Stress and Inflammation Lung Macrophages That Limit Oxidative

Ozone Inhalation Promotes CX3CR1-Dependent Maturation of Resident Lung Macrophages That Limit Oxidative Stress and Inflammation This information is current as of September 27, 2021. Robert M. Tighe, Zhuowei Li, Erin N. Potts, Sarah Frush, Ningshan Liu, Michael D. Gunn, W. Michael Foster, Paul W. Noble and John W. Hollingsworth J Immunol 2011; 187:4800-4808; Prepublished online 19 September 2011; Downloaded from doi: 10.4049/jimmunol.1101312 http://www.jimmunol.org/content/187/9/4800 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/09/21/jimmunol.110131 Material 2.DC1 References This article cites 54 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/187/9/4800.full#ref-list-1

Why The JI? Submit online. by guest on September 27, 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 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts Errata An erratum has been published regarding this article. Please see next page or: /content/196/5/2424.full.pdf

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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ozone Inhalation Promotes CX3CR1-Dependent Maturation of Resident Lung Macrophages That Limit Oxidative Stress and Inﬂammation

Robert M. Tighe,* Zhuowei Li,* Erin N. Potts,* Sarah Frush,* Ningshan Liu,* Michael D. Gunn,*,† W. Michael Foster,* Paul W. Noble,* and John W. Hollingsworth*,†

Inhalation of ambient ozone alters populations of lung macrophages. However, the impact of altered lung macrophage populations on the pathobiology of ozone is poorly understood. We hypothesized that subpopulations of macrophages modulate the response to ozone. We exposed C57BL/6 mice to ozone (2 ppm 3 3 h) or filtered air. At 24 h after exposure, the lungs were harvested and digested and the cells underwent flow cytometry. Analysis revealed a novel macrophage subset present in ozone-exposed mice, + which were distinct from resident alveolar macrophages and identified by enhanced Gr-1 expression [Gr-1 macrophages (Gr-1 Downloaded from Macs)]. Further analysis showed that Gr-1+ Macs exhibited high expression of MARCO, CX3CR1, and NAD(P)H:quinone oxioreductase 1. Gr-1+ Macs were present in the absence of CCR2, suggesting that they were not derived from a CCR2-dependent circulating intermediate. Using PKH26-PCL to label resident phagocytic cells, we demonstrated that Gr-1 Macs were derived from resident lung cells. This new subset was diminished in the absence of CX3CR1. Interestingly, CX3CR1-null mice exhibited enhanced responses to ozone, including increased airway hyperresponsiveness, exacerbated neutrophil influx, accumulation of

8-isoprostanes and protein carbonyls, and increased expression of cytokines (CXCL2, IL-1b, IL-6, CCL2, and TNF-a). Our results http://www.jimmunol.org/ identify a novel subset of lung macrophages, which are derived from a resident intermediate, are dependent upon CX3CR1, and appear to protect the host from the biological response to ozone. The Journal of Immunology, 2011, 187: 4800–4808.

