Aim:
Smoking is a major risk factor for periodontitis and has been shown to impact the oral microbiome, as well as the host. However, studies on the effect of smoking on the transcriptome of healthy normal gingiva are lacking. The purpose of this study was to analyze the effects of smoking on the normal healthy human gingiva transcriptome.

Methods:
Palatal biopsies, in the form of a harvested connective tissue graft, were obtained from periodontally healthy smokers and non-smokers in a prospective, experimental study. Smoking status was determined by self-reporting (≥10cigarettes/day for ≥5 years; never smoker) and verified biochemically (exhaled air CO and serum cotinine levels). Tissue samples were stored frozen until processed for RNA isolation. Following RNA preparation, gene expression was analyzed by next generation sequencing (RNASeq) and real-time PCR. Gene set enrichment/pathway analysis was also performed.

Results:
Tissue specimens were obtained from 24 consented participants. Two smokers were found to have consistently low biochemical values (CO and cotinine) and their samples were excluded. Therefore, transcriptome data were based on samples from 10 smokers and 12 non-smokers. 830 significantly (p<0.05) differentially expressed genes were identified. Of those genes with >2 fold-change (FC) in expression, 98 genes were up-regulated and 151 were down-regulated. CYP1A1 was the most highly up-regulated gene (17.8 FC) and IL36A, DEFB4A, DEFB4B, and SPRR2F were the most highly down-regulated (≥8 FC) genes. Several among the significantly down-regulated genes (e.g., DEFB4A, DEFB4B, CCL20, s100A7A) are reportedly involved in innate immune responses and/or antimicrobial activity.

Conclusions:
Smoking has a significant effect on the gingival transcriptome of normal human gingiva and it seems to negatively impact the ability of the tissues to defend against bacteria; this could partly explain the reported changes in the gingival microbiome of periodontally healthy patients.

Funding:
This study was supported by the authors' institution.