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Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells
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Nicolai S. Bach a,b , Marit Låg a , Johan Øvrevik a,∗ a Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404, Nydalen, N-0403 Oslo, Norway b Department of Biology, Faculty of Mathematics and Natural Sciences, University of Oslo, P.O. Box 1066, Blindern, N-0316 Oslo, Norway
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h i g h l i g h t s • • • • •
We investigated cytokine responses by diesel exhaust particles (DEP) in BEAS-2B cells. DEP induced IL-6 and CXCL8 but not CCL5 in unprimed BEAS-2B cells. DEP induced stronger IL-6/CXCL8 responses but suppressed CCL5 in TLR3-primed cells. Combinatory effects of DEP and TLR3-priming were also observed on MAPKs and NF-B. TLR3-priming may affect susceptibility toward proinflammatory effects of DEP.
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Article history: Received 13 December 2013 Received in revised form 26 March 2014 Accepted 27 March 2014 Available online xxx
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Keywords: Air pollution Diesel exhaust Particulate matter Lung cells Inflammation Cytokines
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1. Introduction
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Inflammation is considered central in the pathology of health effects from airborne particulate matter (PM). Preexisting inflammatory disorders, such as asthma, but also pulmonary infections, appear to be a risk factor of adverse health effects from PM exposure. Thus, to assess whether and how preexisting inflammation may sensitize lung cells toward additional proinflammatory effects of PM, human bronchial epithelial cells (BEAS-2B) were primed with the highly proinflammatory Toll-like receptor 3 (TLR3) ligand, Poly I:C, prior to exposure with diesel exhaust particles (DEP). DEP-exposure alone induced increased gene-expression of interleukin-6 (IL-6) and CXCL8 (IL-8) but did not affect expression of CCL5 (RANTES), while TLR3-priming alone induced expression of IL-6, CXCL8 and CCL5. DEP-exposure exacerbated IL-6 and CXCL8 responses in TLR3-primed cells, while TLR3-induced CCL5 was suppressed by DEP. TLR3-priming and DEP-exposure resulted in possible additive effects on p38 phosphorylation and IB-degradation, while DEP rather suppressed ERK and JNK-activation. However, TLR3-priming elicited a considerable increase in p65-phosphorylation at serine 536 which is known to enhance the transcriptional activity of NF-B. DEP-exposure was unable to induce p65-phosphorylation. Thus TLR3-priming may affect susceptibility toward DEP by activating both shared and complementing pathways required for optimal expression of proinflammatory genes such as IL-6 and CXCL8. The study underscores that primed “sick” cells may be more susceptible toward effects of particle-exposure and respond both stronger and differently compared to unprimed “healthy” cells. © 2014 Published by Elsevier Ireland Ltd.
Urban air particulate matter (PM) is associated with the development or exacerbation of a variety of adverse cardiopulmonary outcomes (Donaldson et al., 2001; Kelly and Fussell, 2011; Sacks et al., 2011). PM has been suggested to contribute to
∗ Corresponding author at: Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404, Nydalen, N-0403 Oslo, Norway. Tel.: +47 21076408; fax: +47 21076686. E-mail address: [email protected] (J. Øvrevik).
the pathogenesis of disease development by promoting chronic low-grade inflammation (Donaldson et al., 2001; Kelly and Fussell, 2011; Salvi and Holgate, 1999). Moreover, preexisting inflammation-related disorders such as chronic obstructive pulmonary disease (COPD) and asthma, but also virus infections, appear to represent susceptibility factors for adverse effects of PM-exposure (Sacks et al., 2011; Wong et al., 2010). Thus preexisting inflammation may possibly sensitize the airways toward additional proinflammatory effects of PM-exposure. The pulmonary epithelium is a physical barrier to the outside environment, and is therefore a primary target of inhaled
http://dx.doi.org/10.1016/j.toxlet.2014.03.021 0378-4274/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Bach, N.S., et al., Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.03.021
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pollutants. In a recent study, we showed that priming bronchial epithelial cells (BEAS-2B) with the highly proinflammatory Tolllike receptor 3 (TLR3) ligand polyinosinic:polycytidylic acid (Poly I:C), sensitized the cells toward additional proinflammatory effects of polycyclic aromatic hydrocarbons (PAHs) from combustionderived pollution (Ovrevik et al., 2013). Poly I:C, which is a synthetic double-stranded RNA analog often used to mimic virus infection, induced release of the neutrophil attracting chemokine CXCL8 (interleukin-8: IL-8) and the eosinophil attractant CCL5 (Regulated upon Activation Normal T-cell Expressed and Secreted: RANTES). Pyrene and pyrene derivatives, at concentrations unable to provoke chemokine responses in unprimed cells, exacerbated CXCL8 and CCL5 responses in TLR3-primed cells (Ovrevik et al., 2013). The mechanisms underlying this increased sensitivity of TLR3-primed cells has not been explored, but conceivably the TLR3-priming and PAH exposure acted in concert on common/shared signaling pathways involved in chemokine regulation, or perhaps more likely, TLR3-priming activated complementary pathways required for maximal chemokine induction by the PAHs. Activation of the redox sensitive transcription factor nuclear factor (NF)-B, appears to be indispensable for CXCL8 expression (Hoffmann et al., 2002). However, NF-B activation alone may have little effect on CXCL8 unless additional pathways are activated. Thus, a combined activation of NF-B along with the three main MAPK pathways ERK1/2, JNK1/2 and p38, may be required for optimal expression of CXCL8. ERK and JNK appears to elicit their effects by regulating additional transcription factors involved in CXCL8 regulation, such as activator protein (AP)-1, while p38 also promote mRNA stabilization (Hoffmann et al., 2002). In addition to their effects on CXCL8, NF-B and MAPKs are known to be involved in regulation of a variety of other proinflammatory genes. The present study aimed to assess whether a preexisting inflammation may represent a susceptibility factor for effects of combustion-derived PM in lung cells. We have previously shown that diesel exhaust particles (DEP), a major component of urban air PM, induce increased expression and release of CXCL8 and IL-6 from BEAS-2B cells (Totlandsdal et al., 2012). Thus, we hypothesized that (1) TLR3-priming would sensitize BEAS-2B cells toward additional proinflammatory effects of DEP-exposure, and (2) that any combinatory effects of TLR3-priming and DEP-exposure could be attributed to effects on MAPK-signaling and/or NF-kB activation.
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2. Materials and methods
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2.1. Reagents
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LHC-9 cell culture medium was from Invitrogen (Carlsbad, CA, USA) and PureColTM collagen from Inamed Biomaterials (Fremont, CA, USA). Polyinosinic:polycytidylic acid (Poly I:C), Ponceau S, phenylmethylsulfonyl fluoride (PMSF), aprotinin, ethylenediaminetetraacetic acid (EDTA), 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) and polyoxyethylene octyl phenyl ether (Triton X-100) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All realtime RT-PCR reagents and TaqMan probes/primers were purchased from Applied Biosystems (Foster City, CA, USA). Bio-Rad DC protein assay was from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Pepstatin A was from Calbiochem (Cambridge, MA, CA, USA). Leupeptin was from Amersham Biosciences (Uppsala, Sweden). Antibodies against phospho- and total ERK1/2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against IB, phospho- and total p65, p38, JNK1/2, were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against -actin, as well as secondary antibodies horseradish peroxidase-conjugated goat-anti-rabbit IgG, were from Sigma–Aldrich Chemical Company (St. Louis, MO, USA). Horseradish peroxidase-conjugated rabbit anti-mouse IgG from Dako (Glostrup, Denmark) was applied. Mild antibody stripping solution® was from Chemicon International (Termecula, CA, USA). All other chemicals used were purchased from commercial sources at the highest purity available.