zone, a gas formed via a chemical reaction between TNF-a, IL-1b, IL-6, and IL-8, which contribute to the biological oxides of nitrogen and volatile organic compounds in the response to ozone (7, 9). Despite the evidence indicating a proin- O presence of sunlight, is a source and cause of environ- flammatory role for macrophages, additional evidence suggests mental lung injury. Inhaled oxidants, such as ozone, are important that macrophages can be protective during ozone-induced lung contributors to a variety of respiratory diseases, including chronic injury (10). Macrophages are important for clearance of both by guest on September 27, 2021 obstructive pulmonary disease, asthma, cystic fibrosis, and adult apoptotic cells and oxidized species generated by ozone, which, respiratory distress syndrome (1). The mechanism of action for the if not cleared, can exacerbate inflammation (10, 11). Just how response to ozone appears to be through a combination of several macrophages can cause both pro- and anti-inflammatory outcomes mechanisms, including the alteration of barrier functions in the remains poorly understood. We hypothesized that individual epithelium (2, 3), the generation of oxidized species such as 5b, macrophage subpopulations within the lung account for the di- 6b-epoxycholesterol found in the epithelial lining fluid (4), and vergent reported functions of macrophages in the setting of ozone the fragmentation of matrix proteins such as hyaluronan to frag- inhalation. ments of low m.w. (5). Inhalation of ozone results in the activation Interest has grown in the identification of macrophage sub- and recruitment of many cell types within the lung (6). populations in the lung and in other organ systems as a means to Macrophages are increased in the airspace after inhalation of understand their diverse function during injury and repair. Given ambient ozone and appear central to the host response to ozone (7, the diverse functional roles of macrophages, it is not surprising that 8). Macrophages can produce proinflammatory cytokines such as subpopulations of macrophages exist that are either recruited from or mature in resident cells in response to environmental challenge. Recent studies have identified distinct macrophage subpopula- *Department of Medicine, Duke University Medical Center, Durham, NC 27710; and †Department of Immunology, Duke University Medical Center, Durham, NC 27710 tions with unique functions in multiple tissues (12). These data also Received for publication May 5, 2011. Accepted for publication August 16, 2011. support plasticity of macrophages in a manner dependent on microenvironment. In the lung, distinct macrophage subpopulations This work was supported by National Institutes of Health Grants ES016126 and ES020426 (to J.W.H.), ES016347 (to W.M.F.), and HL077291 (to P.W.N.). have been characterized in murine models of infectious disease Address correspondence and reprint requests to Dr. John W. Hollingsworth, Division and asthma (13–16). Work from our laboratory identified a lung of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Duke macrophage subpopulation, exudative macrophages (ExMacs), University School of Medicine, 106 Research Drive, Durham, NC 27710. E-mail that are recruited following noninfectious lung injury (17). Sep- address: [email protected] arable macrophage lineages result from the heterogeneity in The online version of this article contains supplemental material. macrophage precursor cells (i.e., monocytes) and the specific Abbreviations used in this article: AHR, airway hyperresponsiveness; AM, alveolar macrophage; BAL, bronchoalveolar lavage; BALF, BAL fluid; ExMac, exudative developmental pathways that are initiated by stimulatory local macrophage; Gr-1 Mac, Gr-1 macrophage; HO-1, heme oxygenase 1; MFI, mean conditions at the time of monocyte differentiation (12). This work fluorescence intensity; NQO1, NAD(P)H:quinone oxioreductase 1; SOD-1, superox- has largely focused upon factors of macrophage recruitment and ide dismutase 1; WT, wild-type. development from circulatory monocytes during pulmonary in- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 jury. However, it is unclear whether resident lung macrophages www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101312 The Journal of Immunology 4801 can extend beyond their primary alveolar membrane defense phe- passed through a 70-mm mesh strainer and centrifuged at 535 3 g at room notype, adapt, and undergo development into unique populations temperature over an 18% nycodenz (Accurate Chemical, Westbury, NY) in the setting of acute lung injury. cushion. Low-density cells were collected, underwent RBC lysis, and then washed 3 2. The following Abs were used: anti–I-A/I-E FITC, anti–I-A/I-E Macrophages are derived either from monocytes or by pro- PE (used in experiments with CX3CR1GFP/WT mice), anti–Ly6-G PE, anti– liferation of local macrophages. Two unique monocyte populations Mac-3 PE, anti–Gr-1 allophycocyanin, and anti-CD11b allophycocyanin- are present in the circulation (18). Constitutive (Gr-1–) monocytes Cy7 (all from BD Pharmingen, Franklin Lakes, NJ); anti-CD11c PE Cy5.5 enter tissues under steady state conditions in the lung and develop and anti-F480 allophycocyanin (eBioscience, San Diego, CA); and anti- MARCO PE (AbD Serotec, Raleigh, NC). Isotype controls were gener- into resident tissue interstitial macrophages and alveolar macro- ated for MARCO using a rat IgG1 negative control (catalog no. MCA1211, phages (AMs) (18–20). This homeostatic mechanism appears to AbD Serotec), which was conjugated to PE fluorochrome using an Ab be dependent on the chemokine receptor CX3CR1 (or the frac- conjugation kit (catalog no. LNK021RPE, AbD Serotec). All staining was talkine receptor). In contrast, inflammatory (Gr-1+) monocytes are performed in PBS with 3% FBS, 10 mM EDTA, 5% normal mouse serum, 5% normal rat serum, and 1% FcBlock (BD Pharmingen). To reduce recruited to the lung during inflammation in a CCR2-dependent ◦ nonspecific binding, the cells were incubated in staining buffer at 4 Cfor10 manner and develop into either an activated macrophage pop- min prior to the addition of Abs. Following staining, the cells were washed 3 ulation known as ExMacs or into monocyte-derived dendritic cells 3 and analyzed using a BD Canto II flow cytometer. Data analysis was (18, 21). The morphology, phenotype, and effector functions of performed using FlowJo (version 8.8.6, Ashland, OR) software. Cell sorting constitutive monocyte-derived macrophages differ markedly from was accomplished with a BD FACSAria cell sorter. those of inflammatory monocyte-derived macrophages (16). We Resident lung phagocytic labeling and others (16, 17) identified that recruited ExMacs, but not res- Phagocytic labeling was based on a protocol from Maus et al. (22). In Downloaded from ident lung macrophages, express high levels of MHCII and co- brief, mice were anesthetized using 1–3% isoflurane. PKH26-PCL (Sigma- stimulatory molecules, stimulate T cell activation, and are a major Aldrich), a red fluorescent dye that labels phagocytic cells, was diluted by source of inflammatory cytokines and chemokines, such as TNF-a, a factor of 20, using the diluent B solution (Sigma-Aldrich). Under sterile CXCL2, CXCL10, and NO synthase 2. Despite this character- conditions, 100 ml diluted PKH26-PCL was administered by retro-orbital ization of recruited macrophages, little is known about the unique injection. The mice were allowed to recover for 24 h, then underwent filtered air or ozone exposure and were euthanized and harvested for flow functional roles of resident lung macrophages in the context of cytometric analysis, as described in the above protocols. In addition to lung noninfectious lung injury. harvest, blood was drawn into a 23-gauge needle with a 1-ml syringe http://www.jimmunol.org/ In the current study, we identify a novel subset of macrophages containing EDTA. The blood underwent RBC lysis using ACK Lysis present in the lung after inhalation of ozone. This subset is sep- Buffer to separate the RBCs from peripheral blood leukocytes. The leukocytes were washed in sterile PBS and then centrifuged at 1600 rpm for 5 arable from resident macrophages and characterized by expression min at 4◦C. The whole-blood leukocytes underwent flow cytometric of unique cell surface markers and gene expression. Using cell analysis for PKH26-PCL. PKH26-PCL was visualized in the PE channel. labeling of resident phagocytic cells, we find that these macrophages are derived from a resident lung intermediate and not Airway physiology a circulating precursor. Finally, we discover that the development Direct measurements of respiratory mechanics in response to methacholine of this macrophage subset is diminished in mice deficient in were made using the flexiVent system (SCIREQ, Montreal, Canada) and CX3CR1. In the setting of CX3CR1 deficiency and reduced reported as total resistance (RT)cmH2O/ml/s. Anesthesia was achieved by guest on September 27, 2021 + with 60 mg/kg pentobarbital sodium injected i.p. Mice were then given a numbers of Gr1 Macs, mice have enhanced response to ozone. In neuromuscular blockade (0.8 mg/kg pancuronium bromide). Mice were conclusion, our data support a novel macrophage subpopulation ventilated with a computer-controlled ventilator (flexiVent) with a tidal that is derived from resident macrophages and can protect the volume of 7.5 ml/kg and a positive end-expiratory pressure of 3 cm H2O. lungs from oxidant-related lung injury. Measurements of respiratory mechanics were made by the forced oscil- lation technique. Response to aerosolized methacholine (0 mg/ml, 10 mg/ ml, 25 mg/ml, and 100 mg/ml) was determined by resistance measure- Materials and Methods ments every 30 s for 5 min, ensuring the parameters calculated had peaked. Mice Total lung capacity breaths were administered after each dose of methacholine to maintain patent airways and return the measurements back to C57BL/6 and CX3CR1GFP/GFP mice were purchased from The Jackson baseline. The resistance measurements were then averaged at each dose GFP/GFP Laboratory (Bar Harbor, ME). CX3CR1 mice were crossed with and graphed (RT cm H2O/ml/s), along with the initial baseline measure- C57BL/6 mice to produce CX3CR1+/GFP mice. CCR22/2 mice were ment. backcrossed on a C57BL/6 background for .10 generations. Experimental groups consisted of 6- to 10-wk-old female mice for all experiments. Bronchoalveolar lavage fluid Animal experiments were conducted in accordance with National Institutes Immediately after pulmonary function measurements, mice were overdosed of Health guidelines and protocols approved by the Animal Care and Use with Nembutal (100 mg/kg) to euthanize them. The chest was opened, the Committee at Duke University. trachea was exposed, and bronchoalveolar lavage (BAL) was performed by Exposure protocol inserting a catheter into the mouse trachea with PE-90 tubing and instilling saline until the lung reached total lung capacity. This procedure was re- C57BL/6, CCR22/2, CX3CR1+/GFP, and CX3CR1GFP/GFP were exposed to peated three times. The total volume returned was the lavage return volume. either filtered air or ozone. Ozone exposure occurred for 3 h at a dose of Cells from the BAL fluid (BALF) were isolated using centrifugation (1500 2 ppm. The ozone was generated by directing 100% oxygen through an rpm, 15 min), and the supernatant was stored at 280˚C for assessment ultraviolet light generator. This was then mixed with the air supply to the of 8-isoprostanes, protein carbonyls, and cytokines. The cells were used for chamber. Temperature and humidity of chamber air were monitored con- cell counts and differentials. tinuously, as was the ozone concentration with an ultraviolet light pho- tometer. The mice receiving filtered air were also placed in chambers for Cytokine measurements 3 h without ozone instillation. The mice were then removed from the Cytokines and chemokines were analyzed using a MILLIPLEX 5-MAP chamber and allowed to recover for 24 h. assay kit (Millipore, Billerica, MA) per the manufacturer’s protocol. This Lung digestion and flow cytometry is a bead-based suspension array using the Luminex technology (Bio-Rad, Hercules, CA, USA), in which fluorescent-coded beads, known as micro- Mice were euthanized 24 h following ozone or filtered air exposure. The spheres, have cytokine capture Abs on the bead surface to bind the pro- lungs were perfused with HBSS to remove residual RBCs from the pul- teins. CX3CL1 was measured from BAL, using a commercially available monary circulation. They were minced and digested for 40 min at 37◦Cin ELISA kit (R&D Systems, Minneapolis, MN) according to the manu- HBSS with 1 mg/ml collagenase A (Roche, Indianapolis, IN) and 0.2 mg/ml facturer’s instructions. The plate was read on a plate reader at a wave- DNase 1 (Sigma-Aldrich, St. Louis, MO). The digestion solution was length of 450 nm. 4802 OZONE MATURES RESIDENT MACROPHAGES

8-Isoprostane measurements expression of both CD11b and CX3CR1. To our surprise, flow cytometric analysis of whole-lung macrophages at 24 h after 8-Isoprostanes were measured in both the BALF supernatant and the cell + supernatant, using purification columns and an enzyme immunoassay kit ozone exposure did not reveal a population of CD11b macro- from Cayman Chemical (Ann Arbor, MI). Samples were briefly diluted 1:2 phages consistent with previously reported ExMacs (Fig. 1A). with column buffer and applied to the purification columns. The sample Rather, we observed a population of autofluorescent+ MHCIIint passed entirely through the column. The column was then washed with CD11chi macrophages, which were Gr-1hi CD11bint. These were column buffer and ultrapure water, and the washes were discarded. Next, 5 low neg ml elution solution was added to the column and allowed to pass through to separate from other AMs, which were Gr-1 CD11b . The Gr- elute the 8-isoprostane. The solution that passed through the column was 1–positive subset was not present in air-exposed mice. AMs and then collected in a 5-ml tube, and the elution solution was evaporated to the Gr-1–positive subset both stained positive for the macrophage dryness, using a stream of dry nitrogen gas to remove all quantities of markers F4/80 and Mac-3 (Supplemental Fig. 1), confirming that organic solvent. The purified samples were then reconstituted with saline and used for the enzyme immunoassay kit (Cayman Chemical), following they were, in fact, macrophages. Graphic representation of the the manufacturer’s protocol. Samples, standards, buffer, bound 8-iso- percentage of Gr-1–positive macrophages as a percentage of total prostane AChE Tracer, and antiserum were added to the plate and in- macrophages (Fig. 1B) and of total cells (Fig. 1C) confirmed this cubated at 40˚C for 18 h. The plate was then washed 5 times with wash identification. This observation suggested that ozone exposure buffer, and Ellman’s reagent (substrate for AChE tracer) was added. After resulted in development of a novel macrophage subset, which was 90 min, the plate was read on a plate reader at a wavelength of 405 nm. The 8-isoprostane concentrations was calculated by plotting the percent defined by increased Gr-1 expression. ratio of standard bound/maximum bound for each of the standards, using Gr-1–positive macrophages are distinct from resident AMs linear and log axes and performing a 4-parameter logistic fit.

