Inhalation of traffic-related particulate matter (e.g., diesel exhaust particles [DEPs]) is associated with acute inflammatory responses in the lung, and it promotes the development and aggravation of allergic airway diseases. We previously demonstrated that exposure to DEP was associated with increased recruitment and maturation of monocytes and conventional dendritic cells (DCs), resulting in TH2 polarization. Monocytes and immature DCs express the G-protein coupled receptor chemR23, which binds the chemoattractant chemerin. Using chemR23 knockout (KO) and corresponding wild-type (WT) mice, we determined the role of chemR23 signaling in response to acute exposure to DEPs and in response to DEP-enhanced house dust mite (HDM)-induced allergic airway inflammation. Exposure to DEP alone, as well as combined exposure to DEP plus HDM, elevated the levels of chemerin in the bronchoalveolar lavage fluid of WT mice. In response to acute exposure to DEPs, monocytes and monocyte-derived DCs accumulated in the lungs of WT mice, but this response was significantly attenuated in chemR23 KO mice. Concomitant exposure to DEP plus HDM resulted in allergic airway inflammation with increased eosinophilia, goblet cell metaplasia, and TH2 cytokine production in WT mice, which was further enhanced in chemR23 KO mice. In conclusion, we demonstrated an opposing role for chemR23 signaling depending on the context of DEP-induced inflammation. The chemR23 axis showed proinflammatory properties in a model of DEP-induced acute lung inflammation, in contrast to anti-inflammatory effects in a model of DEP-enhanced allergic airway inflammation.

It is well accepted that inhalation of traffic-related particulate matter, of which diesel exhaust particles (DEPs) are a main component, is associated with acute inflammatory responses in the lung. In addition, traffic-related particulate matter can contribute to new-onset asthma and the exacerbation of pre-existing asthma (1). Experimental studies in mice showed that exposure to DEP can enhance allergic airway responses, including eosinophilia, goblet cell metaplasia, and TH2 production (2). In addition, in controlled human exposure studies, combined exposure to DEP plus allergens increased allergen-specific Ig levels and induced a TH2 cytokine pattern (2, 3). The mechanistic basis of the inflammatory response in the lung to DEP inhalation and the adjuvant response to DEP on allergen-induced airway inflammation remain incompletely known.

Using a mouse model of DEP-induced acute lung inflammation, we previously demonstrated that exposure to DEP was associated with increased expression of proinflammatory cytokines/chemokines and with the accumulation of neutrophils and monocytes in lung tissue (4). Furthermore, we showed that DEP had a great influence on the biology of conventional dendritic cells (DCs) (5), which are crucial in the induction of asthma. We demonstrated that exposure to DEPs induced the recruitment of monocytes and DCs toward the lung via the G-protein coupled receptors CCR2 and CCR6, increased DC maturation and enhanced DC-induced allergen transport toward the draining lymph nodes, resulting in TH2 polarization. This modulation of DC function could be the mechanistic basis underlying DEP-enhanced allergic airway responses (5, 6).

ChemR23 (also known as chemokine-like receptor 1 [CMKLR1]) is a seven-transmembrane G-protein coupled receptor that is expressed on monocytes, macrophages, NK cells, and conventional and plasmacytoid DCs in humans and mice (79). Its ligand, chemerin, is secreted as a weakly active precursor protein (i.e., prochemerin) that is converted into bioactive chemerin by proteolytic cleavage of its C-terminal (10). This maturation step is mediated by extracellular proteases released by activated macrophages, mast cells, and neutrophils (1113). In addition to chemerin, resolvin E1 (RvE1, an anti-inflammatory lipid, derived from the ω-3 fatty acid eicosapentaenoic acid) is reported as a second putative ligand of chemR23 (14).

Intriguingly, the role of the chemerin/chemR23 axis in inflammation is controversial and seems to have proinflammatory and anti-inflammatory properties depending on the model that is investigated. Previous work from our laboratory demonstrated that exposure to cigarette smoke was associated with increased chemerin levels in the bronchoalveolar lavage fluid (BALF). Moreover, chemR23 knockout (KO) mice were almost completely protected against cigarette smoke–induced lung inflammation (15). In contrast to this proinflammatory role of chemR23 signaling in response to cigarette smoke, chemR23 KO mice showed an increased inflammatory response in a LPS-induced model of acute lung injury (16) and were more susceptible to viral pneumonia (17). In addition, intranasal administration of exogenous chemerin was associated with attenuated allergic airway inflammation and airway hyperresponsiveness (18).

We studied the role of chemR23 signaling in the context of DEP-induced lung responses. First, we examined the chemR23 axis in the murine model of DEP-induced lung inflammation. Next, we investigated the contribution of chemR23 signaling to the adjuvant capacity of DEP in enhancing allergic airway inflammation, using a model of concomitant exposure to DEP and a clinically relevant allergen (e.g., house dust mite [HDM]). We demonstrated that the functional role of chemR23 signaling is dependent on the background inflammatory conditions, which explains the existing controversy in the literature.

Female C57BL/6 mice (6–8 wk old) were purchased from Harlan. Female ChemR23 KO mice (6–9 wk old) and corresponding C57BL/6 wild type (WT) mice (6–9 wk old) were obtained from Deltagen and bred in our animalarium. All in vivo manipulations were reviewed and approved by our local ethical committee (Animal Ethical Committee of the Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium).

DEP (SRM 2975) was obtained from the National Institute for Standards and Technology. For the mouse model of DEP-induced acute lung inflammation, DEPs were suspended in saline (B. Braun Melsungen) containing 0.05% Tween 80 (Invitrogen). Mice were anesthetized with an i.p. ketamine/xylazine injection (Ketamine 1000 CEVA [70 mg/kg; Ceva Sante Animale] and Rompun 2% [7 mg/kg; Bayer]) prior to instillation. The anesthetized mice were instilled intratracheally with 50 μl saline or DEP solution (i.e., 100 μg) on days 1, 4, and 7. On day 9, the animals were sacrificed with a lethal dose of i.p. pentobarbital (Ceva Sante Animale) (46). For the mouse model of DEP-enhanced allergic airway inflammation, HDM (Dermatophagoides pteronyssinus; Greer Laboratories) was suspended in saline. Mice were anesthetized with isoflurane (Abbott Laboratories) prior to instillation. The anesthetized mice were instilled intranasally with 50 μl saline, DEP (25 μg), HDM (1 μg), or combined DEP plus HDM solution on days 1, 8, and 15. On day 17, the animals were sacrificed with a lethal dose of i.p. pentobarbital (Ceva Sante Animale).