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2.2. Culture of cells
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BEAS-2B cells, an immortalized SV40-adenovirus-hybrid (Ad12SV40) transformed human bronchial epithelial cell line was from European Collection of Cell Cultures (ECACC, Salisbury, UK). Cells were grown at 37 ◦ C in a humidified incubator
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with a 5% CO2 atmosphere, where they were passaged twice per week. Cells were cultured in serum-free LHC-9 medium on collagen (PureColTM )-coated culture dishes and flasks. Prior to exposure, cells were plated in 6-well culture dishes, grown to near confluence in serum free LHC-9 medium and exposed as described below.
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2.3. Particles
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DEP (MAPCEL soot) were generated by an unloaded diesel engine (Deutz, 4 cylinder, 2.2 L, 500 rpm) using gas oil as described elsewhere (Totlandsdal et al., 2010). For each experiment, particles were suspended in fresh LHC-9 cell exposure medium (2 mg/ml) and stirred overnight in room temperature before exposure.
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2.4. Exposure of cells
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In all experiments, fresh medium was added the day after seeding and right before exposure and, depending on the experiments, the cells were exposed to 50 or 100 g/ml DEP for 2, 4 and/or 6 h as described under the respective experiments. The controls were added medium that had been subjected to the same stirring procedure as the particle suspensions. In experiments with primed cells, Poly I:C (10 g/ml) was added 30 min prior exposure to DEP, previously optimized in our laboratory.
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2.5. Gene expression analysis by real-time RT-PCR
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Total RNA was isolated using Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA, USA) and reverse transcribed to cDNA on a PCR System 2400 (PerkinElmer, Waltham, MA, USA) using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using pre-designed TaqMan Gene Expression Assays and TaqMan Universal PCR Master Mix and run on ABI 7500 fast (Applied Biosystems). Gene expression of IL-6 (Hs00174131 m1), CXCL8 (Hs00174103 m1) and CCL5 (Hs00174575 m1) were normalized against 18S rRNA (Hs99999901 s1), and expressed as fold change compared to untreated control as calculated by the Ct method (Ct = Ct [Gene of Interest] − Ct[18S]; Ct = Ct [Treated] − Ct [Control]; fold change = 2[−Ct] ).
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2.6. Examination of protein levels by Western blotting
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DEP-induced phosphorylation of MAPK (ERK1/2, JNK1/2, p38) and p65, and degradation of IB were measured by Western blot analysis. After exposure, cell culture medium was removed and the dishes were immediately rinsed with ice-cold PBS, and stored at −70 ◦ C until further processing. Frozen cells were thawed, harvested and sonicated in lysis buffer (20 mM Tris–HCl, pH = 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.4 mM Na-pyrophosphate, 1.0 mM orthovanadate, 1 mM NaF, 21 M leupeptin, 1.5 M aprotinin, 15 M pepstatin A, 1 mM PMFS and 1% TritonX) prior to protein determination using the BioRad DC Protein Assay (BioRad Life Science, CA, USA). Subsequently glycerol, -mercaptoethanol and SDS were added to all samples, and final sample protein concentrations were adjusted by adding more lysis buffer. Proteins (10–20 g/well) from whole-cell lysates were separated by 10–15% SDS-PAGE and blotted onto nitrocellulose membranes. To ensure that the protein levels of each well were equal, Ponceau-staining was used for loading control. The membranes were then probed with antibodies for the respective phosphorylated kinases (p-ERK1/2, p-JNK1/2, p-p38), antibodies for IB or antibodies for phosphorylated p65, prior to incubation with horseradish peroxidase-conjugated secondary antibodies. The blots were developed using the Super-Signal® West Dura chemiluminescence system (Pierce, Perbio Science, Sweden) according to the manufacturer’s instructions. Finally, the membranes were stripped by incubation for 15 min at room temperature with mild antibody stripping solution, and re-probed with -actin, or with the total amount of the respective kinases or p65. Optical quantification of the protein bands was performed by using Image Lab Analysis Software (BioRad).
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Multiple comparisons were analyzed by the Holm–Sidak method. Results are expressed as means ± SEM. All calculations were performed using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA).
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3. Results
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3.1. DEP-induced cytokine and chemokine expression
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As a measure of the proinflammatory effects of DEP-exposure we assessed the expression of IL-6, CXCL8 and CCL5 by realtime PCR. First we explored the time-course of DEP-induced cytokine/chemokine gene expression after 2, 4 and 6 h exposure. DEP (100 g/ml) induced a statistically significant up-regulation of IL-6 mRNA at 2 and 4 h, while CXCL8 was statistically significantly increased at all tested time-points in DEP-exposed cells (Fig. 1).
Please cite this article in press as: Bach, N.S., et al., Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.03.021
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Fig. 1. Time-course of IL-6 and CXCL8 expression in DEP-exposed BEAS-2B cells. Cells were exposed to DEP (100 g/ml) for 2, 4 and 6 h (A and B). IL-6 and CXCL8 expression was measured by real-time RT-PCR. Bars represent means ± SEM of fold increase relative to unexposed cells (n = 3–4). *Statistical significant difference between DEP-exposed and unexposed cells (p < 0.05).
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These results confirm that DEP induce IL-6 and CXCL8 gene expression in BEAS-2B cells, as previously reported (Totlandsdal et al., 2010). However, DEP did not affect CCL5 expression in BEAS-2B cells (data not shown). 3.2. Effect of TLR3-priming on DEP-induced cytokine/chemokine responses Next, we investigated whether TLR3-priming sensitized the cells toward effects of DEP-exposure. Thus, BEAS-2B cells were pretreated with Poly I:C (10 g/ml) for 30 min prior to DEP-exposure (50 and 100 g/ml). TLR3-priming significantly increased IL-6, CXCL8 and CCL5 expression in BEAS-2B cells (Fig. 2A–C). Moreover, in both unprimed and TLR3-primed cells 100 g/ml DEP induced a statistically significant increase IL-6 expression while CXCL8 expression was significantly enhanced from 50 g/ml DEP (Fig. 2A and B). By correcting for background levels of IL-6 and CXCL8 in primed and unprimed control cells, we found that DEP induced statistically significantly higher effects on IL-6 and CXCL8 expression in TLR3-primed vs. unprimed cells (Fig. S1A and B, online supplementary material). In contrast, exposure to DEP significantly down-regulated CCL5 expression in TLR3-primed cells (Fig. 2C). Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet.2014.03.021.
cells, but the difference in phospho-p38 levels of TLR3-primed vs. DEP + TLR-primed were not statistically significant (Fig. 3B). 3.4. Role of the NF-B signaling pathway We also investigated the possible involvement of NF-B in the alteration of DEP-induced cytokine/chemokine responses by TLR3 priming. In the classical NF-B pathway, the p65/p50-dimer is bound in an inactive state in the cytosol by the inhibitor of B (IB). Upon activation, IB is degraded and p65/p50 translocates to the nucleus and binds to target genes (Iwai 2012). DEP alone appeared to induce a weak but not statistically significant degradation of IB at 4 h (Fig. 4A). Phosphorylation of p65 at serine 536 (Ser56), which may increase the transcriptional activity of p65 (Buss et al., 2004; Schmeck et al., 2004) was not affected by DEP (Fig. 4B). In comparison, TLR3-priming alone also appeared to induce a low, but not statistical significant degradation of IB, almost identical to the effect of DEP alone (Fig. 4A). However, in contrast to DEP, TLR3-priming induced a strong increase in p65 Ser536-phosphorylation (Fig. 4B). Of interest, the combination of DEP-exposure and TLR3-priming resulted in an apparently additive increase in IB degradation at 4 h, statistically significantly different from untreated controls (Fig. 4A). However, DEP-exposure did not provide any additional effect on p65 phosphorylation in TLR3-primed cells (Fig. 4B).