We were interested to determine whether unique differences between Downloaded from Protein carbonyl Gr-1–positive macrophages after ozone and resident macrophages Protein carbonyls were measured in both the BALF supernatant and the cell (AMs) could be deﬁned. In addition, we hoped to gain insight into supernatant using an OxiSelect Protein Carbonyl ELISA kit following a potential role of this subset in the host response to ozone lung the manufacturer’s protocol (Cell Biolabs, San Diego, CA). Brieﬂy, BSA injury. Flow cytometry was performed to identify additional cell standards or samples were absorbed onto a 96-well plate for 2 h at 37˚C. The protein carbonyls present in the sample or standard were derivatized to surface markers, which could delineate Gr-1–positive macrophages

DNA hydrazone and probed with an anti-DNP Ab, followed by an HRP- from AMs. Using mice in which an allele for CX3CR1 is replaced http://www.jimmunol.org/ conjugated secondary Ab. The plate was then read at 405 nm by a plate reader. The protein carbonyl content in the sample was determined by comparison with a standard curve that was prepared from predetermined reduced and oxidized BSA standards. Real-time PCR Total RNA was extracted from ﬂow-sorted macrophages using the RNeasy Mini Kit (Qiagen, Valencia, CA), according to the manufacturer’s protocol. Samples were treated with DNase and removal reagents to eliminate DNA

contamination (catalog no. AM1906; Ambion, Austin, TX). RNA samples by guest on September 27, 2021 were then reverse transcribed into cDNA using SuperScript II RT (Invi- trogen, Carlsbad, CA) following the manufacturer’s instructions. All real- time quantitative PCRs were performed using ABI SDS 7500 and SYBR Green reagent (Applied Biosystems, Foster City, CA). Primers were designed using ABI software and were produced by IDT (Coralville, IA). The sequences were as follows: NAD(P)H:quinone oxioreductase 1 (NQO1) forward, 59-GTGGTTTGGGGTGCCAGCCAT-39; NQO1 reverse, 59-AGG- ATGCCACTCTGAATCGGCC-39; CYP1b1 forward, 59-CCAGGCGTCG- CACTTGTAC-39; CYP1b1 reverse, 59-TGGAAAACGTCGCCATAGC-39; superoxide dismutase 1 (SOD-1) forward, 59- GCCCGGCGGATGAAG-39; SOD-1 reverse, 59- CCTTTCCAGCAGTCACATTGC-39; heme oxygenase 1 (HO-1) forward, 59-CAGCCCCACCAAGTTCAAA-39; and HO-1 reverse, 59-TCAGGTGTCATCTCCAGAGTGTTC-39. PCR amplification was performed by the following program: 50˚C, 2 min; 95˚C, 10 min; 95˚C 15 s; 60˚C 1 min, for 40 cycles. Gene expression values were normalized to the housekeeping gene 18S. The data were presented as a fold change FIGURE 1. Flow cytometry of macrophages after ozone challenge. A, compared with 18S. Flow profile of total lung cells isolated 24 h after exposure to filtered air or 2 ppm of ozone. Lungs are digested and then enriched for mononuclear Statistics cells over an 18% nycodenz gradient. Flow cytometry was then performed. Data are expressed as mean 6 SE. The statistical difference between Live cells are gated on the basis of FSC and SSC (R1 Gate). The cells from groups was assessed by the Student t test. A two-tailed p value of ,0.05 R1 are analyzed by CD11c versus MHCII expression to identify individual was considered statistically significant. cell populations. Macrophages are identified as MHCIIint and CD11c+ (Mac). Subgate analysis from the Mac Gate by CD11b and Gr-1 after air exposure identifies macrophages as CD11blow Gr-1low (AM). After ozone Results exposure, in addition to CD11blow Gr-1low macrophages, a population of Ozone exposure results in development of a Gr-1–positive CD11blow Gr-1high macrophages (Gr-1 Macs) develops. B, Graphic rep- subset of macrophages resentation of the number of CD11blow Gr-1high macrophages as a per- int + To further characterize the influx of macrophages after ozone centage of total macrophages (defined as MHCII and CD11c ) after either filtered air or ozone. C, Graphic representation of the number of exposure, we performed multicolor flow cytometric analysis on CD11blow Gr-1high macrophages as a percentage of total cells. The flow whole-lung macrophages, using a previously described technique + plots are from a representative sample of C57BL/6 mice given filtered air (16, 17). Lung macrophages are identified as autofluorescent , or ozone. Data were derived from three or four mice with both exposures int hi MHCII , CD11c by flow cytometry. In previous work, we and are representative of two separate experiments. ***p , 0.0005 be- identified two types of macrophages: resident AMs and ExMacs tween groups of Gr-1high macrophages in either air or ozone exposure (17). ExMacs were separated from AMs by enhanced cell surface groups. The Journal of Immunology 4803 with GFP (CX3CR1GFP/WT) to track CX3CR1 expression, we identified that Gr-1 macrophages (Gr-1 Macs) had enhanced CX3CR1 expression relative to AMs after ozone exposure. This finding was confirmed both by histogram (Fig. 2A) and by a significant difference in mean fluorescence intensity (MFI) (Fig. 2B). We also demonstrated that Gr-1 Macs had enhanced expression of MARCO by histogram analysis (Fig. 2C). This observation was confirmed by a statistically higher MFI in the Gr-1 Mac population (Fig. 2D). MARCO previously has been suggested to have a role in protection of the host from ozone-induced lung injury (10). To further understand the difference between Gr-1 Macs and AMs, we performed flow-sorting experiments. Mice were exposed to ozone, and then whole lungs were processed for flow cytometry. Gr-1 Macs were sorted from AMs. The cells then underwent processing for mRNA analysis. Given the findings of MARCO expression, we used real-time PCR to identify antioxidant gene FIGURE 3. mRNA expression of Gr-1 Macs and resident Macs after ozone exposure. C57BL/6 mice were exposed to 2 ppm of ozone for expression. We did not discover significant difference in expression 3 h, and then the lungs were harvested at 24 h. After digestion and Downloaded from between Gr-1 and AMs for the following genes: thioredoxin re- centrifugation over an 18% nycodenz cushion, the cells are sorted by ductase, glutathione S-transferase A1, CCAAT/enhancer binding flow cytometry. Both AMs and Gr-1 Macs are sorted and then processed protein-b, CSF-1 receptor, glutathione peroxidase 1, runt-related for mRNA analysis. A and B, Enhanced expression of Gr-1 Macs, transcription factor 3, glutathione S-transferase u 1, glutathione S- compared with AMs, after ozone demonstrates enhanced NQO1 ex- transferase M1, and glutathione S-transferase P1 (Supplemental pression (A) and CYP1B1 expression (B). C and D, Reduced expression Fig. 2). However, Gr-1 Macs demonstrated significantly enhanced in Gr-1 Macs over AMs after ozone demonstrates reduced SOD-1 (C)

expression of NQO1 (Fig. 3A) and exhibited a trend toward en- and HO-1 (D). Data are from pooled samples of 15 mice, performed http://www.jimmunol.org/ hanced expression of cytochrome P450, family 1, subfamily B, four times. mRNA was analyzed for each sample as fold change versus , and polypeptide 1 (CYP1B1) (Fig. 3B). Gr-1 Macs had decreased 18S. *p 0.05. expression of HO-1 and SOD-1 (Fig. 3C,3D). Differential cell surface markers and differential mRNA expression support that the source of Gr-1–positive macrophages after ozone was also Gr-1 Macs are, in fact, a unique subset that develops after ozone due to recruitment into the lung. Recruitment of inﬂammatory exposure. In addition, the proﬁle of surface markers and gene monocytes, which mature into ExMacs and monocyte-derived expression suggested that this novel subset of macrophages may dendritic cells, is dependent on the chemokine receptor CCR2 serve a protective role after inhalation of ozone. and its ligand, CCL2. Using C57BL/6 and CCR2-null mice, we

found that no difference could be observed in the number of Gr-1 by guest on September 27, 2021 Gr-1–positive macrophages are derived from a resident lung Macs after ozone exposure in a manner dependent on CCR2 source (Supplemental Fig. 3). This observation suggested that our novel We and others have previously identiﬁed that the recruitment of macrophage subset was not derived from a circulatory in- ExMacs is dependent on recruitment of a CCR2-dependent cir- termediate. On the basis of this result, we considered that Gr-1– culating intermediate. We were therefore interested to determine if positive macrophages could be derived from a resident lung

