazine and ketamine with fibrosis. pulmonary of model Mouse Massachusetts HospitalGeneral Subcommittee on Research Animal Care. committee of the Krembil Research Institute (Universitycare Health Network) and animal (CRCHUM), Centre Research Hospital Montreal of University de protection des animaux (Institutional Animal Protection Committee) of the All animal procedures and protocols were approved by the antibody. anti-ephrin-B2 Comité using blotting western by Institutionnelconfirmed was fibroblasts 7 d. loss ofSuccessful expression ephrin-B2 upon tamoxifen treatment in lung pension (0.1 ml of 10 mg/ml) to delete Cre Ephrinb2 for primers specific using performed was were generated.Genotyping the for hemizygous and crossed with ( Cre-recombinase inducible ( fibroblasts as such cells collagen-expressing in specifically deleted conditionally be could ephrin-B2 Generation of ephrin-B2 conditional-knockout mice. Laboratory Animal Care (AAALAC). (SPF) environment by Association American the for Accreditationcertified of Research Animal Care, and all mice were maintained in a specific pathogen–free tocols were approved by the Massachusetts General Hospital Subcommittee on performed in accordance with National Institute of Health guidelines and pro study. were throughoutthis experiments MD,were used (Frederick, All USA) from National the chased Institute Cancer Frederick(NCI) Mouse Repository Animals. plasma infection. cultured according to the vendor’s protocol. Cell cultures were tested for myco lines. Cell R&D. from purchased was protein TGF- mouse and human Recombinant Sigma-Aldrich. from purchased bol 12-myristate 13-acetate (PMA), 4-hydroxytamoxifen and were fibronectin phor as well as TAPI-0, and GI254023X (Batimastat), BB-94 including tors, inhibi Pharmacological Signaling). Cell HA-tag(C29F4, monoclonal rabbit and Signaling) Cell (#14194, ADAM10 Sigma), HPA008999 and Cruz Santa monoclonal mouse Signaling), Cell D16H11, (clone GAPDH monoclonal rabbit Abcam), (ab34710, I type lagen mouse include: anti- blotting from polyclonal western purchased for used was LPA Antibodies 18:1 Lipids. Polar and Avanti Systems, ephrin-B3 R&D and from ephrin-B2 purchased were ephrin-B1, ephrin-A5, ephrin-A4, ephrin-A2, ephrin-A3, ephrin-A1, PDGF-BB, recombinant human and Mouse USA). of Health, Institutes National Institute, Cancer (National by I.O. Daar shared EphrinB2 WT full-length Plasmids, antibodies and reagents. ONLINE METHODS nature medicine nature conduct histological, immunohistochemical and hydroxyproline analysis. For ERT in vivo mice weremice treated with intraperitoneal injections of tamoxifenthe sus ( ∆ ahgnfe fml C7L6 (- o -ekod mc pur mice 8-week-old) to (6- C57BL/6N female Pathogen-free Human lung fibroblasts (IMR-90) were purchased from ATCC and Juxta-HA Supplementary Table 2 Table Supplementary Efnb2 drug studies with the ADAM10 inhibitor, GI254023X was diluted α EphrinB2 -SMA (clone 1A4; Sigma-Aldrich), rabbit polyclonal col polyclonal rabbit Sigma-Aldrich), 1A4; (clone -SMA , , loxP/loxP EphrinB2 Col1a2 mice Ephrinb2 and the HA-tagged mutants mutants HA-tagged the and ∆ 182–194-HA Col1a2 1 -Cre 7 Skin fibrosis in 6- to 8-week-old mice was was mice 8-week-old to 6- in fibrosis Skin . . Mice homozygous for the floxed -CKO mice), mice carrying a tamoxifen- a carrying mice mice), -CKO Ephrin-B2 plasmids, including HA-tagged ). Three-week-old Three-week-old ). β ERT 2 -actin (AC-15, Sigma), ephrin-B2 (P-20 ephrin-B2 Sigma), (AC-15, -actin Ephrinb2 7 -Cre . Sterile saline was used as control. Mice Six-week-old mice were anesthetized anesthetized were mice Six-week-old allele ( allele and ERT EphrinB2 ) (Jackson Laboratory) were were Laboratory) (Jackson ) or with corn oil (as control) for Efnb2 38, 3 9 . . Mice were euthanized at To generate mice in which loxP/loxP ∆ Efnb2 197–218-HA µ l at 1.2 U per kg body EphrinB2 µ ; ; loxP/loxP l from 10 10 from l Col1a2 were kindly were kindly Efnb2 ∆ ; ; -Cre Ecto-HA Col1a2 cre µ allele g/ml g/ml and ERT β µ -1 -1 , l. ------)

