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Inflammatory Lung DiseaseCharacterize Acute Infection and Chronic Distinct Macrophage Subpopulations

Maxwell, Gary P. Anderson and Margaret L. HibbsMubing Duan, Waichu C. Li, Ross Vlahos, Mhairi J.

ol.1200660http://www.jimmunol.org/content/early/2012/06/10/jimmun

published online 11 June 2012J Immunol 

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The Journal of Immunology

Distinct Macrophage Subpopulations Characterize AcuteInfection and Chronic Inflammatory Lung Disease

Mubing Duan,*,†,‡ Waichu C. Li,* Ross Vlahos,x Mhairi J. Maxwell,*,‡ Gary P. Anderson,x,{

and Margaret L. Hibbs*,‡

Although great progress has been made in delineating lung dendritic cell and lymphocyte subpopulations, similar advances in lung

macrophages (MFs) have been hampered by their intrinsic autofluorescence, cell plasticity, and the complexities of monocyte–MF

compartmentalization. Using spectral scanning, we define alveolar MF autofluorescence characteristics, which has allowed us to

develop an alternative flow cytometry method. Using this methodology, we show that mouse lung MFs form distinct subpopu-

lations during acute inflammation after challenge with LPS or influenza virus, and in chronic inflammatory lung disease conse-

quent to SHIP-1 deletion. These subpopulations are distinguished by differential Mac-1 and CD11c integrin expression rather

than classical M1 or M2 markers, and display differential gene signatures ex vivo. Whereas the resolution of acute inflammation is

characterized by restoration to a homogenous population of CD11chighMac-1neg/low MFs reflective of lung homeostasis, chronic

inflammatory lung disease associated with SHIP-1 deficiency is accompanied by an additional subpopulation of CD11chighMac-1pos

MFs that tracks with lung disease in susceptible genetic background SHIP-12/2 animals and disease induction in chimeric mice.

These findings may help better understand the roles of MF subpopulations in lung homeostasis and disease. The Journal of

Immunology, 2012, 189: 000–000.

Macrophages (MFs) maintain organ homeostasis, facil-itate host defense and wound healing, but also underliethe pathogenesis of many chronic diseases (1). This

functional dichotomy arises from their intrinsic plasticity, causingpolarization into distinct functional phenotypes depending on theenvironmental stimuli. For instance, classically activated or M1-like MFs become more proinflammatory and bactericidal in thepresence of IFN-g and LPS, whereas alternatively activated orM2-like MFs are immunoregulatory, and can promote woundhealing and modify the extracellular matrix through the secretionof proteases and growth factors (2, 3). Because there is complexoverlap between tissue homeostasis, inflammation, and disease

pathogenesis, it is now thought that distinct MF subpopulationsmay exist that subserve their protective or pathogenic roles (1). Aprime example may be in lungs, where residential alveolar MFs(AMFs) are simultaneously implicated to play many homeostatic(4), proinflammatory (5, 6), and pathogenic roles (7, 8).In healthy adult lungs, AMFs exist as the predominant cell type

(98%+) in the alveolar airspaces. There, they sit as a uniform,quiescent, and immunosuppressive population capable of patho-gen phagocytosis and T cell recruitment in the early event of abacterial or viral infection (9, 10). Cell turnover rate is slow (11)and is maintained by constitutively immigrating CCR22 Gr-12

resident monocytes (12, 13). In contrast, CCR2+ Gr-1+ inflam-matory monocytes rapidly migrate into alveolar airspaces afterlung infection and are believed to be the main effectors of acutelung injury and infection-related mortality (6, 14, 15). IncreasedAMFs, however, are correlated with the severity of chronic ob-structive pulmonary disease (COPD) (16), a debilitating chroniccondition characterized by progressive irreversible airflow limi-tation and lung parenchyma destruction. Altered AMF behavior isalso observed in animal models of COPD, chronic lung inflam-mation, and tumorigenesis (7, 8, 17, 18), making the behavior oflung MFs an important field of study.Unfortunately, efforts to identify AMF subsets in the lung mi-

croenvironment have been severely hampered by their intense cellautofluorescence (19), unlike inflammatory lung monocytes (or“exudate” lung MFs), which are less autofluorescent, easier to studyby flow cytometry, and thus better characterized. Altogether, thishas created a gap in our understanding of AMF heterogeneityand their roles in the orchestration of host defense, inflamma-tion, and disease. In this article, we demonstrate that AMF auto-fluorescence emission diminishes above 640 nm, enabling us toestablish a modified flow cytometry method using fluorophore-conjugated Abs that emit at wavelengths (ls) .660 nm. Screen-ing LPS-treated and influenza-infected C57BL/6 mice, we found thatMF heterogeneity to be a prominent component of the acute lungimmune response, distinctly characterized by three MF subpopu-lations present in the alveolar airspaces (CD11chighMac-1neg/low

*Ludwig Institute for Cancer Research, Melbourne, Victoria 3050, Australia;†Department of Surgery, University of Melbourne, Melbourne, Victoria 3010, Aus-tralia; ‡Leukocyte Signaling Laboratory, Department of Immunology, Monash Uni-versity, Central Clinical School, Alfred Medical Research and Education Precinct,Melbourne, Victoria 3004, Australia; xLung Disease Research Group, Department ofPharmacology, University of Melbourne, Melbourne, Victoria 3010, Australia; and{Department of Medicine, Royal Melbourne Hospital, University of Melbourne,Melbourne, Victoria 3010, Australia

Received for publication February 27, 2012. Accepted for publication May 1, 2012.