A tracheal cannula was inserted, and BALF was recovered by instillation of 3 × 300 μl HBSS without Ca2+ or Mg2+ (BioWittaker) supplemented with 1% BSA (for cytokine and chemokine measurements; Sigma-Aldrich) and 6 × 500 μl HBSS without Ca2+ or Mg2+ supplemented with 0.6 mM sodium EDTA (Sigma-Aldrich). The lavage fractions were pooled and total cell counts were performed using a Bürcker chamber (BRAND GMBH).

Pulmonary circulation was rinsed with saline, supplemented with EDTA, to remove the intravascular pool of cells. Lungs or mediastinal lymph nodes (LNs) were minced and incubated in digestion medium (RPMI 1640 supplemented with 5% FCS, 2 mM l-glutamine, 0.05 mM 2-mercaptomethanol [all from Invitrogen], 100 U/ml penicillin with 100 μg/ml streptomycin [Sigma-Aldrich], 1 mg/ml collagenase type 2 [Worthington Biochemical] and 0.02 mg/ml DNase I [grade II from bovine pancreas; Boehringer Ingelheim]) for 45 min at 37°C and 5% CO2. RBCs were lysed using ammonium chloride buffer. Cell counting was performed with a Z2 Coulter counter (Beckman Coulter).

All staining procedures were performed in PBS without Ca2+ or Mg2+ containing 5 mM EDTA and 1% BSA. To minimize nonspecific bindings, BALF cells and lung single-cell suspensions were incubated with anti-CD16/CD32 (clone 2.4G2). Cells were labeled with combinations of CD11c (HL3), MHCII (2G9), CD11b (M1/70), Ly6C (AL-21), Ly6G (1A8), Siglec-F (e50-2440), CD4 (GK1.5), CD8 (53-6.7), CD69 (H1.2F3), CD3 (145-2C11; all BD Biosciences). Data acquisition was performed with a FACSCalibur flow cytometer running CELLQuest software or a LSRFortessa cell analyzer running FACSDiva software (BD Biosciences). FlowJo software was used for data analysis.

Mediastinal LN cells were cultured in culture medium (RPMI 1640 supplemented with 10% FCS, 2 mM l-glutamine, 0.05 mM 2-mercaptomethanol [all from Invitrogen], 100 U/ml penicillin with 100 μg/ml streptomycin [Sigma-Aldrich], 1 mg/ml collagenase type 2 [Worthington Biochemical], and 0.02 mg/ml DNase I [grade II from bovine pancreas; Boehringer Ingelheim]) either alone or supplemented with 3.75 μg/well HDM in round-bottom 96-well plates, and incubated in a humidified 37°C incubator with 5% CO2. After 5 d, supernatants was harvested for protein measurements.

Lungs were fixed in 4% paraformaldehyde and embedded in paraffin (Klinipath). Transversal sections (3 μm) were cut, treated with Ultra V Block (Thermo Scientific), and incubated with polyclonal anti-mouse chemerin Ab (R&D Systems). The goat HRP-Polymer Kit (Biocare Medical) and diaminobenzidine (DakoCytomation) were used for detection. Prochemerin staining was quantified within the airway epithelium in a marked area between the airway lumen and the basement membrane, using KS400 Software (Zeiss) (15). The area with positive staining for prochemerin was normalized to the basement membrane perimeter (Pbm) (nomenclature described in (19)). All airways with Pbm > 2000 μm were excluded. For the visualization of eosinophils or goblet cells, lungs were stained with Congo-Red or periodic acid–Schiff (Klinipath). Quantification was performed (KS400 software) in all airways with Pbm between 800 and 2000 μm.

Chemerin, CCL2, CCL20, IL-4, IL-5, and IL-13 levels in BALF or mediastinal LN culture supernatant were measured using commercially available ELISA kits (R&D Systems). Ig were determined on serum that was collected by retro-orbital bleeding. Total IgE was measured using coated plates and biotinylated polyclonal rabbit anti-mouse IgE (S. Florquin, Université libre de Bruxelles). For detection of HDM-specific IgG1, plates were coated with HDM extract. Serum was added, followed by a HRP-conjugated polyclonal goat anti-mouse IgG Ab (Bethyl Laboratories). Ig values were reported in ODs.

Hematopoietic (CD45+) cells and nonhematopoietic (CD45) lung cells were sorted using an OctoMACS separator and CD45 MicroBeads (according to the manufacturer’s instructions; Miltenyi Biotec). The sorted populations showed >95% purity (data not shown). Next, RNA extraction was performed (miRNeasy Mini Kit; Qiagen), and cDNA (Transcriptor First Strand cDNA synthesis kit; Roche Diagnostics) was obtained following the manufacturer’s instructions. SYBR Green–based RT-PCR reactions (LightCycler 480 SYBR Green I Master; Roche) were performed using a LightCycler 96 system (Roche). Chemerin primer sequences were described previously (15). Data were processed using the standard curve method. Expression of chemerin mRNA was normalized based on the expression of two reference genes (GADPH and HPRT).

Statistical analysis was performed with SPSS, version 22.0. Groups were compared using nonparametric tests (Kruskall–Wallis and Mann–Whitney U) according to standard statistical criteria. Reported values were expressed as mean ± SEM. The p values < 0.05 were regarded as significant.

Chemerin levels in BALF are increased in WT mice exposed to DEP.

We previously identified airway epithelial cells as the major source of (pro)chemerin in lung tissue (15). To examine the localization and expression of (pro)chemerin in response the DEP, we exposed WT mice to saline or DEP (experimental protocol in Fig. 1A), sorted hematopoietic (CD45+) versus structural nonhematopoietic (CD45) cells from lung tissue, and examined the chemerin mRNA expression. Chemerin was mainly expressed in the CD45 cell population, whereas minimal chemerin mRNA expression was observed in the CD45+ cell population. Chemerin mRNA expression in CD45 cells significantly increased in response to exposure to DEP (Fig. 1B). Using immunohistochemistry, we confirmed that the epithelium was the predominant source of (pro)chemerin in the lung. We quantified the (pro)chemerin staining and observed that exposure to DEP was associated with decreased (pro)chemerin staining in the epithelium when compared with control mice (Fig. 1C). To assess whether this was due to increased secretion, we determined (pro)chemerin levels in BALF using ELISA, and found that exposure to DEP elevated the secreted levels of (pro)chemerin (Fig. 1D).

