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. Author manuscript; available in PMC: 2016 Apr 15.
Published in final edited form as: Toxicol Appl Pharmacol. 2015 Feb 25;284(2):236–245. doi: 10.1016/j.taap.2015.02.002

Regulation of ozone-induced lung inflammation and injury by the β-galactoside-binding lectin galectin-3

Vasanthi R Sunil a,*, Mary Francis a, Kinal N Vayas a, Jessica A Cervelli a, Hyejeong Choi a, Jeffrey D Laskin b, Debra L Laskin a
PMCID: PMC4408237  NIHMSID: NIHMS667335  PMID: 25724551

Abstract

Macrophages play a dual role in ozone toxicity, contributing to both pro- and anti-inflammatory processes. Galectin-3 (Gal-3) is a lectin known to regulate macrophage activity. Herein, we analyzed the role of Gal-3 in the response of lung macrophages to ozone. Bronchoalveolar lavage (BAL) and lung tissue were collected 24–72 h after exposure (3 h) of WT and Gal-3−/− mice to air or 0.8 ppm ozone. In WT mice, ozone inhalation resulted in increased numbers of proinflammatory (Gal-3+, iNOS+) and anti-inflammatory (MR-1+) macrophages in the lungs. While accumulation of iNOS+ macrophages was attenuated in Gal-3−/− mice, increased numbers of enlarged MR-1+ macrophages were noted. This correlated with increased numbers of macrophages in BAL. Flow cytometric analysis showed that these cells were CD11b+ and consisted mainly (>97%) mature (F4/80+CD11c+) proinflammatory (Ly6G−Ly6Chi) and anti-inflammatory (Ly6G−Ly6Clo) macrophages. Increases in both macrophage subpopulations were observed following ozone inhalation. Loss of Gal-3 resulted in a decrease in Ly6Chi macrophages, with no effect on Ly6Clo macrophages. CD11b+Ly6G+Ly6C+ granulocytic (G) and monocytic (M) myeloid derived suppressor cells (MDSC) were also identified in the lung after ozone. In Gal-3−/− mice, the response of G-MDSC to ozone was attenuated, while the response of M-MDSC was heightened. Changes in inflammatory cell populations in the lung of ozone treated Gal-3−/− mice were correlated with reduced tissue injury as measured by cytochrome b5 expression. These data demonstrate that Gal-3 plays a role in promoting proinflammatory macrophage accumulation and toxicity in the lung following ozone exposure.

Keywords: Ozone, gal-3, macrophage phenotype, flow cytometry, MDSC

Introduction

Ozone is a highly reactive oxidant present in urban air pollution. The toxicological effects of ozone are attributed to its ability to cause oxidation and peroxidation of membrane lipids and proteins, either directly or indirectly, through the generation of reactive intermediates (Pryor and Church, 1991). This leads to damage of the respiratory epithelium, disruption of alveolar epithelial barrier function, edema and inflammation (Hollingsworth et al., 2007; Al-Hegelan et al., 2011). Evidence suggests that inflammatory macrophages accumulating in the lung in response to ozone-induced injury contribute to pulmonary toxicity [reviewed in (Hollingsworth et al., 2007; Laskin et al., 2011)]. Thus, following exposure to products released from ozone-injured epithelial cells, lung macrophages are classically activated to release cytotoxic/proinflammatory mediators including reactive oxygen and nitrogen species which promote tissue injury. This is supported by findings that blocking proinflammatory macrophages or cytotoxic mediators they release protects against ozone-induced lung injury (Pendino et al., 1995; Kleeberger et al., 2001; Fakhrzadeh et al., 2002; Toward and Broadley, 2002; Fakhrzadeh et al., 2004a; Fakhrzadeh et al., 2004b). Macrophages have also been shown to play a protective role in the lung following ozone exposure, clearing oxidized products and cellular debris (Ishii et al., 1998; Dahl et al., 2007), augmenting lung antioxidant activity, and releasing mediators that suppress inflammation and initiate wound repair (Reinhart et al., 1999; Dahl et al., 2007; Backus et al., 2010). These activities are mediated by distinct subpopulations of macrophages that are alternatively activated (Byers and Holtzman, 2011; Laskin et al., 2011). It appears that the outcome of the pathogenic response to ozone depends in part, on the relative numbers of classically (M1) and alternatively (M2) activated macrophage subpopulations in the lung and their level of activation.

