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Am J Physiol Lung Cell Mol Physiol 290: L1154-L1163, 2006. First published January 6, 2006; doi:10.1152/ajplung.00318.2005
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Diesel exhaust enhances virus- and poly(I:C)-induced Toll-like receptor 3 expression and signaling in respiratory epithelial cells

Jonathan Ciencewicki,1 Luisa Brighton,2 Wei-Dong Wu,2,3 Michael Madden,1,4 and Ilona Jaspers1,2,3

1Curriculum of Toxicology, 2Center for Environmental Medicine, Asthma, and Lung Biology, 3Department of Pediatrics, and 4US EPA-Human Studies Division, University of North Carolina, Chapel Hill, North Carolina

Submitted 19 July 2005 ; accepted in final form 4 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Prior exposure of respiratory epithelial cells to an aqueous-trapped solution of diesel exhaust (DEas) enhances the susceptibility to influenza infections. Here, we examined the effect of DEas on the Toll-like receptor 3 (TLR3) pathway, which is responsible for the recognition of and response to viruses and double-stranded RNA. Flow cytometric and confocal microscopy analyses showed that TLR3 is predominantly expressed in the cytoplasm of respiratory epithelial cells. To examine the effect of DE on TLR3 expression and function, differentiated human bronchial or nasal epithelial cells as well as A549 cells were exposed to DEas and then infected with influenza A or treated with polyriboinosinic acid-polyribocytidylic acid [poly(I:C)], a synthetic form of double-stranded RNA. Exposure to DEas before infection with influenza or stimulation with poly(I:C) significantly upregulated the expression of TLR3. Additionally, preexposure to DEas significantly increased the poly(I:C)-induced expression of IL-6. Overexpression of a dominant-negative mutant form of TNF receptor-associated factor 6 reversed the effects of DEas on poly(I:C)-induced IL-6 expression, suggesting that the response was TLR3 dependent. Similarly, preexposure to DEas significantly increased nuclear levels of interferon regulatory factor 3 and the expression of IFN-beta in response to poly(I:C). Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, was able to abate the effect of DEas on poly(I:C)-induced IFN-beta expression. Together, these results indicate that exposure of respiratory epithelial cells to DEas could potentially alter the response to viral infections by increasing the expression and function of TLR3.

epithelial cells; Toll-like receptors; in vitro; respiratory virus

ADVERSE HEALTH EFFECTS INDUCED by exposure to air pollution have become an increasing problem in many areas worldwide. Although many different emission sources account for the complex air pollution mixture in urban environments, diesel exhaust (DE) has raised a fair amount of concern because it can account for a significant percentage of air pollutants generated by motor vehicles in many places (9, 58). In addition, epidemiological data and experimental studies suggest that DE is a major contributor to adverse health effects associated with exposure to particulate air pollutants (59, 61). Because of their small mass median diameter (0.05–1.0 µm), DE particles are easily respirable and capable of being deposited in the lower airways and alveoli (8, 52). The carbonaceous core of these particles can absorb around 18,000 chemical compounds onto it, including polyaromatic hydrocarbons, which are suspected to play a major role in the adverse health effects induced by exposure to DE (62). DE particulates are also able to travel much greater distances than particles produced by gasoline engines (34). Additionally, it has been shown that exposure to DE and DE particulates increases neutrophil recruitment and pulmonary inflammation in both rodents and humans (23, 36, 38, 44–47, 63).

Furthermore, DE has also been implicated in having adverse effects on host immunity. It has been shown that DE increases cellular responsiveness to histamine (17, 24) and acts as an adjuvant to IgE production (10, 11, 32, 37, 57). In addition, epidemiological evidence has noted an association between ambient particulate matter and pulmonary infections (41), including exacerbation of respiratory symptoms associated with infection (7, 13, 39). Exposure to DE can also enhance the susceptibility to respiratory virus infections. Specifically, our previous studies have demonstrated that exposure to an aqueous-trapped solution of DE (DEas) enhances the susceptibility to influenza infections in human respiratory epithelial cells (19). Furthermore, mice repeatedly exposed to DE showed an increased susceptibility to infection with RSV, as shown by increased inflammatory responses and levels of viral titers after infection with RSV (16). These studies indicate that exposure to DE can increase the susceptibility and exacerbate responses to respiratory virus infections. However, the mechanism by which exposure to DE modulates the host response to respiratory virus infections is not yet clear.

The host's first line of defense against an invading pathogen is the innate immune response. It activates the secondary immune response and keeps the infection under control until the adaptive response is mobilized. An integral role in the innate immune response is played by Toll-like receptors (TLRs). These receptors are members of the superfamily of interleukin-1 receptors (IL-1R) and share homology in the cytoplasmic region referred to as the Toll/IL-1R domain. TLRs recognize conserved pathogen-associated molecular patterns (PAMPs); recognition leads to the production of innate immune defense mediators as well as activation of the adaptive immune response (1, 2, 18). Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), a molecular pattern commonly associated with viral infection. dsRNA stimulates TLR3 signaling, which culminates in the activation of numerous downstream signaling proteins and transcription factors and ultimately results in production of inflammatory cytokines and type I interferons (3, 31).

TLR3 mRNA has been found in human lung, liver, heart, placenta, pancreas, and brain (42). It is also expressed in dendritic cells, intestinal epithelial cells, and airway and respiratory epithelial cells but has not yet been found in monocytes, lymphocytes, T cells, or B cells (6, 14, 33, 50, 60). However, the localization of TLR3 in the cell is still subject to debate. Surface or cytoplasmic expression of TLR3 has been demonstrated in various cell types (14, 30, 31, 50, 53). The most likely explanation for these differences is that localization of TLR3 is cell type specific. Additionally, expression of TLR3 has been shown to be upregulated in dendritic cells as well as in bronchial and pulmonary epithelial cells by viral infection or dsRNA treatment in the form of polyriboinosinic acid-polyribocytidylic acid [poly(I:C)] (14, 30, 43, 53).