This work was supported by the National Health and Medical Research Council(NHMRC) of Australia and the Operational Infrastructure Support Program providedby the Victorian Government, Australia. M.D. is a recipient of a Cancer ResearchInstitute Scholarship from the Peter MacCallum Cancer Centre/Ludwig Institute forCancer Research and the Nick Christopher Scholarship from the Department ofMedicine, University of Melbourne. M.J.M. was supported by a Doherty fellowshipfrom NHMRC. M.L.H. is a Senior Research Fellow of NHMRC.

Address correspondence and reprint requests to Dr. Margaret L. Hibbs, Departmentof Immunology, Alfred Medical Research and Education Precinct, CommercialRoad, Melbourne, VIC 3004, Australia. E-mail address: Margaret.Hibbs@monash.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: AMF, alveolar macrophage; Arg I, arginase I;BAL, bronchoalveolar lavage; BM, bone marrow; COPD, chronic obstructive pul-monary disease; CT, cycle threshold; FSC, forward scatter; iNOS, inducible NOsynthase; LICR, Ludwig Institute for Cancer Research; MF, macrophage; MMP-12, matrix metalloproteinase 12; MT, Masson’s trichrome; qRT-PCR, quantitativeRT-PCR; SSC, side scatter; l, wavelength.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200660

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“residential,” CD11chighMac-1pos “intermediate,” and CD11clow

Mac-1high monocytes). Subpopulations displayed distinct geneprofiles independent of an exclusive M1 or M2 polarization sig-nature, with restoration to the CD11chighMac-1neg/low subpopula-tion marking resolution and the reinstatement of lung homeostasis.Chronic inflammatory lung disease was then investigated usingSHIP-1–deficient mice, where AMF polarization was first iden-tified as an effector of chronic lung disease and tumorigenesis (8).AMF subpopulation deregulation (marked by an additional sub-population of residential CD11chighMac-1pos AMFs) was found tobe a prominent novel feature of SHIP-1–deficient mice and trackedspecifically with lung disease pathogenesis. Altogether, our studiessuggest a novel role for AMF subpopulations and Mac-1 signalingin the regulation of AMF activation, acute infection, and chronicinflammatory lung disease.

Materials and MethodsAnimals

SHIP-12/2 mice (20) were crossed onto the C57BL/6 or BALB/c back-ground for seven generations (21). C57BL/6 or BALB/c mice were bredunder specific pathogen-free conditions by the Ludwig Institute for CancerResearch (LICR, Melbourne, VIC, Australia). BL6.SJL-Prptca mice werepurchased from ARC (Perth, WA, Australia). Animal experiments wereapproved by the Animal Ethics Committees of the LICR/Department ofSurgery and the University of Melbourne in line with National Health andMedical Research Council guidelines.

LPS and Mem71 virus challenge

Ten- to 12-wk-old mice were challenged with a 30 ml transnasal admin-istration of E. coli LPS (10 mg/mouse, serotype O114:B4; Sigma) orMem71 influenza virus (31,000 PFU/mouse, sublethal dose), and eutha-nized at 24 h, 72 h, and day 10. Bronchoalveolar lavage (BAL) cells werecollected as described later. Control mice received 30 ml DMEM.

Acute cigarette smoke exposure

C57BL/6 mice were exposed to cigarette smoke for 4 consecutive days (22),euthanized on day 5, and BAL cells collected. Control mice were housed inidentical chambers that did not receive cigarette smoke.

Creation of CD45.2 bone marrow chimeras

Bone marrow (BM) was extracted aseptically from femurs of 6-wk-olddonor BL6.SJL-Prptca mice (CD45.1 allotype), washed, filtered, andresuspended in 2% FCS + PBS. Four- to 6-wk-old recipient C57BL/6 mice(CD45.2 allotype) received two equal doses of total body irradiation (550rad), separated by 3 h. One hour postirradiation, recipients received 4 3105 BL6.SJL-Prptca donor BM cells through lateral tail vein injection, andmice were then given oral antibiotics for 2 wk postirradiation. Four weekspostirradiation, chimeric mice were challenged with LPS, Mem71 virus, orDMEM; euthanized; and BAL and peripheral blood collected for flowcytometry.

Lung histology

Lungs were inflation fixed in 4% PFA at 25 cmwater pressure, removed, andparaffin embedded. Four-micrometer sections were stained with H&E orMasson’s trichrome (MT). Images were captured using 310 and 320objectives (Nikon Eclipse 90i).

BAL and peritoneal cell collection

Peritoneal cells were obtained from the peritoneum with PBS (5 ml). Thetrachea was cannulated and one 0.4-ml followed by three 0.3-ml aliquots ofPBS sequentially aspirated and retrieved from the lungs to collect BALcells. Cell counts were performed using a nucleocounter (Chemotec). BALcells were resuspended in RBC lysis buffer (0.83% NH4Cl-Tris buffer,37˚C, 3 min) and washed in FACS buffer before FACS analysis and cellsorting.