3.3. Role of the MAPK pathway
4. Discussion
To investigate the mechanisms of the combinatory effects of TLR3-priming and DEP-exposure, we assessed effects on MAPK-signaling in TLR3-primed and unprimed cells, after 2 and 4 h DEP-exposure (100 g/ml). TLR3-priming alone resulted in a weak, but not statistically significant increase in ERK-phosphorylation after 2 h exposure (Fig. 3A). In comparison, p38 and JNK was significantly increased by the TLR3-agonist at both time points (Fig. 3B and C). Previous studies from our group suggest that DEP may induce activation of p38, but not ERK or JNK, in BEAS-2B cells (Alexopoulou et al., 2001; Hashimoto et al., 2000; Takizawa et al., 1999; Totlandsdal et al., 2010). In line with this, DEP did not induce phosphorylation of ERK or JNK (Fig. 3A and C). Instead, DEP rather suppressed ERK-phosphorylation after 4 h in both TLR3-primed and unprimed cells (Fig. 3A). DEP exposure also abrogated the Poly I:C-induced increase in JNK-phosphorylation at 2 h (Fig. 3C). In further coherence with our previous observations, DEP appeared to elicit a certain increase in p38 phosphorylation, in BEAS-2B cells, but this effect was not statistically significant (Fig. 3B). However, DEP exposure enhanced p38 phosphorylation in TLR3-primed cells to a level significantly above that of DEP-exposed unprimed
In the present study, we have investigated whether a preexisting inflammatory stimuli, in the form of TLR3-priming, would sensitize lung cells toward additional proinflammatory effects of DEP. Our results confirm this to a certain extent. However, the tested combination of TLR3-priming and DEP exposure did not result in a straight forward augmentation of all three proinflammatory markers assessed in the BEAS-2B cells. In accordance with previous observations (Ovrevik et al., 2013; Totlandsdal et al., 2012), DEP-exposure alone resulted in up-regulation of IL-6 and CXCL8 expression in BEAS-2B cells, while TLR3-priming by Poly I:C alone induced increased expression of IL-6, CXCL8 as well as CCL5. Of interest DEP exacerbated IL-6 and CXCL8 responses in TLR3-primed cells, and the effects attributable to DEP appeared stronger in primed vs. unprimed cells. Although further studies are required to determine whether DEP and TLR3-priming leads to true synergistic enhancement of IL-6 and CXCL8 release in bronchial epithelial cells, the effects at gene expression levels appeared more than additive. However, in contrast to this exacerbation of IL-6 and CXCL8, DEP-exposure resulted in a strong suppression of CCL5 expression in TLR3-primed cells. Thus, DEP exposure resulted in
Please cite this article in press as: Bach, N.S., et al., Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.03.021
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Fig. 2. Effect of DEP-exposure on cytokine/chemokine expression in TLR3-primed and unprimed BEAS-2B cells. Cells were primed with the TLR3 agonist Poly I:C (10 g/ml) for 30 min prior exposure to DEP (0, 50 and 100 g/ml). IL-6 (A), CXCL8 (B) and CCL5 (C) expression was measured after 4 h using real-time RT-PCR. Bars represent means ± SEM of fold increase relative to unexposed cells (n = 3). *Statistical significant difference between Poly I:C-primed and unprimed cells (p < 0.05). # Statistical significant difference between DEP-exposed and unexposed control cells (p < 0.05).
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both a quantitative and qualitative shift in the proinflammatory response of TLR3-primed cells. The present results suggest that virus-infected cells may be more prone to adverse effects of DEP and react stronger or differently than uninfected, normal cells. In line with this, cigarette smoke condensates have been reported to synergistically enhance the effects of respiratory syncytial virus on CXCL8 and CCL2 (MCP-1) responses in human alveolar A549-cells (Castro et al.,
Fig. 3. Effect of DEP-exposure on MAPK-signaling in TLR3-primed and unprimed BEAS-2B cells. Cells were primed with the TLR3 agonist Poly I:C (10 g/ml) for 30 min prior exposure to DEP (100 g/ml) for 2 and 4 h. Intracellular protein levels of total and phosphorylated ERK, p38, JNK were detected by Western blotting as described under Section 2. The figure displays representative blots of ERK (A), p38 (B) and JNK (C). The graphs depict relative changes quantified by densitometric analysis of multiple Western blots. Bars represent means ± SEM of fold increase relative to unexposed cells (n = 3). *Statistical significant difference from unexposed control (p < 0.05). # Statistical significant difference between DEP-exposed in TLR3-primed vs. unprimed cells (p < 0.05). † Statistical significant difference between TLR3-priming in DEP-exposed vs. unexposed cells (p < 0.05).
2008). Moreover, an epidemiological study carried out by Wong and co-workers (Wong et al., 2010) suggest that influenza infections may be a susceptibility factor for adverse effects of air pollution. However, increased responsiveness toward inhaled pollutants does not seem to be restricted to inflammation by viral infections. Pre-treating cells with TLR2/-4-ligands or cytokines
Please cite this article in press as: Bach, N.S., et al., Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.03.021
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Fig. 4. Effect of DEP-exposure on NF-B signaling in TLR3-primed and unprimed BEAS-2B cells. Cells were primed with the TLR3 agonist Poly I:C (10 g/ml) for 30 min prior exposure to DEP (100 g/ml) 2 and 4 h. Intracellular protein levels of total and phosphorylated p65, as well as IB and -actin were detected by Western blotting as described under Section 2. The figure displays representative blots of IB and -actin (A) as well as p-p65 and total p65 (B). The graphs depict relative changes in IB and p-p65 quantified by densitometric analysis of multiple Western blots. Bars represent means ± SEM of fold increase relative to unexposed cells (n = 3). *Statistical significant difference from unexposed control (p < 0.05). # Statistical significant difference between DEP-exposed in TLR3-primed vs. unprimed cells (p < 0.05).