The respiratory epithelium is a common target for both DE and invading respiratory viruses. Because TLR3 plays such an integral role in the recognition and primary response to viral pathogens, we examined the effects that preexposure to DE has on TLR3 expression and signaling in respiratory epithelial cells. Specifically, we examined whether exposure to DEas enhances TLR3 expression and TLR3-dependent signaling in response to dsRNA or influenza virus infections in human respiratory epithelial cells. The results shown here demonstrate that exposure to DEas significantly increases TLR3 expression and TLR3-mediated innate immune responses in epithelial cells infected with influenza virus or treated with dsRNA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. A549 cells, a human pulmonary type II epithelium-like cell line, were cultured in F12K medium plus 10% FBS and 1% penicillin and streptomycin (all from Invitrogen, Carlsbad, CA). For treatment with DEas and stimulation with poly(I:C), A549 cells were grown in 6- or 24-well plates. When the cells reached ~80% confluence and ~18–24 h before exposure to DEas and stimulation with poly(I:C), the cell culture medium was exchanged for serum-free F12K plus 1.5 µg/ml BSA plus antibiotics. In some experiments, 1 µM wortmannin (Calbiochem, La Jolla, CA) was added 30 min before treatment with DEas. Primary human bronchial epithelial cells were obtained from healthy nonsmoking adult volunteers by cytologic brushing at bronchoscopy. Primary human nasal epithelial cells were obtained from healthy nonsmoking adult volunteers by gently stroking the inferior surface of the turbinate several times with a Rhino-Probe curette (Arlington Scientific, Arlington, TX), which was inserted through an otoscope with a large aperture. The protocols for the acquisition of both primary human bronchial and nasal epithelial cells were reviewed and approved by the University of North Carolina Institutional Review Board, and informed written consent was obtained from all subjects. Both primary human bronchial and nasal epithelial cells were expanded to passage 2 in bronchial epithelial growth medium (Cambrex Bioscience Walkersville, Walkersville, MD) and then plated on collagen-coated filter supports with a 0.4-µm pore size (Trans-CLR; Costar, Cambridge, MA) and cultured in a 1:1 mixture of bronchial epithelial cell basic medium and DMEM-H with SingleQuot supplements (Cambrex), bovine pituitary extracts (13 mg/ml), BSA (1.5 µg/ml), and nystatin (20 U). Upon confluence, all-trans-retinoic acid was added to the medium, and air liquid interface (ALI) culture conditions (removal of the apical medium) were created to promote differentiation. Mucociliary differentiation was achieved 18–21 days after the ALI step.

Generation of A549-TNF receptor-associated factor 6 dominant-negative cells. A549 cells stably transduced with dominant-negative TNF receptor-associated factor 6 (dnTRAF6) were prepared as follows. The sequence encoding a truncated form of TRAF6 was digested out of the plasmid pCIneo-dnTRAF6 (TRAF6289–530), kindly provided by Dr. R. Medhzitov's laboratory (Howard Hughes Medical Institute, Yale University Medical School, New Haven, CT) (5) using EcoRI and SalI restriction enzymes (New England Biolabs, Ipswich, MA) and ligated into EcoRI- and SalI-digested pBabe-puro, which was a kind gift from Dr. A. Baldwin (Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC). The pBabe-puro-dnTRAF6 retroviral vector was transfected into retroviral packaging PT67 cells, and stably transfected cells were selected using 1 µg/ml puromycin (Sigma). As a control, pBabe-puro alone was transfected into PT67 cells. Retroviral-laden supernatants were harvested periodically and stored at –80°C until use. Immediately before use, supernatants were filtered through 0.45-µm filters and supplemented with 8 µg/ml polybrene (Sigma). A549 cells grown to ~50% confluence were transduced using the retrovirus-containing supernatants, and stably transduced A549 cells were selected with 1 µg/ml puromycin for ~2 wk. Expression of dnTRAF6 in stably transduced A549 cells was confirmed by Western blotting.

Exposure to DEas. DEas was generated as described before (27). Briefly, emissions were taken from a Caterpillar diesel engine, model 3304, which was used to power a 113-kW generator. This type of engine was chosen because it is used in nonroad vehicles, which are significant contributors to ambient DE levels and because the projected trend for emissions from nonroad diesel engines is expected to remain at the same level or even increase in the future (59). The DE emissions from this Caterpillar diesel engine were passed through a tubing system with a filter impactor and two impinger tubes (containing 100 ml of PBS each) submerged in an ice bath. Impinger glassware was washed and heated to remove and destroy endotoxin. Of the two impinger tubes, the emissions (at 10 l/min) that entered and remained in the first (primary) tube, but not in the secondary tube, were utilized for the cell exposure studies. Extracts were generated and collected during a 1-h period when the engine was under high load. This type of preparation was chosen because it contains both DE and polar particles and thus water-soluble DE gas-phase components. To determine the mass of the emissions retained within the PBS in an impinger tube, an aliquot was dried overnight at 56°C and corrected for the mass of the PBS contribution (which was determined in a similar manner by overnight drying) and dilution with water from the exhaust. Aliquots of the DEas were kept at –20°C until use. For all cell types used in this study, DEas was added 2 h before infection with influenza or treatment with poly(I:C). Specifically, for the differentiated human nasal and bronchial epithelial cells, DEas was diluted in 200 µl of medium to achieve 22 or 44 µg DEas/cm2 of cell layer and added to the apical side. After the 2-h incubation with DEas, the diluted DEas was removed, and influenza virus or poly(I:C) was diluted in the same volume of medium, which was added to the apical side for 2 h. This dilution was then removed to establish ALI culture conditions again. For the experiments with A549 cells, DEas was diluted in F12K medium-BSA-antibiotics to achieve 25 µg/cm2 and added to the cells. After a 2-h incubation with DEas, poly(I:C) was added to the cells. The effects of exposure to DEas on cell viability were assessed by analyzing cell culture supernatants for lactate dehydrogenase activity using a commercially available kit according to the supplier's instructions (CytoTox 96; Promega, Madison, WI).

