Aryl Hydrocarbon Receptor Protects Lungs from Cockroach Allergen Induced Inflammation by Modulating Mesenchymal Stem Cells

Abstract

Exposure to cockroach allergen leads to allergic sensitization and increased risk of developing asthma. Aryl hydrocarbon receptor (AhR), a receptor for many common environmental contaminants, can sense not only environmental pollutants but also microbial insults. Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the capacity to modulate immune responses. In this study, we investigated whether AhR can sense cockroach allergens and modulate allergen-induced lung inflammation through MSCs. We found that cockroach allergen treated AhR-deficient (AhR^−/−) mice showed exacerbation of lung inflammation when compared to wild-type (WT) mice. In contrast, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an AhR agonist, significantly suppressed allergen-induced mouse lung inflammation. MSCs were significantly reduced in cockroach allergen challenged AhR^−/− mice as compared to WT mice, but increased in cockroach allergen-challenged WT mice when treated with TCDD. Moreover, MSCs express AhR and AhR signaling can be activated by cockroach allergen with increased expression of its downstream genes, cyp1a1 and cyp1b1. Furthermore, we tracked the migration of intravenously injected GFP⁺ MSCs and found that cockroach allergen-challenged AhR^−/− mice displayed less migration of MSCs to the lungs compared to WT. The AhR mediated MSC migration was further verified by an in vitro Transwell migration assay. Epithelial conditioned medium (ECM) prepared from CRE-challenged epithelial cells significantly induced MSC migrations, which was further enhanced by TCDD. The administration of MSCs significantly attenuated cockroach allergen-induced inflammation, which was abolished by TGFβ1 neutralizing antibody. These results suggest that AhR plays an important role in protecting lungs from allergen-induced inflammation by modulating MSC recruitment and their immune-suppressive activity.

INTRODUCTION

Asthma is the most prevalent chronic illness in children, and is a source of increasing clinical and public health concern. Among asthmatic patients, most children and roughly 50% of adults have allergic asthma. Exposure to allergen early in life increases the risk of developing asthma, and cockroach allergen sensitization is a particularly strong risk factor for the development of allergic asthma and asthma morbidity (1, 2). Furthermore, it has been suggested that exposure to indoor pollutants can cause asthma exacerbations and increases the risk of developing asthma (3–8). One intriguing and plausible hypothesis is that the exacerbation of allergy and asthma could be attributable to these environmental pollutants, which often co-exist with allergens. Evidence supporting this hypothesis comes from the observation that diesel exhaust particulates (DEP) exposure has been shown to be positively associated with asthma susceptibility and exacerbation (9–13). For example, prenatal exposure to cockroach allergen was associated with a greater risk of allergic sensitization and this association was greater for children exposed to higher levels of PAHs (12). Furthermore, co-exposure to DEP and house dust mite together can exacerbate allergic sensitization and induce key characteristics of a more severe asthma (9, 13). Together, these findings suggest that exposure to allergens can induce allergic sensitization leading to asthma, which can be further exacerbated by the presence of environmental pollutants. However, the underlying molecular mechanism remains unknown.

Aryl hydrocarbon receptor (AhR) is a multifunctional regulator that senses and responds to environmental stimuli and has been shown to play a role in normal cell development and immune regulation (14). Environmental pollutants such as DEP, polycyclic aromatic hydrocarbon (PAH), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can activate AhR signaling leading to changes in AhR targeted gene transcription (e.g., cytochrome P450 cyp1a1, cyp1b1) and a variety of immunotoxicological effects (15, 16). AhR signaling was found to be critically involved in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD) (17–20) and is increasingly considered as an attractive therapeutic target. Recent discoveries have provided evidence for a previously unidentified pathophysiological function of AhR in that bacterial compounds can act as potential AhR ligands (21, 22). The recognition of these compounds by AhR has been shown to contribute to host defense against invading microbial pathogens. Therefore, AhR not only elicits protection against environmental toxic molecules, but also serves as a sensor for invading pathogenic microbes (AhR-microbiome), suggesting a complex and diverse repertoire of AhR functions. We thus postulated that AhR may either directly mediate environmental allergen-induced immune responses or serve as a link between environmental pollutants and cockroach allergen that could contribute to increased risk of developing allergic diseases. Indeed, findings from our studies demonstrated that AhR was significantly expressed in the airways of asthmatic patients compared to healthy individuals, and that AhR expressed in human lung fibroblasts modulated cockroach allergen-induced cell differentiation and TGFβ1 secretion (23). Furthermore, recent discoveries have demonstrated that AhR controls Th9 (24) and type 1 regulatory T cell (Tr1) (25) differentiation, influences the balance of Tregs and Th17 cells (26, 27), and impacts on γδ+ T cells homeostasis (28), intraepithelial lymphocytes (29), lymphoid follicles (30), dendritic cell function (31), and mast cell differentiation and growth (32, 33). Taken together, these studies suggest that AhR may play a critical role in the development of allergic and inflammatory diseases. However, the regulatory role of AhR in allergen-induced allergic inflammation and underlying mechanisms remain elusive.