FIGURE 1.

Chemerin levels in BALF are increased in WT mice exposed to DEP. Mice were intratracheally exposed to saline (white bar) or 100 μg DEP (black bar). (A) Schematic overview of the model of DEP-induced acute lung inflammation. (B) Chemerin mRNA expression in hematopoietic (CD45+) and structural nonhematopoietic (CD45) lung cells was determined using RT-PCR. (C) Photomicrographs of prochemerin staining in the airway epithelium of mice that were exposed to saline or DEP and quantification of prochemerin staining. Scale bar is shown in the lower left photomicrograph. (D) Chemerin and (E) RvE1 protein levels in BALF were determined by ELISA. Results are expressed as mean ± SEM. n = 8 mice per group. *p < 0.05. EP, endpoints.

FIGURE 1.

Chemerin levels in BALF are increased in WT mice exposed to DEP. Mice were intratracheally exposed to saline (white bar) or 100 μg DEP (black bar). (A) Schematic overview of the model of DEP-induced acute lung inflammation. (B) Chemerin mRNA expression in hematopoietic (CD45+) and structural nonhematopoietic (CD45) lung cells was determined using RT-PCR. (C) Photomicrographs of prochemerin staining in the airway epithelium of mice that were exposed to saline or DEP and quantification of prochemerin staining. Scale bar is shown in the lower left photomicrograph. (D) Chemerin and (E) RvE1 protein levels in BALF were determined by ELISA. Results are expressed as mean ± SEM. n = 8 mice per group. *p < 0.05. EP, endpoints.

Close modal

RvE1 is a second putative ligand of chemR23 (14). Using ELISA, we assessed the RvE1 levels in the BALF and found that these were decreased in response to DEP exposure (Fig 1E).

ChemR23 is required for DEP-induced monocyte and alveolar DC recruitment.

Chemerin/chemR23 signaling can mediate the recruitment of monocytes and (immature) DC (10). To assess the contribution of the chemR23 pathway to the accumulation of monocytes and DC in response to DEP, we exposed WT and chemR23 KO mice to saline or DEP. Upon exposure to DEP, the number of total BALF cells increased in WT mice. This increase was smaller in chemR23 KO mice (Fig. 2A). Exposure to DEP was associated with an accumulation of monocytes in the BALF and lung, and monocyte-derived DC in lung of WT mice, which was severely reduced or absent in chemR23 KO mice (Fig. 2B–D). In addition, DEP exposure increased the number of alveolar DC and their maturation, as demonstrated by increased expression of CD86. These responses were significantly attenuated in chemR23 KO mice compared with WT mice (Fig. 2E, 2F). In contrast, the DEP-induced increase in lung CD11b+ conventional DC and neutrophils in BALF and lung were similar between WT and chemR23 KO mice (Fig. 2G–I). Lung macrophage counts were not affected by DEP exposure and did not differ between WT and chemR23 KO mice (Fig. 2J).

FIGURE 2.

ChemR23 is required for DEP-induced monocyte and alveolar DC recruitment. WT and chemR23 KO mice were exposed to saline (white bars) or DEP (black bars). (A) Total cell number in BALF. (B) Monocytes numbers in BALF (CD11clow, CD11b+, Ly6C+ and Ly6G), (C) percentage of monocytes in lung tissue (CD11clow, CD11b+, Ly6C+, and Ly6G), (D) percentage of lung monocyte-derived DC (CD11chigh, low autofluorescent, CD11b+ and Ly6C+), (E) DC numbers in BALF (CD11chigh, low autofluorescent and MHCII+), (F) mean fluorescence intensity (MFI) of CD86 on alveolar DC, (G) percentage of lung CD11b+ conventional DC (CD11chigh, low autofluorescent, CD11b+ and Ly6C), (H) neutrophils numbers in BALF (CD11clow, CD11b+, Ly6C+ and Ly6G+), (I) percentage of lung neutrophils (CD11clow, CD11b+, Ly6C+, and Ly6G+), and (J) percentage of lung macrophages (CD11chigh, high autofluorescent) were determined by flow cytometry. Results are expressed as mean ± SEM. n = 7–8 mice per group. Representative flow cytometric density plots and gating strategy are shown in Supplemental Fig. 1. *p < 0.05.

FIGURE 2.

ChemR23 is required for DEP-induced monocyte and alveolar DC recruitment. WT and chemR23 KO mice were exposed to saline (white bars) or DEP (black bars). (A) Total cell number in BALF. (B) Monocytes numbers in BALF (CD11clow, CD11b+, Ly6C+ and Ly6G), (C) percentage of monocytes in lung tissue (CD11clow, CD11b+, Ly6C+, and Ly6G), (D) percentage of lung monocyte-derived DC (CD11chigh, low autofluorescent, CD11b+ and Ly6C+), (E) DC numbers in BALF (CD11chigh, low autofluorescent and MHCII+), (F) mean fluorescence intensity (MFI) of CD86 on alveolar DC, (G) percentage of lung CD11b+ conventional DC (CD11chigh, low autofluorescent, CD11b+ and Ly6C), (H) neutrophils numbers in BALF (CD11clow, CD11b+, Ly6C+ and Ly6G+), (I) percentage of lung neutrophils (CD11clow, CD11b+, Ly6C+, and Ly6G+), and (J) percentage of lung macrophages (CD11chigh, high autofluorescent) were determined by flow cytometry. Results are expressed as mean ± SEM. n = 7–8 mice per group. Representative flow cytometric density plots and gating strategy are shown in Supplemental Fig. 1. *p < 0.05.

Close modal

ChemR23 is required for DEP-induced CCL2 and CCL20 production.

CCL2 and CCL20 are important chemokines for the DEP-induced recruitment of monocytes and monocyte-derived DC (6). DEP exposure elevated the levels of CCL2 and CCL20 in the BALF in both WT and chemR23 KO mice. However, the levels of these chemokines were significantly reduced in DEP-exposed chemR23 KO mice (Fig. 3A, 3B).