Spectral scanning of MF autofluorescence

Spectral scanning was performed using a confocal laser-scanning micro-scope (Olympus FV1000). Cells were excited using 473-nm lines froma solid-state laser (Olympus, Tokyo, Japan), and spectral scans (xyl) takenbetween 480 and 680 nm (20-nm steps, 240-mm pinhole). Transmitted

light images were taken after each spectral scan, and cells were identifiedbased on morphology. Settings were consistent between samples withphotobleaching at ,3 and ,7% for C57BL/6 and SHIP-12/2 MFs, re-spectively. Images were captured using 603 water immersion objective(4.833 zoom, 512 3 512 pixels). Virtual l stacks were visualized usingImageJ software, and MF emission spectra was calculated as an average ofthe mean fluorescent intensity versus emission l of $8 MFs.

Flow cytometry analysis and cell sorting

Peritoneal and BAL cells were resuspended in FACS buffer (PBS, 2% FCS,2 mMEDTA), filtered, and incubated with Fc block (0.5 mg/ml, 10 min; BDBiosciences). Cells were stained with 1˚ mAbs (20 min, 4˚C on ice) fol-lowed by streptavidin allophycocyanin-Alexa Fluor 750 where necessary(15 min, 4˚C on ice; Invitrogen). To examine AMFs, we used anti-mouseCD45-FITC (BD Biosciences) to first positively select all leukocytes inBAL, and thus gate out BAL debris. Anti-mouse Ly6g-PE (BD Biosciences)was then used to negatively gate out all neutrophils, and all lymphocyteswere gated out based on forward scatter (FSC) and side scatter (SSC). Thisallowed FACS analysis on the remaining MF/monocyte subpopulationsusing anti-mouse CD11c-allophycocyanin, CD11c-biotin, Ly6c-biotin,class II IA-b–biotin, CD36-allophycocyanin, CD124-biotin, and CD40-biotin (all from BD Biosciences); Mac-1–Pe–Cy7, CD14-PerCP-Cy5.5,and CD23-Pe-Cy7 (eBioscience); and F4/80-biotin and AFS98-biotin(gift from J. Hamilton, University of Melbourne). Data were acquired ona FACSAria (BD Biosciences), which collected $5000 AMF events.Analysis was performed using FlowJo software (Mac V8.7.3). Total BALcell counts were performed using a nucleocounter (Chemotec), with ab-solute cell numbers calculated as total BAL cell number 3 percentage ofcell subpopulation as determined by FACS (using CD45posLy6ghigh forneutrophils, CD45posLy6gneg and FSC versus SSC profile for lymphocytes,and CD45posLy6gpos and FSC versus SSC profile for AMFs).

Sorting of BAL AMFs was performed on individual mouse samples usingthe staining approach described earlier. A total of $250,000 cells/subpopu-lation were sorted for cytospin analysis and quantitative RT-PCR (qRT-PCR).Staining and sorting of cells from LPS and influenza challenged mice wereeither performed on the same day or against vehicle control to enablecomparable results. Sorting purity was $95% for all MF subpopulations.Cytospins of sorted cells were stained with Diff-Quik (Lab Aids) andimages captured at 3 40 oil immersion objective (Nikon Eclipse 90i).

RNA isolation and qRT-PCR

Sorted BAL cells were pelleted, resuspended in 350 ml RLT plus buffer + 2-ME (Qiagen), snap frozen in liquid nitrogen, and stored at 270˚C. RNAwas isolated using the RNeasy Plus Micro Kit (Qiagen), and quality andconcentration were measured (Nanodrop). For independent experiments, aconsistent amount of RNA/sample (200–300 ng) was converted to cDNA(Superscript VILO cDNA Synthesis Kit; Invitrogen) and diluted 1:1.5 inRNase-free H2O. Genes of interest were screened using preoptimized andvalidated TaqMan assays. Reaction mix consisted of 2 ml cDNA, 0.5 mltarget primer, and 5 ml TaqMan Universal PCR Mastermix diluted with2.5 ml RNase-free H2O and run for 40 cycles under fast single-plex con-ditions (ABI 7900ht). Eukaryotic 18S rRNA (Applied Biosystems) was usedas endogenous control for all samples. For each gene target, a no-RT neg-ative control was used to check against nonspecific binding to genomicDNA. Cycle threshold (CT) values were calculated using automatic thresholdanalysis (SDS 2.3 software; Applied Biosystems) and taken as an average ofduplicates. The relative expression of each target gene was calculated usingthe 22DDCT method (normalized to 18S control and relative to DMEM Rcontrol, or LPS R when DMEM R transcript undetectable). Results werecalculated from a total of four independent experiments. All RNA sampleswere prepared together and run on a single plate to ensure that each inde-pendent experiment produced data all directly compared with control R.

Statistical analysis

Statistical analyses were performed using GraphPad Prism V.4.03. Valuesare expressed as mean6 SEM from two or more independent experiments.The unpaired two-tailed Student t test was used for all excepting qRT-PCRdata, which were analyzed using the two-tailed Mann–Whitney U test.