MSCs are multipotent progenitor cells with the ability to self-renew and differentiate into multiple cell types (34, 35). In addition to their stem/progenitor properties, MSCs can sense and control inflammation by moderating the immune response of various immunocytes such as T cells (36, 37), B cells (38), dendritic cells (DCs) (39), alveolar epithelial cells (40), and alveolar macrophages (41). MSCs can suppress lung inflammation (42) and participate in tissue repair and remodeling by differentiating into a number of mature cell types (43–48). Especially, MSCs can control inflammation by balancing the polarization of macrophages toward classically activated (M1, pro-inflammatory) and alternatively activated (M2, anti-inflammatory) macrophages (49, 50). This suggests that understanding the action of MSCs in the airway of allergic asthma could lead to MSC-targeted airway inflammation and remodeling interventions. It is postulated that MSCs can engraft into the lungs after exposure to allergens and play a role in suppressing airway inflammation and repair by differentiating into epithelial cells (mesenchymal-epithelial transition, MET) in early stages, and contribute to progressive fibrosis and pathological remodeling in late stages of asthma. We have recently demonstrated an increased recruitment of MSCs to the airway after exposure to cockroach allergen in our established mouse model of asthma (51) and that MSCs significantly inhibited cockroach allergen-induced inflammatory cytokine secretion.

In this study, we demonstrated, for the first time, that AhR deficiency led to exacerbation of lung inflammation when exposed to cockroach allergen in our well-established asthma mouse model. In contrast, lung inflammation was significantly inhibited when AhR agonist, TCDD, was administered in the mouse model. Moreover, we demonstrated that cockroach allergens can directly induce activation of AhR signaling in bone marrow-derived MSCs, and AhR regulates MSC migration in both in vivo mouse models with administration of GFP⁺ MSCs intravenously and in vitro Transwell migration assay. Furthermore, we observed that the administration of MSCs significantly attenuated allergic inflammation. Our findings suggest that AhR protects lungs from cockroach allergen-induced inflammation though MSCs, and therefore is a potential target for the treatment of allergic diseases such as asthma.

METHODS

Mice

C57BL/6J mice, AhR knock out (AhR^−/−) mice, nestin-GFP-transgenic mice (52), and C57BL/6J-GFP mice (53) were used in this study. All mice were used at 6–9 wks of age, and all experiments used age- and gender-matched controls. All animals were maintained under specific pathogen–free conditions in the animal facility of the Johns Hopkins University School of Medicine. The experimental protocols were reviewed and approved by the Animal Care and Use Committee at Johns Hopkins University School of Medicine.

Cockroach allergen-induced asthma mouse model

Mice were sensitized by intratracheal inhalation of 20 µg cockroach extract (CRE, B46, GREER Laboratories)/mouse in 50µl PBS on days 0–4 and challenged with the same amount of CRE for 4 successive days (days 10 to 13). Control mice received the same volume of PBS. On day 14, mice were sacrificed, bronchoalveolar lavage (BAL) fluid and serum were collected and lungs were harvested for the proposed studies. In some experiments, mice were treated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (10 µg/kg) dissolved in olive oil by oral gavage at indicated time, or injected via the tail vein with purified and cultured bone-marrow-derived MSCs (1×10⁶) in 200 µl PBS with or without IgG1 or TGFβ1 neutralizing antibody (Ab) on day 9 before the stage of CRE challenge.

Analysis of lung inflammation

Detailed methods were described previously (51). Mouse lung were perfused with 20 ml ice-cold PBS injected into right ventricle followed by excision and fixation in 10% neutral buffered formalin. Sections (5µM) were prepared from paraffin-embedded lungs and stained with hematoxylin/ eosin (H&E) and periodic acid-schiff. Images were obtained using a NIKON ECLIPSE Ti-U microscope equipped with DS-Fi2 camera (NIKON, USA). For analysis of BALF, mouse lungs were perfused with 0.8 ml ice-cold Hank’s balanced salt solution twice, and lavage fluids was collected and centrifuged at 1,500 r.p.m. at 4°C for 10 min. Red blood cells in the pellet were lysed by Ammonium-Chloride-Potassium (AcK) lysis buffer, and cellular differential percentage was determined by means of flow cytometry on a FACS Calibur cytometer (BD Biosystems) (51). Lymphocytes were identified as FSC^low/SSC^low and expressing CD3 or CD19. Granulocytes were recognized as SSC^high Gr-1⁺cells; Eosinophils were defined as SSC^high SiglecF⁺ Mac-3⁻ cells. Alveolar macrophages cells were identified as SSC^high SiglecF⁺ Mac-3⁺ cells.

ELISA

Collected BALs were assessed by ELISA for IL4, IL6, IL10, IL13, IL17A, IFNγ, IL22, and TGFβ1 (eBiosciences) according to the manufacturer’s instructions. Results were read with a Bio-Rad Bio-Plex instrument (Bio-Rad Laboratories, Hercules, CA). Serum cockroach allergen-specific IgE and IgG1 were analyzed by ELISA according to our previous published works (51).

