Research paperBenzo(a)pyrene suppresses tracheal antimicrobial peptide gene expression in bovine tracheal epithelial cells
Introduction
The mammalian airway epithelium is equipped with defence mechanisms that protect the lower respiratory tract. Microbes that enter the lower respiratory tract are detected by Toll-like receptors (TLR) on airway epithelial cells, which then signal the cell to produce host defence proteins including β-defensins (Akira et al., 2006; Bals and Hiemstra, 2004; Caswell, 2014). These peptides have bactericidal and immunomodulatory functions that are thought to protect the lower airways from infection and disease (Diamond et al., 2000).
The responsiveness of this system can be impacted by a variety of environmental and host factors. For example, studies in tracheal epithelial cells of cattle have shown that lipopolysaccharide and other TLR agonists upregulate expression of tracheal antimicrobial peptide (TAP, a β-defensin) (Berghuis et al., 2014a), whereas predisposing factors such as stress as modeled by glucocorticoid (Mitchell et al., 2007), viral infection (al-Haddawi et al., 2007), and environmental toxicants such as vanadium in diesel exhaust (Klein - Patel et al., 2006) reduce its expression. Since TAP is capable of in vitro killing of the bacteria that cause pneumonia in cattle (Taha-Abdelaziz et al., 2013), suppression of these peptides is considered a possible reason that these predisposing factors lead to development of pneumonia in cattle (Caswell, 2014).
Cetaceans are negatively impacted by oil spills which, in the case of the Deepwater Horizon spill of 2010, can release millions of gallons of oil into the marine environment. Polycyclic aromatic hydrocarbons (PAHs) are common persistent organic pollutants in crude oil, and are known to have immunosuppressive effects resulting in higher incidence of disease (Allan and Sherr, 2010). Bottlenose dolphins (Tursiops truncatus) affected by the Deepwater Horizon spill were found to have increased prevalence of pneumonia compared to dolphins from areas unaffected by the spill (Schwacke et al., 2014). The mechanisms by which oil exposure predisposes to pneumonia in cetaceans is unknown. We hypothesize that cetaceans within contaminated waters inhale these volatile PAHs when surfacing, and direct effects of these contaminants on airway epithelial cells suppress innate respiratory defences such as β-defensin expression, similar to that described with stress and viral infections in other animals (al-Haddawi et al., 2007; Mitchell et al., 2007).
This study tested the hypothesis that PAHs inhibit the inducible upregulation of TAP gene expression in bovine tracheal epithelial cells. Due to their endangered status, large size, and free-ranging aquatic lifestyle, in vivo studies of innate immunity in cetaceans are limited by current technology and particularly with regards to functionally relevant outcomes of innate immune responses. In vitro analysis of primary cultures of tracheal epithelial cells was used because cells can be exposed to PAHs in a controlled manner, and topical application of PAHs was considered relevant to the scenario of an oil spill event as described above. We have initially modeled these effects in bovine tracheal epithelial cells because of the relatively close evolutionary relationship of cetaceans and ruminants (Yim et al., 2014), the availability of fresh tissue, and the abovementioned comparable effects of diesel exhaust (Klein - Patel et al., 2006), glucocorticoid (Mitchell et al., 2007) and viral infection (al-Haddawi et al., 2007) in this bovine model. The three PAHs (BAP, phenanthrene, and naphthalene) included in this study were chosen based on their inclusion on the Environmental Protection Agency’s list of 16 priority PAHs, their diversity of ring structures (5, 3, and 2 aromatic rings, respectively), their presence in crude oil, and, in the case of BAP, considerable published evidence demonstrating its immunosuppressive effects (Andersson and Achten, 2015; Hylland, 2006; Pampanin and Sydnes, 2013; Reynaud and Deschaux, 2006).
Section snippets
Culture of bovine tracheal epithelial cells
Tracheas from 11 market-weight calves were obtained at slaughter. For each experiment, fresh tracheas were transported in chilled washing solution consisting of phosphate buffered saline (PBS) with 0.1 mg/ml penicillin-streptomycin, 0.5 mg/ml gentamicin, and 10 μg/ml amphotericin B. Using sterile technique, 2 cm2 sections of mucosa were dissected from the trachea, incubated overnight in 50 ml of sterile washing solution containing 0.1% protease (Dispase, Invitrogen, San Diego, CA, USA) at 4 °C,
Effects of lipopolysaccharide and dimethylsulfoxide on tracheal antimicrobial peptide gene expression
In primary cultures of tracheal epithelial cells stimulated with LPS for 16 h, the median normalized ratio of TAP gene expression (relative to GAPDH as the reference gene) was 1.05 (95% confidence interval—lower limit (LL): 0.48, upper limit (UL): 2.28), and the median ratio for non-LPS-stimulated cells was 0.065 (LL: 0.029, UL: 0.147). This corresponded to a 20 ± 17-fold (mean ± SD) increase in TAP gene expression in LPS-stimulated cells compared to non-stimulated cells (P < 0.0001; tracheal
Discussion
Polycyclic aromatic hydrocarbons are persistent organic pollutants that enter the environment from oil spills and other industrial sources. Pneumonia is a common finding in cetaceans exposed to oil spills (Kucklick et al., 2011; White et al., 2016) but the underlying innate immune mechanisms involved in this predilection have not been investigated. Since NF-κB signalling is known to stimulate β-defensin gene expression (Taha-Abdelaziz et al., 2016) and this signalling pathway may be affected by
Conclusion
Of the PAHs examined in this study, it was found that 8 h of exposure to 5 μM of BAP significantly (P < 0.002) suppressed LPS-induced TAP gene expression in bovine tracheal epithelial cells. This level was considered relevant to BAP concentrations found in oil, and coincided with induction of CYP1A1 expression. Other PAHs (naphthalene and phenanthrene) did not consistently suppress TAP gene expression. This furthers our understanding of how organic pollutants may negatively affect innate
Acknowledgements
We thank William Sears for assistance with the statistical analysis.
Funding
This work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (RGPIN-2017-03872, to JLC).
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