&get_box_var;ORIGINAL ARTICLE

The 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 . Importantly, 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, 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

(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

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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 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 , also known as were instilled in the right middle lung macrophages lacking CD71 were CD71, is an integral membrane 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 (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. drivers of pulmonary fibrosis (4). differences in phenotype, function, and To obtain absolute numbers of each Macrophages are strategically positioned at expression of profibrotic genes. Compared leukocyte subtype, these percentages 1 the interface between the airways and the with CD71 macrophages, AMs lacking were multiplied by the total number of environment and are found in close surface expression of CD71 were cells obtained in the lavage fluid.

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Flow Cytometry stained for flow cytometry. Labeled cells (Thermo Fisher) on a ViiA 7 instrument Cells were washed and incubated with near- were acquired on a BD LSR Fortessa II (Life Technologies) with TaqMan primer infrared fixable live/dead stain (Life fluorescence-activated cell sorter (BD sets (Thermo Fisher) for the following Technologies Inc.) as per the manufacturer’s Biosciences). Before acquisition of any data, genes (catalog numbers in parentheses): instructions. Cells were washed before photomultiplier tube voltages were calibrated GAPDH (Hs02758991_g1), TLR1 (Toll-like incubation with human Fc block (BD to the highest signal-to-background ratio receptor 1; Hs00413978_m1), TLR2 Pharmingen, Inc.), and surface staining using the BD Cytometer Setup and Tracking (Hs02621280_s1), TLR3 (Hs01551078_m1), was performed with the following Beads Kit (catalog no. 655051; BD TLR4 (Hs00152939_m1), TLR6 (fluorophore followed by clone in Biosciences). Then, using single stained (Hs01039989_s1), TLR7 (Hs01933259_s1), parentheses) for the analysis of AMs in BAL: compensation beads (UltraComp eBeads; TLR9 (Hs00370913_s1), FcgRIA CD45 (PE [phycoerythrin]-Cy5 [cyanine 5]; Thermo Fisher) and the automated (Hs00174081_m1), ELMO1 (engulfment and HI30), CD11c (PE-Cy7; 3.9), CD14 (BV711; compensation program available in BD cell motility protein 1; Hs00404992_m1), M5E2), CD71 (PE; CY1G4), CD86 FACSDiva software (BD Biosciences), a CD71 (Hs00951083_m1), ACO1 (PE-Dazzle; IT2.2), CD163 (APC compensation matrix was created and ( 1; Hs00158095_m1), IREB2 [allophycocyanin]; GHI/61), CD206 (FITC directly applied to every subsequent sample. (iron-responsive element-binding 2; [fluorescein isothiocyanate]; 15-2), MARCO Data were exported and further analyzed by Hs00386293_m1), SLC40A1 (solute (macrophage receptor with collagenous using FlowJo software. carrier family 40 member 1; structure; APC; PLK-1 [Polo-like kinase 1]), Hs00205888_m1), SLC11A2 (solute carrier family 11 member 2; SR-A (scavenger receptor A; BV421; U23- Immunofluorescence Staining and Hs00167206_m1), HAMP (; 56), and HLA-DR (BV421; L243). Monocyte Imaging Hs00221783_m1), and HMOX (heme and dendritic cell (DC) populations were Cell surface staining was performed using a oxygenase; Hs01558390_m1). Gene analyzed with the following panel: lineage primary CD71 (catalog no. PA5- expression was analyzed using the change- cocktail comprised of CD3 (FITC; HIT3a), 27739; Sigma-Aldrich) or negative control in-threshold comparative cycle threshold CD4 (FITC; OKT4), CD8 (FITC; HIT8a), antibody (rabbit immunoglobulin fraction, method. Fold