(MMP10) Moderates Inflammation by Controlling Macrophage Activation

Stromelysin-2 (MMP10) Moderates Inflammation by Controlling Macrophage Activation

This information is current as Ryan S. McMahan, Timothy P. Birkland, Kate S. Smigiel, of September 26, 2021. Tyler C. Vandivort, Maryam G. Rohani, Anne M. Manicone, John K. McGuire, Sina A. Gharib and William C. Parks J Immunol 2016; 197:899-909; Prepublished online 17 June 2016;

doi: 10.4049/jimmunol.1600502 Downloaded from http://www.jimmunol.org/content/197/3/899

Supplementary http://www.jimmunol.org/content/suppl/2016/06/16/jimmunol.160050 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 72 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/197/3/899.full#ref-list-1

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Stromelysin-2 (MMP10) Moderates Inﬂammation by Controlling Macrophage Activation

Ryan S. McMahan,*,† Timothy P. Birkland,*,‡ Kate S. Smigiel,x Tyler C. Vandivort,*,†,x Maryam G. Rohani,*,‡,x Anne M. Manicone,*,‡ John K. McGuire,*,{ Sina A. Gharib,*,‡ and William C. Parks*,‡,x

Several members of the matrix metalloproteinase (MMP) family control a range of immune processes, such as leukocyte influx and chemokine activity. Stromelysin-2 (MMP10) is expressed by macrophages in numerous tissues after injury; however, little is known of its function. In this study, we report that MMP10 is expressed by macrophages in human lungs from patients with cystic fibrosis and induced in mouse macrophages in response to Pseudomonas aeruginosa infection both in vivo and by isolated resident alveolar and bone marrow–derived macrophages (BMDM). Our data indicates that macrophage MMP10 serves a beneficial function in response to acute infection. Whereas wild-type mice survived infection with minimal morbidity, 50% of Mmp102/2 mice died and Downloaded from all showed sustained weight loss (morbidity). Although bacterial clearance and neutrophil influx did not differ between genotypes, macrophage numbers were ∼3-fold greater in infected Mmp102/2 lungs than in wild-types. Adoptive transfer of wild-type BMDM normalized infection-induced morbidity in Mmp102/2 recipients to wild-type levels, demonstrating that the protective effect of MMP10 was due to its production by macrophages. Both in vivo and in cultured alveolar macrophages and BMDM, expression of several M1 macrophage markers was elevated, whereas M2 markers were reduced in Mmp102/2 tissue and cells. Global gene expression analysis revealed that infection-mediated transcriptional changes persisted in Mmp102/2 BMDM long after they http://www.jimmunol.org/ were downregulated in wild-type cells. These results indicate that MMP10 serves a beneficial role in response to acute infection by moderating the proinflammatory response of resident and infiltrating macrophages. The Journal of Immunology, 2016, 197: 899–909.

