Elsevier

American Journal of Otolaryngology

Volume 40, Issue 1, January–February 2019, Pages 89-92
American Journal of Otolaryngology

Laryngeal inflammatory response to smoke and vape in a murine model

https://doi.org/10.1016/j.amjoto.2018.10.001Get rights and content

Abstract

Purpose

To build a murine model for tobacco smoke and electronic cigarette vapor exposure to characterize the inflammatory and immune responses in the larynx.

Materials and methods

In this pilot study, twenty-four wild-type C57BL/6 mice were divided into four groups: smoke, vapor with nicotine, vapor without nicotine, and air only. Following daily exposure for 4 months, larynges were dissected and processed with cytokine detection arrays. Each laryngeal cytokine level between the four different groups was analyzed statistically by using statistical analysis software (SAS) to calculate the analysis of variance (ANOVA).

Results

IL-4 was the only cytokine found to achieve statistically significant different levels in this study, with elevated levels of IL-4 in the tobacco smoke and vapor with nicotine groups compared to the levels found in the vapor without nicotine and air only groups (p = 0.0418). While statistically non-significant, prominent findings revealed up-regulation of TGF-β2 and TGF-β3 in the smoke group, but near-normal levels of TGF-β2 and TGF-β3 and suppression of IL-10 in the vapor groups (p > 0.05).

Conclusion

The potential utility of the murine model is established for studying the inflammatory and immune effects of tobacco smoke and vapor on the mammalian larynx. IL-4 levels in mice larynges were significantly elevated in the tobacco smoke and vapor with nicotine groups.

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.

Section snippets

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.

References (14)

  • A.M. Glasser et al.

    Overview of electronic nicotine delivery systems: a systematic review

    Am J Prev Med

    (2017)
  • S. Colak et al.

    Targeting TGF-β signaling in cancer

    Trends Cancer

    (2017)
  • V. Uloza et al.

    Characteristics of expression of matrix metalloproteinases (MMP-2 and MMP-9) in glottic squamous cell carcinoma and benign vocal fold lesions

    Clin Experiment Otorhinolaryngology

    (2015)
  • J.C. Luers et al.

    The impact of laryngeal dysplasia on the development of laryngeal squamous cell carcinoma

    Eur Arch Otorhinolaryngol

    (2014)
  • C.E. Steuer et al.

    An update on larynx cancer

    CA Cancer J Clin

    (2017)
  • J. Sanz-Ortega et al.

    3P21, 5Q21, 9P21 and 17P13 Allelic deletions accumulate in the dysplastic spectrum of laryngeal carcinogenesis and precede malignant transformation

    Histol Histopathol

    (2003)
  • F. Linkov et al.

    Early detection of head and neck cancer: development of a novel screening tool using multiplexed immunobead-based biomarker profiling

    Cancer Epidemiol Biomarkers Prev

    (2007)
There are more references available in the full text version of this article.

Cited by (0)

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.

View full text