ResultsSpectral scanning directs new AMF characterization approachusing flow cytometry

Although surface markers exist for the identification of lung den-dritic cell and lymphocyte subpopulations, a standard and consistentapproach to the study of AMFs has yet to be identified due first to

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their intrinsically high autofluorescence. We used confocal mi-croscopy to perform spectral scanning of AMF emission ls. AMFswere significantly more autofluorescent than peritoneal MFs, withemission peaking around the emission ls of traditional fluorophoresFITC and PE, but not allophycocyanin (Fig. 1A, 1B). As expected,flow cytometry using traditional FITC- or PE-conjugated Absproduced only a small shift in positive (i.e., CD11c) staining be-cause of the high background noise from AMF autofluorescence(Fig. 1C). Because this autofluorescence greatly diminishes at l. 660 nm, fluorophores that emitted beyond this l (i.e., allophy-cocyanin, Pe-Cy7, and allophycocyanin-Alexa Fluor 750) were thenput under trial. These produced a clear separation between nega-tive and positive AMF staining, and revealed that AMFs wereCD11chighMac-1neg/low (Fig. 1C), assenting with previous studies ofrat AMFs where differential surface Ag staining could be dis-cretely distinguished using allophycocyanin (23). Although clini-cal study has suggested that basal AMF autofluorescence derivesfrom acquired tobacco tar exposure (24), we show that restingmouse AMFs are intrinsically more autofluorescent compared withperitoneal MFs, further emphasizing the necessity of our method-ological approach.We first tested a panel of M1 andM2 surface markers, but did not

identify distinct AMF subpopulations (Supplemental Fig. 1A).Leukocyte integrins play an important role in monocyte migrationand activation, and are differentially expressed by blood monocytesubsets (25). Thus, we assayed the expression of leukocyte integ-rins CD11a, CD11b (Mac-1), and CD11c in AMFs from controland LPS-treated mice. Only Mac-1 expression was found to bedifferentially distributed in LPS-treated, but not control AMFs(Fig. 1D). Using CD11c and Mac-1 as subpopulation markers, wethen investigated the behavior of AMFs in different respiratoryconditions.

AMF subpopulations identified after LPS and virus challengein mice

To detect AMF populations during acute lung infection, we firststudied C57BL/6 mice 72 h post-LPS and Mem71 influenza viruschallenge, because this is when initial lung neutrophilia subsidesand MFs become prominent. As expected, LPS challenge elicitedan acute inflammatory response with alveolar airspace infiltrationby mononuclear cells, especially near regions of pulmonary bloodvessels (Fig. 2A). BAL MF, neutrophil, and lymphocyte numberswere significantly higher compared with DMEM-treated (i.e.,control) mice (Fig. 2B), as reflected by the altered BAL cell FSCversus SSC (Fig. 2A).We identified three BALMF subpopulations in mice challenged

with LPS or Mem71 influenza virus. In control mice, residentialAMFs were .95% CD11chighMac-1neg/low (subpopulation R), andwere c-fmspos and Ly6cneg (Fig. 2C). After LPS challenge, threeMF subpopulations appeared in BAL: “residential” CD11chigh

Mac-1neg/low AMFs (subpopulation R), CD11chighMac-1pos

AMFs of an “intermediate” phenotype (subpopulation I), and asubpopulation of lung monocytes that were CD11clowMac-1high

(subpopulation Me). All subpopulations were c-fmspos, with onlythe monocyte subpopulation also Ly6cpos (Fig. 2C).A differential MF signature was observed postinfection with

Mem71 (H3N1) influenza virus. A smaller proportion of residentialCD11chighMac-1neg/low AMFs (subpopulation R) was observed,CD11chighMac-1pos intermediate MFs (subpopulation I) werec-fmslow but Ly6cpos, and there was a predominance of CD11cneg/low

FIGURE 1. Spectral scanning directs flow cytometry approach for the

study of AMFs. (A) PMFs and AMFs imaged under transmitted light (top

panels) and at 520–540 nm by a 473-nm laser (bottom panels); repre-

sentative of two independent experiments, n = 4. Scale bar, 10 mm. (B)

Emission spectra of PMFs and AMFs. Gray bars indicate emission

spectrum of fluorophores FITC, PE, and allophycocyanin. Results are

mean 6 SEM, *p , 0.05, **p , 0.0005, ***p , 0.0001; representative of

two independent experiments, n = 4. (C) Flow cytometry of PMFs and

AMFs taken from the same mouse using FITC and PE compared with

allophycocyanin and PE-Cy7; representative of two independent experi-

ments, n = 4. Positive staining in black, and staining with nonbinding

control Abs (i.e., background MF autofluorescence) in gray; representative

of two independent experiments, n = 4. (D) CD11a, CD11b (Mac-1), and

CD11c AMF expression of control and LPS-treated mice. Positive stain-

ing in black, and staining with nonbinding control Abs in gray; repre-

sentative of two independent experiments, n = 4.

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FIGURE 2. Three lung MF subpopulations exist in acute lung inflammation. (A) MT-stained lungs of C57BL/6 mice 72 h post DMEM- (control), LPS-,

and Mem71 virus-challenge (top panels). Red arrows indicate inflammatory cells in alveolar airspaces; black arrow indicates cellular infiltration from

pulmonary vessels. Scale bar, 50 mm. BAL cell size (FSC) and granularity (SSC) from treated mice shown using flow cytometry (bottom panels). (B)

Cellular composition of BAL from control, LPS-, and Mem71-infected mice 72 h postchallenge. Cell differentials determined by flow cytometry. *p ,0.05, **p , 0.005, ***p , 0.0001; n = 6–7 from three independent experiments. (C) Flow cytometry of Mac-1 versus CD11c of BAL MFs in LPS-treated

and Mem71-infected mice. Each subpopulation is gated and further assessed for c-fms and Ly6c expression; n = 6–7 from three independent experiments.