FIGURE 3.

ChemR23 is required for DEP-induced CCL2 and CCL20 production. WT and chemR23 KO mice were exposed to saline (white bars) or DEP (black bars). (A) CCL2, (B) CCL20, (C) and chemerin protein levels in BALF were determined by ELISA. Results are expressed as mean ± SEM. n = 7–8 mice per group. *p < 0.05.

FIGURE 3.

ChemR23 is required for DEP-induced CCL2 and CCL20 production. WT and chemR23 KO mice were exposed to saline (white bars) or DEP (black bars). (A) CCL2, (B) CCL20, (C) and chemerin protein levels in BALF were determined by ELISA. Results are expressed as mean ± SEM. n = 7–8 mice per group. *p < 0.05.

Close modal

The level of the chemR23 ligand, chemerin, in BALF was elevated in WT mice that received DEP as described above. Independent of the exposure, all chemR23 KO mice had increased (pro)chemerin levels when compared with WT controls (Fig. 3C).

Chemerin levels are increased in WT mice coexposed to DEP plus HDM.

Exposure to DEP promotes sensitization toward coinhaled allergens and aggravates asthma (2). To investigate the molecular mechanisms, we set up a model wherein we exposed mice to saline, DEP or HDM alone, or combined DEP and HDM (experimental protocol in Fig. 4A). To examine the adjuvant capacity of DEP optimally, we administered low doses of DEP and HDM that elicited almost no inflammatory or allergic response on their own. In this model, the (pro)chemerin levels in BALF were elevated in WT mice concomitantly exposed to DEP plus HDM, compared with all control groups (Fig. 4B).

FIGURE 4.

Absence of chemR23 aggravates allergic responses in BALF upon combined DEP + HDM exposure. WT and chemR23 KO mice were exposed intranasally to saline (white bar), 25 μg DEP (striped bar), 1 μg HDM (checked bar), or 25 μg DEP plus 1 μg HDM (black bar). (A) Schematic overview of the model of DEP-enhanced allergic airway inflammation. (B) Chemerin and (C) RvE1 protein levels in BALF were determined by ELISA. (D) Monocyte numbers (CD11clow, CD11b+, Ly6C+, and Ly6G), (E) neutrophil numbers (CD11clow, CD11b+, Ly6C+, and Ly6G+), (F) eosinophils, (G) DC numbers (CD11chigh, low autofluorescent and MHCII+), (H) CD4+ T cell numbers (CD3+, CD8, and CD4+), and (I) CD8+ T cell numbers (CD3+, CD4, and CD8+) in BALF were determined by flow cytometry. Eosinophils were determined on cytospins. (J) CCL2 and (K) chemerin protein levels in BALF were determined by ELISA. Results are expressed as mean ± SEM; n = 7–12 mice per group. Lines with branches represent significant differences between DEP plus HDM versus saline, DEP alone, or HDM alone, except in (C) where the line with braches represents differences between saline versus DEP alone, HDM alone, and DEP plus HDM. *p < 0.05. EP, endpoints.

FIGURE 4.

Absence of chemR23 aggravates allergic responses in BALF upon combined DEP + HDM exposure. WT and chemR23 KO mice were exposed intranasally to saline (white bar), 25 μg DEP (striped bar), 1 μg HDM (checked bar), or 25 μg DEP plus 1 μg HDM (black bar). (A) Schematic overview of the model of DEP-enhanced allergic airway inflammation. (B) Chemerin and (C) RvE1 protein levels in BALF were determined by ELISA. (D) Monocyte numbers (CD11clow, CD11b+, Ly6C+, and Ly6G), (E) neutrophil numbers (CD11clow, CD11b+, Ly6C+, and Ly6G+), (F) eosinophils, (G) DC numbers (CD11chigh, low autofluorescent and MHCII+), (H) CD4+ T cell numbers (CD3+, CD8, and CD4+), and (I) CD8+ T cell numbers (CD3+, CD4, and CD8+) in BALF were determined by flow cytometry. Eosinophils were determined on cytospins. (J) CCL2 and (K) chemerin protein levels in BALF were determined by ELISA. Results are expressed as mean ± SEM; n = 7–12 mice per group. Lines with branches represent significant differences between DEP plus HDM versus saline, DEP alone, or HDM alone, except in (C) where the line with braches represents differences between saline versus DEP alone, HDM alone, and DEP plus HDM. *p < 0.05. EP, endpoints.

Close modal

Levels of RvE1 were decreased in WT mice exposed to DEP or HDM alone, or combined DEP plus HDM, when compared with saline-exposed WT mice (Fig. 4C).

Absence of chemR23 aggravates allergic responses in BALF upon combined DEP plus HDM exposure.

To elucidate the role of chemR23 signaling in DEP-enhanced allergic airway inflammation, we exposed WT and chemR23 KO mice to saline, DEP or HDM alone, or combined DEP plus HDM. In this protocol, exposure to sole DEP in WT mice was associated with marginal increases in monocytes and neutrophils in BALF, when compared with WT mice that were exposed to saline (Fig. 4D, 4E). Sole HDM slightly increased the amount of BALF eosinophils, CD4+ T cells, and CD8+ T cells compared with the saline WT group (Fig. 4F, 4H, 4I). In contrast, concomitant exposure to DEP plus HDM greatly enhanced the inflammatory and allergic response in WT mice, with increased numbers of monocytes, eosinophils, DC, CD4+ T cells, and CD8+ T cells in the BALF, when compared with the three WT control groups. This inflammation was further increased in chemR23 KO mice that were exposed to combined DEP plus HDM (Fig. 4D, 4F–I). Of interest, chemR23 KO mice seemed more susceptible to allergic airway inflammation, because chemR23 KO receiving sole HDM had increased monocytes and eosinophils when compared with HDM-exposed WT mice (Fig. 4D, 4F). In line with the data from the model of DEP-induced lung inflammation, the number of BALF monocytes were attenuated in chemR23 KO mice that were exposed to DEP alone, when compared with DEP-exposed WT mice (Fig. 4D).

Levels of CCL2 in BALF were elevated in WT mice exposed to DEP, and they increased further in mice that received concomitant DEP plus HDM. In contrast, chemR23 KO mice had decreased CCL2 levels when compared with their WT controls (Fig. 4J).

