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Loss of Twist1 in the Mesenchymal Compartment Promotes Increased Loss of Twist1 in the Mesenchymal Compartment Promotes Increased Fibrosis in Experimental Lung Injury by Enhanced Expression of CXCL12 This information is current as of October 2, 2021. Jiangning Tan, John R. Tedrow, Mehdi Nouraie, Justin A. Dutta, David T. Miller, Xiaoyun Li, Shibing Yu, Yanxia Chu, Brenda Juan-Guardela, Naftali Kaminski, Kritika Ramani, Partha S. Biswas, Yingze Zhang and Daniel J. Kass J Immunol 2017; 198:2269-2285; Prepublished online 8 Downloaded from February 2017; doi: 10.4049/jimmunol.1600610 http://www.jimmunol.org/content/198/6/2269 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2017/02/07/jimmunol.160061 Material 0.DCSupplemental References This article cites 65 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/198/6/2269.full#ref-list-1 Why The JI? Submit online. by guest on October 2, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Author Choice Freely available online through The Journal of Immunology Author Choice option Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Loss of Twist1 in the Mesenchymal Compartment Promotes Increased Fibrosis in Experimental Lung Injury by Enhanced Expression of CXCL12 Jiangning Tan,* John R. Tedrow,* Mehdi Nouraie,* Justin A. Dutta,* David T. Miller,* Xiaoyun Li,* Shibing Yu,* Yanxia Chu,* Brenda Juan-Guardela,† Naftali Kaminski,† Kritika Ramani,‡ Partha S. Biswas,‡ Yingze Zhang,* and Daniel J. Kass* Idiopathic pulmonary fibrosis (IPF) is a disease characterized by the accumulation of apoptosis-resistant fibroblasts in the lung. We have previously shown that high expression of the transcription factor Twist1 may explain this prosurvival phenotype in vitro. However, this observation has never been tested in vivo. We found that loss of Twist1 in COL1A2+ cells led to increased fibrosis characterized by very significant accumulation of T cells and bone marrow–derived matrix-producing cells. We found that Twist1- Downloaded from null cells expressed high levels of the T cell chemoattractant CXCL12. In vitro, we found that the loss of Twist1 in IPF lung fibroblasts increased expression of CXCL12 downstream of increased expression of the noncanonical NF-kB transcription factor RelB. Finally, blockade of CXCL12 with AMD3100 attenuated the exaggerated fibrosis observed in Twist1-null mice. Tran- scriptomic analysis of 134 IPF patients revealed that low expression of Twist1 was characterized by enrichment of T cell pathways. In conclusion, loss of Twist1 in collagen-producing cells led to increased bleomycin-induced pulmonary fibrosis, which is mediated by increased expression of CXCL12. Twist1 expression is associated with dysregulation of T cells in IPF patients. Twist1 may http://www.jimmunol.org/ shape the IPF phenotype and regulate inflammation in fibrotic lung injury. The Journal of Immunology, 2017, 198: 2269–2285. ne theory of the pathogenesis of idiopathic pulmonary proapoptotic stimuli compared with normal human fibroblasts fibrosis (IPF) suggests that a fibroblast of a very distinct (4). Recent data suggest that signals derived from the extracel- O phenotype, following an unknown injury, accumulates in lular matrix (5) drive the so-called IPF phenotype. Persistence of the lung—a fibroblast that paradoxically demonstrates little apo- fibroblasts in fibrotic lungs may be due to soluble survival factors ptosis (1) despite a microenvironment in the lung that is hostile to (6) or may result from cell-autonomous prosurvival signaling, cell survival (2, 3). Evidence for this theory comes from ex vivo possibly through Akt-related pathways (7, 8). We have previ- fibroblasts derived from IPF lungs that demonstrate resistance to ously shown that the transcription factor Twist1 is one of the most by guest on October 2, 2021 highly expressed transcription factors by gene expression profiling of *Dorothy P. and Richard P. Simmons Center for Interstitial Lung Disease and the IPF lungs and may mediate the pathologic prosurvival phenotype of Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA 15213; †Section of Pulmonary, Critical Care, and Sleep Medicine, fibroblasts in vitro (3). However, in vivo data demonstrating the rel- Department of Internal Medicine, Yale University, New Haven, CT 06520; and evance of Twist1 to human or experimental pulmonary fibrosis have ‡Division of Rheumatology and Clinical