RNA isolation and quantitative real-time PCR analysis

For quantitative real-time PCR analysis (qRT-PCR), RNA from MSCs or single cell suspensions made from lungs of mice was isolated with an RNeasy Plus Mini kit (Qiagen, Valencia, CA), and cDNA templets were synthesized with SuperScript III (Life Technologies). QRT-PCR was performed using SYBR^®Green (AB Applied Biosystems, Foster city, CA) in an ABI Prism 7300 detection system. Data were analyzed using the 2^−ΔΔCT method as described by Livak and Schmittgen (54). The fold change in expression of the target gene relative to the internal control gene (β-actin) was calculated. Primers sequences are available upon request.

Isolation of MSCs

MSCs were isolated from bone marrow of C57BL/6J mice (51). In brief, the isolated cells were cultured with DMEM and 20% fetal bovine serum (FBS, Atlanta Biologicals) at 37°C in a 5% CO2 humidified incubator. After 72 hours, non-adherent cells were removed and adherent cells were cultured for additional 14 days. The adherent cells were retrieved by 0.25% trypsin digestion containing 0.02% ethylenediaminetetraacetic and sorted by markers Sca-1⁺ (D7), CD29⁺ (HMb 1-1), and CD45⁻ (30-F11) from eBioscience, and CD11b⁻ (M1/70) from BD. The sorted cells were enriched by further culture. Using the similar protocol, we isolated GFP⁺ MSCs from C57BL/6J-GFP mice.

Immunofluorescence

For immunofluorescent staining, non-specific binding was blocked using 10% blocking serum in PBS for 1 hour, and the tissue samples were then incubated with primary antibodies to AhR (ab84833, Abcam), nestin (clone 10C2, Abcam), and GFP (clone 4B10, Cell Signaling Technology Inc.) overnight at 4°C. Secondary antibodies conjugated with fluorescence were added, and slides were incubated at room temperature for 1 h. Isotype-matched negative control antibodies (R&D Systems) were used under the same conditions. Nuclei were counterstained with 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Sigma). The sections were mounted with the ProLong Gold Anti-fade Kit (Molecular Probes, Grand Island, NY) and observed by a NIKON ECLIPSE Ti-U microscope equipped with DS-Fi2 camera (NIKON, USA).

Western blotting

Cells were washed twice with ice cold PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Sigma). Protein content was measured with BCA reagent (Pierce). Equivalent protein samples were subjected to SDS-PAGE electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore). After blocking with 5% nonfat dry milk in TBST, the membrane was incubated with AhR primary Abs (Enzo life Science) and visualized with an HRP-conjugated secondary Ab and ECL reagents (Amersham).

In vitro transwell assay

To prepare the conditioned medium (ECM), TC-1 cells, a mouse lung epithelial cell line (55), were cultured with serum-free Dulbecco’s modified Eagle’s medium (DMEM) and treated with cockroach extract (50 µg/ml) with or without TCDD (1.0 nM) or CH122319 (50 nM) for 72 hours; the cultured medium was collected and stored at −80°C. Cell migration was performed in 96-well Transwells (Corning, Inc., Acton, MA). A total of 2×10⁴ BM-MSCs in 50 µl serum-free DMEM were placed in the pre-coated upper chambers with 0.5µg/ml type I collagen (BD Biosciences, San Diego, CA), and 150 µl undiluted ECM was added to the type I collagen coated lower chamber of the transwell. After incubation for 10 hours, cells that migrated to the lower side of the filter were fixed with 10% formaldehyde, stained with hematoxylin (Sigma-Aldrich, St. Louis, MO), and counted using the automated counting of single color image using ImageJ v1.49 (http://imagej.nih.gov/ij/).

In vivo MSC migration by GFP⁺ MSC injection

To examine the MSC migration from blood to the lung, sorted GFP⁺ MSCs (1×10⁶) from C57BL/6J-GFP mice were injected into cockroach allergen sensitized C57BL/6J mice and AhR^−/− mice through tail vein one day before challenge (day 9). After three days of consecutive CRE challenge, mice were sacrificed on day 14, and lung tissues were harvested for the analysis of GFP⁺ cells by immunofluorescent staining. GFP⁺ cells in the lungs were counted using ImageJ v1.49 as above.

Flow cytometric analysis

For the analysis of Tregs and MSCs in lung lymph nodes, the pulmonary hilar lymph nodes were collected and teased apart into a single cell suspension by pressing with the plunger of a 3 ml syringe. Harvested lung tissues were minced and incubated for 45 min at 37°C in DMEM supplemented with 10ug/ml DNase I and 1 mg/ml collagenase D (Sigma Aldrich) in a shaking water bath. After that, the digested lung tissue was passed through a 70-um nylon strainer (BD Biosciences) to obtain single cell suspensions. Red blood cells were lysed by ACK lysis buffer. For Tregs, the cells were first stained with anti-CD4-FITC (RM4–5, eBioscience) and anti-CD25-PE antibodies (PC61.5, eBioscience), followed by intracellular staining with FoxP3-APC (FJK-16s, eBioscience) or APC-conjugated rat IgG2a isotype control (eBioscience). For MSCs, cells were stained with Sca-1, CD29, CD45, and CD11b fluorochrome conjugated antibodies (eBioscience). These samples were analyzed on a FACSCalibur flow cytometer (BD Biosystems).