Infection with influenza or treatment with poly(I:C). Throughout this study, we used influenza A/Bangkok/1/79 (H3N2 serotype), which was propagated in 10-day-old embryonated hen's eggs. The virus was collected in the allantoic fluid and titered by 50% tissue culture infectious dose in Madin-Darby canine kidney cells and hemagglutination as described before (4). Stock virus was aliquoted and stored at –80°C until use. Unless otherwise indicated, for infection of differentiated bronchial or nasal cells, ~3 x 105 cells were infected with 320 hemagglutination units of influenza A Bangkok 1/79. Cells were treated with 100 µg/ml of poly(I:C) (Calbiochem) 2 h after exposure to DEas.

RT-PCR. Total RNA was extracted with TRIzol (Invitrogen) as per the supplier's instruction. First-strand cDNA synthesis and real-time RT-PCR were performed as described previously (20, 21). The sequences for the primers and probes used in this study are as following: for IFN-beta, 5'-FAM-AGCAGCAATTTTCAGTGTCAGAAGCTCCTG-TAMRA-3' (probe), 5'- CAACTTGCTTGGATTCCTACAAAG-3' (sense), and 5'-AGCCTCCCATTCAATTGCC-3' (antisense); for IL-6, 5'-FAM-TGTTACTCTTGTTACATGTCTCCTTTCTCAGGGCT-TAMRA-3' (probe), 5'-GGTACATCCTCGACGGCATCT-3' (sense), and 5'- GTCCCTCTTTGCTGCTTTCAC- 3' (antisense); for TLR3, 5'-FAM-ATGCAGTTCAACAAGCTATTGAACAAAATCTGGA-TAMRA-3' (probe), 5'- ATTAAAAGACCCATTATGCAAAAGATTC- 3' (sense), and 5'-CCTCAAGGAAAACCAATATAATGGA-3' (antisense); for GAPDH, 5'-JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA-3' (probe), 5'-GAAGGTGAAGGTCGGAGTC-3' (sense), and 5'-GAAGATGGTGATGGGATTTC-3' (antisense).

ELISA. Supernatants were analyzed for IL-6 using commercially available ELISA kits as per the supplier's instructions (BD Biosciences, San Diego, CA).

Western blotting. Whole cell lysates were prepared by lysing the cells in RIPA buffer containing 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and protease inhibitors (Cocktail Set III; Calbiochem). Nuclear extracts were prepared as described before (21). Whole cell lysate (20–100 µg) and nuclear extract (20–50 µg) were separated by SDS-PAGE as described before (21). This was followed by immunoblotting using specific antibodies to TLR3 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), interferon regulatory factor 3 (IRF3; 1:1,000; Santa Cruz Biotechnology), phospho-Akt (1:1,000; Cell Signaling, Beverly, MA), or Akt (1:1,000; Santa Cruz Biotechnology). beta-Actin (1:2,000; US Biological, Swampscott, MA) was used as a loading control for TLR3. Antigen-antibody complexes were stained with anti-rabbit or anti-mouse horseradish peroxidase-conjugated antibody (1:4,000; Santa Cruz Biotechnology) and SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). We acquired the chemiluminescent signals using a 16-bit charge-coupled device camera (GeneGnome system; Syngene, Frederick, MD) and visualized using the GeneSnap software (Syngene). Densitometric analysis of the optical densities was performed using GeneTools software (Syngene).

Immunohistochemistry/confocal microscopy. A549 cells were grown on chamber slides (Lab-Tek chamber slides; Nalge Nunc International, Naperville, IL). Cells were prepared as described previously (12) with slight modifications. Briefly, cells were fixed with 4% paraformaldehyde in PBS for 30 min and then permeabilized with 0.5% saponin in 1% BSA in PBS for 30 min. Samples were washed with PBS and then blocked in 1% BSA in PBS for 1 h. Cells were washed in PBS and then treated with anti-TLR3 MAb (TLR3.7) (1:10; Ebioscience, San Diego, CA) overnight at 4°C. A no primary antibody control was also done. Cells were washed in Tris-buffered saline (TBS) and then treated with goat anti-mouse Alexa 488 conjugated secondary antibody (5 µg/ml; Molecular Probes) in TBS-Triton X-100 for 1 h at room temperature. Cells were then washed in TBS and coverslipped with VectaShield with DAPI (Vector Labs, Burlingame, CA). Immunofluorescence was visualized by use of a Zeiss 510 laser scanning confocal microscope at the Michael Hooker Microscopy Core Facility at the University of North Carolina at Chapel Hill.