The levels of chemerin were increased in mice that were coexposed to DEP plus HDM as described above. Independent of the exposure, all chemR23 KO had increased (pro)chemerin levels when compared with WT controls (Fig. 4K).

Absence of chemR23 aggravates allergic responses in lung upon combined DEP plus HDM exposure.

Similarly to the data in the BALF, eosinophils, DCs, and CD4+ T cells accumulated in the lungs of mice that were concomitantly exposed to DEP plus HDM, when compared with WT control groups. Again, this response was further increased in ChemR23 KO mice that received combined DEP plus HDM (Fig. 5A–C). ChemR23 KO mice that received sole HDM had slightly increased eosinophils and DC when compared with HDM-exposed WT mice (Fig. 5A, 5B).

FIGURE 5.

Absence of chemR23 aggravates allergic responses in the lung upon combined DEP plus HDM exposure. WT and chemR23 KO mice were exposed to saline (white bars), DEP (striped bars), HDM (checked bars), or DEP plus HDM (black bars). (A) percentage of eosinophils (CD11clow, CD11b+, and Siglec-F+), (B) percentage of CD11b+ conventional DC (CD11chigh, low autofluorescent, CD11b+, and MHCII+), and (C) percentage of CD4+ T cells (CD3+, CD8, CD4+, and CD69+) in lung tissue were determined by flow cytometry. Representative flow cytometric density plots and gating strategy are shown in Supplemental Fig. 2. (D) Photomicrographs and quantification of Congo Red and (E) periodic acid–Schiff stained sections of the airways of mice that were exposed to saline, DEP, HDM, or DEP plus HDM. Representative photomicrographs from WT mice are shown. Scale bar representative for all images in the panel is shown in the lower left photomicrograph. Results are expressed as mean ± SEM. Lines with branches represent significant differences between DEP plus HDM versus saline, DEP alone, or HDM alone. n = 7–12 mice per group. *p < 0.05.

FIGURE 5.

Absence of chemR23 aggravates allergic responses in the lung upon combined DEP plus HDM exposure. WT and chemR23 KO mice were exposed to saline (white bars), DEP (striped bars), HDM (checked bars), or DEP plus HDM (black bars). (A) percentage of eosinophils (CD11clow, CD11b+, and Siglec-F+), (B) percentage of CD11b+ conventional DC (CD11chigh, low autofluorescent, CD11b+, and MHCII+), and (C) percentage of CD4+ T cells (CD3+, CD8, CD4+, and CD69+) in lung tissue were determined by flow cytometry. Representative flow cytometric density plots and gating strategy are shown in Supplemental Fig. 2. (D) Photomicrographs and quantification of Congo Red and (E) periodic acid–Schiff stained sections of the airways of mice that were exposed to saline, DEP, HDM, or DEP plus HDM. Representative photomicrographs from WT mice are shown. Scale bar representative for all images in the panel is shown in the lower left photomicrograph. Results are expressed as mean ± SEM. Lines with branches represent significant differences between DEP plus HDM versus saline, DEP alone, or HDM alone. n = 7–12 mice per group. *p < 0.05.

Close modal

Histologic analysis revealed peribronchial eosinophilic inflammation and increased goblet cells in WT mice that were exposed to combined DEP plus HDM. In chemR23 KO mice that received DEP plus HDM, this was further increased (Fig. 5D, 5E). Although low-dose HDM on its own had no significant effect on eosinophilia or goblet cell metaplasia in WT mice, numbers of peribronchial eosinophils and goblet cells were elevated in chemR23 KO that received sole HDM (Fig. 5D, 5E).

Absence of chemR23 increases type 2 cytokine production upon combined DEP plus HDM exposure.

To assess type 2 cytokine production in the model of DEP-enhanced allergic inflammation, mediastinal LNs were cultured with HDM and analyzed for cytokine production. LN cells from WT mice that were previously exposed to saline, sole DEP, or low-dose HDM showed no production of type 2 cytokines. However, combined exposure to DEP plus HDM was associated with increased production of IL-4, IL-5, and IL-13 in WT mice. Production of type 2 cytokines was amplified in chemR23 KO mice that received DEP plus HDM (Fig. 6A–C). Exposure to HDM alone was also associated with increased IL-5 and IL-13 levels in chemR23 KO mice in comparison with HDM-exposed WT mice (Fig. 6B, 6C).

FIGURE 6.

Absence of chemR23 increases type 2 cytokine production and Igs upon combined DEP and HDM exposure. WT and chemR23 KO mice were exposed to saline (white bars), DEP (striped bars), HDM (checked bars), or DEP plus HDM (black bars). (A) IL-4, (B) IL-5, and (C) IL-13 protein levels in the supernatant of HDM-restimulated LN cells were determined by ELISA. (D) Total IgE and (E) HDM-specific IgG1 in serum were determined by ELISA. Results are expressed as mean. Lines with branches represent significant differences between DEP + HDM versus saline, DEP alone or HDM alone. n = 7–12 mice per group. *p < 0.05.

FIGURE 6.

Absence of chemR23 increases type 2 cytokine production and Igs upon combined DEP and HDM exposure. WT and chemR23 KO mice were exposed to saline (white bars), DEP (striped bars), HDM (checked bars), or DEP plus HDM (black bars). (A) IL-4, (B) IL-5, and (C) IL-13 protein levels in the supernatant of HDM-restimulated LN cells were determined by ELISA. (D) Total IgE and (E) HDM-specific IgG1 in serum were determined by ELISA. Results are expressed as mean. Lines with branches represent significant differences between DEP + HDM versus saline, DEP alone or HDM alone. n = 7–12 mice per group. *p < 0.05.

Close modal

Total serum IgE and HDM-specific IgG1 were elevated in WT mice that received combined DEP plus HDM. The levels tended to increase in chemR23 KO mice that were exposed to DEP plus HDM (Fig. 6D, 6E).

In this study, we showed that the response to modulation of the chemerin/chemR23 axis is contingent on the specific conditions of DEP-induced airway inflammation. In the model of DEP-induced lung inflammation, chemR23 KO mice had decreased numbers of monocytes and DCs upon DEP-exposure compared with WT mice, suggesting a proinflammatory role for chemR23 signaling. In contrast, in the model of DEP-enhanced allergic airway inflammation, chemR23 KO mice had increased lung eosinophilia, goblet cell metaplasia, and TH2 cytokine production in response to DEP plus HDM compared with WT mice, suggesting an anti-inflammatory role for the chemR23 axis.