Statistical analysis

Data are expressed as the means ± SEM for each group. Statistical significance for normally distributed samples was assessed using an independent two-tailed Student’s t-test or with analysis of Variance by using GraphPad Prism version 5.1 software (GraphPad Software, La Jolla, CA). Differences with P< 0.05 were considered statistically significant.

RESULTS

Deletion of AhR exacerbated cockroach allergen-induced lung inflammation

We have previously demonstrated that AhR plays a role in mediated cockroach allergen-induced immune responses (23). To further examine whether AhR plays a role in mediating cockroach allergen induced allergic inflammation, we have created mouse models of asthma using wild type and AhR^−/− mice (Fig. 1A). Compared with PBS-treated control mice, asthma pathologies were developed in CRE-challenged wild type (WT) and AhR^−/− mice as indicated by significant recruitment of inflammatory cells to the lungs, dense peribronchial infiltrates, and prominent mucus production and goblet hyperplasia (Fig. 1B). When compared with CRE-treated WT mice, CRE-treated AhR^−/− mice displayed exacerbation of peribronchial inflammation and goblet cell hyperplasia. Furthermore, these CRE-treated AhR^−/− mice displayed significantly increased numbers of total inflammatory cells, especially with eosinophils and neutrophils among all analyzed cell types in BAL fluids (Fig. 1C). These sensitized and challenged AhR^−/− mice with CRE also produced higher serum titers of cockroach specific IgE and IgG1 (Fig. 1D). We next examined the levels of Th1/Th2/Th17 and inflammatory cytokines in the mouse BAL fluids (Fig. 1E). As expected, levels of IL-4, IL-13, IL-17, IL-6, IFNγ, and TGFβ1 were significantly increased in CRE-challenged WT and AhR^−/− mice as compared to PBS-treated mice. Interestingly, the levels of IL-4, IL-13, IL-17, and IL-6 were increased, but IFNγ and TGFβ1, were decreased in CRE-treated AhR^−/− mice as compared to WT mice. Given that AhR has been shown to control Treg cell generation (27) that is capable of suppressing inflammation in vivo (56), we specifically examined the percentage of CD4⁺CD25⁺Foxp3⁺ Tregs in the hilar lymph nodes and lung tissues of CRE-treated AhR^−/− and WT mice (Fig. 2A). As shown in Fig. 2B, Tregs were significantly reduced in AhR^−/− mice compared with WT mice. Previously, we documented an increase in the numbers of MSCs in the lungs of our cockroach allergen–induced mouse model of asthma (51). We, therefore, examined the percentage of MSCs (CD45⁻CD11b⁻CD29⁺Sca-1⁺, Fig. 2C) in CRE-treated AhR^−/− and WT mice. Interestingly, MSCs were significantly lower in BAL fluids and lung tissues of CRE-treated AhR^−/− mice as compared to WT mice (Fig. 2D). The reduced MSCs in CRE-treated AhR^−/− mice were further confirmed by co-immunofluorescence staining for both AhR and nestin (marker for MSC) (51, 57) (Fig. 2E). The AhR^−/− mice displayed lower number of nestin⁺ cells in the airway epithelial as compared to CRE-treated WT mice. These data suggest that AhR may protect against cockroach allergen-triggered airway inflammation by regulating Treg and MSC generation and/or migration.

Figure 1.

AhR deficient leads to a more severe allergic inflammation induced by cockroach allergen. (A) Experimental setup for cockroach allergen induced asthma model. Mice were sensitized with 20 µg CRE or saline on days 0 to 4, and challenged with same amount of CRE or PBS on days 10–13. Samples were harvested 24 h after the last challenge. (B) Paraffin tissue sections of lung were stained with H&E (upper panel) and periodic acid-Schiff (PAS, lower panel). Original magnification, 20×. (C) Bronchoalveolar lavage (BAL) differential cell counts of PBS and CRE-challenged WT and AhR^−/− mice were determined by flow cytometry. (D) Serum levels of cockroach allergen specific IgE and IgG1. (E) Levels of cytokines in BALs. Data are representative of three independent experiments (n=4–6 mice/group). Error bars indicate SEM. *P<0.05, **P<0.01.

Figure 2.

Both Tregs and MSCs were diminished in cockroach allergen-treated AhR^−/− mice. (A) Tregs in lung lymphocytes were determined by flow cytometric analysis gating on CD4⁺CD25⁺Foxp3⁺ cells. (B) Percentage of CD4⁺CD25⁺Foxp3⁺cells in lymphonodes and lungs among all analyzed cells. (C) MSCs in lung tissues were determined by flow cytometric analysis gating CD45⁻CD11b⁻CD29⁺Sca-1⁺. (D) Percentage of CD45⁻CD11b⁻CD29⁺Sca-1⁺ cells in BALs and lung tissues among all analyzed cells. (E) Co-localization of AhR and nestin in the airways of cockroach allergen-challenged WT and AhR^−/− mice. Lung sections were stained with anti-AhR (green) and anti-nestin (red) and merged for co-localization (yellow) analysis. Cellular nucleus was stained with DAPI (blue). Original magnification, 40×. Data are representative of three independent experiments (n=4–6 mice/group). Error bars indicate SEM. *P<0.05, **P<0.01.