Flow cytometry. A549 or differentiated bronchial cells were detached by incubating with 0.53 mM EDTA-trypsin at 37°C for 10 min and washed HBSS. For the analysis of TLR3, the cell pellets were resuspended at 106 cells/ml. For intracellular staining of TLR3, cells were permeabilized and fixed by incubating them with Cytoperm/Cytofix (BD Bioscience) as per the supplier's instructions and then washed twice with staining solution containing 1.0% BSA and 0.02% sodium azide in PBS. This step was omitted for cells that were stained for surface expression of TLR3. Cells were then incubated with phycoerthrin (PE)-conjugated anti-TLR3 (TLR3.7) (1 µg/106 cells; Ebioscience) or PE-mouse IgG1 (1 µg/106 cells) for 30 min at 4°C and then washed twice with staining solution. Cells stained for surface expression of TLR3 were then fixed by incubating them with 1% paraformaldehyde for 20 min at 4°C. Data acquisition and analyses were performed on a flow cytometer (FACScan using the Cell Quest software; Becton Dickinson, Mountain View, CA). For each determination, 10,000 cells were examined.

Statistical analysis. Data are expressed as means ± SE of at least three separate experiments. Data from experiments involving primary human nasal or bronchial epithelial cells were analyzed using the Wilcoxon matched-pairs test. Data generated from experiments with A549 cells were analyzed using two-way ANOVA to determine whether there was a significant interaction between the exposure (DEas) and treatment [poly(I:C)], followed by the Tukey-Kramer honestly significant difference post hoc test for multigroup analysis, except for the data in Fig. 7, which were analyzed with the Wilcoxon signed rank test, assuming a theoretical mean of 1.00 for the control group. A value of P <0.05 was considered to be significant.


Figure 7
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  		Fig. 7. Effect of DEas on levels of activated Akt and its role in IFN-beta transcription. A: changes in mRNA were measured 24 h after stimulation with poly(I:C). A549 cells were treated with DMSO or 1 µM wortmannin for 30 min before treatment with 0 or 25 µg/cm2 of DEas. Two hours after treatment with DEas, cells were stimulated with 100 µg/ml poly(I:C). IFN-beta mRNA was quantified using real-time RT-PCR, and values were normalized to GAPDH mRNA. Values are expressed as fold induction over the respective media control. *Significantly different from control, P < 0.05. B: whole cell lysates prepared from A549 cells 1 h after treatment with 0 or 25 µg/cm2 of DEas or stimulation with poly(I:C) were analyzed for levels of phosphorylated Akt. Nitrocellulose membranes were stripped and reprobed using an anti-Akt antibody. Representative immunoblots for phosphorylated (top) and total (bottom) Akt.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
TLR3 expression and localization. The TLRs involved in recognition of nonviral PAMPs are anchored in the cell membrane, with the PAMP-recognizing portion on the extracellular side and the Toll/IL-1R domain on the cytoplasmic side (49). However, the exact cellular location of TLR3 in respiratory epithelial and other cell types is still controversial, with the most recent data indicating that TLR3 is located in cytoplasmic vesicles. Therefore, our first objective was to characterize the expression and localization of TLR3 in respiratory epithelial cells. Using a PE-conjugated anti-TLR3 antibody, we stained both the surface and cytoplasm for TLR3 in A549 cells and subsequently performed flow cytometric analysis to identify TLR3-stained cells. Permeabilization followed by fixation was used to analyze cytoplasmic expression of TLR3, whereas surface expression of TLR3 was assessed in cells that did not receive the permeabilizing treatment. Our results indicate that TLR3 is located predominantly in the cytoplasm with little or no surface expression evident (Fig. 1A). We also confirmed these studies in differentiated human bronchial epithelial cells and observed similar results (data not shown). To further identify cytoplasmic compartments in which TLR3 is localized, we performed indirect immunofluorescent staining of A549 cells for TLR3, followed by visualizing the fluorescent staining using confocal microscopy. Figure 1B indicates that the staining for TLR3 is distributed throughout the cytoplasm, without any staining concentrating along the cell membrane or the nucleus. In addition, the staining pattern seems granular, confirming previous observations of TLR3 being located in cytoplasmic vesicles (12, 30).


Figure 1
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  		Fig. 1. Localization of Toll-like receptor 3 (TLR3). A: surface and cytoplasmic staining for IgG and TLR3 was performed in A549 cells. Levels of TLR3 were measured using flow cytometry and compared with IgG for control. B: A549 cells were immunohistochemically stained for TLR3 (FITC), and nuclei were counterstained with DAPI. Immunofluorescence was visualized using confocal microscopy.

 
Effect of DEas on TLR3 expression. After identifying the localization of TLR3 in our cells, we examined whether the expression of TLR3 was affected in cells exposed to DEas before viral infection or treatment with dsRNA, which are both stimuli that have previously been shown to enhance TLR3 mRNA levels (14, 43, 50, 53). DE has previously been shown to exacerbate inflammatory mediator production in response to RSV in mice (16) and IFN-beta expression in response to influenza in respiratory epithelial cells (19), yet the role of TLR3 in these responses is unknown. Therefore, we wanted to determine whether exposure to DEas could enhance virus-induced expression of TLR3. To do this, we analyzed levels of TLR3 mRNA in differentiated human respiratory epithelial cells exposed to DEas and subsequently infected with influenza. Similar to previous results (53), Figure 2 shows that infection with influenza enhances TLR3 mRNA levels compared with uninfected control cells, although this effect was not statistically significant in the bronchial cells. As expected, exposure to DEas alone did not significantly enhance TLR3 mRNA levels in either differentiated human nasal or bronchial epithelial cells. However, when cells were exposed to DEas before infection with influenza, there was a significant enhancement of the influenza-induced upregulation of TLR3 mRNA levels in bronchial epithelial cells and an increase that was approaching statistical significance in nasal epithelial cells (P = 0.06).


