ABSTRACT
BACKGROUND/AIMS: Nucleotide binding oligomerization domain 1 (NOD1) signal pathway and human ß defensins (hBDs) play crucial roles in innate immune. Cigarette smoke has been confirmed to dampen innate immune in some human tissues, such as oral mucosa. The aim of this study was to evaluate potential effects of smoking on NOD1 signaling and hBDs expression in oral mucosa. METHODS: Tissue specimens of normal oral mucosa were collected from donors undergoing routine surgical treatment. All 20 participants were classified equally as two groups: non-smokers and smokers. By using Western blotting and immunohistochemistry, we investigated differential expression of crucial molecules in NOD1 signal pathway, hBD-1, -2, and -3 in oral mucosa tissues between non-smokers and smokers. Immortalized human oral mucosal epithelial (Leuk-1) cells were treated with various concentrations of cigarette smoke extract (CSE) for 24h. Western blotting and immunofluorescence assays were performed to study CSE-induced alteration of protein expression. Leuk-1 cells were treated with 4% CSE, iE-DAP (NOD1 agonist), CSE + iE-DAP, BAY 11-7082 (NF-κB inhibitor), 4% CSE + BAY 11-7082, respectively. Real-time PCR and ELISA were performed to detect the mRNA levels and secretion of hBD-1, -2, and -3, respectively. RESULTS: The levels of NOD1, NF-κB, hBD-1 and hBD-3 significantly reduced in oral mucosa tissues of smokers compared with non-smokers. The levels of RIP2 (receptor-interacting protein 2), phospho-NF-κB (P-NF-κB) and hBD-2 remarkably enhanced in oral mucosal tissues of smokers. CSE treatment suppressed NOD1 and NF-κB expression and activated RIP2 and P-NF-κB expression in Leuk-1 cells. The mRNA and secretory levels of hBD-1 and -3 were down-regulated by CSE, while the mRNA and secretory level of hBD-2 were up-regulated by CSE. The iE-DAP or BAY 11-7082 treatment reversed the regulatory effects of CSE on levels of hBDs. CONCLUSION: The present study indicated that cigarette smoke could potentially modulate the expression of crucial molecules of NOD1 signal pathway and hBDs in human oral mucosal epithelium. NOD1 signal pathway could play an important role in the regulatory effects of CSE on hBDs levels in oral mucosal epithelial cells.
Subject(s)
Mouth Mucosa/immunology , Nod1 Signaling Adaptor Protein/immunology , Signal Transduction , Smoking/immunology , beta-Defensins/immunology , Adult , Cell Line , Cell Survival , Female , Gene Expression Regulation , Humans , Male , Middle Aged , Mouth Mucosa/metabolism , Mouth Mucosa/pathology , NF-kappa B/analysis , NF-kappa B/genetics , NF-kappa B/immunology , Nod1 Signaling Adaptor Protein/analysis , Nod1 Signaling Adaptor Protein/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/analysis , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/immunology , Smoking/genetics , Smoking/pathology , Young Adult , beta-Defensins/analysis , beta-Defensins/geneticsABSTRACT
BACKGROUND: Cigarette smoke a recognized risk factor for many systemic diseases and also oral diseases. Human beta defensins (HBDs), a group of important antimicrobial peptides expressed by the epithelium, are crucial for local defense and tissue homeostasis of oral cavity. The aim of this study was to evaluate potential effects of whole cigarette smoke (WCS) exposure on the expression and secretion of HBDs by oral mucosal epithelial cells. METHODS: Immortalized human oral mucosal epithelial (Leuk-1) cells were exposed to WCS for various time periods. HBD-1, -2 and -3 expression and subcellular localization were detected by real time qPCR, immunofluorescence assay and confocal microscopy. According to the relative fluorescent intensity, the expression levels of HBD-1, -2 and -3 were evaluated by digital image analysis system. The alteration of HBD-1, -2 and -3 secretion levels was measured by the Enzyme-Linked Immunosorbent Assay. RESULTS: WCS exposure remarkably attenuated HBD-1 expression and secretion while clearly enhanced HBD-2, -3 expression levels and HBD-2 secretion by Leuk-l cells. It appeared that there was no significant effect of WCS exposure on HBD-3 secretion. CONCLUSIONS: WCS exposure could modulate expression and secretion of HBDs by oral mucosal epithelial cells, establishing a link between cigarette smoke and abnormal levels of antimicrobial peptides. The present results may give a new perspective to investigate smoking-related local defense suppression and oral disease occurrence.