changes in mRNA CD19 (FITC; HIB19), CD20 (FITC; 2H7), catalog no. X0936; Dako) followed by goat expression for targeted genes were CD34 (FITC; 561), FceRI (Fce receptor I; antirabbit Alexa Fluor 488 (catalog no. calculated relative to healthy controls. FITC; AER-37); CD45 (BV605; HI30), A-11008; Life Technologies) and Examination of genes using the human CX3CR1 (2A9-1), CCR2 (K036C2), CD36 counterstained with DAPI (catalog no. fibrosis RT2 Profiler PCR Array (PAHS- (5-271), CD11b (AF700; M1/70), CD11c P36941; Life Technologies). To visually 120Z; Qiagen) was conducted following (PE-Cy7; 3.9), CD16 (peridinin chlorophyll confirm/image phagocytosis, 1 3 105 whole the manufacturer’s guidelines. All gene protein-Cy5; B73.1), CD163 (APC; GHI/61), BAL cells were placed in an 8-well chamber expression analysis was performed using CD14 (BV711; M5E2), and CD71 (PE; slide (catalog no. 734-2050; VWR) in live 2 RT Profiler PCR Array Data Analysis CY1G4). All antibodies were purchased from cell imaging solution containing 1% bovine version 3.5 (Qiagen). BioLegend, Inc., with the exception of serum albumin (catalog no. A14291DJ; Life Technologies). Bacterial phagocytosis was HLA-DR (BD Biosciences), SR-A (BD Detection of Iron and Transferrin in captured 2 hours after addition of 100 ml Biosciences), and MARCO (Thermo Fisher). BAL Samples fi of 0.25 mg/ml pHrodo Green S. aureus Surface staining was followed by xation Concentrations of iron and related BioParticles (catalog no. P35382; Life and then permeabilization to allow for were measured by using spectrophotometry Technologies) for 2 hours at 378C, 5% CO , intracellular or intranuclear staining. For 2 kits for the detection of iron (catalog no. with Hoechst 33342 nuclear stain (catalog transferrin uptake and nitric oxide assays, ab83366, Abcam; or catalog no. MAK025, 3 5 no. R37605; Life Technologies) and imaged cells were plated at 1 10 per well and Sigma-Aldrich) and ELISA kits for the using a Leica DM2500 microscope (Leica resuspended in PBS containing either 5 mM detection of transferrin (catalog no. Microsystems), and data were analyzed transferrin (catalog no. P35376; Life ab187391; Abcam) and (catalog no. using ImageJ-win32 software. Staining for Technologies) or 0.1 mMnitricoxide ab108837; Abcam). stain (4-amino-5-methylamino-29,79- iron in cytospin preparations was performed using a Prussian blue test difluororescein diacetate, catalog no. Statistics (catalog no. HT20; Sigma-Aldrich). D23842; Life Technologies) or PBS alone Differences between noncontinuous groups 8 for 30 minutes at 37 C, 5% CO2,and were compared using the Mann-Whitney subsequently stained for flow cytometry. Real-Time PCR U test. Spearman’s correlation was 5 For phagocytosis assay, 2.5 3 10 cells RNA extraction was performed using the used to assess the association between were resuspended in 200 ml of complete RNeasy Plus Micro Kit (Qiagen) according continuous variables. Kaplan-Meier RPMI (RPMI 1620; catalog no. 21870-076, to the manufacturer’s protocol. Total RNA analysis was used to compare time to 10% FBS, 1,000 U/ml penicillin, and 1 was reverse transcribed into cDNA using all-cause mortality between subjects mg/ml streptomycin; Life Technologies) the High-Capacity cDNA Reverse dichotomized above and below median 2 alone or containing 0.125 mg/ml Transcription Kit (Thermo Fisher percentage CD71 . Multivariable Cox Staphylococcus aureus bioparticles Scientific) as per the manufacturer’s proportional regression was used to assess 2 (catalog no. P35367; Life Technologies) for 2 instructions. Real-time PCR reactions were the association between CD71 (expressed hours. Cells were then washed with PBS and performed using Fast qPCR Master Mix as a continuous variable) and plausible

