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The Transferrin Receptor CD71 Delineates Functionally Distinct Airway Macrophage Subsets During Idiopathic Pulmonary Fibrosis Sarah J

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The Transferrin Receptor CD71 Delineates Functionally Distinct Airway Macrophage Subsets During Idiopathic Pulmonary Fibrosis Sarah J &get_box_var;ORIGINAL ARTICLE The Transferrin Receptor CD71 Delineates Functionally Distinct Airway Macrophage Subsets during Idiopathic Pulmonary Fibrosis Sarah J. Allden1,2, Patricia P. Ogger1, Poonam Ghai1, Peter McErlean1, Richard Hewitt1,3, Richard Toshner1,3, Simone A. Walker1, Peter Saunders3, Shaun Kingston3, Philip L. Molyneaux1,3, Toby M. Maher1,3*‡, Clare M. Lloyd1*, and Adam J. Byrne1* 1Inflammation, Repair, and Development Section, National Heart and Lung Institute, Faculty of Medicine, Imperial College, London, United Kingdom; 2UCB Celltech, Slough, United Kingdom; and 3NIHR Respiratory Biomedical Research Unit, Royal Brompton Hospital, London, United Kingdom ORCID IDs: 0000-0003-4199-3355 (P.M.); 0000-0003-4294-0052 (R.H.); 0000-0001-9299-0299 (P.S.); 0000-0003-1301-8800 (P.L.M.); 0000-0001-7192-9149 (T.M.M.); 0000-0001-8977-6726 (C.M.L.); 0000-0003-2736-8174 (A.J.B.). 2 Abstract were enhanced in IPF-BAL, and furthermore, CD71 AMs 1 had an impaired ability to sequester transferrin. CD71 and 2 Rationale: Idiopathic pulmonary fibrosis (IPF) is a devastating CD71 AMs were phenotypically, functionally, and transcriptionally 2 progressive disease with limited therapeutic options. Airway distinct, with CD71 AMs characterized by reduced expression macrophages (AMs) are key components of the defense of the airways of markers of macrophage maturity, impaired phagocytosis, and are implicated in the pathogenesis of IPF. Alterations in iron and enhanced expression of profibrotic genes. Importantly, metabolism have been described during fibrotic lung disease and in proportions of AMs lacking CD71 were independently murine models of lung fibrosis. However, the role of transferrin associated with worse survival, underlining the importance receptor 1 (CD71)-expressing AMs in IPF is not known. of this population in IPF and as a potential therapeutic target. Objectives: To assess the role of CD71-expressing AMs in the IPF lung. Methods: We used multiparametric flow cytometry, gene Conclusions: Taken together, these data highlight how CD71 delineates AM subsets that play distinct roles in IPF expression analysis, and phagocytosis/transferrin uptake assays to 2 delineate the role of AMs expressing or lacking CD71 in the BAL of and furthermore show that CD71 AMs may be an fi patients with IPF and of healthy control subjects. important pathogenic component of brotic lung disease. Measurements and Main Results: There was a distinct increase in proportions of AMs lacking CD71 in patients with IPF compared Keywords: airway macrophages; idiopathic pulmonary fibrosis; with healthy control subjects. Concentrations of BAL transferrin transferrin receptor (Received in original form September 26, 2018; accepted in final form May 2, 2019 ) This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/ licenses/by/4.0/). *These authors contributed equally to this work. ‡T.M.M. is Associate Editor of AJRCCM. His participation complies with American Thoracic Society requirements for recusal from review and decisions for authored works. Supported by a UCB Celltech Studentship (S.J.A.), a National Institute for Health Research (NIHR) Clinician Scientist Fellowship (NIHR reference CS-2013-13- 017 [T.M.M.]), a British Lung Foundation Chair in Respiratory Research (C17-3 [T.M.M.]), a Wellcome Senior Fellowship in Basic Biomedical Science (107059/Z/15/Z [C.M.L.]), a Wellcome Trust award (205949/Z/17/Z [A.J.B.]), and an Asthma UK senior fellowship (AUK-SNF-2017-381 [A.J.B.]). Author Contributions: Conception and design: S.J.A., T.M.M., C.M.L., and A.J.B.; analysis and interpretation: S.J.A., P.P.O., P.G., P.M., R.H., R.T., S.A.W., P.S., S.K., P.L.M., T.M.M., C.M.L., and A.J.B.; and drafting of the manuscript for important intellectual content: S.J.A., T.M.M., C.M.L., and A.J.B. Correspondence and requests for reprints should be addressed to Adam J. Byrne, Ph.D., Inflammation, Repair, and Development Section, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London SW7 2AZ, UK. E-mail: [email protected]. This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org. Am J Respir Crit Care Med Vol 200, Iss 2, pp 209–219, Jul 15, 2019 Copyright © 2019 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201809-1775OC on May 3, 2019 Internet address: www.atsjournals.org Allden, Ogger, Ghai, et al.: CD71 Delineates AM Subsets during IPF 209 ORIGINAL ARTICLE proximity to collagen-producing characterized by reduced expression of At a Glance Commentary myofibroblasts, and they secrete numerous markers of macrophage maturity, profibrotic soluble mediators, chemokines, impaired phagocytosis, and enhanced Scientific Knowledge on the and MMPs (matrix metalloproteases) (4). expression of profibrotic genes. Subject: Alterations in iron AMs in particular have been shown to be Importantly, CD71 AM status was metabolism have been described involved in the