Immunology, Department of Medicine, been lacking. University of Pittsburgh, Pittsburgh, PA 15261 Twist1 is a member of the basic helix–loop–helix family of ORCIDs: 0000-0002-4236-4430 (J.R.T.); 0000-0002-7223-3925 (D.T.M.); 0000- 0001-6947-2901 (Y.Z.); 0000-0001-6597-7830 (D.J.K.). transcription factors and is critical for mesoderm differentiation in Drosophila (9). Twist1 mutations in humans lead to Saethre– Received for publication April 7, 2016. Accepted for publication January 12, 2017. Chotzen syndrome (10), and Twist1 knockout mice die in utero due This work was supported by the Dorothy P. and Richard P. Simmons Center for Interstitial Lung Disease as funded by National Institutes of Health Grants to incomplete neural tube closure (11). Twist1 has multiple func- R01HL095397 and RC2HL101715 (to N.K.), DK104680-01A1 (to P.S.B.), and tions that may be relevant to the pathogenesis of pulmonary fibrosis, R01HL126990 (to D.J.K.). The Human Airway Cell and Tissue Core (supported in part by National Institutes of Health Grant P30 DK072506 and by a Cystic Fibrosis including inhibition of NF-kB inflammatory cells (12, 13). In Foundation research development program grant) provided human lung fibroblasts. cancer, Twist1 expression is a potent prosurvival factor portending a J.T., J.A.D., Y.C., B.J.-G., D.T.M., S.Y., X.L., and K.R. performed experiments; J.T., poor prognosis (14) and contributes to the resistance of cancers to J.R.T., M.N., N.K., P.S.B., Y.Z., and D.J.K. designed the experiments and analyzed chemotherapy-induced apoptosis (14). Related to the cancer phe- data; and J.T., J.R.T., Y.Z., and D.J.K. wrote the manuscript. notype, and perhaps most relevant to fibrosis is the role of Twist1 in Address correspondence and reprint requests to Dr. Daniel J. Kass, University of Pittsburgh, 3459 Fifth Avenue, MUH NW628, Pittsburgh, PA 15213. E-mail address: epithelial–mesenchymal transition (15, 16), a process that has been [email protected] suggested as a source of matrix-producing cells in fibrosis. We The online version of this article contains supplemental material. hypothesized that a strategy to silence expression of Twist1 in fi- Abbreviations used in this article: BAL, bronchoalveolar lavage; CAF, cancer- broblasts would block fibrosis by counteracting the prosurvival associated fibroblast; ChIP, chromatin immunoprecipitation; DLCO, diffusing capac- phenotype and thus promote a fibroblast that is susceptible to ap- ity for carbon monoxide; FVC, forced vital capacity; IPF, idiopathic pulmonary fibrosis; LGRC, Lung Genomics Research Consortium; MIF, macrophage migration optosis. For this study, we tested the hypothesis that transgenic mice inhibitory factor; siRNA, short interfering RNA; a-SMA, a-smooth muscle actin; with loss of Twist1 in the mesenchymal compartment would be Twist1 FL, Twist1 locus flanked by loxP sites; Twist1 WT, wild-type control Twist1. protected from experimental lung fibrosis. These animals, in the This article is distributed under The American Association of Immunologists, Inc., presence of tamoxifen, are engineered to express Cre recombinase Reuse Terms and Conditions for Author Choice articles. in collagen-expressing cells (COL1A2) (17) and to excise the Twist1 Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 locus flanked by loxP sites (Twist1 FL) (18). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600610 2270 Twist1 AND PULMONARY FIBROSIS Materials and Methods for a-SMA and surfactant protein C (Seven Hills Bioreagents, Cincinnati, Ethical statement OH) and CD45 (eBioscience, San Diego, CA). Cy5-conjugated secondary Abs were from Jackson ImmunoResearch Laboratories. Tissue was This work was approved by the Institutional Review Board and the In- mounted with DAPI and anti-fade solution. stitutional Animal Care and Use Committee of the University of Pittsburgh. Sircol assay for acid-soluble collagen Reagents Mouse lungs were perfused through the right ventricle with PBS. The left mAb against Twist (clone 2C1a) was purchased from Abcam (Cambridge, lungs were excised and flash-frozen in liquid nitrogen, followed by MA). Abs to RelB (clone C1E4) and RelA (clone C22B4) were from Cell lyophilization in preparation for collagen determination by the Sircol Signaling Technology (Danvers, MA). Anti-CXCL12 (clone K15C) was collagen assay. Lungs were homogenized in 0.5 M acetic acid with protease
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