Allden, Ogger, Ghai, et al.: CD71 Delineates AM Subsets during IPF 211

ORIGINAL ARTICLE confounding variables (age, sex, baseline online supplement). Because dendritic found no significant alteration in the 1 FVC percent predicted, and baseline DLCO cells and monocytes may also be proportions of CD71 (Figure E1I) or 1 1 2 percent predicted) with all-cause mortality. CD45 CD11c , we performed additional CD71 (Figure E1J) AMs. However, there A backward stepwise selection procedure analysis to assess potential contamination was a decrease in absolute numbers of both 1 2 was used with each variable remaining in within the AM gate; the gating strategy CD71 (Figure E1K) and CD71 AMs the model if P , 0.10. Because of variations employed is outlined in Figure E1B. After (Figure E1L) when we compared aged with 1 1 in sample recovery and cell numbers gating on CD45 CD11c SSChi cells, we younger healthy control subjects. We available for experiments, n values differ selected cells negative for lineage markers further confirmed our findings by using an between some readouts; sample number has (CD3, CD4, CD8, CD19, CD20, CD34, alternate gating strategy to define AMs. 1 been specified in the figure legends. FceRI) and positive for HLA-DR . CD206 has been shown to be highly 2 1 Analysis was performed using Prism Lin HLA-DR cells were separated into expressed in AM populations (22, 24); 1 2 1 1 software (GraphPad Software), MedCalc CD14 CD16 classical monocytes, pregating on CD11c CD206 AMs and 1 1 1 2 statistical software version 18.9, and TIBCO CD14 CD16 intermediate monocytes, subsequently on CD71 / AMs (Figure 2 1 Spotfire software (TIBCO Software). CD14 CD16 nonclassical monocytes, E2A) recapitulated the results shown in 1 1 HLA-DR CD11C myeloid-derived DCs, Figures 1C–1F and E1 (Figures E2B–E2I). 2 1 and CD11c CD123 plasmacytoid DCs, In addition, we performed an analysis of Results and each subtype was expressed as a both innate and adaptive immune cell 1 1 proportion of CD45 CD11c SSchi cells. populations in the IPF BAL (Figure E2J). 2 1 Subject Demographics Lin CD11c cells accounted for around Together, these data indicate that during One hundred ten patients with IPF and 11 1–2% of the AM gate, and DCs and homeostasis, the majority of AMs express control subjects were enrolled in the present monocytes comprised a minor population CD71; however, in the IPF lung, there is a study. Demographic and clinicopathological within these (Figure E1C). Our AM gating distinct expansion of AM populations features are shown in Table 1. Healthy strategy was further confirmed by lacking the transferrin receptor. volunteers had no self-reported history of examination of cytospins of sorted 1 2 lung disease, an absence of infection within CD71 and CD71 AMs (Figure 1B). Defective Transferrin Uptake in the last 6 months, and normal spirometry. Immunohistochemical staining of cytospins 2 fi CD71 AMs FVC percent predicted was signi cantly confirmed the expression of CD71 on AMs 1 Because IPF AMs were marked by a lack of higher in healthy control subjects than in from the CD71 gate and a lack of staining , 2 the transferrin receptor CD71, we next patients with IPF (P 0.05, Mann-Whitney on those sorted from the CD71 gate 2 assessed the concentrations of transferrin in U test). (Figure 1B). The CD71 AM population 1/2 1 the airways and the ability of IPF CD71 was smaller and less granular than CD71 AMs to sequester this complex. Proportions of CD712 AMs Are AMs (Figure E1D). Numbers of both fi 1 2 Signi cantly higher concentrations of Increased in IPF BAL Compared with CD71 and CD71 AMs were increased in transferrin were detected in IPF BAL than Healthy Controls IPF BAL compared with healthy controls in healthy controls (Figure 2A). There was To define whether proportions of CD71- (Figures 1C and 1D). Interestingly, the an inverse correlation between the 1 expressing AMs were altered during IPF, we proportions of CD71 AMs cells were concentrations of transferrin in the airways used a multicolor flow cytometry gating significantly decreased in IPF BAL compared with the proportion of AMs strategy. After exclusion of debris and compared with healthy controls expressing CD71 (Figure 2B). Next, using doublets and selection of live cells, AMs (Figure 1E), with a concomitant increase in flow cytometry, we examined the ability of 1 2 were defined as CD45 cells expressing CD71 AMs during fibrotic lung disease the cells to take up transferrin. Our analysis CD11c (Figure 1A). In this gate, two (Figure 1F). We confirmed these findings in revealed a significant increase in transferrin 1 clear AM populations were apparent, a second validation cohort of 13 patients uptake in the CD71 cells compared with distinguishable by the expression of CD71 with IPF (Figures E1E–E1H). When we AMs lacking CD71 (Figure 2C). Free iron (Figure 1A), based on fluorescence-minus- separated control subjects into younger concentrations (without prior acid one and isotype controls (Figure E1A in the (22–48 yr) or older subjects (.49 yr), we digestion) were also examined but were not

Table 1. Clinical Characteristics of Patients Included in This Study

Healthy IPF (Discovery) IPF (Validation)

Number 11 97 13 Age, yr, mean (min–max) 40 (22–65) 70 (52–91) 68 (38–78) Male sex, n (%) 7 (63%) 71 (73%) 6 (46%) Smoking (ever vs. never), n (%) 4 (36%T) 61 (63%) 9 (69%) FVC, % (6SD) 102.4 (614.5) 74.56 (615.2) 92.14 (617.0) DLCO,%(6SD) NA 46.88 (613.2) 54.45 (617.31) Total cells in BAL, 3106/ml, mean (6SD) 0.126 (60.07) 0.268 (60.23) 0.383 (60.18)

Definition of abbreviations: IPF = idiopathic pulmonary fibrosis; max = maximum; min = minimum; NA = not applicable.