FIGURE 2. Cell surface expression of Gr-1high macrophages. A and B, CD11blow Gr-1low and CD11blow Gr-1high macrophages are analyzed for cell surface expression of CX3CR1, using CX3CR1GFP/WT mice after ozone exposure. Gr-1 Macs (red) have enhanced surface expression of CX3CR1 as compared with AM (blue) by histogram analysis (A) and MFI (B). The black open histogram represents unstained FITC channel autofluorescence, and the green open histogram represents air-exposed macrophages. C and D, Gr-1 Macs and AMs are analyzed for cell surface expression of MARCO. Gr-1 Macs (red) have enhanced cell surface expression for MARCO by histograms (C) and MFI (D), compared with ozone-exposed AMs (blue), isotype control (black), and air-exposed resident macrophages (green). Histograms are a representative sample of C57BL/6 and CX3CR1GFP/WT mice. MFI data are generated from four mice with both filtered air and ozone exposures with two repeats. ***p , 0.0005. 4804 OZONE MATURES RESIDENT MACROPHAGES intermediate. Therefore, we used a technique described by Maus or ozone. Gr-1+ macrophages were then identified. No difference et al. (22) and used by others (23–25), which uses the fluorescent was found in the air-exposed groups (Fig. 5A). However, we ob- dye PKH26-PCL (Sigma-Aldrich) to label resident phagocytic served a significant reduction in the Gr-1 subpopulation after cells in the lung. Mice were given PKH26-PCL via retro-orbital ozone exposure in the CX3CR1-null mice when compared with injection. At 24 h after injection, the mice were exposed to either the WT mice. These data suggest that CX3CR1 was important for ozone or filtered air and processed for flow cytometry. As pre- the maturation of Gr-1+ macrophages. viously demonstrated by Maus et al., this dye does not label cir- Next, we determined the biological effect of CX3CR1 deficiency culating blood cells, as identified by low cell surface expression of and the absence of Gr-1+ macrophages in the context of ozone the phagocytic label (Fig. 4A, solid red histogram). Resident AMs exposure. The role of CX3CR1 in response to ozone was un- (defined as MHCIIint CD11chi Gr-1low CD11blow) successfully known. CX3CL1 is the only known ligand for CX3CR1. We stain with the phagocyte label after either filtered air or ozone assessed the expression of CX3CL1 in the BAL of both WT and exposure (Fig. 4A, blue shaded histogram). Interestingly, Gr-1– CX3CR1-null mice. CX3CL1 was increased in the lavage of WT positive macrophages (defined as MHCIIint CD11chi Gr-1hi ozone-exposed mice, compared with WT air-exposed mice (Fig. CD11bint) also stained for the phagocyte label. To make sure that 5B). CX3CR1-null mice, both ozone exposed and air exposed, had this staining of the phagocytic label was not due to the auto- elevated levels of CX3CL1 in the BAL. This observation is con- fluorescence of macrophages, we compared both non–phagocyte- sistent with prior reports demonstrating the CX3CL1 is increased labeled (open histogram) and phagocyte-labeled (closed histo- in CX3CR1-null mice at baseline owing to failed clearance of the gram) groups for air-exposed AMs (MHCIIint CD11chi) (Fig. 4B), ligand by the receptor (26). On the basis of increased expression

AMs after ozone (MHCIIint CD11chi Gr-1low CD11blow) (Fig. 4C), of CX3CL1 after ozone, we were interested to determine whether Downloaded from and Gr-1–positive macrophages (MHCIIint CD11chi Gr-1hi CX3CR1-null mice had enhanced airway hyperresponsiveness CD11bint) (Fig. 4D). These data conﬁrmed that the phagocyte (AHR) to methacholine challenge. There was no difference in the labeling was not due to autoﬂuorescence. These data further AHR of air-exposed WT mice and that of CX3CR1-null mice support that the Gr-1–positive macrophages present after ozone (Fig. 5C). WT mice exposed to ozone were noted to have en- exposure are derived from a resident lung source and not recruited hanced AHR, compared with air controls. CX3CR1-null mice had

or derived from a circulating intermediate. even further enhanced AHR after ozone exposure when compared http://www.jimmunol.org/

+ with similarly exposed WT mice. This observation demonstrated CX3CR1 deficiency results in a reduction in Gr-1 that ozone-exposed CX3CR1-null mice develop enhanced AHR to macrophages and in enhanced response to ozone methacholine challenge when compared with ozone-exposed WT The previously described labeling of resident lung cells supports mice. On the basis of this observation, we were interested to de- that our macrophage subset was derived from a resident source. Our termine potential mechanisms that could account for this en- present understanding of macrophages during lung injury has fo- hanced AHR. Evidence of enhanced total cells and neutrophils in cused on macrophage differentiation from inflammatory mono- the BAL of CX3CR1-null mice, compared with WT mice, after cytes recruited in a CCR2-dependent fashion. However, maturation ozone, was noted, consistent with enhanced inflammation (Fig. of resident macrophages in the context of injury remains poorly 5D). No significant difference in BAL total protein was observed by guest on September 27, 2021 understood. CX3CR1 has previously been implicated in the between ozone-exposed WT and CX3CR1-null mice (Fig. 5E). maintenance of resident cell populations. As CX3CR1 expression However, evidence of enhanced oxidative stress was present, as was enhanced in Gr-1+ macrophages, we were interested to deter- measured by both 8-isoprostanes and protein carbonyl levels (Fig. mine the effect of CX3CR1 deficiency on the maturation of Gr-1 5F). BAL cytokines, recognized as elevated in the setting of ozone Macs. Flow cytometry was performed on CX3CR1GFP/WT [wild- exposure, were further enhanced in ozone-exposed CX3CR1-null type (WT)] and CX3CR1GFP/GFP (null) after exposure to either air mice (Fig. 5G). CX3CR1 deficiency was associated with enhanced

FIGURE 4. Analysis of macrophages after cell labeling with the dye PKH26-PCL. C57BL/6 mice are given the dye PKH26-PCL or control dilutant via retro-orbital injection 24 h prior to exposure to 3 h of filtered air or 2 ppm of ozone. The mice were then harvested and analyzed by flow cytometry. At the same time, whole blood was obtained from the filtered air- and ozone-exposed mice. A, Histogram analysis of PKH26-PCL expression from whole blood (red histogram), AMs (blue histogram), and Gr-1 Macs (green histograms) after filtered air or ozone. B–D, Histo- gram analysis of phagocytic label with reagent dilutant (control) versus PKH26-PCL for AMs after air exposure (B), for AMs after ozone exposure (C), and for Gr-1 Macs after ozone exposure (D). Data are representative of four mice per exposure with one repeat. The Journal of Immunology 4805