standard protocols of our laboratory as previously described Masson’sor H&E with the stained wereto according skin trichrome and lung 5- paraffin-embedded multiple and formalin Histological analysis. on Research Animal Care. (University Health Research Network) Hospital and Massachusetts Montreal General Hospital of Subcommitteecare (CRCHUM),Centre animal committee Krembil of the Institute Research University the of Committee) Protection Institutionnelanimaux(Institutional Comité protectiondes Animal by the de and biochemical analyses. All animal procedures and protocols were determination approved collagen studies, histological for collected were samples skin for housed andby weeks 2 euthanized CO weremice further ephrin-B2-Fc, or IgG-Fc either with treatment two-week Following weeks. 2 daily for 2 weeks. Control mice received subcutaneous injections of IgG-Fc for body weight per mouse) into a single location on the shaved back of mice once 100 for bleomycin-induced model of skin fibrosis binant ephrin-B2-Fc were performed using the methodology previously reportedIn vivo ImmunoResearch) for 90 min at 4 °C before immediate use. incubated at a 2:1 ratio (wt/wt) with a goat antibodywas systems) against (R&D ephrin-B2-Fc humanhuman or IgG mouse recombinant(Jackson complexes, Generation of ephrin-B2 immune complexes. serum-free DMEM were then added to the apical chambers and exposed to BAL with DiIC12(3) fluorescent dye for 1 h before chemotaxis. cells in 50,000 50 10 at fibronectin with precoated using a 96-Multiwell FluoroBlok Inserting Fibroblast chemotaxis system and invasion assay. (Fisher Scientific; pore size: 8 isolated as previously described fibroblasts. lung mouse primary of Isolation sion assays. Ephrin-B2-depleted BALs were used as chemoattractants in migration and inva bation of overnight BALantibody–coated beads at with ephrin-B2 fluids 4 °C. PBS before resuspension in PBS. Ephrin-B2 was immunoprecipitated by incu col. Beads were washed with PBS and then blocked for 1 h in 2% (w/v) BSA and M-270 Epoxy beads (Life Technologies) according to the manufacturer’s proto beads. (1–2 antibody-coated ephrin-B2 of Preparation Science Inc., SEE112Hu, SEE112Mu) according toand the mousemanufacturer’s BAL fluids and protocol. in human plasma wereDetermination determined by ELISAof (Uscnephrin-B2 Life levels in BAL and plasma. (Pierce) per manufacturer’sKit protocol. Assay Protein (BCA) acid bicinchoninic available commercially a using BAL total protein. Eppendorf tubes for(PGC Scientifics) subsequent analysis. for at min 20 and4 °C supernatants transferred the low-binding to siliconized lavaged with six 0.5-ml aliquots of PBS. BAL samples were centrifuged at 3,000 sis, ephrin-B2 ELISA and total protein concentration determination, lungs were Mouse BAL recovery. using hydroxyproline assay as previously described Hydroxyproline assay. a blinded fashion. shownpreviously as section per fields junction at five randomly selected sites per high-power field in ten high-power uring the distance between the epidermal–dermal junction and the dermal–fat use of photomicrographs (100× magnification) of H&E-stained sections, meas measurement. thickness Dermal µ µ l subcutaneous injections of preclustered ephrin-B2-Fc (100 (100 ephrin-B2-Fc preclustered of injections subcutaneous l g) g) was chemically cross-linked to 2.8 ephrin-B2-Fc injection. Total protein concentration in BAL samples was determined To obtain BAL samples for chemoattractant activity analy Excised skin and lung biopsies were fixed in 10% buffered Collagen content in mouse lungs and skin was measured 3 Subcutaneous injections using mouse recom 8 . Dermal thickness was determined with the the with determined was thickness Dermal µ g/ml (Sigma). Briefly, cells were labeled labeled were cells Briefly, (Sigma). g/ml 2 7 . All investigations were performed in in investigationsperformed were All . µ Fibroblast chemotaxis was assayed m m superparamagnetic Dynabeads 4 µ 0 . . 6- to 8-week-old mice received To generate ephrin-B2 immune m sections of the entire mouse mouse entire the of sections m Primary lung fibroblasts were fibroblasts lung Primary 27, sEphrin-B2 levels in human 38 , 3 Ephrin-B2 antibody antibody Ephrin-B2 9 . doi: 27, 2 10.1038/nm.4419 euthanasia, and euthanasia, 3 9 . µ g per kg kg per g µ m) µ g ------l