In the lung, epithelial cells are the first to encounter inhaled particles and allergens. In response to immunostimulatory Ags, the epithelium can release cytokines and chemokines that direct the recruitment and activation of innate and adaptive immune cells (20). Upon exposure to DEP, we observed decreased (pro)chemerin protein staining in the airway epithelium, which was associated with an increased (pro)chemerin release in the BALF. Similar observations were previously made in our model of cigarette smoke–induced inflammation (15), suggesting that exposure to pollutants can trigger the epithelium to release (pro)chemerin in the alveolar lumen. In addition, we demonstrated a synergistic response between DEP and HDM to induce (pro)chemerin secretion in the BALF.

After proteolytic activation, chemerin attracts chemR23-expressing immune cells, including monocytes and monocyte-derived DCs (10). In chemR23 KO mice, the accumulation of pulmonary monocytes and monocyte-derived DCs was decreased in the model of DEP-induced lung inflammation, suggesting a proinflammatory role for chemR23 signaling, which is similar to previous observations reported by our group in a model of cigarette smoke–induced inflammation (15).

Monocyte-derived DCs are known to stimulate TH2 immunity in response to inhaled allergens (6, 21). Because we observed decreased monocyte and monocyte-derived DC numbers in chemR23 KO mice that were exposed to DEP alone, we were interested in the role of chemR23 signaling in a model of allergic inflammation enhanced by DEP. Intriguingly, we found an increased allergic airway inflammation in chemR23 KO mice that were exposed to concomitant DEP plus HDM, suggesting an anti-inflammatory role of the chemR23 axis in DEP-enhanced allergic airway inflammation. Despite the modest response toward HDM alone in our model, chemR23 KO mice were also more susceptible to (exclusively) HDM-induced allergic airway inflammation, with enhanced eosinophilia and TH2 cytokine production compared with WT mice. Interestingly, in independent models of HDM- or OVA-induced airway inflammation, exogenously administered chemerin was reported to be associated with an anti-inflammatory response (18, 22) suggesting that chemerin mediates its anti-inflammatory properties in models of allergic airway inflammation by signaling via chemR23. The chemerin/chemR23 axis was also found to mediate anti-inflammatory responses in models of viral pneumonia and LPS-induced lung inflammation (16, 17).

Although a direct effect of active chemerin on monocytes has been described (10), it is of interest that chemR23 KO mice had impaired levels of CCL2 and CCL20. We previously showed that monocyte and monocyte-derived DC recruitment in response to DEP is attenuated in mice deficient for CCR2 and CCR6, which are the receptors for CCL2 and CCL20, respectively (6). Possibly, the reduced monocytic response in DEP-exposed chemR23 KO mice is therefore a consequence of the reduced levels of CCL2 and CCL20, rather than representing a direct role for the chemR23 axis in monocyte recruitment. Moreover, the reduced CCL2 and CCL20 levels in the chemR23 KO mice could suggest that chemR23 signaling is required for the upregulation in CCL2 and CCL20 in response to DEP. The fact that chemR23 KO mice receiving concomitant DEP plus HDM also had decreased CCL2 levels compared with WT mice supports the suggestion that chemR23 signaling modulates the production of CCL2. Furthermore, it suggests that the enhanced allergic inflammation and monocyte recruitment that is elicited in the lungs of chemR23 KO mice by DEP plus HDM is independent of CCL2, which is in contrast to the findings of a previous report (18).

The underlying basis of these opposing activities of chemerin/chemR23 signaling remains unclear (23). Various proteases can process prochemerin to bioactive chemerin. Thus, depending on the proteases that are present in the microenvironment, diverse chemerin fragments may be produced with distinct pharmacological properties. Serine proteases (i.e., neutrophil elastase and cathepsin G) that are released upon neutrophil degranulation generate chemerin-157 and chemerin-156 (i.e., prochemerin lacking the last six and seven amino acids, respectively) that are associated with the recruitment of chemR23-expressing Ag presenting cells (10, 11). Because exposure to DEP and cigarette smoke is associated with neutrophil recruitment to the lung and increased expression of neutrophil-derived proteases (4, 15), one can speculate that inhalation of air pollutants promotes a microenvironment that generates proinflammatory peptides of chemerin. On the other hand, mast cell chymase can convert these chemerin-157 and -156 peptides into chemerin-154, which is associated with anti-inflammatory actions on chemR23 (12, 13). Thus, chemerin may be cleaved into diverse anti-inflammatory peptides in response to allergic airway inflammation wherein the activation of mast cells is a prominent feature. A limitation of our study is that we could not distinguish between prochemerin and the bioactive isoforms of chemerin in the two models. To our knowledge, there are no commercially available Abs that efficiently discriminate between prochemerin or the various chemerin isoforms. Nevertheless, such tools are necessary to obtain more insights into the complex biology of chemerin signaling, because prochemerin and the multiple chemerin isoforms can act as antagonists for each other’s activity, and they can all compete for chemR23 binding and ultimate biological response (24). We have attempted to measure chemerin bioactivity in our samples using an aequorin-based calcium assay (17, data not shown). Unfortunately, the chemerin levels were too low to detect any bioactivity, yet this does not exclude that local lung chemerin levels contribute to leukocyte recruitment.

Recently, chemR23 expression was also demonstrated on endothelial cells (25). To explain the opposing roles of the chemerin/chemR23 axis, it was alternatively suggested that chemerin exerted its anti-inflammatory properties via chemR23 that was expressed on nonleukocytic (i.e., endothelial or epithelial) cells (17, 18). In support of this suggestion, we observed that the proinflammatory activity of chemR23 signaling in the model of DEP-induced acute lung inflammation was restricted to hematopoietic cells that were reported to express chemR23 (i.e., monocytes and DCs) (10), although no effects were seen on non-chemR23–expressing cells, such as neutrophils. In contrast, in the model of DEP-enhanced allergic airway inflammation, we observed anti-inflammatory activity for both chemR23 expressing and nonexpressing cells. Although one could suggest that these anti-inflammatory actions could be mediated through release of chemokines by the endothelium or epithelium, experiments with chimeric mice or conditional chemR23 KO should be performed to definitively resolve this issue.