TCDD attenuated cockroach allergen-induced lung inflammation

To further confirm the role of AhR in regulating cockroach allergen-induced lung inflammation, an AhR specific agonist TCDD was administered into asthmatic mice one day prior to cockroach allergen sensitization and challenge (Fig. 3A). We first examined whether TCDD treatment could activate AhR and its downstream genes, cyp1a1, and cyp1b1, in the lungs of CRE-treated mice with or without TCDD. The levels of AhR, cyp1a1 and cyp1b1 were significantly increased in lung tissues from TCDD-treated as compared to untreated CRE-challenged mice (Fig. 3B), suggesting the activation of AhR signaling in TCDD-treated CRE challenged mice. We next examined the role of TCDD in CRE-induced lung inflammation. As shown in Fig. 3C, CRE-triggered peribronchial inflammation and goblet cell hyperplasia was significantly decreased in lungs of CRE-treated mice with TCDD). TCDD alone did not induce lung inflammation. The same was also true for olive oil alone that was used to dissolve TCDD (27) (Data not shown). Furthermore, BAL fluids from TCDD-treated CRE-challenged mice displayed significantly reduced numbers of total inflammatory cells (Fig. 3D), with a most profound reduction in eosinophils but increase in neutrophils. Serum from those mice had significantly lower levels of CRE-specific IgE and IgG1 (Fig. 3E). Similarly, BAL fluids from TCDD-treated CRE-challenged mice had lower levels of IL-4, IL-13, IL-17A, but increased IFN-γ, IL-22, and TGFβ1 (Fig. 3F). Importantly, both Treg cells (Fig. 3G) and MSCs (Fig. 3H) in TCDD treated CRE-challenged mice were significantly increased compared to those untreated CRE-challenged mice. The increased MSCs were further confirmed by immunofluorescence staining for nestin (Fig. 2E). These data suggest that TCDD exposure suppresses allergic immune responses to cockroach allergen in mice, providing additional evidence for the involvement of AhR in mediating allergen-induced allergic inflammation.

Figure 3.

TCDD suppressed cockroach allergen induced lung inflammation. (A) Experimental setup for cockroach allergen induced asthma model. TCDD (10µg/kg) dissolved in olive oil was used to treat mice one day prior to sensitization on day 9. Samples were harvested 24 h after the last challenge. (B) Real-time analyses of the expression of AhR, cyp1a1 and cyp1b1 in lung tissues. Results are expressed as fold change in expression of target genes relative to the internal control gene (β-actin). (C) Paraffin tissue sections of lung were stained with H&E (upper panel) and periodic acid-Schiff (PAS, lower panel). Original magnification, 20×. (D) BAL differential cell counts of CRE-challenged WT mice with or without TCDD treatment or TCDD alone were determined by flow cytometric analysis. (E) Serum levels of cockroach allergen specific IgE and IgG1. (F) Levels of cytokines in BALs. (G) Percentage of CD4⁺CD25⁺Foxp3⁺ cells in lungs among all analyzed cells. (H) Percentage of CD45⁻CD11b⁻CD29⁺Sca-1⁺ cells in lungs among all analyzed cells. (I) Expression of nestin in the airways of cockroach allergen-challenged WT with or without TCDD treatment. Lung sections were stained with anti-nestin (red) and cellular nucleus was stained with DAPI (blue). Original magnification, 40×. Data are representative of two independent experiments (n=4–6 mice/group). Error bars indicate SEM. *P<0.05, **P<0.01.

Cockroach allergens activate AhR signaling in MSCs

Given that MSCs may participate in regulating AhR-mediated allergen induced inflammation, we tested whether AhR signaling can be activated in MSCs after exposure to cockroach allergens. MSCs isolated and cultured from mouse bone marrow (Fig. 4A) were treated with CRE (50 µg/ml), LPS (0.185ng/ml, a mount that is present in CRE), and purified natural Bla g 2 (5µg/ml), and the expression of AhR in MSCs was determined by RT-PCR and Western blot. Similar to LPS, which has been shown to activate AhR signaling (22), cockroach allergens (CRE and Bla g 2) were able to promote AhR expression in MSCs as compared to medium control (Fig. 4B), which was further confirmed by western blot analysis (Fig. 4C) and immunofluorescence staining (Fig. 4D). We next examined whether AhR signaling in MSCs can be directly activated by cockroach allergens. Two AhR downstream genes, cyp1a1 (Fig. 4E) and cyp1b1 (Fig. 4F), were analyzed in MSCs after exposure to CRE by RT-PCR. Compared to un-treated MSCs, significant increase in the expression of both cyp1a1 and cyp1b1 were observed when MSCs were exposed to CRE, LPS, or Bla g 2. However, the increased expression was not seen for MSCs isolated from AhR^−/− mice. Taken together, these data indicate that cockroach allergen contains AhR ligands that directly induce the activation of AhR signaling in MSCs.

Figure 4.