acrophages are critical effector cells of the immune macrophages are induced by infection and proinflammatory Th1 system and play essential, yet distinct, roles in both cytokines, are effective at killing bacteria, and release proin- M promoting and resolving inflammation and in facili- flammatory cytokines, such as IL-1b, IL-12, and TNF-a.M2 tating tissue repair and contributing to its destruction (1). That a macrophages are induced by the Th2 cytokines IL-4 and IL-13 by guest on September 26, 2021 single cell type can serve opposing functions may seem counter- and other factors, release anti-inflammatory factors, such as IL- intuitive, but dramatic phenotypic changes occur when macro- 10, are weakly microbicidal, and promote repair. We recognize, phages respond to local stimuli (1–4). Based on patterns of gene however, that dividing macrophages into M1 and M2 classes and protein expression and function, macrophages are commonly oversimplifies the complex continuum of functional and revers- classified as classically activated (M1) or alternatively activated ible states in which these cells exist (6–9). (M2) cells, as well as a variety of M2 subtypes (1, 4, 5). M1 Several proteins influence macrophage behavior, including some members of the matrix metalloproteinase (MMP) gene family. For example, MMP12 and MMP28, both macrophage products, *Center for Lung Biology, University of Washington, Seattle, WA 98109; either promote or restrict macrophage influx into lung (10, 11), and †Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98105; ‡Department of Medicine, University of Washing- MMP8 promotes M2 polarization (12). Additionally, we reported ton, Seattle, WA 98195; xWomen’s Guild Lung Institute, Cedars–Sinai Medical Cen- that MMP28 and TIMP3, an MMP inhibitor, moderate M1 acti- { ter, Los Angeles, CA 90048; and Department of Pediatrics, University of Washington, vation of macrophages in models of lung infection and fibrosis Seattle, WA 98195 (13, 14). As their name implies, MMPs are thought to degrade ORCIDs: 0000-0003-3489-6127 (R.S.M.); 0000-0003-0475-2923 (T.C.V.); 0000- 0001-6106-8349 (J.K.M.). extracellular matrix proteins, a function that is indeed performed Received for publication March 23, 2016. Accepted for publication May 24, 2016. by some members (15–17). However, matrix degradation is nei- ther the shared nor predominant function of these enzymes. This work was supported by National Institutes of Health Grants HL089455, HL128995, and HL098067 (to W.C.P.), HL093022 (to J.K.M.), and HL116514 (to A.M.M.). Rather, individual MMPs have been shown to regulate specific The microarray experiments–compliant data presented in this article have been sub- immune processes, such as leukocyte influx and activation, anti- mitted to the National Institutes of Health Gene Expression Omnibus (http://www. microbial activity, and restoration of barrier function, typically by ncbi.nlm.nih.gov/geo/query/acc.cgi?token=epkvmssmvhmrlmf&acc=GSE78175) under accession number GSE78175. processing of a range of non–matrix protein substrates (18–25). MMP10 (stromelysin-2) is not expressed in developing or Address correspondence and reprint requests to Dr. William C. Parks, Women’s Guild Lung Institute, Cedars–Sinai Medical Center, 8700 Beverly Boulevard, Los normal adult tissues, including lung (26, 27). However, in both Angeles, CA 90048. E-mail address: [email protected] human conditions and mouse models, MMP10 is induced in re- The online version of this article contains supplemental material. sponse to injury, infection, or transformation in essentially all Abbreviations used in this article: BAL, bronchoalveolar lavage; BMDM, bone marrow– tissues (28–33). In a meta-analysis of gene array experiments derived macrophage; CF, cystic fibrosis; DAB, 3,39-diaminobenzidine; DIG, digoxigenin; involving numerous different host–pathogen interactions, MMP10 iNOS, inducible NO synthase; MHC II, MHC class II; MMP, matrix metalloproteinase. was identified as a common host response gene (34). The wide- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 spread expression of MMP10 among tissues suggests that this www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600502 900 MMP10 SHAPES MACROPHAGE ACTIVATION proteinase serves critical roles in the host response to environ- Immunostaining and in situ hybridization mental insults. As we show in the present study, MMP10 serves Deidentified human lung specimens were obtained with approval of the a protective role in acute infection by moderating the proinflam- University of Washington Institutional Review Board. Sections (5 mm) matory activity of macrophages. were stained with Abs specific for human MMP10 (Abcam, Cambridge, Recently, we reported that macrophage MMP10 promotes the MA) as described (40). Mouse lungs were perfused-fixed with PBS- ability of M2 macrophages to clear scar tissues in normal skin buffered formalin via the trachea under 25-cm pressure for 5 min, incubated in fixative at room temperature for 48 h, dehydrated through wounds by controlling the expression of collagenolytic MMPs, graded ethanol, and embedded in paraffin. Deparaffinized sections (5 particularly MMP13 (35). However, because sterile excision mm) underwent heat-induced (95˚C, 30 min) Ag retrieval in pH 7.0 wounds are not associated with a profound inflammatory re- citrate. Endogenous peroxidase activity was quenched with 3% H2O2 for sponse, the stimulus (i.e., a clean wound) may not have been 10 min. Sections were incubated with 4% normal rabbit serum for 1 h and then overnight at 4˚C with anti-Mac2 Ab (1:500 dilution; clone M3/ sufficiently robust to reveal other MMP10-dependent roles in 38.1.2.8 HL.2, American Type Culture Collection, Manassas VA). Bound macrophage inflammation. Thus, for the present studies, we Ab was detected using a Vectastain Elite ABC (rat IgG) kit (Vector 2 2 challenged Mmp10 / mouse lungs with Pseudomonas aeruginosa, Laboratories, Burlingame, CA) and 3,39-diaminobenzidine (DAB) per- an opportunistic pathogen and common cause of hospital-acquired oxidase substrate and counterstained with hematoxylin. For combined in infections and pneumonia (36) and the major pathogen in lungs of situ hybridization/immunostaining, deparaffinized rehydrated sections were treated with 10 mg/ml proteinase K in RNase-free PBS for 20 min patients with cystic fibrosis (CF) (37). Compared to wild-type mice, at ambient temperature, incubated with 0.1 M triethanolamine in RNase- 2/2 we found that Mmp10 mice were much more susceptible to free water, washed in RNase-free PBS, and then probed overnight at 55˚C airway infection and that MMP10 influenced activation of resident with either a 570-bp sense or antisense digoxigenin (DIG)-labeled RNA and infiltrated macrophages by curbing M1 polarization and probe targeting bases 1050–1620 of murine Mmp10. Probes were labeled Downloaded from using DIG RNA labeling mix and SP6/T7 DIG labeling kit (Roche Di- P. aeruginosa–induced transcriptional changes. Our findings in- agnostics, Indianapolis, IN). After hybridization, slides were washed dicate that MMP10 is a critical cell-autonomous mediator con- three times in 23 SSC/50% formamide for 15 min at 50˚C, once in 23 trolling macrophage activation. SSC at ambient temperature for 20 min, and once in 0.53 SSC for 30 min then developed with anti–DIG-alkaline phosphatase using NBT/BCIP as substrate. Slides were washed with PBS and images were captured using an Olympus BX51 with a DP70 digital camera (Olympus America, Materials and Methods Center Valley, PA). Sections were then processed for immunostaining http://www.jimmunol.org/ Animals with anti–Mac-2 as described above minus Ag retrieval and counter- staining. Mmp102/2 mice and wild-type littermates (C57BL/6J, male and female, 8–16 wk old) were used for these studies. All procedures were approved by Flow cytometry the Office of Animal Welfare at the University of Washington and the Institutional Animal Care and Use Committee at Cedars–Sinai Medical Whole lungs were perfused with 10 ml ice-cold PBS through the left Center. Mmp102/2 mice were generated by targeting exons 3–5 that code ventricle and then harvested, minced into tiny pieces using a razor blade, the catalytic domain as described (38). Mmp102/2 mice breed and appear and digested in RPMI 1640 containing 2.5 mg/ml collagenase type IV (Life normal with similar litter sizes. We also generated Mmp102/2 mice Technologies) for 45 min at 37˚C with shaking. The digestion was stopped (Mmp10tm1Lex/Mmucd) with embryonic stem cells from the Mutant Mouse with addition of FBS to 10%. The lung cell suspension was applied to a 70- Resource and Research Center (https://www.mmrrc.org/catalog/sds.php? mm cell strainer and washed with cold RPMI 1640. The sample was by guest on September 26, 2021 mmrrc_id=11737). These cells were originally generated by Lexicon subjected to RBC lysis (ACK lysing buffer; Thermo Fisher), followed by Pharmaceuticals (The Woodlands, TX), who used a targeting strategy washing in RPMI 1640 and cell counting using a Bio-Rad cell counter. distinct from ours. Lung cells were stained with fluorophore-conjugated Abs and analyzed using a BD LSRFortessa or sorted using a BD FACSAria III (BD Bio- sciences, San Jose, CA). Cell populations in the lungs were identified using Infection model the following combinations of cell surface markers: alveolar macrophages, F4/80+CD11c+Siglec-F+; interstitial macrophages, F4/80+CD11b+CD11c+ Mice were challenged with live P. aeruginosa strain K either by aero- 2 MHC class II (MHC II)+Siglec-F ; Ly6C+ monocytes/macrophages, F4/ solization in a whole animal nebulizer chamber for 30 min to produce a + + + + 2 2 2 5 6 80 Ly6C CD11b CD11c MHC II Siglec-F ; and neutrophils, F4/80 deposition of bacteria between 10 and 10 CFU/left lung or by oro- Ly6G+CD11b+. Sorted cells were kept at 4˚C during sorting, centrifuged, pharyngeal aspiration as described (11, 39). Briefly, bacteria were grown and immediately processed for RNA isolation as described below. Siglec-F in Luria–Bertani agar plates overnight, washed twice, and suspended in sterile PBS. For oropharyngeal aspiration, bacteria were quantified by Ab was from BD Biosciences; all others were from eBioscience (San 8 Diego, CA). OD600 and suspended at 2 3 10 cells/ml, and 50 ml was instilled per mouse. After exposure by either method, the lungs of three to five mice were immediately harvested and homogenized to determine 0 h bacterial Macrophage culture, transfer, and treatments counts (CFU). CFU were determined by plating serial dilutions of left Bone marrow–derived macrophages (BMDM) and alveolar macrophages lungs or spleens homogenized in 1 ml PBS on Luria–Bertani agar plates. were isolated and cultured as described (11, 39). To generate BMDM, Duplicate plates of each dilution were incubated 24–48 h at 37˚Cand marrow cells were differentiated in CSF-1–containing medium for 7 d counted using a Quebec colony counter (Reichert Technologies, Depew, (referred to as M0 macrophages). To generate M1-biased macrophages, NY). M0 BMDM (1.5 3 106/well in six-well plates) were stimulated with 100 ng/ml Escherichia coli 0111:B4 LPS for 24 h or with live P. aeruginosa Sample collection and analyses strain K at a multiplicity of infection of 5 bacteria per macrophage for 1 h before washing away bacteria and then culturing in medium with 75 mg/ml The left lung hilum was ligated, and the left lung and spleen were removed gentamicin to kill live extracellular bacteria. For M2 polarized macro- for determination of bacterial load. Bronchoalveolar lavage (BAL) was phages, BMDM were stimulated with 10 ng/ml each of IL-4 and IL-13 for collected by flushing the right lung via the trachea four times with 0.5 ml 48 h. For adoptive transfer, recipient mice were given 2 3 106 wild-type or saline. The recovered lavage (typically 1.7 ml) was centrifuged at 400 3 g, Mmp102/2 M0 BMDM in 100 ml PBS retro-orbitally 24 h after infection. 4˚C for 10 min. Supernatants were collected and frozen at 270˚C for later For the microarray experiments, M0 BMDM were exposed to P. ae ru gi no sa analysis. Total protein in BAL was quantified using the Pierce BCA strain K as described above. The cells were then cultured in medium protein assay kit (Thermo Scientific, Rockford, IL) and IgM by ELISA containing gentamicin, 100 IU/ml penicillin, and 100 mg/ml streptomycin, (Bethyl Laboratories, Montgomery, TX). Pelleted cells were resuspended and RNA was collected 6 or 24 h later. For alveolar macrophages, cells in in DMEM with 10% FBS, counted in a hemocytometer, and a 100-ml BAL (98% macrophages) from four groups of five naive wild-type mice aliquot was cytospun onto slides and differentially stained with LeukoStat per group were pooled, and 2 3 105 cells were cultured in 24-well plates (Fisher Scientific, Pittsburgh, PA). At least 300 cells were counted per overnight in RPMI 1640 containing 10% FBS and penicillin/streptomycin lavage. Lungs from separate mice were used for RNA isolation or flow before stimulating with LPS or bacteria. The phagocytosis assay is de- cytometry (see below). scribed in the Supplemental Material. The Journal of Immunology 901 mRNA assays mRNA in naive mouse lungs (Fig. 1E). However, at 72 h post- Total RNA was isolated from lungs or macrophages and RT-PCR was infection with P. aeruginosa, we detected a prominent signal for performed and quantified as described (41). Unlabeled PCR primers and Mmp10 mRNA (Fig. 1F) in cells that stained positive for Mac-2, a TaqMan probes (FAM dye labeled) were used to detect mRNAs for Nos2, pan-macrophage marker (11), demonstrating that all cells strongly Ifng, Ccl2, Ccl3, Ccl5, Il6, Tnf, Ccr2, Arg1, Il10, Mrc1, Clec10a, Retnla, positive for Mmp10 mRNA were macrophages (Fig. 1G, 1G9). No Mmp3, 7, 8, 10, 12, and 28, and Hprt (Applied Biosystems, Foster City, specific signal was seen in sections processed with sense RNA CA). The data are expressed as relative quantification, which is the fold change and calculated as 22DDCt. probe (Fig. 1H). These findings demonstrate that MMP10 is not expressed in healthy lung but is induced by macrophages in hu- Cytokine/chemokine assays man and murine lung in response to bacterial infection. Lung samples were homogenized in 2 ml lysis buffer: 150 mM NaCl, MMP10 protects against lethality induced by lung infection 15 mM Tris (pH 7.2), 1 mM MgCl2, 1 mM CaCl2, 1% Triton X-100, and cOmplete mini protease inhibitor cocktail (Roche Molecular Biochemi- To determine the role of MMP10 in the host response to infection, cals, Mannheim, Germany). Homogenates were centrifuged at 10,000 3 g we exposed wild-type and Mmp102/2 mice to live P. aeruginosa for 15 min at 4˚C, and the supernatants were collected. Multiplex reagents by aerosolization or oropharyngeal aspiration. Unchallenged were from R&D Systems (Minneapolis, MN), including the mouse Fluorokine 2/2 MAP kit and analytes for IFN-g, IL-1b, IL-6, IL-10, IL-12, IL-17, TNF-a, Mmp10 mice develop normally and reveal no overt phenotype CXCL1, CXCL2, and CCL2. in any tissue (33, 35, 38). Consistent with our in situ hybridization data (Fig.1E), Mmp10 mRNA levels were very low (average Ct = Microarray analysis 38) in naive wild-type lung, indicating that this transcript was not