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such as TNF-␣ may also enhance the pro-inflammatory effects of PM or PM-derived organic chemicals (Imrich et al., 1999; Inoue et al., 2006; Ning et al., 2004; Steerenberg et al., 1998; Takano et al., 1997). Moreover, both asthma and COPD are considered susceptibility factors for PM-induced disease (Sacks et al., 2011). In striking similarity to our present observations, Devalia et al. (1999) reported that DEP-exposure enhanced CXCL8 responses but suppressed CCL5 in cells from asthmatic patients. CXCL8 is one of the most potent neutrophil recruiting chemokines and is, along with IL-6, often associated with non-allergic inflammation (Fox et al., 2005). CCL5 on the other hand, activates and attracts eosinophils and is involved in allergic inflammation and asthma (Bisset and Schmid-Grendelmeier, 2005). Thus, DEP-exposure may favor an immune response dominated by neutrophils and not by eosinophils. Moreover, it seems likely that any inflammatory stimuli may alter the sensitivity and responsiveness of lung cells toward additional proinflammatory effects of combustion-derived pollutants, and that DEP-exposure may promote both an enhancement
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and a shift in the immune response in individuals with preexisting pulmonary inflammation. MAPK-signaling and NF-B activation are central in regulation of IL-6 and CXCL8 by numerous exposures. Moreover, both DEP and Poly I:C have been shown to induce expression of cytokines and chemokines through up-regulation of MAPK signaling pathways and/or NF-B (Alexopoulou et al., 2001; Hashimoto et al., 2000; Takizawa et al., 1999; Totlandsdal et al., 2010). Thus, it is conceivable that any combinatory effect between DEP-exposure and TLR3-priming could be due to effects on MAPKs and/or NF-B. Previous studies from our research group suggest that DEP induce CXCL8 in BEAS-2B cells through activation p38 and NF-B, but not ERK and JNK. In comparison, DEP-induced IL-6 also appeared to depend on p38 activation, but seemed less affected by NF-B (Totlandsdal et al., 2010). In the present study, we found that DEP-exposure rather suppressed phosphorylation of ERK and JNK. However, we found a weak (although not statistically significant) induction of p38 phosphorylation by DEP alone and DEP also appeared to enhance phospho-p38 in TLR3 primed cells. Moreover, the combination of TLR3-priming and DEP-exposure appeared to result in an additive increase in IB-degradation, which may allow for increased translocation of activated NF-B to the nucleus. Thus it is conceivable that the combinatory enhancement of IL-6 and CXCL8 responses by DEP-exposure and TLR3-priming at least partly may be related to their effects on p38 and NF-B activation. However, additive or synergetic effects on gene expression may not only be due to effects on common/shared signaling pathways. The effects may also be due to one component activating pathways essential for optimal responses, that the other component is unable to evoke. In the present study, TLR3-priming resulted in a considerable increase in p65-phosphorylation at serine 536 (Ser536), which has been reported to be important for transactivation of p65 and transcription of CXCL8 (Buss et al., 2004; Schmeck et al., 2004). DEP-exposure did not affect p65 Ser536-phosphorylation in neither primed nor unprimed cells. Thus, it is possible that the increased phosphorylation level of p65 in TLR3-primed cells may represent a complementary pathway that allows for greater effects of DEP on transcription of CXCL8 and possibly also IL-6. In contrast, lack of Ser536-phosphorylated p65 in unprimed BEAS-2B cells may render the cells with NF-B in a less active state, hence resulting in lower effects of the DEP-exposure. Previous studies suggest that all the three main MAPKs, p38, ERK and JNK, are important for TLR3-mediated induction of CXCL8 in airway epithelial cells, but only JNK seem to be involved in regulation of CCL5 (Berube et al., 2009; Takahashi et al., 2006). It is therefore tempting to speculate that at least the down-regulation of JNK by DEP may be involved in the suppression of CCL5 expression in TLR3-primed cells. Notably, corresponding down-regulations of Poly I:C- and rhinovirus-induced CCL5 and JNK-phosphorylation in BEAS-2B cells have also been reported after exposure with cigarette smoke extracts (Eddleston et al., 2011). However, the down-regulation of CCL5 by DEP seems difficult to attribute to the more moderate effects on JNK, alone. In line with this cigarette smoke-induced suppression of CCL5 also appeared to suppress STAT-activation (Eddleston et al., 2011). Thus, other pathways are likely to be involved in the suppression of TLR3-mediated CCL5 by combustion-derived pollutants. The present study suggests that DEP-exposed immortalized human bronchial epithelial cells respond differently in the presence of a TLR3-agonist. More specifically, DEP induced stronger IL-6 and CXCL8 responses, and attenuated CCL5 expression, in TLR3-primed cells compared to unprimed cells. Thus, DEP-exposure may not only enhance, but also alter pro-inflammatory responses in “inflamed” cell. This strengthens the notion that preexisting inflammation may be a susceptibility factor for PM-induced adverse health effects, and
Please cite this article in press as: Bach, N.S., et al., Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.03.021
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underscores that “healthy models” (cells, animals or humans) may not be sufficient in order to fully evaluate the effects of air pollutants on proinflammatory mediators.
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Conflict of interest
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The authors declare that there are no conflicts of interest. Transparency document
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The Transparency document associated with this article can be found in the online version.
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Acknowledgements
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We thank E. Lilleaas and T. Skuland (Norwegian Inst. of Pub373 lic Health, Oslo Norway) for technical assistance throughout 374 Q2 the study. The work was supported by the Research Council 375 of Norway, through the Environmental Exposures and Health 376 Outcomes-program (grant nos. 185620 and 228143). 372
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References Alexopoulou, L., Holt, A.C., Medzhitov, R., Flavell, R.A., 2001. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413, 732–738. Berube, J., Bourdon, C., Yao, Y., Rousseau, S., 2009. Distinct intracellular signaling pathways control the synthesis of IL-8 and RANTES in TLR1/TLR2, TLR3 or NOD1 activated human airway epithelial cells. Cell Signal. 21, 448–456. Bisset, L.R., Schmid-Grendelmeier, P., 2005. Chemokines and their receptors in the pathogenesis of allergic asthma: progress and perspective. Curr. Opin. Pulm. Med. 11, 35–42. Buss, H., Dorrie, A., Schmitz, M.L., Hoffmann, E., Resch, K., Kracht, M., 2004. Constitutive and interleukin-1-inducible phosphorylation of p65 NF-{kappa}B at serine 536 is mediated by multiple protein kinases including I{kappa}B kinase (IKK)-{alpha}, IKK{beta}, IKK{epsilon}, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor II31-mediated interleukin-8 transcription. J. Biol. Chem. 279, 55633–55643. Castro, S.M., Kolli, D., Guerrero-Plata, A., Garofalo, R.P., Casola, A., 2008. Cigarette smoke condensate enhances respiratory syncytial virus-induced chemokine release by modulating NF-kappa B and interferon regulatory factor activation. Toxicol. Sci. 106, 509–518. Devalia, J.L., Bayram, H., Abdelaziz, M.M., Sapsford, R.J., Davies, R.J., 1999. Differences between cytokine release from bronchial epithelial cells of asthmatic patients and non-asthmatic subjects: effect of exposure to diesel exhaust particles. Int. Arch. Allergy Immunol. 118, 437–439. Donaldson, K., Stone, V., Seaton, A., MacNee, W., 2001. Ambient particle inhalation and the cardiovascular system: potential mechanisms. Environ. Health Perspect. 109 (Suppl. 4), 523–527. Eddleston, J., Lee, R.U., Doerner, A.M., Herschbach, J., Zuraw, B.L., 2011. Cigarette smoke decreases innate responses of epithelial cells to rhinovirus infection. Am. J. Respir. Cell. Mol. Biol. 44, 118–126.