(D) Cytospins of sorted lung MF subpopulations from control, LPS-treated, and Mem71-infected mice. Representative of three independent sorts. Scale

bar, 20 mm. (E) qRT-PCR of sorted lung MF subpopulations for Mac-1, iNOS, Arg-1, MMP-12, IL-6, and IL-10 gene expression. All fold changes relative

to control R (residential MF subpopulation from control mice), except for IL-10, which is calculated relative to LPS R because no IL-10 transcript was

detectable in control R. Results are mean6 SEM, *p, 0.05, **p , 0.01; n = 4 from four independent experiments. Asterisk directly above a bar indicates

p value compared with DMEM R. I, Intermediate; Me, monocyte-like postendotoxin; Mv, monocyte-like postvirus; R, residential.

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Mac-1high monocytes (subpopulation Mv). Unlike LPS challenge,this monocyte subpopulation was Ly6chigh but c-fmslow (Fig. 2C).MFs are believed to polarize into either a classically activated

M1-like or alternatively activated M2-like phenotype (2, 3), al-though how this manifests in the dynamic tissue microenviron-ment remains unclear. To assess this, we used our gating strategyto sort each AMF subpopulation and examined for differences inmorphology and M1/M2 gene expression. Mac-1high (Me and Mv)monocytes were morphologically distinct from both residentialand intermediate BAL MFs, appearing as highly vacuolated cellswith irregular-shaped nuclei (Fig. 2D, Supplemental Fig. 1B). Forgene profiling, we focused on all AMF subpopulations fromLPS-treated mice because LPS is a classical and well-studied M1polarizing stimulus (3), together with subpopulation Mv in Mem71-infected mice because of its differential surface phenotype. Com-pared with residential AMFs (subpopulation R) in control mice,Mac-1 gene expression was significantly upregulated in interme-diate BAL MFs and monocytes (subpopulation I and Me and Mv)as expected (Fig. 2E). Interestingly, both M1 and M2 signatureswere upregulated in BAL monocytes from LPS- and Mem71-challenged mice. Inducible NO synthase (iNOS) was higher in allsubpopulations compared with residential AMFs from controlmice. Upregulation of the classical M2 marker arginase I (Arg I)was simultaneously observed in the intermediate and monocytesubpopulation from LPS-treated mice and in Mem71-infectedmonocytes (Fig. 2E). Both IL-6 (a known M1 MF cytokine)and IL-10 (the classical M2 MF cytokine) were simultaneouslyupregulated in LPS and Mem71 monocyte subpopulations com-pared with residential control. IL-6, but not IL-10, was also slightlybut significantly upregulated in the intermediate AMF subpopu-lation of LPS-treated mice (Fig. 2E). Interestingly, matrix metal-loproteinase 12 (MMP-12) was upregulated in all Mac-1pos BALMF subpopulations present after LPS treatment (Fig. 2E).

Determination of AMF subpopulation origins

We postulated that the CD11chighMac-1pos MFs (subpopulation I)observed during acute inflammation could represent residentialAMFs undergoing a continuum of Mac-1 upregulation. To con-firm the origin of each MF subpopulation, we generated BMchimeras using CD45.1 donors and CD45.2 recipients, which wethen challenged with LPS, Mem71, or DMEM (control). Back-ground lung toxicity was observed, as CD45.2 chimeric mice hadgreater BAL debris and increased BAL MF heterogeneity com-pared with C57BL/6 mice (Supplemental Fig. 1A). Four weekspostirradiation and transfer of CD45.1 BM, 70–90% of BAL MFswere CD45.2+ in control chimeric mice, reflecting low residentialAMF turnover (Fig. 3A, Supplemental Fig. 1C) as previouslyreported (11). In nonirradiated C57BL/6 (i.e., CD45.2 allotypemice), all peripheral blood leukocytes were CD45.2+ (Supple-mental Fig. 1C). In contrast,$95% of circulating blood monocyteswere CD45.1+ 4 wk postirradiation, demonstrating successfulblood leukocyte reconstitution with CD45.1 donor BM (Sup-plemental Fig. 1C).In control, LPS-, andMem71-challenged CD45.2 chimeric mice,

70–90% of all CD11chighMac-1neg/low residential AMFs (sub-population R) were CD45.2+ as expected (Fig. 3A). BAL mono-cytes (subpopulation Me and Mv) in challenged chimeras were$95% CD45.1+, confirming their origin as newly recruited bloodmonocytes (Fig. 3A). Interestingly, in challenged chimeric mice,CD11chighMac-1pos intermediate BAL MFs (subpopulation I) con-tained equal proportions of CD45.1+ and CD45.2+ cells, indicatingthat ∼50% of the subpopulation had originated from residentialAMFs (Fig. 3A).

To determine whether residential AMF Mac-1 upregulationpreceded monocyte recruitment during acute inflammation, wecharacterized BAL MF kinetics 24 h, 72 h, and 10 d post-LPS andMem71 influenza treatment. Twenty-four hours post-LPS, onlyCD11chighMac-1pos intermediates, but not CD11clowMac-1high

FIGURE 3. Identification of lung MF subpopulation origins using ir-

radiated CD45.2 mice reconstituted with BM from CD45.1 donors. (A)

Flow cytometry of BAL MFs from control and challenged chimeric mice

depicting Mac-1 versus CD11c expression. Each lung MF subpopulation

is gated and further assessed for CD45.2 versus CD45.1 expression; n = 6

from two independent experiments. (B) Mac-1 versus CD11c expression of

BAL MFs from LPS- and Mem71 influenza-challenged mice 24 h, 72 h,

and 10 d postchallenge; n = 6 from two independent experiments. I, In-

termediate; Me, monocyte-like postendotoxin; Mv, monocyte-like post-

virus; R, residential.