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. to 1:400 and 1:200, respectively, in blocking solution. Samples were washed washed were Samples solution. blocking in respectively, 1:200, and 1:400 to diluted Scientific) Fisher (Thermo antibodies phalloidin rhodamine and 488 PBS and with forincubated times one hour goat with anti–mouse Fluor Alexa solution). in blocking 1:100 The next day,Aldrich; samples were three washed against antibody primary a with °C at overnight4 hour in blocking solution (10% goat serum; 0.1% triton-X in PBS) andforone blocked PBS, with times incubatedthree washed werethen cells The min. 10–15 TGF- or nM) (20 GI254023X with analysis. Immunofluorescence GE Biosciences, Healthcare). alized by enhanced chemiluminescence and autoradiography (ECL; Amersham with appropriate secondary, HRP-linked antibodies (Pierce). incubated were Proteinsmembranes washing, After were antibodies. visu primary indicated the membranes werein andTBS incubated milk with 5% blocked nonfatwith dry and (Invitrogen), membranes (PDVF) difluoride polyvinylidene onto ferred System or 4–12% SDS-Tris-Glycine Protein Gel. Separated proteins were trans NativePAGE either on separated Proteinwas (Pierce). assay Bis-TrisBCA Gel trifugation (6,000 Inhibitor Cocktail (Thermo ProteinScientific). wereextracts subjected to cen Phosphatase and Protease Halt with supplemented and Scientific) (Thermo Westernanalysis. blot previously described as expressed are controls with values ( tration of Relative nM. 100 transcript abundance of a gene is expressed in in listed primers the using formed GAPDH utpe Qatttv PR ytm Srtgn) s rvosy described previously as (Stratagene) System PCR Quantitative Multiplex reported reverse transcriptioncDNA using iScript Synthesis as previously Kit (BioRad) by generated werecDNAs manufacturer’s and protocol, the to according kits qRT-PCR. and RT-PCR 20 microscopic fields on 25–50 cells per conditions. Filopodia formation. using (60–70% confluency) Lipofectamine 2000 (Thermo Fisher Scientific). formed on humanprimary and mouse lung fibroblastson seeded 6-well plates Transientfection. mutants with ephrin-B2 experiments transfection were per control.and mRNAtrans after were h 48 harvested were Cells levels assessed nonspecific a On-Target as the from used were nontargetingPlussiRNA pool HiPerFect Reagent (Qiagen) at a siRNA/HiPerFect ratio of 1:4 ( using fibroblasts primary mouse humanandinto transfected transiently were were obtained from Dharmacon Inc. (Thermo Scientific). The siRNAs (20 nM) mouse ADAM19 ADAM9 human B2, siRNA and plasmid transfection. EPHB2 test the effect of receptor inhibition, fibroblasts were transfected with siRNA to at for to3 were5 passages Cells h. chemotaxisused 48 and invasion assays. To at read platewas the Tumorand used, was Invasion Scientific) (FisherSystem Experiments were performed in triplicate. For invasion assays, a 96-well BioCoat cells toward any chemoattractant to cells migrated toward DMEM. serum-free Fluoroskan using Ascent reader FLplate (Thermo). fluorescent bottom-reading The a data on were (Ex/Em) expressed nm 544/590 as at the ratio of migrated CO 5% a in °C 37 at h 4 for incubated then was plate The chemoattractants. as Systems) (R&D PDGF-BB orLPA (Avanti Lipids) 18:1 Polar ephrin-B2-Fc, fractions, BAL or doi: 10.1038/nm.4419 Ephb3 , , ∆ was used as reference gene in all qRT-PCR reactions. PCR was per was PCR qRT-PCRreactions. all in gene reference as used was EPHB3 , human human , 3 Ct = Ct 9 , human , . qPCR was performed using fluorogenic SYBR Green and Mx4000 Mx4000 and SYBRGreen fluorogenic using performed was qPCR . MMP1 and mouse mouse and or reference g 2 ) ) at 4 °C, and protein concentrations were determined using ADAM10 EPHB4 EPHB3 , human , atmosphere. Fluorescence of migrated cells was recorded was cells migrated of Fluorescence atmosphere. 