Laryngeal inflammatory response to smoke and vape in a murine model☆
Introduction
Laryngeal squamous cell carcinoma (SCC) accounts for 2%–3% of all malignant tumors and 90% of laryngeal cancers [1,2]. Smoking tobacco is the primary etiological agent for laryngeal dysplasia, and increases the risk of laryngeal cancer by 10 to 30 times more than in non-smokers [3,4]. Using electronic cigarettes, also known as vaping, provides an alternative mode of nicotine delivery by producing an inhaled aerosol instead of smoke [5]. While the effects of smoke on the larynx have been well-established, the effects of vape are unknown.
Approximately 60% of patients present with advanced laryngeal cancer at diagnosis [3]. However, literature has shown promising results for the early detection of head and neck cancer in patients using serum biomarkers [6,7]. Specifically, serum levels of IL-6, IL-8, IL-10, TGF-β and TNF-α levels have been found to be increased in patients with laryngeal SCC compared to healthy controls [8]. While cytokine serum levels have previously been studied, laryngeal cytokine levels in laryngeal SCC or pre-malignancy have not previously been described in the literature to the best of our knowledge. Similar to the idea behind potentially using serum biomarkers for the early detection of head and neck cancers, identification of elevated local inflammatory and immune biomarkers from laryngeal biopsies could serve as a prognostic factor for risk of future malignancy in patients. The goal of this pilot study is to demonstrate a murine model of tobacco smoke and electronic cigarette vapor exposure to characterize the inflammatory and immune responses in the mammalian larynx.
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Materials and methods
Following approval by our institutional animal care and use committee, twenty-four wild-type C57BL/6 mice were divided into four groups: tobacco smoke, vapor with nicotine, vapor without nicotine, and air only. In our pilot study, the number of animals required for significant cytokine expression was based on standard statistical methods using the InStat statistics program for the Macintosh. Based on prior work studying lungs in C57BL/6 mice exposed to tobacco smoke [9], the standard deviation
Results
IL-4 was significantly elevated in the tobacco smoke group and the vapor with nicotine group (p = 0.0418) [Fig. 3]. TGF-β2 and TGF-β3 were non-statistically significantly elevated in the tobacco smoke group. Vapor with and without nicotine both resulted in suppression of IL-10, although this was also not statistically-significant. Besides IL-4, concentration levels between the four groups for the remaining cytokines were all statistically non-significant, and concentration levels of 4 out of
Discussion
Cigarette smoke has been shown to promote continuous TH17 cell mediated lung inflammation through double-stranded cleavage of nuclear DNA, resulting emphysema in mice [9]. TH17 cell differentiation from naïve T helper cells is induced by TGF-β and is inhibited by IL-4 [10]. Thus, the TH17 and TGF-β cytokine detection kits were chosen for this study to analyze inflammatory and immune response to tobacco smoke and vapor exposure in mice larynges. The TH17 cytokine detection kit included IL-4. In
Conclusion
Significantly elevated levels of IL-4 and upregulation of TGF-β, while statistically non-significant, demonstrate the efficacy of the murine model for evaluating inflammatory, immune, and possibly carcinogenic effects of smoke and vape on the mammalian larynx. While tobacco smoke and vapor both appear to produce an inflammatory response in the murine model, further research with appropriate power is needed to reach statistical significance.
Acknowledgements
This research was supported in part by a Head & Neck Cancer Seed grant from the Dan L. Duncan Cancer Center at Baylor College of Medicine. We also greatly appreciate Lizhen Song and Monica Jeongsoo Hong in the lab of Dr. Kheradmand for their assistance with specimen processing.
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This study was funded in part by a Head & Neck Cancer development grant from the Dan L. Duncan Cancer Center at Baylor College of Medicine. There are no conflicts of interest. This has not been published elsewhere.
This study was performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act (7 U.S.C. et seq.); the animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine.
This paper was presented in part as a poster at the American Academy of Otolaryngology – HNSF Annual Meeting, Chicago, IL, September 10–13, 2017.