Total RNA from BMDM was purified using RNeasy mini kits (Qiagen, expressed by any resident cell, including alveolar and interstitial Downloaded from Hilden, Germany). All RNA samples were of high quality as assessed by macrophages in unchallenged lung. In response to P. aeruginosa an Agilent 2100 bioanalyzer, with RNA integrity number $ 9.8. Each infection, expression of Mmp10 mRNA was induced in wild-type RNA sample was converted to cDNA, labeled, and hybridized to GeneChip mouse gene 2.0 ST arrays (Affymetrix, Santa Clara, CA) fol- lungs, peaked 3 d postinfection, and remained elevated at 7 d lowing the manufacturer’s protocols. The whole-genome GeneChip mouse postinfection (Fig. 2A). The pattern and extent of MMP10 ex- gene 2.0 ST is comprised of 35,240 transcripts including 26,515 unique pression were equivalent between the two infection protocols and genes. Hybridized arrays were scanned with an Affymetrix GeneChip 3000 mirrored the 0–24 h induction response we reported in mice in- http://www.jimmunol.org/ scanner, and image generation and feature extraction were performed using Affymetrix GeneChip operating software. All arrays passed the manu- fected by nasal inoculation with P. aeruginosa strain PA51673, CF facturer’s quality specifications with respect to background and percentage clinical isolate (38). In contrast, expression of other MMPs in- present call rates. Gene expression levels were estimated from probe increased modestly (MMP3, MMP12), if at all (MMP28), or tran- tensities using the robust multiarray analysis method with quantile nor- siently (MMP7, MMP8) (Supplemental Fig. 1A). MMP8, which is malization and background adjustment (42). Differential gene expression produced by many cell types and is particularly abundant in between wild-type and Mmp10-null samples at each time point was determined using a Bayesian implementation of the parametric t test (43), neutrophils, quickly increased and then fell (Supplemental Fig. and multiple hypothesis testing was controlled using false discovery rate 1B), likely reflecting the rapid influx and efflux of neutrophils analysis (q value) (44). A q value of ,0.01 was used to identify sig- in this model (11). Thus, among MMPs, MMP10 revealed a potent nificant differential gene expression. Functional enrichment analysis and sustained response to infection. by guest on September 26, 2021 of differentially expressed genes was performed with WebGestalt Whereas infection with P. aeruginosa led to no mortality in program (45) based on multiple publicly available resources includ- 2/2 ing Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. wild-type mice, half of Mmp10 mice had died by 48 h after 2 2 Enrichment p values were determined using a Fisher exact test and P. aeruginosa exposure (Fig. 2B). Among the survivors, Mmp10 / corrected for multiple testing using the Benjamini–Hochberg method mice were more susceptible to infection than were wild-type mice , (46) with a significance adjusted p value of 0.01. Minimal infor- as manifested by a reproducible and significant delay in their ability mation about microarray experiments–compliant data have been submitted to the National Institutes of Health Gene Expression Om- to regain body weight (Fig. 2C). Lethality and delayed weight re- 2/2 nibus (accession no. GSE78175; http://www.ncbi.nlm.nih.gov/geo/ covery in Mmp10 mice coincided with the marked upswing in query/acc.cgi?token=epkvmssmvhmrlmf&acc=GSE78175). Mmp10 expression in wild-type mice (Fig. 2A). Additionally, we observed greater mortality (Supplemental Fig. 1C) and delayed Statistical analysis weight gain (data not shown) in Mmp10tm1Lex/Mmucd mice generated Statistical analysis was performed using two-way ANOVA with a with embryonic stem cells in which the Mmp10 locus was targeted Bonferroni posttest where appropriate. Data are presented as means 6 by a gene-trap approach. Thus, the phenotypes we observed in our SEM, with a p value # 0.05 considered statistically significant. Mor- 2/2 tality data were analyzed by the Kaplan–Meier log-rank test. Prism 5 Mmp10 mice were not due to an off-target artifact. These data software (GraphPad Software, La Jolla, CA) was used for all statistical indicate that MMP10 serves a critical and protective role in the host analyses. response to infection. Lethality and morbidity in Mmp102/2 mice were not due to impaired bacterial clearance. By 48 h, both wild-type and Mmp102/2 Results mice had effectively cleared the infection (Fig. 2D). Additionally, MMP10 is expressed in infected lungs bacteremia, as measured by the recovery of live bacteria from We stained de-identified samples of CF lungs, all colonized with spleen (Fig. 2E), and spleen weights (data not shown) before and P. aeruginosa, from transplant recipients (n = 5) and normal human during infection did not differ between genotypes. Furthermore, lungs (n = 3) for MMP10 protein using a human-specific Ab. In all infection-induced increase in lung permeability, a marker of acute CF samples, signal for MMP10 was seen in most mononuclear cells lung injury, also did not differ between genotypes (Supplemental (Fig. 1B, 1C). The large, pale nuclei of these cells and their ac- Fig. 1D). We did not see any indication (edema, hemorrhage, cumulation within interstitial areas are characteristic features of inflammation) that other tissues (liver, intestines) were involved macrophages. No signal for MMP10 was detected in normal human (data not shown). lung, including resident alveolar macrophages (Fig. 1A). Whereas mortality of Mmp102/2 mice was more pronounced We used in situ hybridization coupled with immunostaining to when P. aeruginosa was administered by aerosolization ($50%; identify the cell sources of Mmp10 mRNA expression in mouse Fig. 2B, Supplemental Fig. 1C) compared with oropharyngeal as- lung. Similar to human lung, we detected no signal for Mmp10 piration (#14%; n . 90 mice/genotype from several experiments; 902 MMP10 SHAPES MACROPHAGE ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. MMP10 is expressed in the lungs of patients with CF and from mice infected with P. aeruginosa.(A–C) Sections of normal or CF human lungs were stained for MMP10 protein (DAB, brown) and countered stained with hematoxylin. (A) MMP10 was not detected in alveolar macrophages (arrow), airway epithelium (AE), or elsewhere in normal human lung. (B) MMP10 protein is expressed strongly in mononuclear cells; weak signal was seen in some AE. (C) Higher magnification shows a nest of mononuclear cells with strong MMP10 expression. (D) No specific signal was seen in CF sections processed with preimmune serum (Cnt Ab). (A, C,andD) Original magnification 3200. (B) Original magnification 3100. (E–H) Wild-type mice were infected with P. aeruginosa (PA) by oropharyngeal aspiration and controls (Cnt) received an equal volume of saline. Lungs were harvested 72 h later, and sections were probed for Mmp10 mRNA by hybridization with DIG anti-sense (AS) or sense (S) RNA probes. Images were collected, and sections were stained with Mac-2 Ab (DAB, brown). (E) No signal for Mmp10 mRNA was seen in lung sections from control mice. (F) Signal for Mmp10 mRNA was detected in mononuclear cells (arrows) in infected lungs, whereas weak signal was observed in AE cells. Higher magnification (3400) images show Mmp10 RNA expression before (G)andafter costaining with Mac-2 Ab (G9). Arrows indicate examples of cells positive for both Mmp10 mRNA (G) and Mac-2 protein (G9). (H)Thesectionserialtothatin (G) was hybridized with sense RNA and showed no signal. Arrows indicate the same cells highlighted in (G)and(G9). data not shown), morbidity as gauged by degree of weight loss and died due to altered or excessive inflammation. We examined BAL delay in weight recovery was similar by either method of P. aeruginosa and lung sections at various times after infection. Consistent with infection. Thus, to minimize a potential survival bias, our subse- the similar rate of bacterial clearance (Fig. 2D), neutrophils, which quent in vivo data were obtained with mice infected by oropha- are critical for clearance of P. aeruginosa, were the predominant ryngeal aspiration. cell in BAL after infection (.95% of total cells), and their