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FIGURE 4. Lung MF subpopulations exist in SHIP-12/2 mice with spontaneous lung disease. (A) MT-stained lungs of C57BL/6, acute cigarette smoke-

exposed, and C57BL/6 SHIP-12/2 mice (top panels). Red arrow indicates myeloid infiltrates in SHIP-12/2 alveolar airspaces; black arrow indicates regions

of collagen deposition. Scale bar, 50 mm. FSC versus SSC of BAL from respective mice (bottom panel). (B) BAL cell differentials as determined by flow

cytometry. *p, 0.05, **p, 0.005, ***p, 0.0001; n = 7 from two independent experiments. (C) Mac-1 versus CD11c expression of BAL MFs. Each MF

subpopulation is further analyzed for c-fms and Ly6c expression; n = 6 from two independent experiments. (D) Transmitted light image (top panels) and

autofluorescence emission (middle panels) of BAL MFs. Respective BAL MF emission spectra (bottom panel). Results are mean 6 SEM; *p , 0.005,

**p , 0.0005, ***p , 0.0001; representative of two independent experiments, n = 4. Scale bar, 10 mm. (E) Cytospins of sorted BAL MF subpopulations.

Representative of three independent sorts. Scale bar, 20 mm. (F) qRT-PCR of sorted C57BL/6 and SHIP-12/2 BAL MF subpopulations for Mac-1, iNOS,

Arg-1, MMP-12, IL-6, and IL-10 gene expression. Fold changes relative to DMEM R of Fig. 2E, except for IL-10, which is (Figure legend continues)

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monocytes, were additionally observed in BAL, suggesting thatresidential AMF Mac-1 upregulation occurs before monocyte re-cruitment during acute lung inflammation (Fig. 3B). By day 10,resolution of all lung MFs to a single CD11chighMac-1neg/low resi-dential AMF phenotype had occurred in LPS-treated mice. Thiscontrasted greatly with influenza infection, where four discrete MF-like subpopulations still existed, further highlighting differences inMF subpopulation regulation during lung disease (Fig. 3B).

Identification of AMF subpopulations in chronic lung disease

SHIP-1 is a hematopoietic-specific 59 inositol phosphatase that re-presses the antiapoptotic and proinflammatory PI3K pathway (20,26), which is of particular importance in MFs. M2 AMF polari-zation was first identified in SHIP-12/2 mice that spontaneouslydeveloped lung disease (8), making SHIP-12/2 mice an invaluablemodel for examining MF-mediated mechanisms underlying chroniclung disease. To examine whether AMF subpopulations werea hallmark of SHIP-12/2 lung disease, we studied C57BL/6SHIP-12 /2 mice that spontaneously developed severe lunginflammation marked by the infiltration of large myeloid cells(Fig. 4A). Increased collagen deposition and alveolar wall thickeningwas frequently observed in SHIP-12/2 lung, especially surroundingregions of MF consolidation (Fig. 4A). SHIP-12/2 BAL cells weregreatly heterogeneous as marked by their FSC versus SSC (Fig. 4A),with MF, neutrophil, and lymphocyte numbers significantly greatercompared with age-matched C57BL/6 controls (Fig. 4B).Instead of global M2 MF polarization, we observed AMF

subpopulation deregulation in C57BL/6 SHIP-12/2 mice (Fig. 4C),which had residential-like CD11chighMac-1neg/low AMFs (subpop-ulation Ri) and an extra subpopulation of CD11chighMac-1pos AMFs(subpopulation Rii), and no lung monocyte involvement. Bothsubpopulations were Ly6cneg, a characteristic of nonrecruitedresidential tissue MFs, and c-fmslow compared with C57BL/6 res-idential AMFs (Fig. 4C). Interestingly, SHIP-12/2 AMFs weremore autofluorescent than C57BL/6 AMFs, with a more diffuseincreased cytoplasmic signal observed (Fig. 4D). SHIP-12/2

Mac-1pos AMFs (subpopulation Rii) were larger, highly vacu-olated, and often multinucleated compared with C57BL/6 andSHIP-12/2 Mac-1neg/low AMFs (Fig. 4E). Cell sorting showedsignificant upregulation of iNOS and Arg I in both SHIP-12/2

AMF subpopulations (Fig. 4F). Interestingly, high MMP-12expression segregated with only subpopulation Rii (Fig. 4F).Acute cigarette smoke exposure was studied alongside C57BL/6

SHIP-12/2 mice as a negative control for the specificity of AMFsubpopulation formation in pathological disease. Although ciga-rette smoke is the major epidemiological cause of COPD and lungcancer, models of acute cigarette exposure do not model the de-velopment of chronic lung disease (27), although neutrophilic lunginflammation is observed (22). In C57BL/6 mice, acute cigarettesmoke exposure did not induce AMF Mac-1 upregulation despitean increase in BAL neutrophil and MF numbers, increased AMFautofluorescence, and AMF granularity. This further indicates thatAMF subpopulations may correlate with selective immunologicaland pathological processes, rather than nonspecifically in all in-flammatory lung conditions (Fig. 4A–D).