FIGURE 5. Ozone exposure in CX3CR1-null mice. C57BL/6 and CX3CR1GFP/GFP (CX3CR1-null) mice were exposed to filtered air or 2 ppm of ozone. A, Flow cytometric analysis of Gr- 1 Macs in WT (open box) and CX3CR1- null mice (closed box) after filtered air (FA) and ozone as a percentage of total cells. B, CX3CL1 protein expression was analyzed by ELISA in BAL from WT and CX3CR1-null mice after filtered air (open box) and ozone (closed box) exposure. C, AHR after increasing doses of methacholine in WT and CX3CR1-null mice after either filtered air or ozone exposure for 3 h. D, Total cells and neutrophils (PMNs) from BAL cell count Downloaded from differentials in WT (open box) and CX3CR1-null mice (closed box) 24 h after filtered air and ozone exposure. E– G, Analysis of total protein (E), 8-isoprostanes and protein carbonyls (F), and cytokines (CXCL2, IL-1b, IL-6, CCL2 http://www.jimmunol.org/ and TNF-a)(G) in BAL from WT (open box) and CX3CR1-null mice (closed box) after filtered air and ozone. Data are from four to six mice per group (WT- FA, WT-ozone, CX3CR1-null–FA, and CX3CR1-null–ozone) with one repeat. *p , 0.05, **p , 0.005. by guest on September 27, 2021

AHR, oxidative stress, and proinflammatory cytokines. Together after ozone exposure. However, this observation remains some- these findings suggest that CX3CR1-dependent Gr-1+ macro- what inconsistent and may be dependent on the intensity and phages are protective to the lung after ozone exposure. duration of exposure, as other groups have demonstrated that ozone exposure results in a decrease in macrophage-derived Discussion cytokines (29–31). Macrophages can also serve a protective role Our results demonstrate that inhalation of ozone results in the in the setting of ozone-induced lung injury. Work by Dahl et al. development of a novel macrophage subset characterized by en- (10) showed that macrophage expression of both MARCO and hanced expression of the cell surface marker Gr-1. These mac- scavenger receptor AI and AII was critical to protection against rophages are not recruited from a circulating intermediate in a ozone injury in the lung via scavenging of oxidized lipid moieties. manner dependent on CCR2, but rather appear to be derived from These ostensibly conflicting observations suggest that individual a resident lung source. We demonstrate that this novel macrophage macrophage subpopulations may be present after ozone exposure subset has enhanced cell surface expression of CX3CR1 and and have divergent roles in the development of the host response. MARCO and a unique mRNA expression profile when compared Heightened interest in macrophage heterogeneity has evolved with other resident macrophages. The development of this novel as a result of our understanding that macrophages have a variety subset of macrophages is dependent on the chemokine receptor of functions dependent on the local cytokine microenvironment CX3CR1. Furthermore, CX3CR1-null mice have enhanced bi- to which they are exposed (12, 32). We previously showed that ological response to inhaled ozone. Our observations, in total, after both infectious and noninfectious lung injury ExMacs are support a novel macrophage subset, which appears to protect the recruited from a blood-borne intermediate (16, 17). We anticipated lung from the biological response to ambient ozone. recruitment of ExMacs into the lung after ozone exposure. To our Ambient ozone remains a significant health burden, which can surprise, no significant accumulation of CD11b+ ExMacs was exacerbate chronic respiratory conditions such as asthma. Mac- noted at 24 h after inhalation of ozone. In addition, no CCR2- rophages appear to be critical in the response to inhaled ozone. dependent recruitment of macrophages was observed. This finding Pendino et al. (7) demonstrated that when the macrophage in- suggests that ExMacs are not recruited to the lung in the setting of hibitor gadolinium chloride was provided to rats prior to ozone ozone exposure. This lack of recruitment could be an issue of the exposure, these rats were protected from lung injury, as measured timing after injury or may reflect the difference in the severity of by decreased lavage fluid protein levels, inflammatory cells, the injury models between ozone and bleomycin or influenza. cytokines, and NO-inducible NO synthase. We and others (27, 28) In this study, we identified a macrophage subset that was not have identified macrophage-derived mediators present in the lung dependent on CCR2 and was delineated from other macrophages 4806 OZONE MATURES RESIDENT MACROPHAGES by expression of Gr-1, not CD11b. The Gr-1 epitope is expressed on recruited to the lung that were dependent on CCR2, which sug- the two molecules Ly-6G and Ly-6C. The expression of Ly-6G is gested that our macrophage subset may be derived from a resident restricted to granulocytes; therefore, Gr-1+/Ly-6G+ cells are de- lung population. Using a labeling technique described by Maus fined as neutrophils (33). Excluding neutrophils, Gr-1+ expression et al. (22) and used by others (23–25), we determined that Gr-1– within the lung has been identified with monocytes, with positive macrophages were, in fact, derived from a resident lung monocyte-derived dendritic cells, and, under certain injury con- intermediate. To our knowledge, this is the first report of a resident ditions, with ExMacs (16, 17). It is generally accepted that mac- lung subset identified by flow cytometry after inhalation of ozone. rophages and dendritic cells derived from monocytes in the setting Prior reports have noted that resident lung monocytes and mac- of inflammation have increased expression of Gr-1, which reflects rophages have gene expression profiles different from those of this recent differentiation from monocytes. In this setting, all of recruited cells (40). Other work supports that resident lung mac- these cells are also characterized by being CD11b+ and dependent rophages contribute to alveolar cytokine release and recruitment on CCR2 for their migration. Several other descriptions of pul- of neutrophils and monocytes into the alveolar space after expo- monary macrophages with low levels of Gr-1 expression have sure to LPS (41–43). These findings suggest that resident lung been made. Gr-1dull macrophages, which also express CD11c, and macrophages are critical for the host response to inflammation low levels of both CD11b and F4/80 were identified after pul- but that they remain poorly characterized in the setting of in- monary infection with Streptococcus pneumoniae and were re- flammation. It is also clear that some macrophages function to sponsible for early production of TNF-a (34). Recent work limit pulmonary inflammation by clearance of inflammatory described Gr-1dim/Ly-6Gdim cells, which were also low in mediators such as oxidized lipid species and apoptotic cells (10, expressing for F4/80 (35). The presence of these cells was asso- 44, 45). To better understand the complex functional role of lung Downloaded from ciated with the progression of Mycobacterium tuberculosis pul- macrophages after environmental challenges, delineation of monary infection. Although several descriptions of Gr-1 Macs and macrophage subpopulations in the lung will be necessary. their function have been made, it remains unclear if a common We made the interesting discovery that Gr-1 Macs had enhanced cell exists among the various descriptions. Gr-1 Macs are asso- CX3CR1 expression. CX3CR1 expression on blood monocytes ciated with a proinflammatory, not protective, function in these defines them as constitutive monocytes important for the de-

