title: Netrin-1 Regulates Fibrocyte Accumulation in the Decellularized Fibrotic Sclerodermatous Lung Microenvironment and in Bleomycin-Induced Pulmonary Fibrosis.
abstract: Fibrocytes are collagen-producing leukocytes that accumulate in patients with systemic sclerosis (SSc; scleroderma)-related interstitial lung disease (ILD) via unknown mechanisms that have been associated with altered expression of neuroimmune proteins. The extracellular matrix (ECM) influences cellular phenotypes. However, a relationship between the lung ECM and fibrocytes in SSc has not been explored. The aim of this study was to use a novel translational platform based on decellularized human lungs to determine whether the lung ECM of patients with scleroderma controls the development of fibrocytes from peripheral blood mononuclear cells.
  
  
  
  We performed biomechanical evaluation of decellularized scaffolds prepared from lung explants from healthy control subjects and patients with scleroderma, using tensile testing and biochemical and proteomic analysis. Cells obtained from healthy controls and patients with SSc-related ILD were cultured on these scaffolds, and CD45+pro-ColIα1+ cells meeting the criteria for fibrocytes were quantified. The contribution of the neuromolecule netrin-1 to fibrosis was assessed using neutralizing antibodies in this system and by administering bleomycin via inhalation to netrin-1(+/-) mice.
  
  
  
  Compared with control lung scaffolds, lung scaffolds from patients with SSc-related ILD showed aberrant anatomy, enhanced stiffness, and abnormal ECM composition. Culture of control cells in lung scaffolds from patients with SSc-related ILD increased production of pro-ColIα1+ cells, which was stimulated by enhanced stiffness and abnormal ECM composition. Cells from patients with SSc-related ILD demonstrated increased pro-ColIα1 responsiveness to lung scaffolds from scleroderma patients but not enhanced stiffness. Enhanced detection of netrin-1-expressing CD14(low) cells in patients with SSc-related ILD was observed, and antibody-mediated netrin-1 neutralization attenuated detection of CD45+pro-ColIα1+ cells in all settings. Netrin-1(+/-) mice were protected against bleomycin-induced lung fibrosis and fibrocyte accumulation.
  
  
  
  Factors present in the lung matrices of patients with scleroderma regulate fibrocyte accumulation via a netrin-1-dependent pathway. Netrin-1 regulates bleomycin-induced pulmonary fibrosis in mice. Netrin-1 might be a novel therapeutic target in SSc-related ILD.
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Huanxing
authors.initials: H
authors.lastname: Sun
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Hongyi
authors.initials: H
authors.lastname: Pan
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Xiaosong
authors.initials: X
authors.lastname: Chen
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Jenna L
authors.initials: JL
authors.lastname: Balestrini
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: TuKiet T
authors.initials: TT
authors.lastname: Lam
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Jean E
authors.initials: JE
authors.lastname: Kanyo
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Anne
authors.initials: A
authors.lastname: Eichmann
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Mridu
authors.initials: M
authors.lastname: Gulati
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Wassim H
authors.initials: WH
authors.lastname: Fares
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Hanwen
authors.initials: H
authors.lastname: Bai
authors.affiliation: Medical University of South Carolina, Charleston.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Feghali-Bostwick
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Ye
authors.initials: Y
authors.lastname: Gan
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Xueyan
authors.initials: X
authors.lastname: Peng
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Meagan W
authors.initials: MW
authors.lastname: Moore
authors.affiliation: University of Michigan, Ann Arbor.
authors.forename: Eric S
authors.initials: ES
authors.lastname: White
authors.affiliation: Yale University School of Engineering, New Haven, Connecticut.
authors.forename: Parid
authors.initials: P
authors.lastname: Sava
authors.affiliation: Yale University School of Engineering, New Haven, Connecticut.
authors.forename: Anjelica L
authors.initials: AL
authors.lastname: Gonzalez
authors.affiliation: Yale University Program of Computational Biology and Bioinformatics, New Haven, Connecticut.
authors.forename: Yuwei
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Niklason
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Erica L
authors.initials: EL
authors.lastname: Herzog
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D001696:Biomechanical Phenomena; D001761:Bleomycin / Q000633:toxicity; D016022:Case-Control Studies; D002454:Cell Differentiation; D003094:Collagen / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D005355:Fibrosis; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006579:Heterozygote; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D007963:Leukocytes, Mononuclear; D008168:Lung / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D017563:Lung Diseases, Interstitial / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D008855:Microscopy, Electron, Scanning; D009414:Nerve Growth Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000075388:Netrin-1; D040901:Proteomics; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012595:Scleroderma, Systemic / Q000150:complications / Q000378:metabolism*; D054457:Tissue Scaffolds; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000903:Antibiotics, Antineoplastic; D057134:Antibodies, Neutralizing; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; C000617351:NTN1 protein, human; D009414:Nerve Growth Factors; C000617353:Ntn1 protein, mouse; D025521:Tumor Suppressor Proteins; D001761:Bleomycin; D000075388:Netrin-1; D003094:Collagen; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human