Fox, S.E., Lu, W., Maheshwari, A., Christensen, R.D., Calhoun, D.A., 2005. The effects and comparative differences of neutrophil specific chemokines on neutrophil chemotaxis of the neonate. Cytokine 29, 135–140. Hashimoto, S., Gon, Y., Takeshita, I., Matsumoto, K., Jibiki, I., Takizawa, H., Kudoh, S., Horie, T., 2000. Diesel exhaust particles activate p38 MAP kinase to produce interleukin 8 and RANTES by human bronchial epithelial cells and N-acetylcysteine attenuates p38 MAP kinase activation. Am. J. Respir. Crit. Care Med. 161, 280–285. Hoffmann, E., Dittrich-Breiholz, O., Holtmann, H., Kracht, M., 2002. Multiple control of interleukin-8 gene expression. J. Leukoc. Biol. 72, 847–855. Imrich, A., Ning, Y.Y., Koziel, H., Coull, B., Kobzik, L., 1999. Lipopolysaccharide priming amplifies lung macrophage tumor necrosis factor production in response to air particles. Toxicol. Appl. Pharmacol. 159, 117–124. Inoue, K., Takano, H., Yanagisawa, R., Hirano, S., Kobayashi, T., Ichinose, T., Yoshikawa, T., 2006. Effects of organic chemicals derived from ambient particulate matter on lung inflammation related to lipopolysaccharide. Arch. Toxicol. 80, 833–838. Kelly, F.J., Fussell, J.C., 2011. Air pollution and airway disease. Clin. Exp. Allergy 41, 1059–1071. Ning, Y., Tao, F., Qin, G., Imrich, A., Goldsmith, C.A., Yang, Z., Kobzik, L., 2004. Particle-epithelial interaction: effect of priming and bystander neutrophils on interleukin-8 release. Am. J. Respir. Cell. Mol. Biol. 30, 744–750. Ovrevik, J., Refsnes, M., Holme, J.A., Schwarze, P.E., Lag, M., 2013. Mechanisms of chemokine responses by polycyclic aromatic hydrocarbons in bronchial epithelial cells: sensitization through toll-like receptor-3 priming. Toxicol. Lett. 219, 125–132. Sacks, J.D., Stanek, L.W., Luben, T.J., Johns, D.O., Buckley, B.J., Brown, J.S., Ross, M., 2011. Particulate matter-induced health effects: who is susceptible? Environ. Health Perspect. 119, 446–454. Salvi, S., Holgate, S.T., 1999. Mechanisms of particulate matter toxicity. Clin. Exp. Allergy 29, 1187–1194. Schmeck, B., Zahlten, J., Moog, K., van, L.V., Huber, S., Hocke, A.C., Opitz, B., Hoffmann, E., Kracht, M., Zerrahn, J., Hammerschmidt, S., Rosseau, S., Suttorp, N., Hippenstiel, S., 2004. Streptococcus pneumoniae-induced p38 MAPK-dependent phosphorylation of RelA at the interleukin-8 promotor. J. Biol. Chem. 279, 53241–53247. Steerenberg, P.A., Zonnenberg, J.A., Dormans, J.A., Joon, P.N., Wouters, I.M., van, B.L., Scheepers, P.T., Van, L.H., 1998. Diesel exhaust particles induced release of interleukin 6 and 8 by (primed) human bronchial epithelial cells (BEAS 2B) in vitro. Exp. Lung Res. 24, 85–100. Takahashi, N., Yamada, T., Narita, N., Fujieda, S., 2006. Double-stranded RNA induces production of RANTES and IL-8 by human nasal fibroblasts. Clin. Immunol. 118, 51–58. Takano, H., Yoshikawa, T., Ichinose, T., Miyabara, Y., Imaoka, K., Sagai, M., 1997. Diesel exhaust particles enhance antigen-induced airway inflammation and local cytokine expression in mice. Am. J. Respir. Crit. Care Med. 156, 36–42. Takizawa, H., Ohtoshi, T., Kawasaki, S., Kohyama, T., Desaki, M., Kasama, T., Kobayashi, K., Nakahara, K., Yamamoto, K., Matsushima, K., Kudoh, S., 1999. Diesel exhaust particles induce NF-kappa B activation in human bronchial epithelial cells in vitro: importance in cytokine transcription. J. Immunol. 162, 4705–4711. Totlandsdal, A.I., Cassee, F.R., Schwarze, P., Refsnes, M., Lag, M., 2010. Diesel exhaust particles induce CYP1A1 and pro-inflammatory responses via differential pathways in human bronchial epithelial cells. Part Fibre Toxicol. 7, 41. Totlandsdal, A.I., Herseth, J.I., Bolling, A.K., Kubatova, A., Braun, A., Cochran, R.E., Refsnes, M., Ovrevik, J., Lag, M., 2012. Differential effects of the particle core and organic extract of diesel exhaust particles. Toxicol. Lett. 208, 262–268. Wong, C.M., Thach, T.Q., Chau, P.Y., Chan, E.K., Chung, R.Y., Ou, C.Q., Yang, L., Peiris, J.S., Thomas, G.N., Lam, T.H., Wong, T.W., Hedley, A.J., 2010. Part 4. Interaction between air pollution and respiratory viruses: time-series study of daily mortality and hospital admissions in Hong Kong. Res. Rep. Health Eff. Inst., 283–362.
Please cite this article in press as: Bach, N.S., et al., Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.03.021
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Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells
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Nicolai S. Bach a,b , Marit Låg a , Johan Øvrevik a,∗ a Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404, Nydalen, N-0403 Oslo, Norway b Department of Biology, Faculty of Mathematics and Natural Sciences, University of Oslo, P.O. Box 1066, Blindern, N-0316 Oslo, Norway
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We investigated cytokine responses by diesel exhaust particles (DEP) in BEAS-2B cells. DEP induced IL-6 and CXCL8 but not CCL5 in unprimed BEAS-2B cells. DEP induced stronger IL-6/CXCL8 responses but suppressed CCL5 in TLR3-primed cells. Combinatory effects of DEP and TLR3-priming were also observed on MAPKs and NF-B. TLR3-priming may affect susceptibility toward proinflammatory effects of DEP.
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Article history: Received 13 December 2013 Received in revised form 26 March 2014 Accepted 27 March 2014 Available online xxx
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Keywords: Air pollution Diesel exhaust Particulate matter Lung cells Inflammation Cytokines
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1. Introduction
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Inflammation is considered central in the pathology of health effects from airborne particulate matter (PM). Preexisting inflammatory disorders, such as asthma, but also pulmonary infections, appear to be a risk factor of adverse health effects from PM exposure. Thus, to assess whether and how preexisting inflammation may sensitize lung cells toward additional proinflammatory effects of PM, human bronchial epithelial cells (BEAS-2B) were primed with the highly proinflammatory Toll-like receptor 3 (TLR3) ligand, Poly I:C, prior to exposure with diesel exhaust particles (DEP). DEP-exposure alone induced increased gene-expression of interleukin-6 (IL-6) and CXCL8 (IL-8) but did not affect expression of CCL5 (RANTES), while TLR3-priming alone induced expression of IL-6, CXCL8 and CCL5. DEP-exposure exacerbated IL-6 and CXCL8 responses in TLR3-primed cells, while TLR3-induced CCL5 was suppressed by DEP. TLR3-priming and DEP-exposure resulted in possible additive effects on p38 phosphorylation and IB-degradation, while DEP rather suppressed ERK and JNK-activation. However, TLR3-priming elicited a considerable increase in p65-phosphorylation at serine 536 which is known to enhance the transcriptional activity of NF-B. DEP-exposure was unable to induce p65-phosphorylation. Thus TLR3-priming may affect susceptibility toward DEP by activating both shared and complementing pathways required for optimal expression of proinflammatory genes such as IL-6 and CXCL8. The study underscores that primed “sick” cells may be more susceptible toward effects of particle-exposure and respond both stronger and differently compared to unprimed “healthy” cells. © 2014 Published by Elsevier Ireland Ltd.
Urban air particulate matter (PM) is associated with the development or exacerbation of a variety of adverse cardiopulmonary outcomes (Donaldson et al., 2001; Kelly and Fussell, 2011; Sacks et al., 2011). PM has been suggested to contribute to
∗ Corresponding author at: Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404, Nydalen, N-0403 Oslo, Norway. Tel.: +47 21076408; fax: +47 21076686. E-mail address: [email protected] (J. Øvrevik).