experimental models. None of the prior studies that we are aware velopment and replenishment of resident niches of monocytes and http://www.jimmunol.org/ of have identified a Gr-1–positive macrophage population from macrophages (18). CX3CR1 is expressed on monocytes, macro- resident cells. This observation suggests that the Gr-1 Macs we phages, mucosal DCs, NK cells, CD8 T cells, and CD4 T cells speak of are different from those previously described. It remains (18, 46, 47). CX3CR1 has only one known ligand, CX3CL1 (also unknown whether Gr-1 Macs have functional implications or known as fractalkine). Despite the characterization that blood represent a useful marker for identifying subpopulations of mye- monocytes express CX3CR1 and data from other organ systems loid cells. that suggest a critical role for the chemokine axis in inflammation The appearance of this novel subpopulation after ozone sug- (48–50), until recently there has been a dearth of information on gested that it may have unique functions when compared with the effect(s) this receptor/ligand axis exercises on the recruitment previously described macrophages. We demonstrated that Gr-1 of cells into the lung during lung injury. CX3CL1 is constitutively by guest on September 27, 2021 Macs had enhanced cell surface expression of both CX3CR1 expressed on lung macrophages (51), bronchial epithelium (52), and MARCO. The high level of expression of MARCO suggested and pulmonary endothelium (53). Multiple stimuli, including IFN-g that these macrophages may be protective in the response to ozone, (52), chronic cigarette smoke (54), and allergen challenge (55), based on prior observations that MARCO served to scavenge will cause increased pulmonary CX3CL1 expression. We identi- oxidized lipid species produced after ozone exposure to the epi- fied that levels of CX3CL1 were increased in the lavage fluid of thelial lining fluid (10). Drawing on this finding, we hypothesized ozone-challenged C57BL/6 mice when compared with filtered air that the expression profiles of Gr-1 Macs and the Gr-1–negative controls. The precise effect of CX3CL1 after inhalation of ozone lung macrophages would each be unique. After sorting, real-time remains unknown. Our findings suggest that CX3CL1 may con- PCR analysis revealed that these Gr-1 macrophages had enhanced tribute to the maturation of Gr-1 Macs. Prior work demonstrated expression of NQO1 and reduced expression of both SOD-1 and that increased CX3CL1 appeared to be associated with the re- HO-1. NQO1 is a detoxifying 2-electron reductase that can act as cruitment of CX3CR1+ mononuclear phagocytes during chronic an antioxidant through regeneration of antioxidant forms of smoke exposure (54). A follow-up study revealed that CX3CL1, in ubiquinone and vitamin E quinine. Toxicology studies identify fact, failed to recruit significant numbers of CX3CR1+ cells into a cytoprotective role for NQO1. For example, NQO1 detoxifies the airspaces, but rather was required for amplifying a subset of quinones derived from the oxidation of phenolic metabolites of CD11b-expressing macrophages, which were spatially confined to benzene (36), and the absence of NQO1 results in increased the interstitium and were a source of IL-6 and TNF-a (51). This toxicity to menadione (37). These results established a role for description of cells most consistently fits with prior descriptions of NQO1 in protection against quinone toxicity. Myeloid expression ExMacs (16, 17). We demonstrated the presence of a CX3CR1+ of NQO1 is both inducible by the antioxidant response element macrophage population from the whole lung that did not have NF-erythroid factor 2 and associated with resistance to oxidant- enhanced CD11b expression but instead had enhanced Gr-1. Gr-1+ induced cell injury (38, 39). However, NQO1 has previously been macrophages also seem to be dependent on CX3CR1 for their reported to function as either an antioxidant or a pro-oxidant, development after ozone. Gr-1+macrophages appear to be a depending on the quinine substrate and cell type. The high level unique macrophage subset when compared with circulatory- of expression of both MARCO and NQO1 is suggestive of a pro- derived phagocytes present in the lung following exposure to tective phenotype for this subpopulation of macrophages. cigarette smoke (54). Much of the work on macrophage subpopulations in the lung has We found that CX3CR1 deficiency was associated with en- focused on recruited cells from the bone marrow. Inflammatory hanced ozone-induced lung inflammation. This observation is in monocytes are recruited into the lung in a manner dependent on apparent conflict with prior reports demonstrating protection from CCR2 and then appear to differentiate into exudative macrophages the development of emphysema in a model of chronic smoke (16, 22). In the present work we did not identify macrophages exposure (51) and protection from allergic airways disease (56). The Journal of Immunology 4807