Bone marrow-derived MSCs express AhR and can be activated by cockroach allergens. (A) MSCs were isolated from mouse bone marrow, cultured and identified by flow cytometric analysis gating CD45⁻CD11b⁻CD29⁺Sca-1⁺. (B) Real-time PCR analysis of AhR expression in MSCs. Results are expressed as fold change in expression of AhR relative to the internal control β-actin. Data are expressed as mean ± SEM of two independent experiments. (C) Western blot analysis of AhR expression in cell lysates from MSCs treated with the same conditions in (B) (upper figure). Results were expressed as relative levels of AhR to the expression of total actin (lower figure). Data represent results from two independent experiments. (D) AhR expression in MSCs after treatment with CRE. Cultured and treated MSCs were stained intracellularly with anti-AhR (green). Nucleus was stained by DAPI (blue). Original magnification, 20×. Figures represent results from two independent experiments. (E–F) Real-time analyses of cyp1a1 (E) and cyp1b1 (F) expression in MSCs from WT and AhR^−/− mice. Results are expressed as fold change over medium in expression of target genes to the internal control β-actin. Data are expressed as mean ± SEM of two independent experiments. *P<0.05, **P<0.01.

AhR regulates MSCs migration

To test whether AhR can regulate cockroach allergen induced MSC migration to the lung tissue, we tracked MSC migration in the mice with GFP⁺ MSCs intravenously injection in our mouse model of asthma as previously described (51). After mice were injected with 1×10⁶ GFP⁺ MSCs though the tail vein on day 9 prior to CRE challenge (Fig. 5A), significantly increased GFP⁺ MSCs were detected in the lungs of CRE-challenged WT-mice compared to PBS-treated controls. Notably, the migrated GFP⁺ MSCs were significantly decreased in the lungs of CRE-challenged AhR^−/− mice (Fig. 5B and C). To explore the underlying mechanisms, we collected epithelium-conditioned medium (ECM) from cultured mouse lung epithelial cells (TC-1) stimulated with medium, CRE, CRE with TCDD or CH122319 (AhR antagonist), TCDD, and CH122319, respectively, for 72 hours. MSC migration assay was performed in which ECM was placed in the bottom chamber and MSCs were placed in the upper chamber of a Transwell. Significantly greater numbers of migrated MSCs were noted in the group that received CRE-challenged ECM relative to unchallenged group. TCDD significantly promoted CRE-challenged ECM induced MSC migration (Fig. 5D and E). In contrast, we saw a non-statistically significant reduction in the migration of MSCs for the CRE with CH122319-challenged ECM. No significant MSC migration was observed for either TCDD or CH122319-challenged ECM alone.

Figure 5.

AhR regulates MSCs migration in mouse asthma model and an in vitro transwell assay. (A) Experimental setup for cockroach allergen induced asthma model. GFP⁺ MSCs (1×10⁶) were administered intravenously on day 9. (B) Immunofluorescence analysis of injected GFP⁺ MSCs in the airways of CRE-challenged or saline-treated mouse models with WT and AhR^−/− mice. Original magnification, 20×. (C) The numbers of injected GFP⁺ MSCs were counted using ImageJ v1.49. Data represent results from two independent experiments with 4–6 mice/group. (D) Transwell assays for MSCs migration induced by the conditioned medium (ECM) from TC-1 treated with CRE alone or with TCDD or CH122319. Migrated cell to the lower side of the filter were fixed with 10% formaldehyde, stained with hematoxylin. (E) Number of migrated MSCs was carried out using the automated counting of single color image using ImageJ v1.49. (F) Levels of active TGFβ1 in ECM that was used for Transwell assay were measured by ELISA. Data in (E) and (F) are expressed as mean ± SEM from two independent experiments. *P<0.05, **P<0.01.

Previously, we demonstrated that TGFβ1 is a primary factor to recruit MSCs to the airways in mouse models of asthma (51). We, therefore, postulate that TGFβ1 may also a mediator in AhR-induced MSC migration in the current model system. Indeed, significantly higher levels of active TGFβ1 were detected in ECMs prepared from CRE-challenged TC-1 cells compared with those from the resting cells. Furthermore, the concentrations of TGFβ1 in the ECMs were further increased in the group that received both CRE and TCDD (Fig. 5F). While no statistically significant was observed, there was a reduction in active TGFβ1 level in CRE-challenged ECM with CH122319. Taken together, these findings suggest that AhR activation is crucial in controlling cockroach allergen induced MSC migration into the lung tissues, and this process may be mediated through the control of TGFβ1 release.

MSCs attenuated cockroach allergen-induced lung inflammation through TGFβ1

MSCs have been shown to ameliorate allergic airway inflammation in mouse models of asthma (58–60). Thus, it is likely that the exacerbated inflammation in AhR^−/− mice may be due to the insufficient number of MSCs present in the lungs. To test this hypothesis, AhR^−/− mice were administered MSCs intravenously before CRE challenge (Fig. 6A). Significantly diminished peribronchial inflammation and goblet cell hyperplasia were found compared to AhR^−/− mice without MSC treatment (data not shown). Moreover MSCs administration significantly reduced the BAL total cell count (Fig. 6B), and the numbers of airway eosinophils and neutrophils (Fig. 6C). Furthermore, MSCs administration significantly suppressed the production of IL-4, IL-13, and IL-17 with increased levels of IFN-γ in the BAL fluids (Fig. 6D). To test the role of TGFβ1 in this event, TGFβ1 neutralizing Ab or control Ab (IgG1) was administrated on day 9 prior to allergen challenge (Fig. 6A). The attenuated lung inflammation caused by MSCs, including the total cell count in BAL fluids (Fig. 6B), airway eosinophils and neutrophils (Fig. 6C), and cytokine secretion (Fig. 6D), was significantly reversed. These observations suggest that MSCs attenuate AhR-mediated lung inflammation, and TGFβ1 may be a critical factor for MSC immune suppression.