Galectin-3 (Gal-3) is a β-galactoside-binding lectin important in diverse biological processes including cell proliferation, adhesion, differentiation and regulation of the immune system (Perillo et al., 1998; Liu, 2005; Liu and Hsu, 2007). Gal-3 is expressed by activated macrophages, and it acts in an autocrine and paracrine manner to promote macrophage release of proinflammatory mediators including tumor necrosis factor, IL-12, CCL3 and CCL4, as well as reactive nitrogen species generated via inducible nitric oxide synthase (iNOS) (Nishi et al., 2007; Papaspyridonos et al., 2008; Jeon et al., 2010; Dragomir et al., 2012a; Dragomir et al., 2012b). In the present studies, mice with a targeted disruption of the gal-3 gene were used to analyze its role in ozone-induced macrophage activation and tissue injury. We found that loss of Gal-3 resulted in decreased accumulation of proinflammatory/cytotoxic macrophages in the lung following ozone intoxication. This was correlated with increases in anti-inflammatory/wound repair macrophages and M-MDSC, and reduced lung injury. These data provide support for a role of Gal-3 and proinflammatory/cytotoxic macrophages in the pathogenic response of mice to ozone. Identifying specific macrophage subpopulations involved in ozone toxicity and proteins regulating their activity may lead to the development of new therapeutic approaches for reducing inflammatory lung injury.

Materials and methods

Animals and exposures

Female specific pathogen-free C57Bl6/J wild type (WT) and B6.Cg-Lgals3<tm1Poi>/J (Gal-3−/−) mice (8–11 weeks; 17–22 g) were obtained from The Jackson Laboratories (Bar Harbor, ME). Animals were housed in filter-top microisolation cages and maintained on food and water ad libitum. All animals received humane care in compliance with the institution’s guidelines, as outlined in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. Animals were exposed to ultra-pure air (Messer Gas, Allentown, PA) or 0.8 ppm ozone for 3 h in a whole body Plexiglas chamber. Ozone was generated from oxygen gas via an ultraviolet light ozone generator (Gilmont Instruments, Barrington, IL) and mixed with air. Concentrations inside the chamber were monitored using a Photometric ozone analyzer (model 400E, Teledyne Instruments, City of Industry, CA).

Sample collection

Animals were euthanized 24–72 h after exposure by intraperitoneal injection of Nembutal (200 mg/kg). BAL was collected by slowly instilling and withdrawing 1 ml of PBS into the lung three times through a cannula in the trachea. BAL fluid was centrifuged (300 × g, 8 min), supernatants collected, aliquoted, and stored at −80°C until analysis. Cell pellets were resuspended in 1 ml PBS and viable cells counted on a hemocytometer using trypan blue dye exclusion. The lung was then removed and instilled five times with additional PBS (1 ml) while massaging the tissue. Lavage fluid was centrifuged (400 × g, 6 min, 4°C), the cell pellet resuspended in 1 ml PBS and combined with the first BAL lavage cell suspension. Cells were washed twice with PBS and resuspended in 200 μl staining buffer (PBS, 2% FCS and 0.02% sodium azide). Total protein was quantified in cell-free BAL using a BCA Protein Assay kit (Pierce Biotechnologies Inc., Rockford, IL) with bovine serum albumin as the standard. Samples (25 μl) from 8 mice per treatment group were analyzed in triplicate and evaluated at 560 nm on a Vmax MAXline™ microplate reader (Molecular Devices, Sunnyvale, CA).