RvE1 is a second putative ligand of chemR23 (14). In models of experimental asthma, exogenous administration of RvE1 could both impair the development and promote the resolution of OVA-induced airway inflammation (2628). One can therefore speculate that the opposing role of chemR23 signaling in our experiments was due to preferential binding of chemR23 to chemerin (in the model of acute DEP-induced inflammation) or RvE1 (in the model of DEP-enhanced allergic airway inflammation). Although a confounding role for RvE1 cannot be excluded, the decreased RvE1 levels in both models suggest that the proinflammatory and anti-inflammatory properties of the chemR23 axis in the models are not attributable to RvE1. Indeed, (exogenously administered) chemerin on its own is reported to have both proinflammatory and anti-inflammatory activities (16, 18, 29).

Given the anti-inflammatory role of the chemerin/chemR23 axis in diverse lung disease models, including models of allergic airway inflammation, chemerin or chemR23 agonists are proposed as novel candidate therapeutics for treatment of asthma (18, 22). On the other hand, blocking chemR23 is also suggested as therapeutic intervention in disease models in which the chemR23 axis has proinflammatory properties (15). Our data highlight the complexity of chemerin/chemR23 signaling and the opposing activities depending on the inflammatory conditions. To consider the chemerin/chemR23 axis as a therapeutic target for lung diseases, further research is needed into the mechanisms mediating the proinflammatory versus anti-inflammatory roles of chemR23.

We thank Greet Barbier, Indra De Borle, Ann Neesen, Katleen De Saedeleer, Anouck Goethals, Marie-Rose Mouton, and Lien Coelembier for technical assistance, and Dr. Parmentier (Université Libre de Bruxelles, Brussels, Belgium) for help with the aequorin-based calcium assay to detect the chemoattractant activity of chemerin.

This work was supported by the Interuniversity Attraction Poles Programme/Belgian Federal Science Policy P7/30 and the Fund for Scientific Research–Flanders (FWO-Vlaanderen). S.P. is a postdoctoral researcher for FWO-Vlaanderen.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BALF

bronchoalveolar lavage fluid

DC

dendritic cell

DEP

diesel exhaust particle

HDM

house dust mite

KO

knockout

LN

lymph node

Pbm

basement membrane perimeter

RvE1

resolvin E1

WT

wild type.