2/2 numbers did not differ between genotypes (Fig. 3B). Lymphocyte Increased macrophage inﬂux in lungs of Mmp10 mice numbers, which increased in response to infection, also did not Because lethality in Mmp102/2 mice was not associated with differ signiﬁcantly between genotypes (data not shown). In con- impaired bacterial clearance, we predicted that Mmp102/2 mice trast, we detected 3-fold more macrophages in BAL (Fig. 3C) at The Journal of Immunology 903

did not differ between genotypes (Fig. 3C). Flow cytometry of CD45+ cells from nonlavaged lung demonstrated that numbers of alveolar (F4/80+CD11c+Siglec-F+) and interstitial (F4/80+CD11b+ CD11c+MHC II+Siglec-F2) macrophages did not differ between naive wild-type and Mmp102/2 mice and that the influx of macrophages into lungs of both genotypes was predictably due to infiltrated cells (F4/80+Ly6C+CD11b+CD11c+MHC II2Siglec-F2; data not shown). Similarly, we observed a 2-fold increase in BAL macrophages in Mmp10tm1Lex/Mmucd mice after infection (data not shown). The number and differential of circulating leukocytes did not differ between wild-type and Mmp102/2 mice (data not shown). Additionally, we reported no difference in the ability of wild-type and Mmp102/2 macrophages to migrate toward serum or other chemotactic stimuli (35), indicating that MMP10 does not affect the migratory machinery of macrophages. We also assessed the expression of various chemokines and cytokines that could affect macrophage influx or that have proinflammatory activity. We

found no differences in the levels for TNF-a, CXCL1/2, IL-6, or Downloaded from IL-17 in BAL or lung homogenates (data not shown). In contrast, we found elevated expression of CCL2/MCP1, a potent macrophage chemokine (47, 48), in Mmp102/2 lungs (see Supplemental Fig. 3A), a ﬁnding that was mirrored in isolated macrophages (see FIGURE 2. MMP10 is induced in P. aeruginosa–infected mouse lung below).

and protects against the lethal effects of lung infection. Wild-type and http://www.jimmunol.org/ Mmp102/2 mice were infected with P. aeruginosa by oropharyngeal as- MMP10 expression by macrophages piration (A and C) or by aerosolization (B, D, and E). (A) Wild-type mice Macrophages that influx into the lung, or any tissue, in response to were sacrificed at 0 (uninfected), 1, 2, 3, 4, and 7 d postinfection (n =5 infection or injury are distinct from the resident population (49, mice per time point). RNA was isolated and processed for RT-PCR for 50). We found that both resident and recruited macrophages Mmp10 mRNA. Data are presented as relative quantification (RQ). The expressed MMP10 at fairly similar levels after infection (data not number above the 3-d time point is the average Ct value for those samples. For the 0 d samples, the Ct was .35. (B) Survival was significantly re- shown). We then assessed whether macrophages in culture reduced in Mmp102/2 after infection (n = 11 wild-type, 10 Mmp102/2 mice; spond as these cells did in vivo. Consistent with our in vivo data, Kaplan–Meier log-rank test, x2 = 5.775, p = 0.0163). (C) Surviving Mmp10 mRNA was not expressed by alveolar macrophages Mmp102/2 (n = 15) mice had delayed recovery of body weight after in- (Fig. 4B) or bone marrow (Fig. 4C) freshly isolated from naive by guest on September 26, 2021 fection compared with wild-type mice (n = 13). *p # 0.0288 by two-way wild-type mice. Upon CSF-1–mediated differentiation into mac- paired ANOVA between genotypes. (D) Clearance of bacteria from lungs rophages (M0), MMP10 was induced in bone BMDM (Fig. 4C, did not differ between genotypes. (E) Septicemia as measured by live Supplemental Fig. 2A). MMP10 was not induced by marrow cells bacteria counts in spleen also did not differ between genotypes. cultured with GM-CSF (Supplemental Fig. 2A). As GM-CSF promotes differentiation into dendritic cells (5), these negative 48 h postinfection, coincident with the upswing in MMP10 ex- data indicate further that macrophages are the source of MMP10. pression (Fig. 2A), and even more in tissue sections from Mmp102/2 Expression of MMP10 by both alveolar macrophages and BMDM mice (Fig. 3E). At 4 and 24 h postinfection, before the significant was stimulated with LPS and even more so by exposure to live increase in MMP10 expression, the number of macrophages in BAL P. aeruginosa (Fig. 4B, 4C), reaching levels that were quite similar

FIGURE 3. Increased macrophage inﬂux in Mmp102/2 lungs. Mmp102/2 and wild-type mice (n = 6–8 per genotype per time point) were infected with P. aeruginosa, andBALandlungtissuewerecollected4and48hlater. In BAL samples, total cell (A)andneutrophil(B)counts did not differ between genotypes. (C) Macrophage numbers in BAL did not differ at 4 h postinfection, but at 48 h, ∼3-fold more macrophages were detected in Mmp102/2 BAL compared with wild-type. *p = 0.0045 by unpaired t test. (D) Sections of wild-type (WT) and Mmp102/2 lungs collected at 48 h postinfection were stained for macrophages with Mac-2 Ab. Scale bars, 500 mm. 904 MMP10 SHAPES MACROPHAGE ACTIVATION