AMF subpopulations are disease dependent in SHIP-12/2

mice

Chronic lung disease is background dependent in SHIP-12/2 mice(21); with lung pathology manifest in the C57BL/6 but not BALB/c

background at 12 wk (Fig. 5A). Although deletion of SHIP-1 pro-duces a rare subpopulation of Mac-1pos AMFs with dendrite-likeprocesses in BALB/c mice, large, morphologically activatedMac-1pos AMFs are only a characteristic of C57BL/6 SHIP-12/2

mice (Fig. 5B, 5C), thus tracking with lung disease rather thanintrinsic SHIP-1 deletion itself.

AMF subpopulations track with spontaneous lung diseaseinduction

We further followed AMF subpopulation deregulation using anovel model of spontaneous lung disease induction. CD45.1+

BL6.SJL-Ptprca chimeric mice were transplanted with CD45.2+

C57BL/6 or SHIP-12/2 BM to study whether hematopoieticSHIP-1 deletion was sufficient to spontaneously induce lung dis-ease. We found residential AMF replacement varies between paperand wood-chip bedding, with wood-chip bedding greatly exacer-bating residential AMF replacement. To ensure complete replace-ment by donor AMFs, we specifically housed all disease inductionexperiments on wood-chip bedding, to ascertain that any lungpathology would be a result of recipient and not remaining donorAMFs. Six weeks posttransplant, residential AMFs replacementwas now 80–85% CD45.1+ in C57BL/6 chimeras and .99%CD45.1+ in SHIP-12/2 chimeras, with no changes in the residentialCD11chighMac-1neg/low phenotype or lung pathology observed (Fig.5D, 5E). Eight to 10 mo posttransplantation, when symptoms ofillness were first identified, spontaneous granulomatous lung diseasewas observed in all SHIP-12/2 but not C57BL/6 chimeras, andaccompanied by Mac-1high AMF subpopulation formation (Fig.5D, 5F). Interestingly, SHIP-12/2 chimeras did not develop thesame lung disease phenotype as C57BL/6 SHIP-12/2 mice, pos-sibly because of their retention of a subset of CD45.1+ SHIP-1+/+

blood lymphocytes (Supplemental Fig. 1D).

DiscussionIn this article, to our knowledge, we present the first findings seg-regating residential MF heterogeneity into distinct subpopulationsduring acute lung inflammation and chronic inflammatory lungdisease. Currently, identification of putative tissue MF subsets isof great interest, because it is now believed that an imbalance ofhomeostatic versus pathogenic MF subsets may be driving thepathogenesis of inflammation and disease (1). Recently, differ-ential F4/80 and CD11c surface expression has been used todiscriminate distinct MF and dendritic cell subsets in the colon,which shift in balance during gut homeostasis and induced in-flammatory disease (28). In lung, although significant advanceshave been made in the characterization of dendritic cell subsets(29), inflammatory lung monocytes and blood-derived exudateMFs (14), the intense autofluorescence of residential AMFs hasrendered the global study of lung MF heterogeneity highly dif-ficult. In this study, we used Mac-1 and CD11c surface expressionas a clear segregator of residential as well as emigrating monocyteand lung MF subsets.In healthy lungs at rest, CD11chighMac-1neg/low residential

AMFs (subpopulation R) maintain immunological homeostasisthrough constant removal of innocuous foreign Ags found in thelocal lung microenvironment (30) and through suppression ofinflammatory T cell responses through regulation of pulmonaryDC activity and migration (10). LPS challenge orchestrates twoMF-related changes in alveolar airspaces. First, a subpopulationof residential AMFs upregulate Mac-1 expression (subpopulation

calculated relative to LPS R of Fig. 2E. Results are mean6 SEM; *p, 0.05, **p, 0.01, ***p, 0.005; n = 4 from four independent experiments. Asterisk

directly above a bar indicates p value compared with DMEM R.

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FIGURE 5. Mac-1pos AMF subpopulation tracks with lung disease independent of SHIP-1 deletion. (A) MT-stained lungs from 12-wk-old BALB/c,

BALB/c SHIP-12/2, C57BL/6, and C57BL/6 SHIP-12/2 mice. Red arrows indicate myeloid infiltrates in C57BL/6 SHIP-12/2 alveolar airspaces. Scale

bar, 20 mm. (B) Flow cytometry of Mac-1 versus CD11c expression of BAL MFs. Representative of three independent experiments; n = 3–6. (C) Cytospins

of sorted BAL MF subpopulations. Representative of two independent sorts. Scale bar, 20 mm. (D) MT-stained lungs from respective chimeric mice. Red

arrows indicate myeloid infiltrates. Scale bar, 100 mm. (E) Mac-1 versus CD11c, and CD45.2 versus CD45.1 expression of 6-wk-old chimeric mice.

Representative of two independent experiments; n = 3–8 mice. (F) Mac-1 versus CD11c, and CD45.2 versus CD45.1 expression of 10-mo-old chimeric

mice. Representative of two independent experiments; n = 3–8 mice.