Flow Cytometry

Cells were incubated for 10 min at 4°C with anti-mouse CD16/32 (1:200; Biolegend, San Diego, CA) to block nonspecific binding, and then with FITC-conjugated anti-mouse CD11b (1:200; Biolegend), PE-conjugated anti-mouse Ly6C (1:200; Biolegend), PE/Cy7-conjugated anti-mouse F4/80 (1:200; Biolegend), AF 700-conjugated anti-mouse CD11c (1:200, Biolegend), and AF 647-conjugated anti-mouse Ly6G (1:200; Biolegend) antibodies for 30 min at 4°C, followed by eFluor 780-conjugated fixable viability dye (1:1000; eBioscience, San Diego, CA). Cells were analyzed on a Gallios Flow Cytometer (Beckman Coulter, Brea, CA), and data analyzed using Beckman Coulter Kaluza version 1.2 software. The gating strategy and cell populations characterized are shown in Fig. 1.

Fig. 1.

Fig. 1

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Flow chart of gating strategy for identifying lung cell populations. Viable cells staining negatively (CD11b−) or positively (CD11b+) for CD11b were analyzed for expression of Ly6G. Ly6G− and Ly6G+ cells were then analyzed for expression of Ly6C, followed by F4/80 and CD11c. Ly6G+ cells were analyzed for expression of Ly6C followed by F4/80. G-MDSC, granulocytic myeloid derived suppressor cells; M-MDSC, monocytic myeloid derived suppressor cells.

Immunohistochemistry

The lung was inflated in situ via the trachea with PBS containing 3% paraformaldehyde. After 4 h on ice, the tissue was transferred to 50% ethanol. Tissue sections (4 μm) were prepared, deparaffinized with xylene (4 min, x 2), followed by decreasing concentrations of ethanol (100%–50%) and finally, water. After antigen retrieval using citrate buffer (10.2 mM sodium citrate, 0.05% Tween 20, pH 6.0) and quenching of endogenous peroxidase with 3% H2O2 for 15 min, sections were incubated for 2 h at room temperature with 10–100% goat serum to block nonspecific binding. This was followed by overnight incubation at 4°C with rabbit IgG or rabbit polyclonal anti-Gal-3 (1:2000; R&D Systems, Minneapolis, MN), anti-iNOS (1:750; Abcam, Cambridge, MA), anti-mannose receptor (MR)-1 (1:1000; Abcam), or anti-cytochrome b5 (1:250; Abcam) antibodies. Sections were then incubated with biotinylated secondary antibody (Vector Labs, Burlingame, CA) for 30 min at room temperature. Binding was visualized using a Peroxidase Substrate Kit DAB (Vector Labs). Random sections from three mice per treatment group were analyzed by light microscopy using an Olympus BX51 microscope (Olympus America Inc., Center Valley, PA).

Statistical analysis

All experiments were repeated at least 3 times. Data were analyzed using student’s t-test and 2-way ANOVA; a p value of <0.05 was considered statistically significant.

Results

In previous studies we demonstrated that both proinflammatory/cytotoxic M1 macrophages and anti-inflammatory/wound repair M2 macrophages accumulate in the lung following ozone exposure (Sunil et al., 2012). To assess the role of Gal-3 in the accumulation of these macrophage subpopulations in the lung, we used Gal-3−/− mice. In air exposed WT mice, low level Gal-3 staining was evident in lung epithelium and Type II cells, but not in macrophages (Fig. 2). Treatment of mice with ozone resulted in a time related increase in numbers of Gal-3+ macrophages in the lung, with no effect on epithelial cells. As expected, Gal-3 staining was not detectable in lungs of air or ozone exposed Gal-3−/− mice (Fig. 2). Following ozone exposure, an increase in iNOS+ M1 macrophages was observed in the lungs of WT mice at 24 h and 48 h (Fig. 3). At these times, iNOS was also evident in Type II epithelial cells. By 72 h post ozone, iNOS expression was reduced to levels below control. We also observed increased numbers of MR-1+ M2 macrophages in the lung at 24 h and 48 h after ozone (Fig. 4). Whereas ozone-induced increases in iNOS+ macrophages, as well as Type II cells, were reduced in Gal-3−/− mice relative to WT mice, numbers of MR-1+ macrophages were increased. These cells were larger than MR-1+ macrophages in lungs of WT mice and most prominent at 24 h and 48 h post-exposure (Fig. 4). Small MR-1+ macrophages were also noted in air exposed Gal-3−/− mice, but not in WT mice. Increases in MR-1+ macrophages in ozone-exposed Gal-3−/− mice were correlated with increases in BAL cell number (Fig. 5). Differential analysis revealed that these cells were >97% macrophages.