1
Guarnieri
M.
,
Balmes
J. R.
.
2014
.
Outdoor air pollution and asthma.
Lancet
383
:
1581
1592
.
2
Maes
T.
,
Provoost
S.
,
Lanckacker
E. A.
,
Cataldo
D. D.
,
Vanoirbeek
J. A.
,
Nemery
B.
,
Tournoy
K. G.
,
Joos
G. F.
.
2010
.
Mouse models to unravel the role of inhaled pollutants on allergic sensitization and airway inflammation.
Respir. Res.
11
:
7
.
3
Diaz-Sanchez
D.
,
Garcia
M. P.
,
Wang
M.
,
Jyrala
M.
,
Saxon
A.
.
1999
.
Nasal challenge with diesel exhaust particles can induce sensitization to a neoallergen in the human mucosa.
J. Allergy Clin. Immunol.
104
:
1183
1188
.
4
Provoost
S.
,
Maes
T.
,
Pauwels
N. S.
,
Vanden Berghe
T.
,
Vandenabeele
P.
,
Lambrecht
B. N.
,
Joos
G. F.
,
Tournoy
K. G.
.
2011
.
NLRP3/caspase-1-independent IL-1beta production mediates diesel exhaust particle-induced pulmonary inflammation.
J. Immunol.
187
:
3331
3337
.
5
Provoost
S.
,
Maes
T.
,
Willart
M. A.
,
Joos
G. F.
,
Lambrecht
B. N.
,
Tournoy
K. G.
.
2010
.
Diesel exhaust particles stimulate adaptive immunity by acting on pulmonary dendritic cells.
J. Immunol.
184
:
426
432
.
6
Provoost
S.
,
Maes
T.
,
Joos
G. F.
,
Tournoy
K. G.
.
2012
.
Monocyte-derived dendritic cell recruitment and allergic T(H)2 responses after exposure to diesel particles are CCR2 dependent.
J. Allergy Clin. Immunol.
129
:
483
491
.
7
Zabel
B. A.
,
Allen
S. J.
,
Kulig
P.
,
Allen
J. A.
,
Cichy
J.
,
Handel
T. M.
,
Butcher
E. C.
.
2005
.
Chemerin activation by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades.
J. Biol. Chem.
280
:
34661
34666
.
8
Vermi
W.
,
Riboldi
E.
,
Wittamer
V.
,
Gentili
F.
,
Luini
W.
,
Marrelli
S.
,
Vecchi
A.
,
Franssen
J. D.
,
Communi
D.
,
Massardi
L.
, et al
.
2005
.
Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin.
J. Exp. Med.
201
:
509
515
.
9
Parolini
S.
,
Santoro
A.
,
Marcenaro
E.
,
Luini
W.
,
Massardi
L.
,
Facchetti
F.
,
Communi
D.
,
Parmentier
M.
,
Majorana
A.
,
Sironi
M.
, et al
.
2007
.
The role of chemerin in the colocalization of NK and dendritic cell subsets into inflamed tissues.
Blood
109
:
3625
3632
.
10
Wittamer
V.
,
Franssen
J. D.
,
Vulcano
M.
,
Mirjolet
J. F.
,
Le Poul
E.
,
Migeotte
I.
,
Brézillon
S.
,
Tyldesley
R.
,
Blanpain
C.
,
Detheux
M.
, et al
.
2003
.
Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids.
J. Exp. Med.
198
:
977
985
.
11
Wittamer
V.
,
Bondue
B.
,
Guillabert
A.
,
Vassart
G.
,
Parmentier
M.
,
Communi
D.
.
2005
.
Neutrophil-mediated maturation of chemerin: a link between innate and adaptive immunity.
J. Immunol.
175
:
487
493
.
12
Guillabert
A.
,
Wittamer
V.
,
Bondue
B.
,
Godot
V.
,
Imbault
V.
,
Parmentier
M.
,
Communi
D.
.
2008
.
Role of neutrophil proteinase 3 and mast cell chymase in chemerin proteolytic regulation.
J. Leukoc. Biol.
84
:
1530
1538
.
13
Cash
J. L.
,
Hart
R.
,
Russ
A.
,
Dixon
J. P.
,
Colledge
W. H.
,
Doran
J.
,
Hendrick
A. G.
,
Carlton
M. B.
,
Greaves
D. R.
.
2008
.
Synthetic chemerin-derived peptides suppress inflammation through ChemR23.
J. Exp. Med.
205
:
767
775
.
14
Arita
M.
,
Bianchini
F.
,
Aliberti
J.
,
Sher
A.
,
Chiang
N.
,
Hong
S.
,
Yang
R.
,
Petasis
N. A.
,
Serhan
C. N.
.
2005
.
Stereochemical assignment, antiinflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1.
J. Exp. Med.
201
:
713
722
.
15
Demoor
T.
,
Bracke
K. R.
,
Dupont
L. L.
,
Plantinga
M.
,
Bondue
B.
,
Roy
M. O.
,
Lannoy
V.
,
Lambrecht
B. N.
,
Brusselle
G. G.
,
Joos
G. F.
.
2011
.
The role of ChemR23 in the induction and resolution of cigarette smoke-induced inflammation.
J. Immunol.
186
:
5457
5467
.
16
Luangsay
S.
,
Wittamer
V.
,
Bondue
B.
,
De Henau
O.
,
Rouger
L.
,
Brait
M.
,
Franssen
J. D.
,
de Nadai
P.
,
Huaux
F.
,
Parmentier
M.
.
2009
.
Mouse ChemR23 is expressed in dendritic cell subsets and macrophages, and mediates an anti-inflammatory activity of chemerin in a lung disease model.
J. Immunol.
183
:
6489
6499
.
17
Bondue
B.
,
Vosters
O.
,
de Nadai
P.
,
Glineur
S.
,
De Henau
O.
,
Luangsay
S.
,
Van Gool
F.
,
Communi
D.
,
De Vuyst
P.
,
Desmecht
D.
,
Parmentier
M.
.
2011
.
ChemR23 dampens lung inflammation and enhances anti-viral immunity in a mouse model of acute viral pneumonia.
PLoS Pathog.
7
:
e1002358
.
doi:10.1371/journal.ppat.1002358
18
Zhao
L.
,
Yang
W.
,
Yang
X.
,
Lin
Y.
,
Lv
J.
,
Dou
X.
,
Luo
Q.
,
Dong
J.
,
Chen
Z.
,
Chu
Y.
,
He
R.
.
2014
.
Chemerin suppresses murine allergic asthma by inhibiting CCL2 production and subsequent airway recruitment of inflammatory dendritic cells.
Allergy
69
:
763
774
.
19
Bai
A.
,
Eidelman
D. H.
,
Hogg
J. C.
,
James
A. L.
,
Lambert
R. K.
,
Ludwig
M. S.
,
Martin
J.
,
McDonald
D. M.
,
Mitzner
W. A.
,
Okazawa
M.
, et al
.
1994
.
Proposed nomenclature for quantifying subdivisions of the bronchial wall.
J. Appl. Physiol. (1985).
77
:
1011
1014
.
20
Lambrecht
B. N.
,
Hammad
H.
.
2013
.
Asthma: the importance of dysregulated barrier immunity.
Eur. J. Immunol.
43
:
3125
3137
.
21
Plantinga
M.
,
Guilliams
M.
,
Vanheerswynghels
M.
,
Deswarte
K.
,
Branco-Madeira
F.
,
Toussaint
W.
,
Vanhoutte
L.
,
Neyt
K.
,
Killeen
N.
,
Malissen
B.
, et al
.
2013
.
Conventional and monocyte-derived CD11b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen.
Immunity
38
:
322
335
.
22
Doyle
J. R.
,
Krishnaji
S. T.
,
Zhu
G.
,
Xu
Z. Z.
,
Heller
D.
,
Ji
R. R.
,
Levy
B. D.
,
Kumar
K.
,
Kopin
A. S.
.
2014
.
Development of a membrane-anchored chemerin receptor agonist as a novel modulator of allergic airway inflammation and neuropathic pain.
J. Biol. Chem.
289
:
13385
13396
.
23
Bondue
B.
,
Wittamer
V.
,
Parmentier
M.
.
2011
.
Chemerin and its receptors in leukocyte trafficking, inflammation and metabolism.
Cytokine Growth Factor Rev.
22
:
331
338
.
24
Rourke
J. L.
,
Dranse
H. J.
,
Sinal
C. J.
.
2013
.
Towards an integrative approach to understanding the role of chemerin in human health and disease.
Obes. Rev.
14
:
245
262
.
25
Kaur
J.
,
Adya
R.
,
Tan
B. K.
,
Chen
J.
,
Randeva
H. S.
.
2010
.
Identification of chemerin receptor (ChemR23) in human endothelial cells: chemerin-induced endothelial angiogenesis.
Biochem. Biophys. Res. Commun.
391
:
1762
1768
.
26
Haworth
O.
,
Cernadas
M.
,
Yang
R.
,
Serhan
C. N.
,
Levy
B. D.
.
2008
.
Resolvin E1 regulates interleukin 23, interferon-gamma and lipoxin A4 to promote the resolution of allergic airway inflammation.
Nat. Immunol.
9
:
873
879
.
27
Aoki
H.
,
Hisada
T.
,
Ishizuka
T.
,
Utsugi
M.
,
Kawata
T.
,
Shimizu
Y.
,
Okajima
F.
,
Dobashi
K.
,
Mori
M.
.
2008
.
Resolvin E1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma.
Biochem. Biophys. Res. Commun.
367
:
509
515
.
28
Flesher
R. P.
,
Herbert
C.
,
Kumar
R. K.
.
2014
.
Resolvin E1 promotes resolution of inflammation in a mouse model of an acute exacerbation of allergic asthma.
Clin. Sci.
126
:
805
814
.
29
Lin
Y.
,
Yang
X.
,
Yue
W.
,
Xu
X.
,
Li
B.
,
Zou
L.
,
He
R.
.
2014
.
Chemerin aggravates DSS-induced colitis by suppressing M2 macrophage polarization.
Cell. Mol. Immunol.
11
:
355
366
.

The authors have no financial conflicts of interest.

Supplementary data