3 Filopodial protrusions at the cell front were analyzed from 9 . – Ct Cells and tissues were harvested, lysed in RIPA in buffer lysed harvested, were tissues and Cells Total RNA was extracted using Qiagen extraction extraction Qiagen using extracted was RNA Total Ephb4 before addition of BAL as a chemoattractant. , human , target MMP2 , human human , ∆ ). Relative changes in transcript levels compared mRNA wereOn-Target Pools andPlusSmart Cells were plated on glass coverslips, treated treated coverslips, glass on plated were Cells ∆ The siRNA duplexes targeting human ephrin- Ct values ( values Ct β Supplementary Table 2 Supplementary , human , EPHB4 as described and fixed with 4% PFAfor 4% with fixed and described as ADAM12 , mouse ephrin-B2, mouse mouse ephrin-B2, mouse , MMP7 ∆ ∆ Ct = = Ct , human human , α , human , -SMA (clone 1A4, Sigma- 1A4, (clone -SMA ∆ Ct treated ADAM17 at a final concen final a at MMP14 – – µ g/ ∆ µ Ct l). l). siRNAs , human human , control , human , Ephb2 ∆ ) as as ) 3 Ct 9 ------, . approved by the INER Ethics Committee and the MGH Institutional Review Review Institutional MGH the and described Committee Ethics INER the byapproved previously as (MGH), Hospital General Nacional de Enfermedades Respiratorias (INER), Mexico and the Massachusetts fiber-optic bronchoscopy. Bronchoscopies were performed at both the Instituto volunteer donors were recovered after instillation of sterile 0.9%samples. BALsaline Human by flexible and immediately frozen and stored at −80 °C until analysis. fuged at 1,500 CTAD (citrate-theophylline-adenosine-dipyridamole). Whole blood was centri informed consent. was obtainedvenipuncture via Blood into tubes containing obtained through the Partners Institutional Review Board. All subjects provided was collection Approval plasma for disease. lung chronic of history a without to recruit controls. Controls were at least 50 years of age and were nonsmokers Partners Healthcare System Research Study Volunteer Program (RSVP)Association was used Society Respiratory Japanese (ERS), Society (ATS), EuropeanRespiratory Society Thoracic American statementthe sus of consen joint recent 2011 the on based criteria diagnostic IPF satisfy to had subjects IPF inclusion, study For Hospital. General Massachusetts the at care Human plasma samples. confocal microscope (Zeiss). LSM780 Zeiss a with DAPI(Vectorlabs). acquired with Mediumwere Images MountingVECTASHIELD Antifade with mounted and PBS with times three 41. 40. 39. 38. sonable request. A Data availability. reported as mean are Data distribution. normal a displayed data All groups. between different cally significant. * P The 5.0. Software GraphPad Prism using times survival in differences for test log-rank ANOVAor two-way conditions, effects mental condition overall for analyzed by unpaired Student ofweight body equal and genotype before treatments.data were Experimental No animals were excluded from analysis. Animals were distributed into groups inhibitor compared with WT control mice, accepting a type I error rate of 0.05. amount of fibrosis present in the in reduction 50% a detect to power 80% an on based estimated were sizes Sample mice. of response fibrotic the in variability inherent the for account and lungdermal fibrosis produced mice, in of extent the on inhibition ADAM10 or fibroblasts in ephrin-B2 of depletion basis of our previous studies. In each experiment evaluating the effects of geneticanalysis. Statistical at −70 °C until use. low-binding Eppendorf tubes, and supernatants from both institutions were natantskept that were collected at MGH were immediately transferred to siliconized super BAL participants. all from obtained was consent informed and Board, values obtainedvalues are and indicatedstatistilegends figures in the figure when