Fig. 2.

Fig. 2

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Effects of ozone on Gal-3 expression in the lung. Histologic sections, prepared 24–72 h after exposure of WT and Gal-3−/− mice to air or ozone, were stained with antibody to Gal-3. Binding was visualized using a peroxidase DAB substrate kit. Arrows indicate alveolar macrophages. One representative section from 3–5 mice per treatment group is shown (Original magnification, x600).

Fig. 3.

Fig. 3

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Effects of loss of Gal-3 on ozone-induced increases in iNOS+ macrophages in the lung. Histologic sections, prepared 24–72 h after exposure of WT and Gal-3−/− mice to air or ozone, were stained with antibody to iNOS. Binding was visualized using a peroxidase DAB substrate kit. Arrows indicate alveolar macrophages. One representative section from 3–5 mice per treatment group is shown (Original magnification, x600).

Fig. 4.

Fig. 4

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Effects of loss of Gal-3 on ozone-induced increases in MR-1+ macrophages in the lung. Histologic sections, prepared 24–72 h after exposure of WT and Gal-3−/− mice to air or ozone, were stained with antibody to MR-1. Binding was visualized using a peroxidase DAB substrate kit. Arrows indicate alveolar macrophages. One representative section from 3–5 mice per treatment group is shown (Original magnification, x600).

Fig. 5.

Fig. 5

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Effects of ozone on bronchoalveolar lavage (BAL) cells and protein content in WT and Gal-3−/− mice. BAL was collected 24–72 h after exposure of mice to air or ozone. Upper panel: Viable cells were enumerated by trypan blue due exclusion. Lower panel: Cell-free supernatants were analyzed in triplicate for protein using a BCA protein assay kit. Bars are the mean ± SE (n = 8 mice per treatment group). *Significantly different (p <0.05) from air-exposed animals; #Significantly different (p<0.05) from WT animals.

To further characterize lung macrophages responding to ozone and to assess the effects of loss of Gal-3, we used techniques in flow cytometry. In these experiments, cells were first analyzed for expression of CD11b, a beta2-integrin expressed on infiltrating myeloid cells (Fullerton et al., 2013). This was followed by analysis of the granulocytic marker, Ly6G (Lee et al., 2013), the macrophage activation marker, Ly6C (Zimmermann et al., 2012; Epelman et al., 2014), and the mature alveolar macrophage markers, F4/80 and CD11c (Grundy and Sentman, 2005; Zaynagetdinov et al., 2013; Ji et al., 2014) (Fig. 1). Consistent with previous studies (Garn et al., 2006; Matthews et al., 2007; Zaslona et al., 2014), resident alveolar macrophages from air exposed mice were identified as CD11b−CD11c+F4/80+Ly6G−Ly6Clo (Fig. 1). These cells were largely unaffected by ozone (data not shown). Conversely, ozone caused a significant increase in CD11b+ infiltrating myeloid cells in the lung; these cells were comprised of monocytic Ly6G− and granulocytic Ly6G+ subpopulations (Table 1 and Fig. 1). In both WT and Gal-3−/− mice, the majority of the infiltrating cells (>97%) were Ly6G− macrophages. Ozone exposure resulted in an increase in both Ly6G− and Ly6G+ cells in WT, as well as Gal-3−/− mice. Greater numbers of granulocytic Ly6G+ cells were observed in Gal-3−/− mice, when compared to WT mice, 72 h post ozone exposure (Table 1).

Table 1.