Figure 2
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  		Fig. 2. Effect of aqueous-trapped solution of diesel exhaust (DEas) and influenza A on TLR3 mRNA levels. Changes in mRNA were measured 24 h after infection with influenza A. Differentiated nasal (A) or bronchial (B) epithelial cells were treated with 0 or 44 µg/cm2 of DEas for 2 h before infection with 320 hemagglutination units (HAU)/105 cells of influenza A. TLR3 mRNA was quantified using real-time RT-PCR. Values are normalized to GAPDH mRNA. *Significantly different from influenza-infected cells, and #significantly different from media control (P < 0.05).

 
We repeated these experiments with poly(I:C), a synthetic form of dsRNA, instead of the influenza virus, to induce TLR3 expression (Fig. 3) in A549 cells, a human respiratory epithelial cell line. Figure 3A shows that, although not significant, treatment with poly(I:C) alone causes a modest upregulation of TLR3 mRNA in A549 cells, whereas exposure to DEas alone had no observable effect on TLR3 mRNA levels in this cell type. However, when cells are preexposed to DEas before stimulation with poly(I:C), there is a significant interaction between the exposure and treatment, resulting in a significant upregulation of TLR3 mRNA levels above all other groups. To determine whether the enhanced TLR3 mRNA levels correspond to enhanced TLR3 protein expression, we analyzed TLR3 protein levels in whole cell lysates by Western blotting. Figure 3B shows that the upregulation of TLR3 seen at the mRNA level in cells exposed to DEas before stimulation with poly(I:C) was in fact translated into increased TLR3 protein expression in A549 cells. The densitometric readings that were taken (expressed as fold induction over the media control) show that exposure of cells to DEas before stimulation with dsRNA enhanced TLR3 expression. Together, these results demonstrate that exposure to DEas significantly enhances virus or dsRNA-induced TLR3 expression in different human respiratory epithelial cell types.


Figure 3
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  		Fig. 3. Effect of DEas and polyriboinosinic acid:polyribocytidylic acid [poly(I:C)] on TLR3 mRNA levels. Changes in mRNA were measured 24 h after stimulation with poly(I:C). A: A549 cells were treated with 0 or 25 µg/cm2 of DEas 2 h before stimulation with 100 µg/ml of poly(I:C). TLR3 mRNA was quantified using real-time RT-PCR. Values are normalized to GAPDH mRNA. *Significantly different from all other groups, P < 0.01. B: whole cell lysates prepared from A549 cells treated with 0 or 25 µg/cm2 of DEas before stimulation with 100 µg/ml poly(I:C) were analyzed for TLR3 protein levels. Nitrocellulose membranes were stripped and reprobed with anti-beta-actin antibody. Optical densitometry readings for TLR3 were normalized to beta-actin levels and shown as fold induction over the media control (numbers on top) to facilitate comparison.

 
DEas increases poly(I:C)-induced inflammatory mediator production. Stimulation of TLR3 by dsRNA or viral infection results in the activation of two separate signaling pathways, one culminating in the activation of interferon regulatory factors (IRFs) and the production of type I IFNs and the other one culminating in the activation of NF-{kappa}B and the production of proinflammatory mediators. Production of inflammatory mediators is a common response of respiratory epithelial cells to viral infections and can be exacerbated by a number of environmental pollutants (16, 51). After observing an upregulation of TLR3 expression caused by prior exposure to DEas, we determined whether exposure to DEas also enhanced TLR3-mediated proinflammatory mediator production by analyzing the expression of IL-6 in cells exposed to DEas and stimulated with poly(I:C). Using real-time RT-PCR, we analyzed IL-6 mRNA levels 24 h after exposure to DEas and stimulation with poly(I:C) in A549 cells. Figure 4A shows that levels of IL-6 mRNA were elevated, although not significantly, in response to treatment with poly(I:C) alone. Exposure to DEas alone had no effect on IL-6 mRNA levels. When cells were exposed to DEas before treatment with poly(I:C), IL-6 mRNA levels were significantly increased above the medium control despite the fact that there was not a significant interaction between exposure and treatment. To validate the IL-6 mRNA levels, we also measured IL-6 protein levels in cell culture supernatants and observed a similar overall effect (Fig. 4B). Specifically, there was a significant interaction between exposure and treatment, which resulted in a significant increase in secretion of IL-6 above all other groups in cells exposed to DEas before treatment with poly(I:C). These findings, along with our mRNA data, show that exposure to DEas increases the TLR3-mediated inflammatory response.


Figure 4
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  		Fig. 4. Effect of DEas on IL-6 levels. Changes in IL-6 mRNA (A) and protein (B) levels were measured 24 h after stimulation with poly(I:C). A549 cells were treated with 0 or 25 µg/cm2 of DEas 2 h before stimulation with 100 µg/ml of poly(I:C). IL-6 mRNA was quantified using real-time RT-PCR, and values were normalized to GAPDH mRNA. IL-6 protein levels were measured in cellular supernatants by ELISA. *Significantly different from all groups, P < 0.01. #Significantly different from media control, P < 0.05.