the pathogenesis of disease development by promoting chronic low-grade inflammation (Donaldson et al., 2001; Kelly and Fussell, 2011; Salvi and Holgate, 1999). Moreover, preexisting inflammation-related disorders such as chronic obstructive pulmonary disease (COPD) and asthma, but also virus infections, appear to represent susceptibility factors for adverse effects of PM-exposure (Sacks et al., 2011; Wong et al., 2010). Thus preexisting inflammation may possibly sensitize the airways toward additional proinflammatory effects of PM-exposure. The pulmonary epithelium is a physical barrier to the outside environment, and is therefore a primary target of inhaled
http://dx.doi.org/10.1016/j.toxlet.2014.03.021 0378-4274/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Bach, N.S., et al., Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.03.021
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pollutants. In a recent study, we showed that priming bronchial epithelial cells (BEAS-2B) with the highly proinflammatory Tolllike receptor 3 (TLR3) ligand polyinosinic:polycytidylic acid (Poly I:C), sensitized the cells toward additional proinflammatory effects of polycyclic aromatic hydrocarbons (PAHs) from combustionderived pollution (Ovrevik et al., 2013). Poly I:C, which is a synthetic double-stranded RNA analog often used to mimic virus infection, induced release of the neutrophil attracting chemokine CXCL8 (interleukin-8: IL-8) and the eosinophil attractant CCL5 (Regulated upon Activation Normal T-cell Expressed and Secreted: RANTES). Pyrene and pyrene derivatives, at concentrations unable to provoke chemokine responses in unprimed cells, exacerbated CXCL8 and CCL5 responses in TLR3-primed cells (Ovrevik et al., 2013). The mechanisms underlying this increased sensitivity of TLR3-primed cells has not been explored, but conceivably the TLR3-priming and PAH exposure acted in concert on common/shared signaling pathways involved in chemokine regulation, or perhaps more likely, TLR3-priming activated complementary pathways required for maximal chemokine induction by the PAHs. Activation of the redox sensitive transcription factor nuclear factor (NF)-B, appears to be indispensable for CXCL8 expression (Hoffmann et al., 2002). However, NF-B activation alone may have little effect on CXCL8 unless additional pathways are activated. Thus, a combined activation of NF-B along with the three main MAPK pathways ERK1/2, JNK1/2 and p38, may be required for optimal expression of CXCL8. ERK and JNK appears to elicit their effects by regulating additional transcription factors involved in CXCL8 regulation, such as activator protein (AP)-1, while p38 also promote mRNA stabilization (Hoffmann et al., 2002). In addition to their effects on CXCL8, NF-B and MAPKs are known to be involved in regulation of a variety of other proinflammatory genes. The present study aimed to assess whether a preexisting inflammation may represent a susceptibility factor for effects of combustion-derived PM in lung cells. We have previously shown that diesel exhaust particles (DEP), a major component of urban air PM, induce increased expression and release of CXCL8 and IL-6 from BEAS-2B cells (Totlandsdal et al., 2012). Thus, we hypothesized that (1) TLR3-priming would sensitize BEAS-2B cells toward additional proinflammatory effects of DEP-exposure, and (2) that any combinatory effects of TLR3-priming and DEP-exposure could be attributed to effects on MAPK-signaling and/or NF-kB activation.
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2. Materials and methods
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LHC-9 cell culture medium was from Invitrogen (Carlsbad, CA, USA) and PureColTM collagen from Inamed Biomaterials (Fremont, CA, USA). Polyinosinic:polycytidylic acid (Poly I:C), Ponceau S, phenylmethylsulfonyl fluoride (PMSF), aprotinin, ethylenediaminetetraacetic acid (EDTA), 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) and polyoxyethylene octyl phenyl ether (Triton X-100) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All realtime RT-PCR reagents and TaqMan probes/primers were purchased from Applied Biosystems (Foster City, CA, USA). Bio-Rad DC protein assay was from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Pepstatin A was from Calbiochem (Cambridge, MA, CA, USA). Leupeptin was from Amersham Biosciences (Uppsala, Sweden). Antibodies against phospho- and total ERK1/2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against IB, phospho- and total p65, p38, JNK1/2, were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against -actin, as well as secondary antibodies horseradish peroxidase-conjugated goat-anti-rabbit IgG, were from Sigma–Aldrich Chemical Company (St. Louis, MO, USA). Horseradish peroxidase-conjugated rabbit anti-mouse IgG from Dako (Glostrup, Denmark) was applied. Mild antibody stripping solution® was from Chemicon International (Termecula, CA, USA). All other chemicals used were purchased from commercial sources at the highest purity available.
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2.2. Culture of cells
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BEAS-2B cells, an immortalized SV40-adenovirus-hybrid (Ad12SV40) transformed human bronchial epithelial cell line was from European Collection of Cell Cultures (ECACC, Salisbury, UK). Cells were grown at 37 ◦ C in a humidified incubator
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with a 5% CO2 atmosphere, where they were passaged twice per week. Cells were cultured in serum-free LHC-9 medium on collagen (PureColTM )-coated culture dishes and flasks. Prior to exposure, cells were plated in 6-well culture dishes, grown to near confluence in serum free LHC-9 medium and exposed as described below.
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2.3. Particles
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DEP (MAPCEL soot) were generated by an unloaded diesel engine (Deutz, 4 cylinder, 2.2 L, 500 rpm) using gas oil as described elsewhere (Totlandsdal et al., 2010). For each experiment, particles were suspended in fresh LHC-9 cell exposure medium (2 mg/ml) and stirred overnight in room temperature before exposure.
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In all experiments, fresh medium was added the day after seeding and right before exposure and, depending on the experiments, the cells were exposed to 50 or 100 g/ml DEP for 2, 4 and/or 6 h as described under the respective experiments. The controls were added medium that had been subjected to the same stirring procedure as the particle suspensions. In experiments with primed cells, Poly I:C (10 g/ml) was added 30 min prior exposure to DEP, previously optimized in our laboratory.
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2.5. Gene expression analysis by real-time RT-PCR
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Total RNA was isolated using Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA, USA) and reverse transcribed to cDNA on a PCR System 2400 (PerkinElmer, Waltham, MA, USA) using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using pre-designed TaqMan Gene Expression Assays and TaqMan Universal PCR Master Mix and run on ABI 7500 fast (Applied Biosystems). Gene expression of IL-6 (Hs00174131 m1), CXCL8 (Hs00174103 m1) and CCL5 (Hs00174575 m1) were normalized against 18S rRNA (Hs99999901 s1), and expressed as fold change compared to untreated control as calculated by the Ct method (Ct = Ct [Gene of Interest] − Ct[18S]; Ct = Ct [Treated] − Ct [Control]; fold change = 2[−Ct] ).
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2.6. Examination of protein levels by Western blotting
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DEP-induced phosphorylation of MAPK (ERK1/2, JNK1/2, p38) and p65, and degradation of IB were measured by Western blot analysis. After exposure, cell culture medium was removed and the dishes were immediately rinsed with ice-cold PBS, and stored at −70 ◦ C until further processing. Frozen cells were thawed, harvested and sonicated in lysis buffer (20 mM Tris–HCl, pH = 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.4 mM Na-pyrophosphate, 1.0 mM orthovanadate, 1 mM NaF, 21 M leupeptin, 1.5 M aprotinin, 15 M pepstatin A, 1 mM PMFS and 1% TritonX) prior to protein determination using the BioRad DC Protein Assay (BioRad Life Science, CA, USA). Subsequently glycerol, -mercaptoethanol and SDS were added to all samples, and final sample protein concentrations were adjusted by adding more lysis buffer. Proteins (10–20 g/well) from whole-cell lysates were separated by 10–15% SDS-PAGE and blotted onto nitrocellulose membranes. To ensure that the protein levels of each well were equal, Ponceau-staining was used for loading control. The membranes were then probed with antibodies for the respective phosphorylated kinases (p-ERK1/2, p-JNK1/2, p-p38), antibodies for IB or antibodies for phosphorylated p65, prior to incubation with horseradish peroxidase-conjugated secondary antibodies. The blots were developed using the Super-Signal® West Dura chemiluminescence system (Pierce, Perbio Science, Sweden) according to the manufacturer’s instructions. Finally, the membranes were stripped by incubation for 15 min at room temperature with mild antibody stripping solution, and re-probed with -actin, or with the total amount of the respective kinases or p65. Optical quantification of the protein bands was performed by using Image Lab Analysis Software (BioRad).