Effects of ozone on infiltrating CD11b+ myeloid cells

Time after ozone (h) Monocytic (Ly6G−) Granulocytic (Ly6G+)
WT Gal-3−/− WT Gal-3−/−
Air 11.7 ± 1.9 17.0 ± 5.7 0.2 ± 0.05 0.4 ± 0.1
24 h 45.3 ± 3.8* 56.7 ± 8.6* 2.1 ± 0.6 3.2 ± 0.9*
48 h 70.4 ± 12.6* 16.0 ± 0.7# 7.5 ± 2.4* 3.7 ± 1.0*
72 h 62.0 ± 9.1* 80.7 ± 8.4* 5.4 ± 1.1* 11.9 ± 1.3 *, #
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Cells, collected 24–72 h after exposure of WT and Gal-3−/− mice to air or ozone, were immunostained with antibodies to CD11b and Ly6G and analyzed by flow cytometry as described in the Materials and Methods. Data are the number of CD11b+ cells x 103 negative (Ly6G−) or positive (Ly6G+) for Ly6G. Values are mean ± SE (n=4–14 mice/treatment group).

*

Significantly different (p<0.05) from Air.

#

Significantly different (p<0.05) from WT.

Further analysis of the Ly6G− cells revealed that they consisted of two major subpopulations: proinflammatory Ly6Chi and anti-inflammatory Ly6Clo macrophages; most of these cells were mature lung macrophages, expressing F4/80+ and CD11c+. Treatment of WT mice with ozone resulted in an increase in both Ly6Chi and Ly6Clo macrophages (Fig. 6). While proinflammatory Ly6Chi macrophages increased rapidly (within 24 h) and remained elevated for at least 72 h, increases in anti-inflammatory Ly6Clo macrophages were not evident until 72 h post ozone. Loss of Gal-3 resulted in a significant decrease in numbers of proinflammatory Ly6Chi macrophages in the lung at all post exposure times, with no significant effects on anti-inflammatory Ly6Clo macrophages (Fig. 6).

Fig. 6.

Fig. 6

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Effects of ozone on mature proinflammatory and anti-inflammatory macrophage subpopulations in the lung. BAL cells, collected 24–72 h after exposure of WT and Gal-3−/− mice to air or ozone, were stained with antibodies to CD11b, Ly6G, Ly6C, F4/80 and CD11c or appropriate isotypic controls, and analyzed by flow cytometry. Upper panel: Mature (F4/80+CD11c+) CD11b+Ly6G−Ly6Chi proinflammatory macrophages; Lower panel: Mature (F4/80+CD11c+) CD11b+Ly6G−Ly6Clo anti-inflammatory macrophages. Bars are the mean ± SE (n = 4–14 mice per treatment group). *Significantly different (p <0.05) from air-exposed animals; #Significantly different (p<0.05) from WT animals.

Flow cytometric analysis of CD11b+Ly6G+ infiltrating granulocytic cells revealed that they also expressed high levels of Ly6C, a characteristic feature of myeloid derived suppressor cells (MDSC) (Kong et al., 2013). These cells were found to consist of F4/80− granulocytic (G) and F4/80+ monocytic (M) subpopulations (Fig. 1). In WT mice, a time related increase in both subpopulations was observed in the lung beginning 24 h after ozone and persisting for at least 72 h (Fig. 7). While loss of Gal-3 resulted in an attenuated response of G-MDSC to ozone at 48 h and 72 h, a heightened response was observed in the M-MDSC at 24 h and 72 h (Fig. 7).

Fig. 7.

Fig. 7

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Effects of loss of Gal-3 on MDSC. BAL cells, collected 24–72 h after exposure of WT and Gal-3−/− mice to air or ozone, were stained with antibodies to CD11b, Ly6G, Ly6C and F4/80 or appropriate isotypic controls, and analyzed by flow cytometry. Upper panel: CD11b+Ly6G+Ly6C+F4/80− granulocytic (G) MDSC; Lower panel: CD11b+Ly6G+Ly6C+F4/80+ monocytic (M) MDSC. Bars are the means ± SE (n = 4–14 mice per treatment group). *Significantly different (p <0.05) from air-exposed animals; #Significantly different (p<0.05) from WT animals.