monocytes (51). In M0- and M2-polarized BMDM, we saw no difference in the expression of M1 and M2 markers between wild- type and Mmp102/2 BMDM (Supplemental Fig. 4, Table II), with one notable exception. IFN-g expression was elevated ∼10-fold in Mmp102/2 M0 BMDM compared with wild-type M0 cells (Supplemental Fig. 4A), suggesting that in the absence of MMP10 macrophages are primed toward a proinflammatory phenotype. We did observe significant MMP10-dependent effects in M1- polarized macrophages (Table II). In particular, the induction of FIGURE 4. MMP10 is expressed by both resident and infiltrated mac- CCL2 in response to P. aeruginosa was markedly greater in 2/2 rophages. (A) Alveolar macrophages were isolated from BAL of naive Mmp10 alveolar macrophages and BMDM compared with wild-type mice, cultured for 24 h, and exposed to 100 ng/ml LPS for 24 h wild-type cells (Fig. 6). Additionally, expression of mRNAs for or to live P. aeruginosa at a multiplicity of infection of 5:1 for 1 h and then the M1 markers iNOS, IFN-g, and CCL5 and the ability to washed and refed as described under Materials and Methods. RNA was phagocytize E. coli particles, an M1 property, were significantly collected 24 h later, and Mmp10 expression was determined by RT-PCR. elevated in M1-polarized Mmp102/2 macrophages (Supplemental The numbers above the bars are the average Ct values (n = cells from four Fig. 4A). Expression of IL-6 did not differ between genotypes B mice per condition). ( ) RNA was isolated from fresh wild-type bone (Supplemental Fig. 4B, Table I). Furthermore, expression of the marrow (d0), from cells following differentiation to macrophages in cul- M2 markers arginase-1 and IL-10 was stimulated in P. aeruginosa– ture for 7 d (M0), and from M0 macrophages exposure to LPS or Downloaded from P. aeruginosa (n = marrow from three mice per condition) treated BMDM, a response that was significantly diminished in Mmp102/2 BMDM (Supplemental Fig. 4C). CD206 (mannose receptor) was downregulated in M1-polarized wild-type BMDM 2/2 between cell types and comparable to the levels detected for and significantly more so in Mmp10 BMDM (Supplemental Mmp10 mRNA in lung in response to airway infection (see Fig. Fig. 4C, Table I). The levels of other M2 markers, including CCR2 (Supplemental Fig. 4D) and resistin-like a (35), did not differ 2A). Taken together, these findings indicate that both resident al- 2/2 veolar and infiltrated macrophages express similar levels of between M1-polarized wild-type and Mmp10 BMDM. Col- http://www.jimmunol.org/ MMP10 in response to infection. lectively, these data indicate that MMP10 functions to moderate several characteristic responses of M1 macrophage activation. Rescue of Mmp102/2 phenotype MMP10 moderates bacteria-induced transcriptional changes We performed adoptive transfer experiments to determine whether in macrophages wild-type macrophages could ameliorate morbidity in infected Mmp102/2 mice. Indeed, adoptive transfer of wild-type BMDM To gain insight into potential mechanisms by which MMP10 influences the macrophage response to bacterial infection, we per- 24 h postinfection with P. aeruginosa resulted in significantly 2/2 more rapid recovery of weight loss (used as a surrogate for mor- formed global gene expression analysis. Wild-type and Mmp10 bidity) comparable to that seen in wild-type mice (Fig. 5). BMDM were exposed to P. aeruginosa for 1 h, and cells were by guest on September 26, 2021 Mmp102/2 BMDM did not affect weight loss in either wild-type or Mmp102/2 recipients (Fig. 5). These findings demonstrate that macrophages are the key cells mediating the MMP10-dependent phenotypes in response to P. aeruginosa lung infection. MMP10 regulates macrophage activation We assessed whether MMP10 influences the state of macrophage activation by measuring the in vivo expression of M1 and M2 markers (5). Compared to levels in wild-type mice, we found elevated expression of mRNAs for the M1 markers IFN-g, inducible NO synthase (iNOS), and CCL2 (Supplemental Fig. 3A) and reduced expression levels of M2 markers arginase-1 and IL-10 in infected Mmp102/2 lungs (Supplemental Fig. 3C). In contrast, IL-12a, an M1 marker, was reduced in Mmp102/2 lungs (Supplemental Fig. 3A), and protein levels of TNF-a and IL-1b did not differ between genotypes (Supplemental Fig. 3B). Addi- tionally, as we found in a skin wound model (35), expression of the M2 marker resistin-like a (Retnla, FIZZ1) was not affected by MMP10 (Supplemental Fig. 3D). Overall, the in vivo phenotypes we saw in Mmp102/2 mice, as summarized in Table I, support the idea that MMP10 mitigates the proinflammatory activity of macrophages. We next assessed whether MMP10 plays a cell-autonomous role in regulating macrophage polarization. We treated wild-type and Mmp102/2 M0 BMDM with live P. aeruginosa to stimulate M1 FIGURE 5. Adoptive transfer of wild-type macrophages improves body differentiation or with IL-4 and IL-13 for M2 differentiation and weight recovery. Wild-type and Mmp102/2 mice (n = 6–7 mice per group) assessed expression of various markers. Expression of MMP10 by were exposed to P. aeruginosa by oropharyngeal aspiration and 24 h later M0 macrophages was stimulated by M1 agonists (Fig. 4C, were injected retro-orbitally with vehicle (100 ml saline) or with 2 3 106 Supplemental Fig. 2B) but was not further elevated by M2 ago- wild-type or Mmp102/2 BMDM. Mice were weighed and monitored daily nists (Supplemental Fig. 2B), consistent with findings in human for 7 d after infection. *p # 0.03 between genotypes by unpaired t test. The Journal of Immunology 905

Table I. Summary of in vivo phenotypes 1386 were upregulated and 2505 were downregulated at 24 h. We applied functional analysis using WebGestalt to identify biological Response in Mmp102/2 processes enriched within the subset of persistently upregulated Process/Factors Mice Relative to Wild-Types genes (Fig. 7D). We found that the absence of Mmp10 in P. aeruginosa– Neutrophil influx No D exposed BMDM was associated with persistent activation of a Bacterial clearance No D wide repertoire of pathways mapping to immunity, inflamma- ↑ Lethality, morbidity tion, apoptosis, and cell signaling. In contrast, functional anal- Macrophage influx ↑ M1 markers ysis of the persistently downregulated genes showed enrichment iNOS, IFN-g, CCL2a ↑ of processes involved in metabolism, transcription, and trans- IL-12a ↓ lation. CCL3,a IL-1b,b TNF-ab No D M2 markers IL-10, Arg1 ↓ Discussion Retnla No D In this study, we report on the expression and function of mac- aNot necessarily a consensus M1 marker. rophage MMP10 in the host response to infection. Our findings b Assessed by protein levels in BAL (Supplemental Fig. 2). indicate that MMP10 is not expressed in healthy, unchallenged No D, no significant difference between genotypes. human and mouse lung but is markedly induced in both resident and infiltrated macrophages following infection with P. aeruginosa. harvested at 6 and 24 h later to assess acute and persistent gene With a combination of in vivo and primary macrophage models, Downloaded from expression changes. We identified many genes differentially we demonstrate that MMP10 protects mice from morbidity and expressed (q , 0.01) compared with untreated controls at 6 h mortality induced by acute P. aeruginosa lung infection. By postinfection in both wild-type and Mmp102/2 BMDM. As ex- assessing the expression of various markers and overall gene ex- pected, P. aeruginosa infection caused a robust transcriptional pression, our findings indicate that MMP10 functions to drive the response in both wild-type (12,672 genes) and Mmp102/2 (15,164 activation of macrophages from proinflammatory (M1-like) cells genes) BMDM. The vast majority of differentially expressed into immunosuppressive (M2-like) cells. Our findings complement genes (n = 10,286) were common between wild-type and observations we reported with others showing greater mortality http://www.jimmunol.org/ 2 2 Mmp102/2 BMDM (Fig. 7A). Moreover, the direction and pattern and delayed weight gain in Mmp10 / mice following acute liver of differential expression of the common genes were similar in injury (52) and more macrophages biased toward M1 activation 2 2 wild-type and Mmp102/2 cells, indicating that P. aeruginosa in- and more tissue injury in Mmp10 / mice subjected to acute 2 2 fection was the dominant cause of early transcriptional changes colon injury (33). Furthermore, Mmp10 / mice are much more (Fig. 7B). Functional enrichment analysis of the smaller subsets of susceptible to the lethal effects of acute lung injury caused by genes that were differentially expressed only in wild-type or bleomycin toxicity than are wild-type animals (T.C. Vandivort, Mmp102/2 at 6 h did not reveal coherent phenotypes (data not T.P. Birkland, and W.C. Parks, unpublished observations). Thus,