Figure 6.

MSCs attenuated cockroach allergen-induced exacerbation of lung inflammation through TGFβ1. (A) Schematic of experimental protocol for mouse models of asthma. MSCs (1×106, i.v), TGFβ1 neutralizing antibody (Ab, i.p), and IgG1 (i.p) were administered on day 9. (B) Total cells in BALs and (C) Differential cell counts in BALs collected from CRE-challenged AhR^−/− mice and CRE-challenged AhR^−/− mice with MSCs+IgG1 or MSCs+TGFβ1 Ab. (D) Levels of cytokine in BALs were analyzed by ELISA. Data are expressed as mean ± SEM from two independent experiments (n=4–6 mice/group). **P<0.01.

DISCUSSION

Recent increasing evidence to suggest that AhR is involved in disease tolerance and can also serve as a sensor of bacterial compounds, which contain AhR ligands, playing a protective role against invading microbial pathogens (21, 22). Thus, the focus of the current research has been shifted from the role of AhR in the xenobiotic pathway towards its mode of action in response to physiological ligands with different signaling cascades (61, 62). These novel rationales prompted us to consider the notion that cockroach allergens may contain AhR ligands, which can directly activate AhR signaling and contribute to allergen-induced inflammation. In this study, we specifically focused on the role of AhR in mediating cockroach allergen-induced lung inflammation.

Previously, our group was the first to demonstrate that AhR signaling is activated by cockroach allergen in fibroblasts and plays a crucial role in modulating cockroach allergen-induced immune responses through controlling active TGFβ1 release (23). In this study, we provided evidence that active AhR signaling may have beneficial effects in cockroach allergen-induced lung inflammation. We found that, compared to wild-type mice, cockroach allergen treated AhR^−/− mice showed exacerbation of lung inflammation, including significant recruitment of eosinophils and neutrophils, prominent mucus production, increased cockroach allergen specific IgE and IgG1, and higher levels of Th2/Th17 cytokines IL4, IL13, and IL17, with decreasing Th1 cytokine IFN-γ in the BAL. Of interest, TGFβ1, which we have shown to be able to mobilize MSCs, was significantly reduced in cockroach allergen challenged AhR^−/− mice. To further confirm these findings, the AhR agonist TCDD was utilized to treat mice before cockroach allergen challenge. As expected, TCDD treatment led to an attenuated lung inflammation with decreased Th2/Th17 cytokines IL4, IL13, and IL17, but increased Th1 cytokines IFN-γ, and TGFβ1 in BAL. IL22 has been suggested to be crucial in a protective effect of AhR signaling on inflammation (63); we have measured IL22 and showed that it was increased in TCDD treated mice. These results supported an important role for TCDD in suppressing lung inflammation and were consistent with several previous studies demonstrating a suppression of allergic airway inflammation in mouse asthma models by AhR ligands (64–67). Of these, FICZ administration reduced the pulmonary eosinophilia that normally follows OVA challenge in sensitized mice and coincidentally reduced expression levels of the Th2 cytokines IL4 and IL5 in lung tissue (65). Studies in AhR^−/− mice demonstrated that exposure to cigarette smoke enhanced neutrophil influx and secretion of inflammatory cytokine IL6, prostaglandin E2, and increased tissue damage (68). Similar effects for AhR on skin diseases were also found. Skin inflammation was stronger in AhR^−/− mice compared with their wild-type littermates in an imiquimod mouse model of psoriasis, and activation of AhR by the administration of FICZ before imiquimod treatment remarkably reduced skin inflammation (69). Our findings reveal that AhR has a protective effect against cockroach allergen-induced inflammation. However, AhR has been suggested to have the janus-faced role in regulating immunological responses with either ameliorating or conversely aggravating inflammatory diseases (27, 61, 70), depending on the nature of the ligands, participating cell types, inflammatory status, and diseases. Thus, further investigation is needed into the role of AhR in regulating allergic inflammation in asthma.

Several major cell types that are involved in innate and adaptive immunity have been linked to AhR mediated inflammatory processes, including epithelial cells (71, 72), fibroblasts (73), macrophages (74), dendritic cells(DCs) (31), eosinophils (65), and mast cells (32). For instance, macrophages express AhR and can be activated by TCDD to produce modulatory chemokines or release pro-inflammatory cytokines (69). Activated AhR signaling in DCs promotes expression of immune-suppressive enzyme IDO that may contribute to the immunosuppression (31). Our previous studies demonstrated that AhR deficient mast cells respond poorly to stimulation owing to defective calcium signaling and mitochondrial function, thus, suggesting a critical role for AhR in mast cells at barrier organs like lung and skin (32). Additionally, AhR has been linked to controlling differentiation of Tregs that were critical in immunosuppression (27, 75). In our study, Tregs were decreased in CRE-challenged AhR^−/− mice, but increased in CRE-treated mice after TCDD treatment, suggesting Tregs may play a role in AhR mediated lung inflammation. It is also worth noting that AhR regulates immunity through modulation of Th17 cells (26) and innate lymphoid cells (76).