In further studies we determined if changes in lung macrophage subpopulations in Gal-3−/− mice were correlated with alterations in markers of ozone-induced oxidative stress and injury. Treatment of WT mice with ozone resulted in a marked upregulation of cytochrome b5 expression, an indicator of acute lung injury and oxidative stress (Rodriguez-Ariza et al., 2005; Menoret et al., 2012) (Fig. 8). This was most notable in macrophages and epithelial cells 24 h and 48 h post exposure. Loss of Gal-3 significantly attenuated this response. Ozone exposure also resulted in increases in BAL protein content indicating alveolar-epithelial barrier dysfunction (Bhalla, 1999); however, this was largely unaffected by loss of Gal-3 (Fig. 5).

Fig. 8.

Fig. 8

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Effects of ozone on cytochrome b5 protein expression in WT and Gal-3−/− mice. Histologic sections, prepared 24–72 h after exposure of mice to air or ozone, were stained with antibody to cytochrome b5. Binding was visualized using a peroxidase DAB substrate kit. One representative section from 3–5 mice per treatment group is shown (Original magnification, x600).

Discussion

Macrophages have been shown to be important in promoting both acute and chronic inflammation, as well as in suppressing inflammatory responses and inducing wound repair (Hollingsworth et al., 2007; Laskin et al., 2011). Evidence suggests that these diverse activities are mediated by distinct subpopulations of macrophages that appear sequentially in tissues in response to injury (Byers and Holtzman, 2011; Laskin et al., 2011). Consistent with this notion are previous reports that, initially, proinflammatory/cytotoxic M1 macrophages, and subsequently anti-inflammatory/wound repair M2 macrophages, accumulate in the lung after ozone, and that these cells contribute to tissue injury and repair, respectively (Pendino et al., 1994; Koike et al., 1998; Hollingsworth et al., 2007; Tighe et al., 2011; Sunil et al., 2012). The present studies identify a novel population of macrophages in the lung after exposure to ozone that is important in promoting proinflammatory signaling and tissue injury. These cells express Gal-3, a carbohydrate-binding lectin, known to regulate macrophage activation (Perillo et al., 1998; Liu, 2005; Liu and Hsu, 2007; Nishi et al., 2007). These findings are important, as they provide new mechanistic insights into the pathogenesis of ozone-induced lung injury.

Gal-3 is thought to regulate macrophage functioning via its interaction with CD98, a membrane glycoprotein involved in promoting inflammation and fibrosis (Dong and Hughes, 1997; Lopez et al., 2006; Maldonado et al., 2011; Mackinnon et al., 2012). Gal-3 has been shown to play a role in regulating inflammation and in the pathophysiology of emphysema and pulmonary fibrosis, as well as in acute hepatotoxicity, chronic kidney disease and cancer, pathologies associated with excessive inflammatory macrophage activity (Nishiyama et al., 2000; Henderson et al., 2008; Dragomir et al., 2012a; Dragomir et al., 2012b; Mukaro et al., 2013; O’Seaghdha et al., 2013; Fortuna-Costa et al., 2014). We found that ozone exposure resulted in a time related increase in Gal-3+ macrophages in the lung. Similar increases in Gal-3+ macrophages have been observed in the lung after exposure to other pulmonary toxicants including nitrogen mustard, radiation and ultrafine particles (Kasper and Hughes, 1996; Andre et al., 2006; Malaviya et al., 2012; Van der Meeren et al., 2014). Increased Gal-3 expression has also been noted in lungs of humans with inflammatory diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis (Nishi et al., 2007; Pilette et al., 2007). These findings suggest that upregulation of Gal-3 may represent a common pathway for promoting proinflammatory/cytotoxic activation of macrophages in diverse pulmonary pathologies.