We suspect that the discrepancy is related to the different models. through scavenging of oxidized lipids by macrophage receptors MARCO and SR-AI/II. J. Clin. Invest. 117: 757–764. Allergen challenge is likely to affect the adaptive immune system, 11. Ishii, Y., K. Hashimoto, A. Nomura, T. Sakamoto, Y. Uchida, M. Ohtsuka, whereas the acute response to ozone is predominantly dependent S. Hasegawa, and M. Sagai. 1998. Elimination of neutrophils by apoptosis on the innate immune system (6). Chronic smoke exposure ap- during the resolution of acute pulmonary inflammation in rats. Lung 176: 89–98. 12. Gordon, S., and P. R. Taylor. 2005. Monocyte and macrophage heterogeneity. pears to cause chronic inflammation, which may affect different Nat. Rev. Immunol. 5: 953–964. cell types and mechanisms. Cigarette smoke seems to stimulate or 13. Careau, E., and E. Y. Bissonnette. 2004. Adoptive transfer of alveolar macro- induce a CX3CR1-dependent recruitment of macrophages from phages abrogates bronchial hyperresponsiveness. Am. J. Respir. Cell Mol. Biol. 31: 22–27. circulating intermediates, and a similar process does not appear to 14. Maus, U., J. Huwe, R. Maus, W. Seeger, and J. Lohmeyer. 2001. Alveolar JE/ evolve in our acute ozone exposure model. Our results provide MCP-1 and endotoxin synergize to provoke lung cytokine upregulation, se- strong support that, following exposure to environmental toxicants quential neutrophil and monocyte influx, and vascular leakage in mice. Am. J. Respir. Crit. Care Med. 164: 406–411. such as ozone, CX3CR1 is protective and, in addition, contributes 15. Taut, K., C. Winter, D. E. Briles, J. C. Paton, J. W. Christman, R. Maus, to maturation of resident macrophages. R. Baumann, T. Welte, and U. A. Maus. 2008. Macrophage turnover kinetics in the lungs of mice infected with Streptococcus pneumoniae. Am. J. Respir. Cell In summary, we identify a novel macrophage subset in the lungs Mol. Biol. 38: 105–113. of mice after inhalation of ozone. This unique macrophage subset 16. Lin, K. L., Y. Suzuki, H. Nakano, E. Ramsburg, and M. D. Gunn. 2008. CCR2+ is characterized by enhanced Gr-1 expression and enhanced cell monocyte-derived dendritic cells and exudate macrophages produce influenza- induced pulmonary immune pathology and mortality. J. Immunol. 180: 2562–2572. surface expression of both MARCO and CX3CR1. These mac- 17. Tighe, R. M., J. Liang, N. Liu, Y. Jung, D. Jiang, M. D. Gunn, and P. W. Noble. rophages have a unique mRNA expression profile when compared 2011. Recruited exudative macrophages selectively produce CXCL10 following with residual resident macrophages. The mRNA profile is char- non-infectious lung injury. Am. J. Respir. Cell Mol. Biol. February 17, 2011. 18. Geissmann, F., S. Jung, and D. R. Littman. 2003. Blood monocytes consist of acterized by enhanced NQO1 expression, which is suggestive of a two principal subsets with distinct migratory properties. Immunity 19: 71–82. Downloaded from detoxification phenotype. This macrophage subpopulation appears 19. Landsman, L., C . Varol, and S. Jung. 2007. Distinct differentiation potential of to be derived from resident lung phagocyte cells, not from a cir- blood monocyte subsets in the lung. J. Immunol. 178: 2000–2007. 20. Landsman, L., and S. Jung. 2007. Lung macrophages serve as obligatory in- culating intermediate. Finally, we establish that the presence of this termediate between blood monocytes and alveolar macrophages. J. Immunol. macrophage subpopulation is dependent on CX3CR1. Further- 179: 3488–3494. 21. Tsou, C. L., W. Peters, Y. Si, S. Slaymaker, A. M. Aslanian, S. P. Weisberg, more, following ozone, CX3CR1 adds a protective element to lung M. Mack, and I. F. Charo. 2007. Critical roles for CCR2 and MCP-3 in monocyte defenses, because in the absence of CX3CR1 both biological mobilization from bone marrow and recruitment to inflammatory sites. J. Clin. http://www.jimmunol.org/ (neutrophilic inflammation, oxidant stress, and proinflammatory Invest. 117: 902–909. 22. Maus, U., S. Herold, H. Muth, R. Maus, L. Ermert, M. Ermert, N. Weissmann, cytokine production) and physiological (AHR) phenotypes asso- S. Rosseau, W. Seeger, F. Grimminger, and J. Lohmeyer. 2001. Monocytes ciated with ozone are enhanced. Together these observations recruited into the alveolar air space of mice show a monocytic phenotype but support the existence of a novel subpopulation of protective de- upregulate CD14. Am. J. Physiol. Lung Cell. Mol. Physiol. 280: L58–L68. 23. Janssen, W. J., L. Barthel, A. Muldrow, R. E. Oberley-Deegan, M. T. Kearns, toxifying lung macrophages that we now term ToxMacs. We C. Jakubzick, and P. M. Henson. 2011. Fas determines differential fates of speculate that ToxMacs limit the biological and physiological resident and recruited macrophages during resolution of acute lung injury. Am. J. responses to ozone owing to their ability to scavenge oxidized Respir. Crit. Care Med. March 31, 2011. 24. Wilson, A. A., G. J. Murphy, H. Hamakawa, L. W. Kwok, S. Srinivasan, species and/or manage oxidant balance. Improved understanding A. H. Hovav, R. C. Mulligan, S. Amar, B. Suki, and D. N. Kotton. 2010. of macrophage subpopulations may provide insights into potential Amelioration of emphysema in mice through lentiviral transduction of long-lived by guest on September 27, 2021 treatments to protect against oxidant-induced lung injury or dis- pulmonary alveolar macrophages. J. Clin. Invest. 120: 379–389. 25. Jennings, J. H., D. J. Linderman, B. Hu, J. Sonstein, and J. L. Curtis. 2005. ease. Monocytes recruited to the lungs of mice during immune inflammation ingest apoptotic cells poorly. Am. J. Respir. Cell Mol. Biol. 32: 108–117. 26. Cardona, A. E., M. E. Sasse, L. Liu, S. M. Cardona, M. Mizutani, C. Savarin, Disclosures T. Hu, and R. M. Ransohoff. 2008. Scavenging roles of chemokine receptors: The authors have no financial conflicts of interest. chemokine receptor deficiency is associated with increased levels of ligand in circulation and tissues. Blood 112: 256–263. 27. Garantziotis, S., Z. Li, E. N. Potts, J. Y. Lindsey, V. P. Stober, V. V. Polosukhin, T. S. Blackwell, D. A. Schwartz, W. M. Foster, and J. W. Hollingsworth. 2010. References TLR4 is necessary for hyaluronan-mediated airway hyperresponsiveness after 1. Ciencewicki, J., S. Trivedi, and S. R. Kleeberger. 2008. Oxidants and the ozone inhalation. Am. J. Respir. Crit. Care Med. 181: 666–675. pathogenesis of lung diseases. J. Allergy Clin. Immunol. 122: 456–468, quiz 28. Pendino, K. J., R. L. Shuler, J. D. Laskin, and D. L. Laskin. 1994. Enhanced 469–470. production of interleukin-1, tumor necrosis factor-alpha, and fibronectin by rat 2.Foster,W.M.,andP.T.Stetkiewicz.1996. Regional clearance of solute from the lung phagocytes following inhalation of a pulmonary irritant. Am. J. Respir. Cell respiratory epithelia: 18-20 h postexposure to ozone. J. Appl. Physiol. 81: 1143–1149. Mol. Biol. 11: 279–286. 3. Kehrl, H. R., L. M. Vincent, R. J. Kowalsky, D. H. Horstman, J. J. O’Neil, 29. Devlin, R. B., K. P. McKinnon, T. Noah, S. Becker, and H. S. Koren. 1994. W. H. McCartney, and P. A. Bromberg. 1987. Ozone exposure increases re- Ozone-induced release of cytokines and fibronectin by alveolar macrophages and spiratory epithelial permeability in humans. Am. Rev. Respir. Dis. 135: 1124–1128. airway epithelial cells. Am. J. Physiol. 266: L612–L619. 4. Pulfer, M. K., and R. C. Murphy. 2004. Formation of biologically active oxy- 30. Becker, S., M. C. Madden, S. L. Newman, R. B. Devlin, and H. S. Koren. 1991. sterols during ozonolysis of cholesterol present in lung surfactant. J. Biol. Chem. Modulation of human alveolar macrophage properties by ozone exposure 279: 26331–26338. in vitro. Toxicol. Appl. Pharmacol. 110: 403–415. 5. Garantziotis, S., Z. Li, E. N. Potts, K. Kimata, L. Zhuo, D. L. Morgan, 31. Janic, B., T. M. Umstead, D. S. Phelps, and J. Floros. 2005. Modulatory effects R. C. Savani, P. W. Noble, W. M. Foster, D. A. Schwartz, and of ozone on THP-1 cells in response to SP-A stimulation. Am. J. Physiol. Lung J. W. Hollingsworth. 2009. Hyaluronan mediates ozone-induced airway hyper- Cell. Mol. Physiol. 288: L317–L325. responsiveness in mice. J. Biol. Chem. 284: 11309–11317. 32. Mosser, D. M., and J. P. Edwards. 2008. Exploring the full spectrum of mac- 6. Al-Hegelan, M., R. M. Tighe, C. Castillo, and J. W. Hollingsworth. 2011. rophage activation. Nat. Rev. Immunol. 8: 958–969. Ambient ozone and pulmonary innate immunity. Immunol. Res. 49: 173–191. 33. Hestdal, K., F. W. Ruscetti, J. N. Ihle, S. E. Jacobsen, C. M. Dubois, W. C. Kopp, 7. Pendino, K. J., T. M. Meidhof, D. E. Heck, J. D. Laskin, and D. L. Laskin. 1995. D. L. Longo, and J. R. Keller. 1991. Characterization and regulation of RB6-8C5 Inhibition of macrophages with gadolinium chloride abrogates ozone-induced antigen expression on murine bone marrow cells. J. Immunol. 147: 22–28. pulmonary injury and inflammatory mediator production. Am. J. Respir. Cell 34. Hatta, M., N. Yamamoto, A. Miyazato, N. Ishii, K. Nakamura, K. Inden, Mol. Biol. 13: 125–132. T. Aoyagi, H. Kunishima, Y. Hirakata, K. Suzuki, et al. 2010. Early production 8. Hotchkiss, J. A., J. R. Harkema, D. T. Kirkpatrick, and R. F. Henderson. 1989. of tumor necrosis factor-alpha by Gr-1 cells and its role in the host defense to Response of rat alveolar macrophages to ozone: quantitative assessment of pneumococcal infection in lungs. FEMS Immunol. Med. Microbiol. 58: 182–192. population size, morphology, and proliferation following acute exposure. Exp. 35. Lyadova, I. V., E. N. Tsiganov, M. A. Kapina, G. S. Shepelkova, V. V. Sosunov, Lung Res. 15: 1–16. T. V. Radaeva, K. B. Majorov, N. S. Shmitova, H. J. van den Ham, 9. Arsalane, K., P. Gosset, D. Vanhee, C. Voisin, Q. Hamid, A. B. Tonnel, and V. V. Ganusov, et al. 2010. In mice, tuberculosis progression is associated with B. Wallaert. 1995. Ozone stimulates synthesis of inflammatory cytokines by intensive inflammatory response and the accumulation of Gr-1 cells in the lungs. alveolar macrophages in vitro. Am. J. Respir. Cell Mol. Biol. 13: 60–68. PLoS ONE 5: e10469. 10. Dahl, M., A. K. Bauer, M. Arredouani, R. Soininen, K. Tryggvason, 36. Moran, J. L., D. Siegel, and D. Ross. 1999. A potential mechanism underlying S. R. Kleeberger, and L. Kobzik. 2007. Protection against inhaled oxidants the increased susceptibility of individuals with a polymorphism in NAD(P)H: 4808 OZONE MATURES RESIDENT MACROPHAGES