 
To ensure that the effect of DEas on poly(I:C)-induced IL-6 expression was mediated by TLR3 and not by other TLR3-independent pathways, such as dsRNA-activated protein kinase-dependent activation of NF-{kappa}B and production of inflammatory cytokines (40), we examined IL-6 production in cells overexpressing mutant dnTRAF6 lacking the NH2-terminal zinc binding structures (5). Although several other signaling pathways converge at TRAF6 to subsequently activate NF-{kappa}B and enhance inflammatory cytokine production, TRAF6 is also one of the main signaling proteins involved in the TLR3-mediated inflammatory response (22). With the use of retroviral expression vectors, A549 cells were stably transduced with the control vector or a vector expressing a truncated form of TRAF6 (5). Both cell lines were exposed to DEas and stimulated with poly(I:C), and IL-6 mRNA levels were quantified 24 h after stimulation with poly(I:C) by real time RT-PCR. Figure 5 shows that cells transduced with a control vector responded similarly to those shown in Fig. 4A, whereby IL-6 mRNA levels were significantly increased above control in cells exposed to DEas before treatment with poly(I:C) despite the absence of a significant interaction effect. However, when cells expressing the dominant-negative form of TRAF6 were exposed to DEas before treatment with poly(I:C), there was not a significant upregulation of IL-6 mRNA levels, as seen in cells expressing the control vector. Additionally, the IL-6 levels observed in the dnTRAF6 cells treated with DEas + poly(I:C) were significantly decreased compared with control cells treated in the same manner. Interestingly, IL-6 expression was not completely abrogated in cells expressing dnTRAF6, which could be mediated by the endogenously expressed wild-type form of TRAF6 or TRAF6-independent pathways. Nevertheless, these results show that the effects of DEas on poly(I:C)-induced IL-6 production were largely dependent on TRAF6, an integral component of the TLR3 signaling pathway.


Figure 5
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  		Fig. 5. Effect of dominant-negative TNF-associated factor 6 (dnTRAF6) on DEas-induced effects on IL-6 levels. A549 cells were stably transduced with a control vector or a truncated form of TRAF6 (dnTRAF6) and treated with 0 or 25 µg/cm2 of DEas 2 h before stimulation with 100 µg/ml of poly(I:C). Changes in IL-6 mRNA levels were measured 24 h after stimulation with poly(I:C) using real-time RT-PCR. Values were normalized to GAPDH mRNA. #Significantly different from media control, P < 0.001. @Significantly different from dnTRAF6-DEas + poly(I:C) group, P < 0.05.

 
DEas enhances the poly(I:C)-induced IFN-beta expression. After elucidating the effects of DEas on poly(I:C)-induced IL-6 expression, we turned our attention to the IFN branch of the TLR3 pathway. For this part of the study, we examined how exposure to DEas affects IFN-beta expression after stimulation with poly(I:C). IFN-beta is a type I interferon with anti-viral properties, which is produced early on during viral infections (25, 55). We used the same exposure and treatment regimen as before and then quantified levels of IFN-beta mRNA 24 h after treatment with poly(I:C) using real-time RT-PCR (Fig. 6A). No significant increase in mRNA levels was seen in cells exposed to DEas or poly(I:C) alone. However, there was a significant interaction between exposure and treatment, which resulted in a significant increase in IFN-beta mRNA levels in cells exposed to DEas before treatment with poly(I:C), indicating that TLR3-mediated IFN-beta production is also enhanced by exposure to DEas.


Figure 6
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  		Fig. 6. Effect of DEas on IFN-beta transcription. A549 cells were treated with 0 or 25 µg/cm2 of DEas 2 h before stimulation with 100 µg/ml poly(I:C). A: changes in IFN-beta mRNA levels were measured 24 h after stimulation with poly(I:C). IFN-beta mRNA was quantified using real-time RT-PCR, and values were normalized to GAPDH mRNA. *Significantly different from all other groups, P < 0.05. B: nuclear extracts prepared 4 h after stimulation with poly(I:C) were examined for interferon regulatory factor 3 (IRF3) levels by Western blotting. Representative immunoblot for nuclear IRF3 is shown.

 
TLR3-mediated expression of IFN-beta is dependent on IRF3, which is the main transcription factor controlling IFN-beta transcription (28, 35, 54). To determine whether exposure to DEas enhances poly(I:C)-induced IFN-beta expression at the transcriptional level, we determined the nuclear levels of IRF3. A549 cells were exposed and treated as before, and Western blotting was used to assay levels of IRF3 in nuclear fractions harvested 4 h after stimulation with poly(I:C) (Fig. 6B). Treatment with DEas or poly(I:C) alone resulted in a mild increase in the levels of nuclear IRF3. Similar to the IFN-beta mRNA levels, when cells were exposed to DEas before stimulation with poly(I:C), nuclear IRF3 levels were greater compared with cells only stimulated with poly(I:C), indicating that exposure to DEas enhances poly(I:C)-induced activation of IRF3. Together, these results show how preexposure to DEas can upregulate IFN-beta levels through enhanced activation of IRF3.