regulation of the ECM via associated with shortersurvivalinpatients during fibrotic lung disease and in secretion of MMPs or by direct uptake of with IPF, highlighting this pathway as a murine models of lung fibrosis. collagen (5, 6). potential prognostic factor or therapeutic However, how the role of transferrin Iron is an essential nutrient for target. Some of the results of these studies receptor 1 (CD71)-expressing airway numerous cellular processes; however, have been reported previously in the form macrophages contributes to the excess iron can also be harmful because it of an abstract (23). pathogenesis of idiopathic pulmonary acts as a catalyst in the formation of free fibrosis is not known. radicals from reactive oxygen species via the Fenton reaction (7). Iron content is Methods What This Study Adds to the therefore tightly regulated at the intra- and Field: We found that there was an extracellular levels. In addition to limiting Collection of BAL Samples increase in proportions of airway toxicity from iron overload, regulation of All patients and control subjects provided macrophages lacking CD71 in patients free iron serves as an innate immune written informed consent to participate in with idiopathic pulmonary fibrosis mechanism against invading pathogens, the study, which was approved by a Royal compared with healthy control limiting the availability for use by Brompton Hospital ethics committee 2 – subjects. CD71 airway macrophages pathogenic microorganisms (8 10). (10/HO720/12). Bronchoscopies were were characterized by reduced Alterations in iron metabolism have been performed with subjects under a light expression of markers of macrophage described in chronic lung diseases such as sedation with midazolam in combination maturity, impaired phagocytosis, and chronic obstructive pulmonary disease with local anesthesia with lidocaine. Four – – enhanced expression of profibrotic (8, 11), asthma (12 14), and IPF (15 17). 60-ml aliquots of warmed sterile saline genes. Proportions of airway Transferrin receptor 1, also known as were instilled in the right middle lung macrophages lacking CD71 were CD71, is an integral membrane protein that lobe and aspirated by syringe, and lavage independently associated with worse mediates the uptake of diferric transferrin aliquots collected after each instillation survival, underlining the importance complexes via receptor-mediated were pooled for each patient. Volume and of this population in idiopathic endocytosis (18). At steady state, the BAL appearance were recorded for all pulmonary fibrosis and as a potential majority of iron in the circulation is bound samples. therapeutic target. to transferrin, which limits iron-catalyzed free radical production and facilitates delivery to target cells (19). CD71 has been BAL Processing Idiopathic pulmonary fibrosis (IPF) is a shown to be highly expressed on human BAL samples were processed and stained devastating disease with an unknown AMs in multiple contexts; alterations in on the day of sample collection. Whole etiology characterized by deposition of numbers of CD71-expressing AMs have BAL was strained through a 70-mm excess extracellular matrix (ECM) and the been reported in sarcoidosis and sterile strainer and subsequently destruction of lung architecture leading to hypersensitivity pneumonitis (20), as well centrifuged (700 3 g; 5 min; 48C), and compromised gas exchange (1). IPF is the as in ILD cohorts (21). CD71 expression pellets were subjected to red blood cell most common form of interstitial lung has also been shown to be induced in AMs lysis (155 mM NH4Cl, 10 mM KHCO3, disease (ILD), with more than 5,000 new in murine models of lung fibrosis (22). 0.1 mM ethylenediaminetetraacetic acid, cases diagnosed each year in the United However, the contribution of CD71- pH 7.3) for 10 minutes before washing Kingdom (2). Treatment options are limited expressing AMs to the development of IPF and resuspension in complete media for IPF, and existing therapies slow but do is not known. (RPMI with 10% fetal calf serum, 2 mM not reverse disease progression (2). There is In this study, we sought to assess the L-glutamine, 100 U/ml penicillin- thereforeanurgentneedtounderstandthe role of CD71-expressing AMs in IPF. We streptomycin). Cells were counted and fundamental mechanisms involved in IPF to found that in IPF airways, there was a pelleted onto glass slides by devise new therapeutic approaches. distinct increase in proportions of AMs cytocentrifugation (5 3 104 cells/slide). Airway macrophages (AMs) play lacking CD71 compared with healthy Differential cell counts were performed essential roles in the maintenance of normal control airways. The concentration of BAL by Wright-Giemsa–stained cytospin. homeostasis of the airway and in host transferrin was enhanced in IPF BAL, and Percentages of eosinophils, 2 defense, repair, surfactant processing, and furthermore, CD71 AMs had an impaired lymphocytes/mononuclear cells, 1 inflammatory cascades (3). In particular ability to take up transferrin. CD71 and neutrophils, and macrophages were 2 AMs have been shown to be important CD71 AMs were defined by clear determined from a total of 400 cells.
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