quinone oxidoreductase 1 (NQO1) to benzene toxicity. Proc. Natl. Acad. Sci. 47. Foussat, A., A. Coulomb-L’Hermine, J. Gosling, R. Krzysiek, I. Durand- USA 96: 8150–8155. Gasselin, T. Schall, A. Balian, Y. Richard, P. Galanaud, and D. Emilie. 2000. 37. Radjendirane, V., P. Joseph, Y. H. Lee, S. Kimura, A. J. Klein-Szanto, Fractalkine receptor expression by T lymphocyte subpopulations and in vivo F. J. Gonzalez, and A. K. Jaiswal. 1998. Disruption of the DT diaphorase production of fractalkine in human. Eur. J. Immunol. 30: 87–97. (NQO1) gene in mice leads to increased menadione toxicity. J. Biol. Chem. 273: 48. Suzuki, F., T. Nanki, T. Imai, H. Kikuchi, S. Hirohata, H. Kohsaka, and 7382–7389. N. Miyasaka. 2005. Inhibition of CX3CL1 (fractalkine) improves experimental 38. Zhu, H., Z. Jia, L. Zhang, M. Yamamoto, H. P. Misra, M. A. Trush, and Y. Li. autoimmune myositis in SJL/J mice. J. Immunol. 175: 6987–6996. 2008. Antioxidants and phase 2 enzymes in macrophages: regulation by Nrf2 49. Lesnik, P., C. A. Haskell, and I. F. Charo. 2003. Decreased atherosclerosis in signaling and protection against oxidative and electrophilic stress. Exp. Biol. CX3CR1-/- mice reveals a role for fractalkine in atherogenesis. J. Clin. Invest. Med. (Maywood) 233: 463–474. 111: 333–340. 39. Ross, D., D. Siegel, D. G. Schattenberg, X. M. Sun, and J. L. Moran. 1996. Cell- 50. Inoue, A., H. Hasegawa, M. Kohno, M. R. Ito, M. Terada, T. Imai, O. Yoshie, specific activation and detoxification of benzene metabolites in mouse and human M. Nose, and S. Fujita. 2005. Antagonist of fractalkine (CX3CL1) delays the bone marrow: identification of target cells and a potential role for modulation of initiation and ameliorates the progression of lupus nephritis in MRL/lpr mice. apoptosis in benzene toxicity. Environ. Health Perspect. 104(Suppl 6): 1177–1182. Arthritis Rheum. 52: 1522–1533. 40. Srivastava, M., S. Jung, J. Wilhelm, L. Fink, F. Bu¨hling, T. Welte, R. M. Bohle, 51. Xiong, Z., A. S. Leme, P. Ray, S. D. Shapiro, and J. S. Lee. 2011. CX3CR1+ W. Seeger, J. Lohmeyer, and U. A. Maus. 2005. The inflammatory versus con- lung mononuclear phagocytes spatially confined to the interstitium produce stitutive trafficking of mononuclear phagocytes into the alveolar space of mice is TNF-a and IL-6 and promote cigarette smoke-induced emphysema. J. Immunol. associated with drastic changes in their gene expression profiles. J. Immunol. 186: 3206–3214. 175: 1884–1893. 52. Fujimoto, K., T. Imaizumi, H. Yoshida, S. Takanashi, K. Okumura, and K. Satoh. 41. Maus, U. A., S. Janzen, G. Wall, M. Srivastava, T. S. Blackwell, J. W. Christman, 2001. Interferon-gamma stimulates fractalkine expression in human bronchial W. Seeger, T. Welte, and J. Lohmeyer. 2006. Resident alveolar macrophages are epithelial cells and regulates mononuclear cell adherence. Am. J. Respir. Cell replaced by recruited monocytes in response to endotoxin-induced lung in- Mol. Biol. 25: 233–238. flammation. Am. J. Respir. Cell Mol. Biol. 35: 227–235. 53. Balabanian, K., A. Foussat, P. Dorfmu¨ller, I. Durand-Gasselin, F. Capel, 42. Blackwell, T. S., L. H. Lancaster, T. R. Blackwell, A. Venkatakrishnan, and L. Bouchet-Delbos, A. Portier, A. Marfaing-Koka, R. Krzysiek, A. C. Rimaniol, J. W. Christman. 1999. Chemotactic gradients predict neutrophilic alveolitis in et al. 2002. CX(3)C chemokine fractalkine in pulmonary arterial hypertension. endotoxin-treated rats. Am. J. Respir. Crit. Care Med. 159: 1644–1652. Am. J. Respir. Crit. Care Med. 165: 1419–1425. Downloaded from 43. Hollingsworth, J. W., II, D. N. Cook, D. M. Brass, J. K. Walker, D. L. Morgan, 54. McComb, J. G., M. Ranganathan, X. H. Liu, J. M. Pilewski, P. Ray, W. M. Foster, and D. A. Schwartz. 2004. The role of Toll-like receptor 4 in en- S. C. Watkins, A. M. Choi, and J. S. Lee. 2008. CX3CL1 up-regulation is as- vironmental airway injury in mice. Am. J. Respir. Crit. Care Med. 170: 126–132. sociated with recruitment of CX3CR1+ mononuclear phagocytes and 44. Borges, V. M., R. W. Vandivier, K. A. McPhillips, J. A. Kench, K. Morimoto, T lymphocytes in the lungs during cigarette smoke-induced emphysema. Am. J. S. D. Groshong, T. R. Richens, B. B. Graham, A. M. Muldrow, L. Van Heule, Pathol. 173: 949–961. et al. 2009. TNFalpha inhibits apoptotic cell clearance in the lung, exacerbating 55. Rimaniol, A. C., S. J. Till, G. Garcia, F. Capel, V. Godot, K. Balabanian, acute inflammation. Am. J. Physiol. Lung Cell. Mol. Physiol. 297: L586–L595. I. Durand-Gasselin, E. M. Varga, G. Simonneau, D. Emilie, et al. 2003. The

45. Teder, P., R. W. Vandivier, D. Jiang, J. Liang, L. Cohn, E. Pure´,P.M.Henson,and CX3C chemokine fractalkine in allergic asthma and rhinitis. J. Allergy Clin. http://www.jimmunol.org/ P. W. Noble. 2002. Resolution of lung inflammation by CD44. Science 296: 155–158. Immunol. 112: 1139–1146. 46. Imai, T., K. Hieshima, C. Haskell, M. Baba, M. Nagira, M. Nishimura, 56. Mionnet, C., V. Buatois, A. Kanda, V. Milcent, S. Fleury, D. Lair, M. Langelot, M. Kakizaki, S. Takagi, H. Nomiyama, T. J. Schall, and O. Yoshie. 1997. Y. Lacoeuille, E. Hessel, R. Coffman, et al. 2010. CX3CR1 is required for airway Identification and molecular characterization of fractalkine receptor CX3CR1, inflammation by promoting T helper cell survival and maintenance in inflamed which mediates both leukocyte migration and adhesion. Cell 91: 521–530. lung. Nat. Med. 16: 1305–1312. by guest on September 27, 2021 The Journal of Immunology

Corrections

Tighe, R. M., Z. Li, E. N. Potts, S. Frush, N. Liu, M. D. Gunn, W. M. Foster, P. W. Noble, and J. W. Hollingsworth. 2011. Ozone inhalation promotes CX3CR1-dependent maturation of resident lung macrophages that limit oxidative stress and inﬂammation. J. Immunol. 187: 4800–4808.

Following an inquiry at Duke University, we have been informed that FlexiVent data provided to us by the animal pulmonary physiology laboratory may have been unreliable. The data in Figs. 1–4 are not affected. We have replicated the studies in Fig. 5A–F, and the new data support the originally published findings. We were unable to repeat the experiments in Fig. 5G; therefore, to maintain the accuracy of the scientific record, a corrected Fig. 5 and figure legend, representing the new data, are shown below.

FIGURE 5. Ozone exposure in CX3CR1-null mice. C57BL/6 (WT) and CX3CR1GFP/GFP (CX3CR1-null) mice were exposed to filtered air or 2 ppm of ozone for 3 h and then underwent analysis 12–24 h after exposure. A, Flow cytometric analysis of Gr-1 Macs in WT (open box) and CX3CR1-null mice (closed box) as a percentage of total cells at 24 h after exposure to filtered air (FA) or ozone. B, CX3CL1 protein expression was analyzed by ELISA in BAL from WT and CX3CR1-null mice 24 h after filtered air (open box) or ozone (closed box) exposure. C, AHR after increasing doses of methacholine in WT and CX3CR1-null mice 24 h after filtered air or ozone exposure. D, Total cells and neutrophils (PMNs) from BAL cell count differentials in WT (open box) and CX3CR1-null mice (closed box) 24 h after filtered air or ozone exposure. E, Analysis of total protein in BAL from WT (open box) and CX3CR1-null mice (closed box) 24 h after filtered air or ozone exposure. F, Analysis of cytokines by multiplex from concentrated BAL fluid in WT (open box) and CX3CR1-null mice (closed box) 12 h after filtered air or ozone exposure. Data for AHR are from n 5 7WTFA,n 5 7 CX3CR1-null FA, n 5 12 WT O3, and n 5 14 CX3CR1-null O3 (#p , 0.05 for WT or CX3CR1-null O3 versus FA control, *p , 0.05 for WT versus CX3CR1-null O3 exposed, &p , 0.05 for WT versus CX3CR1-null FA exposed). Data for other experiments are from three to eight mice per group (WT-FA, WT-ozone, CX3CR1-null–FA, and CX3CR1-null–ozone). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600002