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Multiple comparisons were analyzed by the Holm–Sidak method. Results are expressed as means ± SEM. All calculations were performed using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA).
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3.1. DEP-induced cytokine and chemokine expression
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As a measure of the proinflammatory effects of DEP-exposure we assessed the expression of IL-6, CXCL8 and CCL5 by realtime PCR. First we explored the time-course of DEP-induced cytokine/chemokine gene expression after 2, 4 and 6 h exposure. DEP (100 g/ml) induced a statistically significant up-regulation of IL-6 mRNA at 2 and 4 h, while CXCL8 was statistically significantly increased at all tested time-points in DEP-exposed cells (Fig. 1).
Please cite this article in press as: Bach, N.S., et al., Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.03.021
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Fig. 1. Time-course of IL-6 and CXCL8 expression in DEP-exposed BEAS-2B cells. Cells were exposed to DEP (100 g/ml) for 2, 4 and 6 h (A and B). IL-6 and CXCL8 expression was measured by real-time RT-PCR. Bars represent means ± SEM of fold increase relative to unexposed cells (n = 3–4). *Statistical significant difference between DEP-exposed and unexposed cells (p < 0.05).
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These results confirm that DEP induce IL-6 and CXCL8 gene expression in BEAS-2B cells, as previously reported (Totlandsdal et al., 2010). However, DEP did not affect CCL5 expression in BEAS-2B cells (data not shown). 3.2. Effect of TLR3-priming on DEP-induced cytokine/chemokine responses Next, we investigated whether TLR3-priming sensitized the cells toward effects of DEP-exposure. Thus, BEAS-2B cells were pretreated with Poly I:C (10 g/ml) for 30 min prior to DEP-exposure (50 and 100 g/ml). TLR3-priming significantly increased IL-6, CXCL8 and CCL5 expression in BEAS-2B cells (Fig. 2A–C). Moreover, in both unprimed and TLR3-primed cells 100 g/ml DEP induced a statistically significant increase IL-6 expression while CXCL8 expression was significantly enhanced from 50 g/ml DEP (Fig. 2A and B). By correcting for background levels of IL-6 and CXCL8 in primed and unprimed control cells, we found that DEP induced statistically significantly higher effects on IL-6 and CXCL8 expression in TLR3-primed vs. unprimed cells (Fig. S1A and B, online supplementary material). In contrast, exposure to DEP significantly down-regulated CCL5 expression in TLR3-primed cells (Fig. 2C). Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet.2014.03.021.
cells, but the difference in phospho-p38 levels of TLR3-primed vs. DEP + TLR-primed were not statistically significant (Fig. 3B). 3.4. Role of the NF-B signaling pathway We also investigated the possible involvement of NF-B in the alteration of DEP-induced cytokine/chemokine responses by TLR3 priming. In the classical NF-B pathway, the p65/p50-dimer is bound in an inactive state in the cytosol by the inhibitor of B (IB). Upon activation, IB is degraded and p65/p50 translocates to the nucleus and binds to target genes (Iwai 2012). DEP alone appeared to induce a weak but not statistically significant degradation of IB at 4 h (Fig. 4A). Phosphorylation of p65 at serine 536 (Ser56), which may increase the transcriptional activity of p65 (Buss et al., 2004; Schmeck et al., 2004) was not affected by DEP (Fig. 4B). In comparison, TLR3-priming alone also appeared to induce a low, but not statistical significant degradation of IB, almost identical to the effect of DEP alone (Fig. 4A). However, in contrast to DEP, TLR3-priming induced a strong increase in p65 Ser536-phosphorylation (Fig. 4B). Of interest, the combination of DEP-exposure and TLR3-priming resulted in an apparently additive increase in IB degradation at 4 h, statistically significantly different from untreated controls (Fig. 4A). However, DEP-exposure did not provide any additional effect on p65 phosphorylation in TLR3-primed cells (Fig. 4B).
3.3. Role of the MAPK pathway
4. Discussion
To investigate the mechanisms of the combinatory effects of TLR3-priming and DEP-exposure, we assessed effects on MAPK-signaling in TLR3-primed and unprimed cells, after 2 and 4 h DEP-exposure (100 g/ml). TLR3-priming alone resulted in a weak, but not statistically significant increase in ERK-phosphorylation after 2 h exposure (Fig. 3A). In comparison, p38 and JNK was significantly increased by the TLR3-agonist at both time points (Fig. 3B and C). Previous studies from our group suggest that DEP may induce activation of p38, but not ERK or JNK, in BEAS-2B cells (Alexopoulou et al., 2001; Hashimoto et al., 2000; Takizawa et al., 1999; Totlandsdal et al., 2010). In line with this, DEP did not induce phosphorylation of ERK or JNK (Fig. 3A and C). Instead, DEP rather suppressed ERK-phosphorylation after 4 h in both TLR3-primed and unprimed cells (Fig. 3A). DEP exposure also abrogated the Poly I:C-induced increase in JNK-phosphorylation at 2 h (Fig. 3C). In further coherence with our previous observations, DEP appeared to elicit a certain increase in p38 phosphorylation, in BEAS-2B cells, but this effect was not statistically significant (Fig. 3B). However, DEP exposure enhanced p38 phosphorylation in TLR3-primed cells to a level significantly above that of DEP-exposed unprimed
In the present study, we have investigated whether a preexisting inflammatory stimuli, in the form of TLR3-priming, would sensitize lung cells toward additional proinflammatory effects of DEP. Our results confirm this to a certain extent. However, the tested combination of TLR3-priming and DEP exposure did not result in a straight forward augmentation of all three proinflammatory markers assessed in the BEAS-2B cells. In accordance with previous observations (Ovrevik et al., 2013; Totlandsdal et al., 2012), DEP-exposure alone resulted in up-regulation of IL-6 and CXCL8 expression in BEAS-2B cells, while TLR3-priming by Poly I:C alone induced increased expression of IL-6, CXCL8 as well as CCL5. Of interest DEP exacerbated IL-6 and CXCL8 responses in TLR3-primed cells, and the effects attributable to DEP appeared stronger in primed vs. unprimed cells. Although further studies are required to determine whether DEP and TLR3-priming leads to true synergistic enhancement of IL-6 and CXCL8 release in bronchial epithelial cells, the effects at gene expression levels appeared more than additive. However, in contrast to this exacerbation of IL-6 and CXCL8, DEP-exposure resulted in a strong suppression of CCL5 expression in TLR3-primed cells. Thus, DEP exposure resulted in
Please cite this article in press as: Bach, N.S., et al., Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.03.021
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Fig. 2. Effect of DEP-exposure on cytokine/chemokine expression in TLR3-primed and unprimed BEAS-2B cells. Cells were primed with the TLR3 agonist Poly I:C (10 g/ml) for 30 min prior exposure to DEP (0, 50 and 100 g/ml). IL-6 (A), CXCL8 (B) and CCL5 (C) expression was measured after 4 h using real-time RT-PCR. Bars represent means ± SEM of fold increase relative to unexposed cells (n = 3). *Statistical significant difference between Poly I:C-primed and unprimed cells (p < 0.05). # Statistical significant difference between DEP-exposed and unexposed control cells (p < 0.05).