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A detected in IPF or healthy BAL 200K 5 supernatants (data not shown); however, 105 10 we detected iron-containing AMs by 200K 4 4 Prussian blue staining of cytospin 10 10 1 150K preparations (Figure 2D), and CD71 AMs

3 3 appeared to sequester more iron than their 10 100K 10 2 CD71 counterparts. We next examined 0 50K 0 the expression of genes involved in iron 3 3 1/2

−10 SSC-A −10 FSC-H homeostasis in CD71 AMs derived SSC-A 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K −103 0 103 104 105 from patients with IPF. IRP1 (iron FSC-A FSC-A Live/Dead regulatory protein 1), which binds and stabilizes CD71 mRNA (25), was 5 5 5 1 10 10 10 overexpressed in CD71 compared with 2 CD71 AMs (Figure 2E), whereas IRP2 104 4 4 10 10 showed similar expression levels between both cells (Figure 2F). Additional genes 3 3 3 10 10 10 involved in iron homeostasis were 0 0 0 examined, including HMOX1 (heme

SSC-A 3 3 3 −10 SSC-A −10 −10 oxygenase [decycling] 1), SSC-A (SLC40A1), and HAMP, revealing no −103 0 103 104 105 −103 0 103 104 105 −103 0 103 104 105 fi CD71 CD11C CD45 signi cant differences in expression of these factors compared with CD71-expressing – B C AMs (Figures E3A E3C). Number of CD71+ AMs 1 1.5 CD71 AMs Are Phenotypically * 2 Distinct from CD71 AMs 2 CD71– AMs Because CD71 AMs were elevated in IPF 1.0 and demonstrate defective iron- sequestering abilities, and because iron )/mL BAL fluid 6 status is known to influence macrophage 0.5 activation (26), we next contrasted the CD71+ AMs expression of macrophage phenotypic Cells (×10 1/2 0.0 markers in CD71 AM populations. y h Cells were pregated on either lt IPF 1 1 1 a SSchiCD45 CD11c CD71 or He 1 1 2 SSchiCD45 CD11c CD71 cells, and subsequently, we assessed the proportion of D E F 1 2 Number of CD71– AMs CD71+ AMs (%) CD71– AMs (%) CD71 or CD71 AMs that were positive for the marker of interest (Figure 3A). We ** **** 0.25 **** 100 50 found that multiple cell surface proteins associated with macrophage activation were 0.20 80 40 1 expressed at higher levels in CD71 AMs 2 0.15 60 30 compared with the CD71 population.

)/mL BAL fluid fi

6 These included the high-af nity scavenger %AMs 0.10 40 %AMS 20 receptor for the Hb–haptoglobin complex 0.05 20 10 CD163 (Figure 3B) and CD86 (Figure 3C).

Cells (×10 Of note, HLA-DR has been proposed to 0.00 0 0 identify mature resident AMs in the hy IPF lt IPF IPF a airways (22, 27) and was found to be ealthy 1 Healthy He H elevated in CD71 AMs (Figure 3D); conversely, the monocyte marker CD14 was 2 2 Figure 1. Proportions of CD71 airway macrophages (AMs) are increased in idiopathic significantly increased in CD71 AMs pulmonary fibrosis (IPF) BAL compared with healthy controls. (A) Gating strategy for flow 1 2 compared with CD71 AMs (Figure 3E). cytometric analysis of BAL CD71-expressing AMs. (B) Wright-Giemsa staining of sorted CD71 AMs (upper left) and CD711 AMs (lower left) and immunofluorescence staining for CD71 in sorted CD712 AMs (upper right) and CD711 AMs (lower right). (C and D) Numbers of CD711 (C) CD71 Delineates Functionally Distinct and CD712 (D) AMs in healthy and IPF BAL. (E and F) Proportions of CD711 (E)andCD712 (F) AM Populations AMs in healthy and IPF BAL. n = 11 healthy controls; n = 97 IPF. Data are presented as AMs play an important role in the detection mean 6 SD. *P , 0.05, **P , 0.01, and ****P , 0.0001, Mann-Whitney U test. FSC = forward and elimination of pathogens, and defects in scatter; SSC = side scatter. iron sequestration may permit inappropriate

