Nicotine reduces TNF-α expression through a α7 nAChR/MyD88/NF-ĸB pathway in HBE16 airway epithelial cells.
ABSTRACT To explore the signaling mechanism associated with the inhibitory effect of nicotine on tumor necrosis factor (TNF)- α expression in human airway epithelial cells.
HBE16 airway epithelial cells were cultured and incubated with either nicotine or cigarette smoke extract (CE). Cells were then transfected with α1, α5, or α7 nicotinic acetylcholine receptor (nAChR)-specific small interfering RNAs (siRNAs). The effects of nicotine on the production of proinflammatory factors TNF-α, in transfected cells were analyzed. Furthermore, we assayed the expression levels of myeloid differentiation primary response gene 88 (MyD88) protein, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 protein, NF-κB activity and NF-κB inhibitor alpha (I-κBα) expression in cells after treatment with nicotine or α7 nAChR inhibitor, α -bungarotoxin (α-BTX).
The production of TNF-α was lower in cells pretreated with nicotine before lipopolysaccharide (LPS) stimulation, compared with LPS-only-treated cells. In contrast, in α7 siRNA-transfected cells incubated with nicotine and LPS, TNF-α expression was higher than that in non-transfected cells or in α1 or α5 siRNA-transfected cells. Addition of MyD88 siRNA or the NF-κB inhibitor pyridine-2,6-dithiocarboxylic acid (PDTC) also reduced TNF-α expression. Furthermore, we found that nicotine decreased MyD88 protein, NF-κB p65 protein, NF-κB activity and phospho-I-κBα expression induced by CE or LPS. The inhibitor α-BTX could reverse these effects.
Nicotine reduces TNF-α expression in HBE16 airway epithelial cells, mainly through an α7 nAChR/MyD88/NF-κB pathway.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: The alpha 7 nicotinic receptor (α7nAChR) is expressed by oral keratinocytes. α7nAChR activation mediates anti-inflammatory responses. The objective of this study was to determine if α7nAChR activation inhibited pathogen-induced interleukin-8 (IL-8) expression by oral keratinocytes. Periodontal tissue expression of α7nAChR was determined by real-time PCR. OKF6/TERT-2 oral keratinocytes were exposed to Porphyromonas gingivalis in the presence and absence of a α7nAChR agonist (PHA-543613 hydrochloride) alone or after pre-exposure to a specific α7nAChR antagonist (α-bungarotoxin). Interleukin-8 (IL-8) expression was measured by ELISA and real-time PCR. Phosphorylation of the NF-κB p65 subunit was determined using an NF-κB p65 profiler assay and STAT-3 activation by STAT-3 in-cell ELISA. The release of ACh from oral keratinocytes in response to P. gingivalis lipopolysaccharide was determined using a GeneBLAzer M3 CHO-K1-bla cell reporter assay. Expression of α7nAChR mRNA was elevated in diseased periodontal tissue. PHA-543613 hydrochloride inhibited P. gingivalis-induced expression of IL-8 at the transcriptional level. This effect was abolished when cells were pre-exposed to a specific α7nAChR antagonist, α-bungarotoxin. PHA-543613 hydrochloride downregulated NF-κB signalling through reduced phosphorylation of the NF-κB p65-subunit. In addition, PHA-543613 hydrochloride promoted STAT-3 signalling by maintenance of phosphorylation. Furthermore, oral keratinocytes upregulated ACh release in response to P. gingivalis lipopolysaccharide. These data suggest that α7nAChR plays a role in regulating the innate immune responses of oral keratinocytes.Agents and Actions 03/2014; · 1.59 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: B-cells are prominent immune cells in established periodontitis lesions. Tumour necrosis factor superfamily (TNFSF) cytokines play roles in supporting B-cell function as well as bone re-modelling. The influence of smoking on factors that support B-cell function in periodontitis remains unclear. To investigate plasma concentrations of TNF (TNSF1A), soluble receptor activator of nuclear-factor Kappa-B ligand (sRANKL/TNFSF11), a proliferation-inducing ligand (APRIL/TNFSF13), B-cell activating factor (BAFF/TNFSF13B) and Osteoprotegerin (OPG/TNFRSF11B) in smokers and non-smokers with and without chronic periodontitis Plasma concentrations of TNFSF and OPG were evaluated in 200 systemically healthy subjects divided into four groups: non-smokers with periodontitis (n = 101), smokers with periodontitis (n = 55), healthy non-smokers (n = 27) and healthy smokers (n = 17). Periodontitis patients had significantly higher plasma sRANKL, TNF, APRIL and BAFF and lower OPG than healthy subjects (p < 0.01). TNF and sRANKL were significantly greater in smokers with periodontitis (p = 0.011, p = 0.001) and OPG concentrations significantly lower (p = 0.001), whereas APRIL or BAFF were little changed. Plasma APRIL, BAFF, sRANKL and TNF correlated with probing depth and clinical attachment loss. TNFSF cytokines correlate with periodontitis disease severity. However, only TNF, sRANKL and OPG levels were altered by cigarette smoking. APRIL and BAFF appear as good indicators of disease severity.Journal Of Clinical Periodontology 09/2013; 40(9):875-82. · 3.69 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The vagus nerve can control inflammatory response through a 'cholinergic anti-inflammatory pathway', which is mediated by the α7-nicotinic acetylcholine receptor (α7nAChR) on macrophages. However, the intracellular mechanisms that link α7nAChR activation and pro-inflammatory cytokine production remain not well understood. In this study, we found that miR-124 is upregulated by cholinergic agonists in LPS-exposed cells and mice. Utilizing miR-124 mimic and siRNA knockdown, we demonstrated that miR-124 is a critical mediator for the cholinergic anti-inflammatory action. Furthermore, our data indicated that miR-124 modulates LPS-induced cytokine production by targeting signal transducer and activator of transcription 3 (STAT3) to decrease IL-6 production and TNF-α converting enzyme (TACE) to reduce TNF-α release. These results also indicate that miR-124 is a potential therapeutic target for the treatment of inflammatory diseases.Cell Research advance online publication 27 August 2013; doi:10.1038/cr.2013.116.Cell Research 08/2013; · 10.53 Impact Factor