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both a quantitative and qualitative shift in the proinflammatory response of TLR3-primed cells. The present results suggest that virus-infected cells may be more prone to adverse effects of DEP and react stronger or differently than uninfected, normal cells. In line with this, cigarette smoke condensates have been reported to synergistically enhance the effects of respiratory syncytial virus on CXCL8 and CCL2 (MCP-1) responses in human alveolar A549-cells (Castro et al.,
Fig. 3. Effect of DEP-exposure on MAPK-signaling in TLR3-primed and unprimed BEAS-2B cells. Cells were primed with the TLR3 agonist Poly I:C (10 g/ml) for 30 min prior exposure to DEP (100 g/ml) for 2 and 4 h. Intracellular protein levels of total and phosphorylated ERK, p38, JNK were detected by Western blotting as described under Section 2. The figure displays representative blots of ERK (A), p38 (B) and JNK (C). The graphs depict relative changes quantified by densitometric analysis of multiple Western blots. Bars represent means ± SEM of fold increase relative to unexposed cells (n = 3). *Statistical significant difference from unexposed control (p < 0.05). # Statistical significant difference between DEP-exposed in TLR3-primed vs. unprimed cells (p < 0.05). † Statistical significant difference between TLR3-priming in DEP-exposed vs. unexposed cells (p < 0.05).
2008). Moreover, an epidemiological study carried out by Wong and co-workers (Wong et al., 2010) suggest that influenza infections may be a susceptibility factor for adverse effects of air pollution. However, increased responsiveness toward inhaled pollutants does not seem to be restricted to inflammation by viral infections. Pre-treating cells with TLR2/-4-ligands or cytokines
Please cite this article in press as: Bach, N.S., et al., Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.03.021
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Fig. 4. Effect of DEP-exposure on NF-B signaling in TLR3-primed and unprimed BEAS-2B cells. Cells were primed with the TLR3 agonist Poly I:C (10 g/ml) for 30 min prior exposure to DEP (100 g/ml) 2 and 4 h. Intracellular protein levels of total and phosphorylated p65, as well as IB and -actin were detected by Western blotting as described under Section 2. The figure displays representative blots of IB and -actin (A) as well as p-p65 and total p65 (B). The graphs depict relative changes in IB and p-p65 quantified by densitometric analysis of multiple Western blots. Bars represent means ± SEM of fold increase relative to unexposed cells (n = 3). *Statistical significant difference from unexposed control (p < 0.05). # Statistical significant difference between DEP-exposed in TLR3-primed vs. unprimed cells (p < 0.05).
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such as TNF-␣ may also enhance the pro-inflammatory effects of PM or PM-derived organic chemicals (Imrich et al., 1999; Inoue et al., 2006; Ning et al., 2004; Steerenberg et al., 1998; Takano et al., 1997). Moreover, both asthma and COPD are considered susceptibility factors for PM-induced disease (Sacks et al., 2011). In striking similarity to our present observations, Devalia et al. (1999) reported that DEP-exposure enhanced CXCL8 responses but suppressed CCL5 in cells from asthmatic patients. CXCL8 is one of the most potent neutrophil recruiting chemokines and is, along with IL-6, often associated with non-allergic inflammation (Fox et al., 2005). CCL5 on the other hand, activates and attracts eosinophils and is involved in allergic inflammation and asthma (Bisset and Schmid-Grendelmeier, 2005). Thus, DEP-exposure may favor an immune response dominated by neutrophils and not by eosinophils. Moreover, it seems likely that any inflammatory stimuli may alter the sensitivity and responsiveness of lung cells toward additional proinflammatory effects of combustion-derived pollutants, and that DEP-exposure may promote both an enhancement
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and a shift in the immune response in individuals with preexisting pulmonary inflammation. MAPK-signaling and NF-B activation are central in regulation of IL-6 and CXCL8 by numerous exposures. Moreover, both DEP and Poly I:C have been shown to induce expression of cytokines and chemokines through up-regulation of MAPK signaling pathways and/or NF-B (Alexopoulou et al., 2001; Hashimoto et al., 2000; Takizawa et al., 1999; Totlandsdal et al., 2010). Thus, it is conceivable that any combinatory effect between DEP-exposure and TLR3-priming could be due to effects on MAPKs and/or NF-B. Previous studies from our research group suggest that DEP induce CXCL8 in BEAS-2B cells through activation p38 and NF-B, but not ERK and JNK. In comparison, DEP-induced IL-6 also appeared to depend on p38 activation, but seemed less affected by NF-B (Totlandsdal et al., 2010). In the present study, we found that DEP-exposure rather suppressed phosphorylation of ERK and JNK. However, we found a weak (although not statistically significant) induction of p38 phosphorylation by DEP alone and DEP also appeared to enhance phospho-p38 in TLR3 primed cells. Moreover, the combination of TLR3-priming and DEP-exposure appeared to result in an additive increase in IB-degradation, which may allow for increased translocation of activated NF-B to the nucleus. Thus it is conceivable that the combinatory enhancement of IL-6 and CXCL8 responses by DEP-exposure and TLR3-priming at least partly may be related to their effects on p38 and NF-B activation. However, additive or synergetic effects on gene expression may not only be due to effects on common/shared signaling pathways. The effects may also be due to one component activating pathways essential for optimal responses, that the other component is unable to evoke. In the present study, TLR3-priming resulted in a considerable increase in p65-phosphorylation at serine 536 (Ser536), which has been reported to be important for transactivation of p65 and transcription of CXCL8 (Buss et al., 2004; Schmeck et al., 2004). DEP-exposure did not affect p65 Ser536-phosphorylation in neither primed nor unprimed cells. Thus, it is possible that the increased phosphorylation level of p65 in TLR3-primed cells may represent a complementary pathway that allows for greater effects of DEP on transcription of CXCL8 and possibly also IL-6. In contrast, lack of Ser536-phosphorylated p65 in unprimed BEAS-2B cells may render the cells with NF-B in a less active state, hence resulting in lower effects of the DEP-exposure. Previous studies suggest that all the three main MAPKs, p38, ERK and JNK, are important for TLR3-mediated induction of CXCL8 in airway epithelial cells, but only JNK seem to be involved in regulation of CCL5 (Berube et al., 2009; Takahashi et al., 2006). It is therefore tempting to speculate that at least the down-regulation of JNK by DEP may be involved in the suppression of CCL5 expression in TLR3-primed cells. Notably, corresponding down-regulations of Poly I:C- and rhinovirus-induced CCL5 and JNK-phosphorylation in BEAS-2B cells have also been reported after exposure with cigarette smoke extracts (Eddleston et al., 2011). However, the down-regulation of CCL5 by DEP seems difficult to attribute to the more moderate effects on JNK, alone. In line with this cigarette smoke-induced suppression of CCL5 also appeared to suppress STAT-activation (Eddleston et al., 2011). Thus, other pathways are likely to be involved in the suppression of TLR3-mediated CCL5 by combustion-derived pollutants. The present study suggests that DEP-exposed immortalized human bronchial epithelial cells respond differently in the presence of a TLR3-agonist. More specifically, DEP induced stronger IL-6 and CXCL8 responses, and attenuated CCL5 expression, in TLR3-primed cells compared to unprimed cells. Thus, DEP-exposure may not only enhance, but also alter pro-inflammatory responses in “inflamed” cell. This strengthens the notion that preexisting inflammation may be a susceptibility factor for PM-induced adverse health effects, and
Please cite this article in press as: Bach, N.S., et al., Toll like receptor-3 priming alters diesel exhaust particle-induced cytokine responses in human bronchial epithelial cells. Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j.toxlet.2014.03.021
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underscores that “healthy models” (cells, animals or humans) may not be sufficient in order to fully evaluate the effects of air pollutants on proinflammatory mediators.
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Conflict of interest
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The authors declare that there are no conflicts of interest. Transparency document
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The Transparency document associated with this article can be found in the online version.
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Acknowledgements
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We thank E. Lilleaas and T. Skuland (Norwegian Inst. of Pub373 lic Health, Oslo Norway) for technical assistance throughout 374 Q2 the study. The work was supported by the Research Council 375 of Norway, through the Environmental Exposures and Health 376 Outcomes-program (grant nos. 185620 and 228143). 372
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