In accord with our earlier studies (Pendino et al., 1995; Fakhrzadeh et al., 2002; Groves et al., 2013), following ozone exposure, we observed increased numbers of both iNOS+ and MR-1+ macrophages in the lung, markers of proinflammatory and anti-inflammatory macrophages, respectively. For both iNOS+ and MR-1+ macrophage subpopulations, maximal accumulation was evident 24–48 h post exposure. The fact that ozone-induced increases in MR-1+ macrophages were greater in Gal-3−/− mice, when compared to WT mice, suggests a more prominent induction of tissue repair processes in these mice. Gal-3 has previously been shown to upregulate iNOS expression in brain macrophages (Jeon et al., 2010). The present studies suggest a similar regulatory role for Gal-3 in lung macrophage iNOS expression. This is based on our observation that loss of Gal-3 resulted in reduced numbers of iNOS+ macrophages in the lung. Findings that decreases in iNOS+ macrophages in Gal-3−/− mice were correlated with increases in MR-1+ macrophages support the idea that the pathogenic response to ozone involves a balance in the activity of M1 and M2 macrophages. Increased numbers of MR-1+ macrophages in lungs of Gal-3−/− mice following ozone exposure may contribute to reduced lung injury in these mice. This is supported by our findings that in Gal-3−/− mice, MR-1+ macrophages are enlarged, relative to cells in WT mice, and foamy in appearance, indicating robust tissue repair activity (Boven et al., 2006). These data suggest that in the absence of Gal-3, there is a shift in the balance of macrophage subpopulations towards an M2 phenotype resulting in more rapid tissue repair.

Cytochrome b5 is a highly conserved protein that participates in the oxidation of endogenous and xenobiotic substances (Vergeres and Waskell, 1995; Schenkman and Jansson, 2003; Menoret et al., 2012). It is rapidly upregulated in the lung after enterotoxin inhalation and is thought to be an early marker of acute lung injury, inflammation and oxidative stress (Menoret et al., 2012). Consistent with ozone-induced oxidative stress and lung injury, we found that cytochrome b5 expression was upregulated in the lung after ozone. The observation that this response was reduced in Gal-3−/− mice, are in accord with a proinflammatory/cytotoxic role of Gal-3 in ozone-induced injury. In contrast, loss of Gal-3 had no significant effect on ozone-induced increases in BAL protein indicating that proinflammatory mediators generated by Gal-3+ macrophages do not contribute to alveolar epithelial barrier dysfunction.

In contrast to the lack of effects of Gal-3 on BAL protein, BAL macrophage number was increased in Gal-3−/− mice, relative to WT mice, following ozone exposure. To determine if this was due to a shift in the phenotype of cells accumulating in the lung, we characterized the cells using techniques in flow cytometry. In WT and Gal-3−/− mice, ozone exposure resulted in increased numbers of infiltrating (CD11b+) monocytic (Ly6G−) and granulocytic (Ly6G+) inflammatory cells in the lung. The majority of the cells were Ly6G−, consistent with the observation that macrophages are the predominant cell population recovered in BAL (Ji et al., 2014). Whereas there were no major differences between WT and Gal-3−/− mice in the response of Ly6G− monocytic cells to ozone, maximum increases in Ly6G+ granulocytic cells were delayed in Gal-3−/− mice relative to WT mice. These differences likely reflect the unique phenotype of the responding cell populations. This is supported by our findings of heterogeneity within the Ly6G− and Ly6G+ cell populations. Thus, while Ly6G− cells consisted of mature (F4/80+CD11c+) proinflammatory Ly6Chi and anti-inflammatory Ly6Clo macrophages, Ly6G+ cells consisted of F4/80+ monocytic and F4/80− granulocytic Ly6C+ MDSC. Each of these cell populations responded distinctly to ozone and was differentially affected by loss of Gal-3. Whereas in WT mice, Ly6Chi macrophages increased rapidly in the lung and persisted for at least 72 h following ozone exposure, the appearance of Ly6Clo macrophages was delayed for 72 h. This is similar to our immunohistochemistry data which showed sequential accumulation of proinflammatory iNOS+ and anti-inflammatory MR-1+ macrophages in the lung after ozone. Further analysis of lung macrophages revealed that in WT mice, most of the Ly6Chi proinflammatory cells were F4/80+CD11c+ indicating a mature phenotype. Loss of Gal-3 resulted in a decrease in proinflammatory Ly6Chi cells in the lung, which correlated with reduced numbers of proinflammatory iNOS+ macrophages. These data are consistent with reports that Ly6Chi macrophages express iNOS (Dragomir et al., 2012a), and provide further support for the proinflammatory actions of Gal-3. In contrast to the effects of loss of Gal-3 on Ly6Chi cells, there were no major effects on anti-inflammatory Ly6Clo macrophages, despite immunohistochemical evidence of increased numbers of MR-1+ macrophages in the lung after ozone. These data are in accord with the idea that there are multiple phenotypically distinct populations of anti-inflammatory/wound repair macrophages that respond to tissue injury (Alber et al., 2012; Aggarwal et al., 2014; Wang et al., 2014).