shown). across organ systems, MMP10 appears to play a beneficial role in by guest on September 26, 2021 Because gene expression is a temporally dynamic process, we mitigating the deleterious responses to injury and infection by postulated that the role of Mmp10 in regulating transcriptional promoting the conversion of proinflammatory macrophages to- programs induced by bacterial challenge may be revealed by ex- ward immunosuppressive cells. Our findings support the general amining a later time point. Thus, we focused on the large set of concept that in response to acute injury or infection macrophage common differentially expressed genes at 6 h and assessed activation evolves from potentially detrimental M1-biased cells to whether their levels remained significantly altered at 24 h post- suppressive M2-biased macrophages (6, 39, 50). 2 2 infection. Of the 10,286 common genes, 5,609 remained differ- Although more macrophages emigrated into Mmp10 / lungs in entially expressed 24 h postinfection (Fig. 7C). Notably, we response to infection, we found no difference in the ability of 2 2 observed that most (3981) genes that remained differentially wild-type and Mmp10 / macrophages to migrate toward serum expressed at the 24 h time point were unique to Mmp102/2 or wound-tissue homogenate (35), indicating that MMP10 does BMDM, implying that MMP10 plays a distinct role in moderating not affect the migratory machinery of macrophages. With others 2 2 the postinfection transcriptional response. Of the 3981 genes that (53), we reported that Mmp10 / macrophages have an impaired remained differentially expressed only in Mmp102/2 BMDM, ability to migrate over fibronectin and to invade into Matrigel.

Table II. Relative expression of macrophage activation markers

Response in Mmp102/2 Macrophagesa

Pathway Factor M0b M1 Activatedc M2 Activatedd M1 markers or function IFN-g ↑↑ ↑ iNOS, CCL5 nd ↑ nd CCL2 No D or nd ↑ No D Phagocytosis ↑ IL-1b, IL-12a No D or nd ↓ No D IL-6, TNF-a No D No D No D M2 markers Arg1, CD206, IL-10 No D ↓ No D CCR2, Retnla No D No D No D aRelative to expression levels in wild-type BMDM. Arrow indicates about a 2-fold or greater difference between genotypes (p , 0.01). bBMDM 7 d in culture after marrow harvest. cBMDM were exposed to live P. aeruginosa for 1 h, and RNA was isolated 24 h later. dBMDM were treated with IL-4 and IL-13 for 24 h. nd, not detected in either genotype; No D, no difference between genotypes. 906 MMP10 SHAPES MACROPHAGE ACTIVATION

possess the enzymes needed to invade through the artificial setting of Matrigel. We conclude that MMP10 does not affect macrophage migration per se in physiologic settings. An open question is whether increased morbidity and mortality in Mmp102/2 animals was due to more macrophage numbers, to a bias toward M1 activation, or a combination of these two processes. We propose that altered activation of both resident and infiltrated macrophages toward an M1, proinflammatory state led 2/2 2 2 to worse outcomes for infected Mmp10 mice and this delete- FIGURE 6. Elevated expression of CCL2 by Mmp10 / macrophages. (A) Alveolar macrophages (n = cells from four mice/genotype) and (B) rious effect was augmented by the presence of more M1-biased BMDM (n = marrow from eight mice per genotype) from naive wild-type cells. Consistent with this conclusion, Johnston et al. (39) reported and Mmp102/2 mice were infected with P. aeruginosa, and expression of that the macrophage phenotypes begin to evolve toward an M2 CCL2 was assessed by RT-PCR. *p , 0.05 between genotypes by un- state ∼48 h after lung infection with P. aeruginosa, and D’Alessio paired t test. et al. (60) recently demonstrated that therapeutically promoting macrophage differentiation toward M2 cells (via instillation of Although these findings are seemingly opposed to our in vivo IL-4) markedly reduces lethality and morbidity in response to acute observations, there are two important caveats with these in vitro P. aeruginosa infection. In Mmp102/2 mice and macrophages, we studies. First, it is not clear whether macrophages migrate over found increased expression levels of M1 markers and decreased

fibronectin on their way into and through the interstitial space. levels of M2 markers (Tables I, II), such as the increased and Downloaded from Although fibronectin would be present in the area, it appears that prolonged expression of CCL2 (MCP-1), which was also seen in other matrix components, particularly versican and hyaluronan injured Mmp102/2 colons (33). CCL2, a potent macrophage (54), serve as the interstitial substrata that macrophages follow as chemokine, is expressed predominantly by resident lung macro- they migrate through tissue (55, 56). Second, Matrigel is a highly phages (47, 48), and overexpression of this factor may account for dense material that does not mirror the more porous architecture the excess macrophage inflammation seen in Mmp102/2 mice.

of interstitial matrix (57, 58). In fact, Matrigel does not contain The diminished expression of IL-10 and Arg1, both M2 markers, http://www.jimmunol.org/ interstitial matrix components; it is comprised of misassembled in infected Mmp102/2 lungs and BMDM provides further evi- basement membrane components (57). Thus, unlike how they dence that MMP10 broadly affects macrophage polarity to mod- move through the interstitium in vivo, macrophages may require erate inﬂammation. proteases to burrow through a thick plug of Matrigel in a culture Neutrophil inﬂux, which accounted for ∼95% of the cells re- dish. As we reported that MMP10 activates matrix-remodeling covered from BAL, and the levels of CXCL1 and CXCL2, critical programs in macrophages (35, 59), Mmp102/2 cells may not neutrophil chemokines, did not differ between wild-type and by guest on September 26, 2021

FIGURE 7. Global gene expression analysis of wild- type and Mmp102/2 BMDM in response to P. aeruginosa exposure. BMDM from wild-type and Mmp102/2 mice were exposed to P. aeruginosa for 1 h a multiplicity of infection of 5:1. Infection medium was removed, and cells were cultured in medium containing antibiotics until cells were harvested for RNA collection 6 or 24 h later. (A) Bacteria exposure induced significant transcriptional changes in both wild-type and Mmp102/2 BMDM 6 h after challenge. (B) Heat map of the genes commonly differentially expressed in both wild-type and Mmp102/2 BMDM shows similar patterns of up- and downregulation 6 h postinfection. (C) Of the genes that were commonly differentially expressed at 6 h, many genes remained significantly changed at 24 h postinfection exclusively in Mmp102/2 BMDM. (D) Functional analysis of genes exclusively upregulated in Mmp102/2 at 24 h reveals significant enrichment in immune and inflammatory signaling pathways. The Journal of Immunology 907