Allden, Ogger, Ghai, et al.: CD71 Delineates AM Subsets during IPF 213

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A Transferrin in BAL B CD71+ AMs v BAL Transferrin C AM Transferrin Uptake * 20000 100 100 **** p < 0.001 r = –0.38 80 80

15000 AMs + 60 60 10000 40 40

5000 CD71+ AMs (%)

ng/mL in BAL fluid 20 20 % of Transferrin 0 0 0 1000 10000 100000 IPF Transferrin (ng/ml) Healthy CD71– CD71+

D E ACO1 (IRP1) F IREB2 (IRP2) ns CD71– CD71+ 300 * 80

200 60 CT CT 100 40   - - 2 2

0 20

–100 0

CD71– CD71+ CD71– CD71+ Figure 2. Defective transferrin uptake in CD712 airway macrophages (AMs). (A) Concentrations of transferrin in the BAL of patients with idiopathic pulmonary fibrosis (IPF) or of healthy control subjects (n = 7 healthy control subjects; n = 31 patients with IPF). (B) Correlation of proportions of BAL CD711 AMs with concentrations of transferrin in the airways (n = 67 patients with IPF). (C) Comparison of transferrin uptake in IPF-CD712 and IPF-CD711 AMs. (D) Staining (Prussian blue) for iron in sorted CD712 or CD711 AMs. Arrows indicate iron-positive AMs. (E and F) analysis of sorted CD712 or CD711 AMs is shown for ACO1 (n = 10 patients with IPF) (E) and IREB2 (n = 6 patients with IPF) (F). GAPDH was used as a housekeeping gene. Data are presented as mean 6 SD. *P , 0.05 and ****P , 0.0001, Mann-Whitney U test. ns = not significant.

pulmonary bacterial growth. Because the MARCO (also known as SR-A6) and CD712 AMs Have Profibrotic microbiome in patients with IPF has been CD204 or SR-A, have been shown to play Characteristics reported to be altered (28), and because a key role in AM phagocytosis (31, 32). To further examine the role of CD71- increased bacterial burden correlates with Percentages of AMs expressing CD206 expressing AMs during IPF, we used a PCR clinical decline (29), we next examined the (Figure 4D), MARCO (Figure 4E), and array that interrogates 84 genes involved in 1 2 ability of CD71 / AMs to detect and SR-A (Figure 4F) were increased in the remodeling cascade (Figures 5A and 1 2 eliminate pathogens. Assessment of CD71 AMs compared with CD71 5B). Figure 5A highlights significantly unstimulated cells revealed a significant AMs. Analysis of genes involved in differentially expressed genes, showing increase in the presence of nitric oxide in phagocytosis were also quantified. ELMO, CCL2, CCL3, IL-10, and CCR2 to be 2 1 2 CD71 cells compared with CD71 which is involved in antibody-mediated significantly increased in CD71 cells, (Figure 4A). In addition, the ability of phagocytosis, and FCgRIB did not differ whereas CEBPB (CCAAT/enhancer- 1 2 1 2 CD71 / cells to phagocytose bacteria in CD71 / AMs(FiguresE4AandE4B). binding protein-b), IL1A, TIMP2 1 (S. aureus) was assessed; CD71 TLRs are critical in the detection of (metalloproteinase inhibitor 2), and ITGB5 displayed an enhanced phagocytosis pathogen-associated molecular patterns and ITGB8 (-b5 and -b8, 2 1 ability compared with the CD71 AMs and damage-associated molecular respectively) were increased in the CD71 (Figures 4B and 4C). We assessed the patterns. The expression of TLR genes cells. Figure 5B highlights genes that 2 1 expression of several receptors known to was examined in CD71 and CD71 showed at least a fourfold increase in 2 1 be involved in AM phagocytosis of S. cells. Expression of TLR2 (Figure 4G) and CD71 compared with CD71 cells. aureus; CD206 (also known as the TLR3 (Figure 4H), but not of TLR1, 4, 6, Fibrosis-related genes upregulated in 2 1 ) has been implicated in 7, or 9 (Figures E4C–E4G), was CD71 AMs compared with CD71 cells 2 the phagocytic uptake of pathogens (30). significantly increased in CD71 AMs are shown in green, and those 1 2 Macrophage scavenger receptors, such as compared with CD71 . downregulated in CD71 AMs compared

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A 105 95.6 105 99.4105 100105 46.7