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I). Shortly afterward, inflammatory blood monocytes (subpopu-lation M) are recruited into alveolar airspaces where they mediatebactericidal activity directly through phagocytosis and NO pro-duction, and indirectly through the amplification of the local in-flammatory response. Although recruitment of inflammatory lungmonocytes has been well studied (11, 13, 31), to our knowledge,we are the first to identify their simultaneous expression of bothM1- and M2-like MF markers ex vivo. This is significant becausethe current doctrine tends to regard M1 and M2 MFs as differ-entially polarized MF subtypes that mediate distinct functions inthe tissue microenvironment (32, 33), trending the identificationof a M2 MF based solely on its expression of M2 markers such asArg I and IL-10. Because lung MFs exist in a microenvironmentthat is dynamic and complex, ex vivo characterization of func-tionally distinct subpopulations using flow cytometry may allowus to better understand the role of AMFs in homeostasis anddisease.Monocytes are key effector cells in inflammation and host de-

fense (14). Interestingly, we also discriminated key differences inthe lung monocyte subpopulation postinfection with Mem71 in-fluenza A virus compared with LPS treatment. First, LPS-mediatedacute lung inflammation is resolved by day 10, whereas Mem71infection triggers a longer lasting complex MF recruitment circuitin lung. Second, infiltrating monocytes are more dominant inMem71-infected compared with LPS-treated mice, with the sub-population Mv monocytes expressing higher levels of iNOS, IL-6,Arg I, and IL-10. Mem71 is a type A influenza strain of inter-mediate virulence that, given at sublethal dosages, induces a lunginflammatory response that peaks at day 3 and is resolved by day10 (34). In contrast, infection with high-fatality influenza A strains(i.e., avian H5N1/97) leads to patient death from acute respiratorydistress and multiple organ dysfunction, postulated to be caused byaberrant cytokine production from virus-exposed lung MFs (5,35). Interestingly, CCR22/2 mice infected with the highly virulentPR8 influenza A strain virus have decreased lung monocyte re-cruitment and are protected against morbidity and mortalityindependent of virus clearance efficiency (6, 14), clearly high-lighting the significant contributions of lung monocytes in acutelung disease. In our model of nonlethal influenza infection, weobserved the highest levels of iNOS and IL-6, but also Arg I andIL-10, in subpopulation Mv. Arg I suppresses NO productionthrough competition for arginine substrate with iNOS (36), and itssimultaneous upregulation may help regulate NO levels to preventNO-mediated pneumonitis and mortality after influenza infection(37). IL-10 deactivates MF proinflammatory cytokine productionin vitro (38). Although IL-10 production by effector T cells isimplicated in the suppression of influenza-induced inflammationand lethality (39), we show that the CD11cneg/lowMac-1high Mvsubpopulation already produces IL-10 during the peak innate re-sponse. Thus, we postulate that lung monocytes may exhibit bothanti-inflammatory and proinflammatory mechanisms in vivo toprotect from influenza-induced cytokine dysfunction and mortality.In particular, it will be greatly interesting to examine whether in-fluenza fatality occurs alongside a global imbalance of the lungMF subpopulations or a shift in the M2 , M1 simultaneous char-acteristics of recruited lung monocytes.Because it has been known for some time that increased AMF

numbers clinically correlate with COPD severity (16, 40), animalmodels of chronic inflammatory lung disease are of great interest.Although large vacuolated AMFs are found in almost all animalmodels of COPD (7, 17, 41, 42), no single pathological AMFsubpopulation has yet been identified or associated with diseasepathogenesis. In this article, we report the presence of a subpop-ulation of constitutively Mac-1pos residential AMFs in C57BL/6

SHIP-12/2 mice that spontaneously develop COPD-like lung dis-ease. Our findings implicate this Mac-1pos AMF subset as theagents of disease rather than SHIP-1 deletion itself, because BALB/c SHIP-12/2 mice that lack disease do not have this prominentMac-1pos AMF subpopulation. Interestingly, high MMP-12 ex-pression tracks specifically with the Mac-1pos AMF subpopulationin C57BL/6 SHIP-12/2 mice. Downstream MMP-12 upregulationis a common feature of all COPD animal models (7, 17, 41, 42),implicating its role as a major COPD effector. Clinically, a minorSNP allele in MMP-12 that reduces its protease activity is protec-tive against COPD onset in smokers (43). Because MMP-12 ex-pression is also transiently upregulated in Mac-1pos AMFs afterLPS treatment, we postulate that deregulation of SHIP-1 signalingin a susceptible genetic background creates spontaneous AMFheterogeneity wherein chronic AMF Mac-1, and hence MMP-12,expression may be responsible for the lung disease pathogenesis.In summary, we have used a novel flow cytometry approach to

identify and characterize AMF subpopulation heterogeneity inboth acute lung inflammation and chronic lung disease. We reportMac-1 as a marker of activated residential AMFs and show that exvivo lung MFs adopt both M1 and M2-like characteristics duringacute inflammation and disease. Specifically, we show that AMFsubpopulations are a feature of and track with the development ofchronic inflammatory lung disease. This study paves the way forelucidating the role of AMF subpopulations in lung disease path-ogenesis, and the possibility of selectively targeting pathologicalAMF subpopulations to attenuate lung disease.

AcknowledgmentsWe thank V. Feakes, H.J. Seow, J. Jones, S. Bozinovski, and S. Cody for

excellent technical assistance and the LICR animal facility staff, especially

A. Naughton, M. Asquith, and N. Grixti, for excellent animal assistance.

DisclosuresThe authors have no financial conflicts of interest.

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