MDSC consist of a heterogeneous population of hematopoietic monocytic and granulocytic precursors with immunosuppressive and anti-inflammatory activity (Nagaraj and Gabrilovich, 2012). MDSC have been implicated in wound healing, a process thought to be mediated via the release of TGF-β and IL-10 (Chambers et al., 2013; Sarkar et al., 2013). Under inflammatory conditions, MDSC expand and develop into G-MDSC or M-MDSC, which are thought to contribute to protection against unrestrained inflammation (Chambers et al., 2013; Sarkar et al., 2013). Following ozone inhalation, we identified both G-MDSC and M- MDSC in the lungs of WT mice. MDSC have been shown to play a key role in the resolution of acute inflammation following spinal cord injury and in suppressing autoimmune disease in the central nervous system (Yi et al., 2012; Saiwai et al., 2013), and they may similarly be important in limiting ozone-induced inflammation and injury. Loss of Gal-3 was associated with a decrease in G-MDSC at 48 h and 72 h post ozone, but an increase in M-MDSC, which was most notable at 72 h. These findings suggest that in the absence of Gal-3, M-MDSC play a more prominent role in the resolution of tissue injury.

In summary, the present studies identify multiple monocytic subpopulations accumulating in the lung following ozone inhalation, and demonstrate a role for Gal-3 in promoting proinflammatory macrophage activation which contributes to pulmonary toxicity. These studies also demonstrate that there are increased numbers of inflammatory cells displaying a M-MDSC phenotype in the lungs of ozone-treated Gal-3−/− mice, which we speculate contribute to the resolution of tissue injury. Elucidating specific subpopulations of inflammatory cells responding to ozone and their function may lead to development of novel approaches for mitigating pulmonary toxicity.

Highlights.

  • Multiple monocytic-macrophage subpopulations accumulate in the lung after ozone inhalation

  • Galectin-3 plays a proinflammatory role in ozone-induced lung injury

  • In the absence of gal-3, inflammatory cells with a myeloid derived suppressor cell phenotype contribute to tissue repair

Acknowledgments

This work was supported by NIH grants ES004738, AR055073, ES007148 and ES005022.

Abbreviations

Gal-3

galectin-3

BAL

bronchoalveolar lavage

iNOS

inducible nitric oxide synthase

MR-1

mannose receptor-1

MDSC

myeloid derived suppressor cell

WT

wild type

M

monocytic

G

granulocytic

Footnotes

Conflict of interest

The authors declare no conflict of interest.

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Contributor Information

Mary Francis, Email: maryfranrutgers@gmail.com.

Kinal N. Vayas, Email: kinalv5@gmail.com.

Jessica A. Cervelli, Email: j.cervelli@pharmacy.rutgers.edu.

Hyejeong Choi, Email: choi@eohsi.rutgers.edu.

Jeffrey D. Laskin, Email: jlaskin@eohsi.rutgers.edu.

Debra L. Laskin, Email: laskin@eohsi.rutgers.edu.

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