104 104 104 104 AMs + SSC SSC SSC 103 103 SSC 103 103

0 0 0 0 CD71 Gated on Non-debris, single, live, –103 –103 –103 –103 CD45+CD11c+SSChi cells –103 0 103 104 105 –103 0 103 104 105 –103 0 103 104 105 –103 0 103 104 105 105 CD163 CD86 HLA-Dr CD14

104

3 9.61 88.0 5 5 5 5 59.9 SSC-A 10 10 34.7 10 74.1 10 92.9 10 0 4 4 4 4 3 10 10 10 10

–10 AMs – SSC SSC SSC 3 SSC 3 3 3 –103 0 103 104 105 10 10 10 10 0 0 0 0 CD71 CD71 –103 –103 –103 –103

–103 0 103 104 105 –103 0 103 104 105 –103 0 103 104 105 –103 0 103 104 105 CD163 CD86 HLA-Dr CD14

B CDE CD163 CD86 HLA-DR CD14 **** **** **** **** 100 100 100 100

50 50 50 50 % CD71+/– AMS % CD71+/– AMS % CD71+/– AMS % CD71+/– AMS

0 0 0 0

CD71– CD71+ CD71– CD71+ CD71– CD71+ CD71– CD71+ Figure 3. CD711 and CD712 airway macrophages (AMs) are phenotypically distinct. (A) Flow cytometric gating strategy for the analysis of AM phenotypic markers. Data are shown as a percentage of either CD711 AMs expressing a marker of interest (highlighted in red) or CD712 AMs expressing a marker of interest (highlighted in blue). (B–E) Proportions of idiopathic pulmonary fibrosis (IPF)-CD711 and IPF-CD712 AMs expressing CD163 (n =17 patients with IPF) (B), CD86 (n = 12 patients with IPF) (C), HLA-DR (n = 38 patients with IPF) (D), and CD14 (n = 38 patients with IPF) (E). Data are presented as mean 6 SD. ****P , 0.0001 by Mann-Whitney U test. SSC = side scatter.

1 with CD71 AMs are shown in red. To assess AMs were associated with survival, with a pathogens. Although iron is required for 2 the clinical impact of CD71 status, we next hazard ratio of 0.9639 (95% confidence normal cellular functions, inappropriately 2 asked whether the percentage of CD71 AMs interval, 0.9317–0.9972; P = 0.0338) and high concentrations may accumulate in the was related to survival in patients with IPF. 1.0372 (95% confidence interval, lung through inhaling iron-containing Kaplan-Meier survival analyses showed a 1.0137–1.0612; P =0.0018),forsubjectswith particulate matter such as pollutants, significant relationship (P = 0.0085) between IPF dichotomized above and below the cigarette smoke, or siderophores (19, 33). 2 CD71 status and survival when subjects were median for proportion of CD71 AMs, It is therefore critical that AMs are dichotomized to those above and below the respectively. capable of efficiently sequestering and median (Figure 5C). There was no correlation processing excess iron in the lung without 2 between proportions of CD71 AMs and inappropriate activation. Our data indicate baseline disease severity (measured by FVC Discussion that during IPF, there is an expansion of 2 fi and DLCO), suggesting that CD71 AM status AMs, which are de ned by defective is an independent determinant of outcome in AMs are key components of lung immunity transferrin uptake, functional and IPF. In a Cox proportional hazards model, and provide the first line of defense phenotypic immaturity, and expression of 2 both baseline FVC and percentage CD71 against inhaled particulates and invading profibrotic factors. Together, our data

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ACB D NO+ AMs Phagocytosis CD206

25 *** 150 *** **** 100 20 100 15 50 10 50 %CD71+/– AMs 5 %CD71+/– AMs %CD71+/– AMs

0 0 0

CD71– CD71+ CD71– CD71+ CD71– CD71+

E F G TLR2 H TLR3 MARCO SR-A 60 100 *** 200 * 10 ** ** 8 150 40 6 CT 50 CT 100   - - 2 2 4 20 50 %CD71+/– AMs %CD71+/– AMs 2