Role of Akt in IFN-beta production. Because we observed both an increase in IFN-beta mRNA levels and increased nuclear levels of IRF3 in cells exposed to DEas and subsequently stimulated with poly(I:C), we examined upstream components of the TLR3-mediated interferon pathway. Specifically, we examined the role of the phosphatidylinositol 3-kinase (PI3-kinase) pathway in the DEas-induced upregulation of IFN-beta. Evidence suggests that Akt, the downstream component of PI3-kinase, is responsible for phosphorylating IRF3, which is required for complete activation of the transcription factor (48). Before exposure to DEas and stimulation with poly(I:C), cells were pretreated with wortmannin to specifically inhibit activation of PI3-kinase and thus Akt. These cells were then analyzed for IFN-beta mRNA levels by real-time RT-PCR. Figure 7A shows that, in cells pretreated with the vehicle control, exposure to DEas before treatment with poly(I:C) significantly increased levels of IFN-beta mRNA. However, when cells were pretreated with the PI3-kinase inhibitor wortmannin, IFN-beta mRNA levels were not significantly enhanced, supporting Akt's role in IFN-beta gene transcription. To further support a role for Akt in the effects of DEas on poly(I:C)-induced IFN-beta expression, we analyzed the levels of phosphorylated and thus activated Akt in whole cell lysates by Western blotting (Fig. 7B). Exposure to DEas increased the levels of phosphorylated Akt compared with control and poly(I:C)-treated cells. Together, these results suggest that the effects of DEas on poly(I:C)-induced IFN-beta expression are at least partially mediated by the effects of DEas on Akt activity.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have recently demonstrated that exposure of respiratory epithelial cells to DEas increases the susceptibility to influenza virus infections (19). Similarly, exposure to DE has previously been shown to alter the response to and exacerbate respiratory symptoms associated with viral infection in mice (15, 16). However, the mechanism(s) by which these effects occur has not yet been fully elucidated. TLR3 has emerged as a key receptor mediating proinflammatory mediator and type I IFN production in response to viral infections (29, 49). On the basis of current knowledge and previous findings in our laboratory, we hypothesized that exposure to DEas modifies the TLR3-mediated viral response pathway. We show evidence here that exposure to DEas before infection with influenza A or stimulation with the dsRNA synthetic analog poly(I:C) significantly upregulates TLR3 expression in respiratory epithelial cells obtained from different regions of the human respiratory tract. This upregulation of TLR3 resulted in increased inflammatory cytokine and type I IFN response, as demonstrated by increased levels of IL-6 and IFN-beta, respectively. Together, these results provide compelling evidence for the ability of DE to alter the innate immune defense response against viral infection through an increase in TLR3 expression and signaling.

The cellular localization of TLR3 is still the subject of debate, and both cytoplasmic and surface expressions have been documented and appear to depend, at least in part, on the cell type (12, 14, 30, 31, 50, 53). Using flow cytometry as well as confocal microscopy, we demonstrate here that, in respiratory epithelial cells, TLR3 is located in the cell cytoplasm and most likely in endoplasmic vesicles, as has been suggested previously (12, 30). Because uptake of RNA viruses, such as influenza A, depends on the host cell's endosomes, it seems plausible that virus-induced signaling mediating the innate immune defense response should originate from the endosome. It has been shown previously that TLR3 levels can be upregulated by viral infection in respiratory epithelial cells (14, 43, 53). We observed similar effects when primary, human, differentiated bronchial and nasal epithelial cells were infected with influenza A virus, although a significant increase was not observed in bronchial cells. Additionally, when A549 cells were stimulated with poly(I:C) to mimic the viral by-product dsRNA, we also observed an increase in TLR3 mRNA levels (see Fig. 3). This effect, although not significant, mirrors what has been seen previously (14, 50) and may represents part of the epithelial cells’ anti-viral response mechanism to enhance the production and release of mediators aimed at preventing neighboring cells from becoming infected with the invading pathogen. However, more interestingly, prior exposure to DEas caused a significant upregulation of virus or dsRNA-induced TLR3 mRNA levels that was two to four times greater than what was seen with influenza or poly(I:C) alone. This effect was observed in our primary cells derived from the nasal and bronchial epithelium as well as in our cell line derived from the alveolar region of the lung, suggesting that this is an effect that occurs throughout the respiratory tract. Given the fact that the pulmonary epithelium is the primary target for both inhaled vehicle emissions, i.e., DE, as well as many viral infections, the interaction of DE exposure with a viral infection leading to an upregulation of TLR3, as demonstrated here, could significantly alter the host's response to many other respiratory viral infections.

Stimulation of TLR3 culminates in the activation of two separate signaling cascades: a TRAF6-dependent pathway that mediates the activation of NF-{kappa}B and the expression of proinflammatory mediators, such as IL-6, and a TANK-binding kinase 1-dependent pathway that mediates the activation of IRF3 and the expression of type I IFNs. Our data demonstrate that prior exposure to DEas caused a significant increase in both the mRNA and protein levels of the proinflammatory cytokine IL-6 in response to stimulation with poly(I:C). This response was hindered in A549 cells stably transduced with dnTRAF6, which had significantly lower IL-6 mRNA levels than that shown in cells transduced with a control vector. The IL-6 response was not completely abrogated in the dnTRAF cells because these cells still exogenously express the wild-type form of TRAF6 and TLR3-independent pathways that may contribute to the inflammatory response. Nevertheless, these data further support the notion that enhanced IL-6 expression in cells exposed to DEas before stimulation with poly(I:C) was more than likely dependent on TLR3. Inflammatory mediators, such as IL-6, are produced by infected cells to orchestrate an antiviral defense response aimed at clearing the invading pathogen. However, excessive inflammation is detrimental to the host, causing tissue injury. Enhancement of the IL-6 response in an infected host could lead to increased inflammation, recruitment and activation of immune cells, and fever, thereby increasing the morbidity of a respiratory infection. Thus enhanced expression and function of TLR3 in cells exposed to DEas before viral infection are likely to increase lung injury. Using a mouse in vivo model, we aim to examine whether the effects of DE exposure on influenza-induced TLR3 expression and function can also be observed in vivo and whether greater inflammation and injury of the lung can be observed under these conditions.