In this study, we reported a novel function of AhR in regulating allergen-induced lung inflammation through controlling MSC recruitment and function. MSCs have a remarkable capacity to modulate immune responses and change the progression of different inflammatory diseases through the release of a variety of bioactive molecules (77–79). Recent studies have suggested that MSCs ameliorate allergic airway inflammation in mouse models of asthma (58–60). This study illustrated that MSCs were decreased in CRE-challenged AhR^−/− mice with exacerbated lung inflammation, but increased in TCDD-treated CRE-challenged mice with ameliorated lung inflammation. Furthermore, the exacerbated lung inflammation in CRE-challenged AhR^−/− mice was significantly decreased after the administration of MSCs intravenously. Taken together, these results suggest that MSCs may be crucial in AhR-mediated allergen-induced lung inflammation. However, we recognized that the fold-change in MSCs does not correspond to the changes in lung inflammation in our mouse models, but these migrated MSCs may amplify the innate and adaptive immune responses through interaction with epithelial cells, DCs, macrophages and/or other cells by producing immune-regulatory factors, and subsequently modulate the progression of inflammation.

To determine the biologic link between AhR and MSCs, we first examined the activation of AhR signaling in cockroach allergen-challenged MSCs. We found that AhR signaling in MSCs can be activated by cockroach allergens (CRE, Bla g 2) as determined by an increased expression of AhR and its downstream genes, cyp1a1 and cypab1. As suggested, LPS contaminants in CRE may induce AhR activation (80–82), therefore, we measured the level of LPS within CRE and examined LPS-induced AhR activation using same amount of LPS reported. Furthermore, we also performed experiments using LPS-removed CRE (Data not shown). Results from the above-described experiments suggest that, in addition to LPS, cockroach allergens may contain AhR ligands, which activate AhR signaling in MSCs.

Next, we investigated whether AhR regulates the recruitment of MSCs to the lungs. Indeed, there were significantly reduced MSCs in BAL fluids as well as lung tissues of CRE-treated AhR^−/− mice as compared to WT mice. Moreover, less recruitment of GFP⁺ MSC were seen in the lungs of AhR^−/− mice as compared to WT mice when GFP⁺ MSCs were injected via tail veil in the mouse asthma model. Furthermore, our ex vivo study using the ALI-ECM-based cell migration system demonstrated that AhR agonist TCDD can potentiate CRE-induced MSC migration. Taken together, these data suggest that AhR may be crucial in controlling MSC migration. Future work is to explore the detailed mechanisms underlying AhR controlling MSC migration.

Our previous study (51) and the results from the present work suggest that TGFβ1 may be a critical factor mediating AhR-regulated MSC migration. We hypothesized that TGFβ1 released from damaged/repairing epithelium in response to allergens recruits MSCs in early stages, whereas in later stages, CRE can penetrate damaged epithelial cells and activate AhR signaling in MSCs, thus modulating MSC function at the site of injury. Consistent with this hypothesis, we found that TGFβ1 was increased in mice after CRE challenge, but decreased in AhR^−/− mice as compared to WT mice. Moreover, TGFβ1 was increased in ECM from CRE-challenged epithelial cells, which was further potentiated by TCDD. Interestingly, it seems there was a strong correlation between levels of TGFβ1 in ECMs and degrees of ECM-induced MSC migration. Furthermore, TGFβ1 neutralizing antibody reversed, at least in part, the inflammation suppression by the administration of MSCs. We hypothesized that TGFβ1 may regulate either AhR-mediated MSC migration or inflammatory mediator release from MSCs or both. It was suggested that there is a cross-talk between AhR and TGF-β1 signaling in various experimental models (23, 83–85), but the molecular mechanism is still poorly understood. Furthermore, other factors, including those released from CRE-challenged epithelium, may also be involved in MSC migration and MSC immune suppression.

Taken together, our studies have demonstrated that AhR may have a protective effect against cockroach allergen-induced inflammation. MSCs express AhR and can be activated by cockroach allergens. Furthermore, AhR regulates MSC recruitment to the lung tissues, and TGFβ1 may be crucial in the AhR-regulated MSC migration and their immune-suppressive activity. Further investigations will focus on identifying the major components in cockroach extract that trigger the activation of AhR signaling, the cross-talk between AhR and TGFβ1 signaling, and mechanisms regarding the role of activated AhR in controlling MSC function (e.g., release of modulatory chemokines or proinflammatory cytokines). Findings from these studies will provide basis for further investigation into the role of AhR signaling in mediating co-exposure of environmental chemicals with allergen induced exacerbation of allergic sensitization and asthma.

Abbreviations

AhR

Aryl hydrocarbon receptor

MSC

Mesenchymal stem cell

TGFβ-1

Transforming growth factor β-1

ECM

Epithelial conditioned medium

CRE

Cockroach extract

TCDD

2,3,7,8-Tetrachlorodibenzo-p-dioxin

BAL

Bronchoalveolar lavage

Treg

Regulatory T cell

DEP

Diesel exhaust particulate