ah, G. Raghu, Kapoor, M. D. Lagares, Tager,A.M. Crit. Care Med. Care Crit. management. and diagnosis for guidelines evidence-based fibrosis: skinfibrosis. Rheum. Arthritis tobleomycin-induced susceptibility in increased results fibroblasts formation. myofibroblast and fibrosis (2008). 45–54 to lung injury by mediating fibroblast recruitment and vascular leak. vascular and recruitment fibroblast mediating by injury lung to 4 1 as determined by two investigatorsby two determined as et al. t al. et g et al. et t al. et for 15 min to obtain plasma, which was then placed in aliquots

Data are available from the corresponding authors upon rea P ± Loss of peroxisome proliferator-activated receptor gamma in mouse 183

An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary idiopathic statement: ATS/ERS/JRS/ALAT official An The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis LPA1pulmonary receptor links acid lysophosphatidic The Life Sciences Reporting Summary for Reporting Summary Life Sciences < 0.05, ** s.d. 60 niiin f oa ahso kns peet eprmna lung experimental prevents kinase adhesion focal of Inhibition Sample sizes were determined by power analysis on the the on analysis power by determined were sizes Sample , 2822–2829 (2009). 2822–2829 , , 788–824 (2011). 788–824 , BAL samples from individuals with IPF and healthy healthy and IPF with individuals from samples BAL Subjects with IPF were identified from those receiving Ephrinb2 P ′ s < 0.01, *** t -test for differences between each of the experi -CKO mice or mice treated with ADAM10 Arthritis Rheum. Arthritis P < 0.001 was considered significantly n 4

1 ≥ and Latin American Thoracic Thoracic American Latin and 8 mice per group per 8 mice wereto used 4 1 (

Supplementary Table Supplementary 1 64 3 8 , 1653–1664 (2012). 1653–1664 , . These studies were were studies These . nature medicine nature is available. Am. J. Respir. J. Am. Nat. Med. Nat.

14 ). ). ------,

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 nature research | life sciences reporting summary a. State the source of each eukaryotic cell line used. Human lung fibroblasts (IMR-90) were purchased from ATCC and cultured according to vendor’s protocol.

b. Describe the method of cell line authentication used. Cell lines were authenticated by the ATCC.

c. Report whether the cell lines were tested for Cell lines were not tested for mycoplasma contamination. mycoplasma contamination.

d. If any of the cell lines used are listed in the database No commonly misidentified cell lines were used. 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 Adult sex- and age-matched C57BL/6 mice at 6-8 weeks of age were purchased materials used in the study. 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 pro2(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.

Policy information about studies involving human research participants 12. Description of human research participants Describe the covariate-relevant population Human BAL samples. We recovered BAL samples from individuals with IPF and characteristics of the human research participants. 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 Nature Medicine: doi:10.1038/nm.4419