As indicated above, stimulation of TLR3 also results in type I IFN expression. Similar to the effects seen on IL-6 expression, our data demonstrated that there is a significant interaction between DEas exposure and treatment with poly(I:C), resulting in significantly greater IFN-beta mRNA levels. We were also able to show DEas-induced increases in the nuclear translocation of the transcription factor IRF3, which mediates IFN-beta transcription. In addition, previous studies have shown that activation of the protein kinase Akt is needed to fully activate IRF3 (28, 35, 48, 54). Akt is activated by the upstream kinase PI3-kinase, which is recruited and activated by TLR3 through phosphorylated tyrosine residues on the cytoplasmic domain of TLR3 (48). Results shown here indicate that exposure to DEas, but not stimulation with poly(I:C), increases levels of phosphorylated and thus activated Akt. Additionally, when Akt activity was inhibited in A549 cells before exposure to DEas and stimulation with poly(I:C), there was a reduction in IFN-beta mRNA levels compared with the vehicle control. Together, these results suggest that the effects of DEas on virus-induced production of IFN-beta are mediated by DEas-enhanced Akt activity. It is possible that the effects of DEas on Akt activity are not entirely dependent on TLR3 because there are several signaling pathways that lead to phosphorylation of Akt and activation of PI3-kinase. This notion is supported by our data showing enhanced levels of phosphorylated Akt in cells exposed to DEas alone and by previous data demonstrating that, in mouse epidermal cells, exposure to DE particulates enhanced the levels of phosphorylated Akt and activated NF-{kappa}B in a PI3-kinase-dependent manner (26). Thus exposure to DEas could potentially affect virus-induced IFN-beta expression via two possibly interdependent pathways: 1) through enhancement of the virus-induced expression and activity of TLR3 and 2) through enhancement of IRF3 activity by activation of PI3-kinase. Future analysis of these pathways and their interaction may provide a greater understanding of the mechanisms involved.

Furthermore, it is possible that production and release of IFN-beta activate a positive feedback loop, resulting in increased TLR3 expression and further IFN-beta production. Specifically, stimulation with the type I IFNs (IFN-{alpha} and IFN-beta) resulted in enhanced expression of TLR3 and increased poly(I:C)-induced signaling in A549 cells (56). Our group (19) has previously shown that treatment with DEas significantly enhances influenza-induced IFN-beta expression in both primary respiratory epithelial cells and A549 cells. Similarly, we show here that treatment with DEas significantly enhances the poly(I:C)-induced IFN-beta response and that this response may be caused by DEas-induced activation of Akt and subsequent activation of IRF3 (see Figs. 6 and 7). On the basis of the findings published by Tissari et al. (56), it seems plausible that the enhanced expression of IFN-beta in cells exposed to DEas and stimulated with poly(I:C) activates such a positive feedback loop, resulting in the increased expression of TLR3. Consequently, IFN-beta-dependent enhanced expression of TLR3 would result in increased binding of dsRNA and therefore enhanced TLR3 signaling, which in turn would culminate in increased inflammatory mediator and IFN production, as was observed here.

Together, the results presented here describe a mechanism by which exposure to an air pollutant, such as DE, can alter the innate immune defense responses against viral infections in respiratory epithelial cells, namely, through an increase in the expression of TLR3. By increasing TLR3 levels, DEas enhances part of the innate immune response that is thought to provide a bridge between the innate and adaptive immune responses against viral infections (1, 2, 18). Within epithelial cells, this translates into increased recruitment and activation of inflammatory and immune cells, which can have detrimental consequences if left unchecked. Additionally, the increased immune signaling by epithelial cells may also result in increased dendritic cell activation, leading to increased antigen presentation and adaptive immune activation, possibly resulting in an autoimmune or allergic reaction. Similarly, the enhanced TLR3 expression and signaling seen in epithelial cells may also occur within dendritic cells, leading to similar consequences. On the other hand, because TLR3-dependent signaling culminates in mediator production whose purpose it is to limit and aid in clearing the infection, the increased TLR3 expression and signaling observed here may be beneficial to the host and help in fighting off the invading pathogen. Enhanced TLR3 expression may lead to quicker activation and mobilization of innate and adaptive immunity components, resulting in a greater anti-viral response. Further studies are currently underway to determine whether the effects of DE on virus-induced TLR3 expression and function observed in respiratory epithelial cells in vitro will translate into beneficial or detrimental effects in vivo.

Because epidemiological evidence has noted an association between exposure to ambient particulate matter and pulmonary infections (41), including exacerbation of respiratory symptoms associated with infection (7, 13, 39), the results presented here combined with previous findings in our laboratory (19) provide a potential mechanism by which exposure to particulate air pollutants, such as DE, enhances the susceptibility and response to respiratory virus infections. It will be of great interest and public concern to elucidate further mechanisms involved in these processes and to identify the effects of air pollutants on other aspects of host defense responses against respiratory viruses.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The project described was supported by Grant ES-013611 from the NIEHS and by grants from the EPA (CR829522) and the American Chemistry Council (5-45974) (all I. Jaspers) and also by the EPA-University of North Carolina Curriculum in Toxicology Training agreement (T829472) (J. Ciencewicki).


   ACKNOWLEDGMENTS
 
We thank Dr. James Samet for critical review of this manuscript, Wenli Zhang for technical assistance, Dr. Neil Alexis for help with the flow cytometric analyses, Robert Silbajoris for help with the real time RT-PCR, and Dr. Larry Kupper for helpful advice regarding the statistical analysis of the data.

The content in this manuscript is solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Environmental Health Sciences (NIEHS). This publication has not been formally reviewed by the American Chemistry Council. The views expressed in this document are solely those of the authors. Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency (EPA) through cooperative agreement CR829522 [GenBank] with the Center for Environmental Medicine, Asthma, and Lung Biology, it has not been subjected to the Agency's required peer and policy review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. Jaspers, Univ. of North Carolina, CB# 7310, 104 Mason Farm Rd., Chapel Hill, NC 27599 (e-mail: Ilona_jaspers{at}med.unc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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