0 0 0 0

CD71– CD71+ CD71– CD71+ CD71– CD71+ CD71– CD71+ Figure 4. CD711 and CD712 airway macrophages (AMs) are functionally distinct. (A) Comparison of nitric oxide (NO) expression in idiopathic pulmonary fibrosis (IPF)-CD711 and IPF-CD712 AMs (n = 23 patients with IPF). (B) Phagocytosis assay of IPF-CD711 and IPF-CD712 AMs (n =23 patients with IPF). (C) Representative staining of AMs after phagocytosis assay showing phagocytosed bacteria (green) and DAPI (blue). (D–F) Proportions of IPF-CD711 and IPF-CD712 AMs expressing CD206 (n = 39 patients with IPF) (D), MARCO (n = 7 patients with IPF) (E), and SR-A (n = 7 patients with IPF) (F). (G and H) Expression of TLR2 (n = 10 patients with IPF) (G) and TLR3 (n = 10 patients with IPF) (H) in CD711/2 AMs. GAPDH was used as a housekeeping gene. Data are presented as mean 6 SD. *P , 0.05, **P , 0.01, ***P , 0.001, and ****P , 0.0001, Mann-Whitney U test. identify a novel population of AMs that regarding AM functional subsets in the high expression of the classical monocyte mark disease progression during IPF and human lung and, indeed, during IPF. CD71 activation marker CD14, in addition to identify the CD71 pathway as a potential may delineate a population of protective expression of CCL2 and its receptor CCR2, 1 target for therapeutic intervention during resident AMs, whereas monocyte-derived compared with the CD71 population. 2 fibrosing lung disease. AMs are defined by a lack of CD71 Similarly, there was an increase in the Multiple recent reports have indicated expression and drive pathology during IPF, expression of chemokine receptor CXCR4 2 that lung-resident AMs maintain their analogous to AMs found in murine models and chemokine CCL3 (MIP-1a) in CD71 populations via proliferation in situ during of fibrosing lung disease (13). CD71 has cells. Examination of morphology of AMs homeostasis (34–37) and also that, during been described as a marker of mature from patients with IPF indicated that 2 ongoing inflammatory responses, macrophages (40, 41), with increased CD71 cells were smaller and less granular 1 monocytes are recruited to the lung and expression reported in AMs in a murine than CD71 AMs. Taken together, this 2 develop into AM-like cells (36, 38). model of fibrosis (42), in addition to suggests that CD71 cells may have been Misharin and colleagues recently showed increased expression on ILD AMs (21). We recruited into the lung and may have that monocyte-derived rather than fetally found enhanced expression of multiple played a role in further recruitment of derived tissue-resident AMs were essential markers that have been described as monocyte-derived cells. However, we for the development of pulmonary fibrosis markers of mature AM phenotypes, such as cannot discount the possibility that the 1 in murine models, whereas deletion of HLA-DR and CD206 on CD71 AMs local environmental milieu during IPF leads 2 tissue-resident AMs had no effect on the compared with the CD71 AM population. to the downregulation of CD71 in tissue- 2 disease (39). However, little is known Conversely, CD71 AMs were defined by resident AMs.

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CD71+CD71– Interestingly, multiple fibrotic Scatter plot CD71– vs CD71+ ABP1P2P3P1P2P3 pathways appeared to be activated in BCL2 2 SERPINE1 CD71 AMs. Of note, expression of MMP2 MMP9 0.6 2 TIMP1 ) and MMP9 was increased in CD71 cells VEGFA 1 Ct