Mmp102/2 mice in response to infection. Similarly, Koller et al. MMP10 is beneficial by moderating the proinflammatory activity (33) found no difference in neutrophil numbers between wild-type of M1-biased macrophages and by stimulating the ability of M2- and Mmp102/2 mice during the first 14 d after colon injury. biased macrophages to remodel scar tissue. However, in a chronic However, they did find that elevated neutrophil counts persisted in setting, MMP10-driven matrix remodeling could be excessive and Mmp102/2 mice at 21 d after injury, at which time their numbers detrimental, as suggested in our smoke-exposure studies. Still, an had dropped in wild-type colons. Although sustained neutrophil important and shared conclusion among these models is that influx may have been secondary to worse injury of Mmp102/2 MMP10 functions to control macrophage behavior. colons, it is also possible that a bias toward M1 macrophages Significant differences in expression of M1 and M2 markers maintained neutrophil influx that, in turn, led to more tissue were only observed between infected (M1-biased) wild-type and damage. In our lung infection model, the lack of difference in Mmp102/2 macrophages but not between M0 or M2 cells (Table II). neutrophil numbers and indices of acute lung injury between ge- Consistent with these data, transcriptomic analysis did not reveal notypes indicates that neutrophils did not contribute to worse significant enrichment of any inflammation-related pathways outcomes in Mmp102/2 mice. A potential caveat to this conclu- in genes differentially expressed between M0 wild-type and sion is whether MMP10 influences the state of neutrophil acti- Mmp102/2 BMDM (data not shown). Thus, the effects of vation. For example, in other studies, we found that MMP7 MMP10, at least in terms of polarization, are more significant in shedding of syndecan-1/CXCL1 complexes from injured lung M1-activated macrophages. However, MMP10 does shape some epithelium permits both neutrophil migration through the mucosal functional properties of M2-biased macrophages, such as their barrier and their subsequent activation (20, 61). Without this ability to clear collagen (35). The P. aeruginosa–induced tran- 2 2 2 2 processing mechanism, Mmp7 / mice are markedly protected scriptional changes between wild-type and Mmp10 / BMDM Downloaded from from the lethal effects of neutrophil-mediated lethality in response were quite similar at 6 h postinfection but diverged significantly at to acute lung injury (20). However, we propose that the similar 24 h. Nearly 4000 of the genes that were differentially expressed rate of bacterial clearance in wild-type and Mmp102/2 mice does in both genotypes at 6 h remained significantly changed only in not support an MMP10-dependent role in neutrophil activation. Mmp102/2 BMDM at 24 h. These data indicate that MMP10 Although MMP10 can also be expressed by injured/infected functions to broadly moderate or quench the temporal transcrip-

epithelium, including in lung (38), we do not think that the en- tional response induced by bacteria in infiltrating macro- http://www.jimmunol.org/ zyme produced by these cells contributed to the key phenotypes phages. The genes that remained persistently upregulated only in and responses we report in the present study. Indeed, adoptive Mmp102/2 macrophages at 24 h were significantly enriched for transfer of wild-type macrophages into Mmp102/2 recipients re- inflammation and immune-related pathways, consistent with our duced postinfection morbidity to levels seen in wild-types. Simi- observations in the infected Mmp102/2 lung. larly, using bone marrow transplantation, Koller et al. (33) In summary, our data demonstrate a novel role for MMP10 in concluded that the lack of MMP10 in macrophages led to worse moderating lung inflammation by altering the balance of M1/M2 tissue damage in a model of acute colon injury. Furthermore, the transcripts and broadly suppressing immune-related pathways in macrophage polarization changes we observed in vivo (Table I) macrophages. Importantly, the patterns of MMP10 expression are were largely duplicated with isolated macrophages in culture conserved between human and mouse lung and macrophages. The by guest on September 26, 2021 (Table II). These findings support the conclusion that MMP10 is a critical functions of MMP10 in lung inflammation are largely at- cell-autonomous regulator of macrophage activation. In contrast, tributable to infiltrated macrophages and may therefore be relevant in the function of epithelial MMP10 remains unanswered. In skin other organs and inflammatory conditions. Given the importance of wound models, we found no re-epithelialization phenotype in macrophage polarization in the initiation and resolution of inflam- Mmp102/2 mice (35) or in mice overexpressing active MMP-10 in mation, further study of MMP10 function is warranted in other basal keratinocytes (32). disease models, especially those involving bacterial infection. Al- We recently contributed to a multicenter genome-wide associ- though beyond the scope of this study, identification of the physi- ation study that identified MMP10 as a candidate gene for chronic ologic substrates cleaved by MMP10 will be essential to determine obstructive pulmonary disease (59). Using a model of chronic (6 the mechanisms by which MMP10 regulates macrophage polarization. mo) exposure to cigarette smoke, we found that Mmp102/2 mice Schlage et al. (73) have identified multiple MMP10 substrates are fully resistant to the development of emphysema. Additionally, in primary mouse fibroblasts, but we predict that macrophage- MMP10 is produced by macrophages from human smokers with derived MMP10 may cleave a limited and distinct set of substrates, emphysema (62) and is one of two genes whose expression cor- some of which may only be expressed in an inflamed microenvi- relates with reduced lung function (i.e., forced expiratory volume ronment. Determining the relevant MMP10 cleavage events in the in 1 s) in smokers (63). These findings indicate that macrophage lung is a significant challenge but a priority for our future work. MMP10 contributes to disease progression in emphysema, which is seemingly opposed to the protective role for this MMP in acute Acknowledgments infection we reported in the present study. However, there are We thank Drs. Ann Chen and Gail Deutsch for help acquiring human lung important differences between these models, especially with re- specimens, Ying Wang for maintaining mouse strains, Dr. Elaine Raines for spect to macrophage biology. Macrophages that function early in providing Mac-2 Ab, and Fred Farin, Theo Bammler, and Jesse Thai for inflammation are functionally distinct from those that function late assistance with microarray experimental design and processing. in inflammation or in a persistent inflammatory response (4, 6, 50, 64, 65), and this concept has been demonstrated in models of liver Disclosures (66), vascular (67), cardiac (68), and kidney disease (69). Addi- The authors have no financial conflicts of interest. tionally, whereas acute infection and injury bias toward an M1 phenotype (4), cigarette smoke promotes expansion of M2 macrophages (70). Macrophages are considered to be the destructive References cell in emphysema (71, 72), and our studies in skin wounds in- 1. Gordon, S., and P. R. Taylor. 2005. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5: 953–964. dicate that MMP10 promotes expression of matrix-degrading 2. Mantovani, A., A. Sica, and M. Locati. 2005. Macrophage polarization comes of proteinases in M2 macrophages (35). Thus, in an acute setting, age. Immunity 23: 344–346. 908 MMP10 SHAPES MACROPHAGE ACTIVATION

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