CCL2  0.1 compared with CD71 AMs, suggesting a * - MYC CEBPB2 CCL3 role in transforming growth factor-b * TIMP2 MMP2 –0.4 activation in the fibrotic lung (4). ** IL10 ** CCR2 Additional remodeling genes were CXCR4 –0.9 THBS1 2 ACTA2 ITGB5 increased in CD71 cells, including VEGFA MMP14 ITGB8 TGFBR1 –1.4 IL-1A (vascular endothelial growth factor A) and PLAU TGIF1 CCL2 SERPINE1 (serine protease inhibitor family SP1 –1.9 TIMP1 CCL3 SMAD4 E member 1) encoding PAI-1 (plasminogen SMAD7 VEGFA STAT1 –2.4 activator inhibitor 1). PAI-1 inhibits the **CEBPB enzyme uPA (urokinase plasminogen TGFBR2 Log10 (CD71+ (Avg n=3) 2ˆ MMP2 CCR2 AKT1 –2.9 ILK activator), which is responsible for the SMAD2 IL-10 TGFB1 cleavage of plasminogen to form plasmin, IL1A **TIMP2 which in turn mediates the degradation of ** 0.1 0.6 ENG –3.4 –2.9 –2.4 –1.9 –1.4 –0.9 –0.4 the ECM (43). The transcription factor SERP1NA1 2 STAT6 -Ct MYC was increased in CD71 cells. MYC TNF Log10 (CD71– (Avg n=3) 2ˆ ) ITGB1 plays a key role in cell cycle progression and ITGB5 ** ITGB8 has been shown to be critical in **ITGAV NFKB1 differentiating monocytes into osteoclasts, a JUN THBS1 specialized bone tissue macrophage, IL1B suggesting a possible role for MYC SERP1NH1 2 differentiation in CD71 cells (44). BCL2 4 Fold Change *p<0.05, **p<0.01 (B-cell lymphoma 2) is also increased in Magnitude of gene expression 2 CD71 cells, which plays a role exerting min avg max antiapoptotic effects (45). Iron sequestration by AMs represents a protective innate immune mechanism that limits available free iron concentrations, C which are used by pathogenic CD71-High microorganisms. Recent work has indicated 100 fi CD71-Low that there is a signi cant difference between 90 the microbiota in subjects with IPF and healthy control subjects (28) and, 80 furthermore, that alterations in the 70 pulmonary bacterial burden are related to exacerbations of the disease (29, 46). The 60 observed differences in TLR expression, 50 nitric oxide production, phagocytotic 1 2 ability, and transferrin uptake in CD71 / % survival 40 P=0.0085 populations suggest a discordant ability to 30 eliminate infectious agents in these 20 populations. Although CD71 AMs have impaired transferrin uptake, and although 10 there is an inverse correlation between the 0 concentrations of transferrin in the airways 0 365 730 1095 and the proportion of AMs expressing Days CD71, it should be noted that there were 1 2 numerically more CD71 AMs in the BAL Figure 5. CD71 airway macrophages (AMs) have profibrotic characteristics. (A) Heat map of patients with IPF than in healthy representation of fibrosis array of sorted CD711/2 idiopathic pulmonary fibrosis (IPF)-AMs. (B) 1 2 individuals. It is therefore likely that other Scatterplot highlighting differentially expressed genes in CD71 and CD71 IPF-AMs. Plot shows 2 1 mechanisms contribute to the increased fibrosis-related genes upregulated in CD71 AMs compared with CD71 cells in green and those downregulated in CD712 AMs compared with CD711 AMs in red. Genes with statistically significant transferrin load observed in IPF, including and greater than twofold change compared with control are highlighted. (C) Kaplan-Meier survival plot increased vascular leak and exposure of for subjects with IPF dichotomized above (High) and below (Low) the median for proportion of CD712 airway cells to Hb as well as synthesis of AMs. Individuals with an increased proportion of CD712 AMs have worse survival than patients with a transferrin by immune or stromal cells low proportion of CD712 AMs (n = 97 patients with IPF). (such as airway epithelial cells). Alternative

Allden, Ogger, Ghai, et al.: CD71 Delineates AM Subsets during IPF 217

ORIGINAL ARTICLE phagocytosis mechanisms, such as mannose bound iron–clearing mechanisms in all clinical relevance because proportions of 2 detection–linked phagocytosis and tissues of the body, including binding by CD71 AMs are expanded in patients with increased scavenger receptor expression transferrin and phagocytosis by progressive fibrotic disease compared with (MARCO and SR-A), may explain why a macrophages, are therefore rapid, highly those presenting with a nonprogressive 1 greater proportion of CD71 cells efficient, and tightly regulated. In the lung, disease course. These data suggest that iron phagocytosed bacteria, because they the majority of residual free iron is likely metabolism and CD71-expressing AMs expressed significantly more mannose sequestered by bacteria, resulting in may be both a novel marker of poor 2 receptor than CD71 cells. It is of interest unmeasurable concentrations and reflecting prognosis during IPF and a potential that iron could be readily detected in AMs the importance of iron clearance therapeutic target. n but that there were minimal concentrations mechanisms in the lung. in lavage supernatants (without prior acid In summary, our findings indicate that Author disclosures are available with the text treatment). These results are perhaps not during IPF, there is an expansion of a of this article at www.atsjournals.org. surprising, given that free iron is both an population of AMs with impaired Acknowledgment: The authors acknowledge important cofactor required for replication transferrin receptor expression and defects the support of the flow cytometry facility at the by many species of pathogenic bacteria and in components of free iron uptake and South-Kensington campus and in particular Jane a potent oxidizing agent. Non–protein- bacterial phagocytosis. Our work is of Srivastava and Dr. Jessica Rowley.

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