Nicotine reduces TNF-α expression through a α7 nAChR/MyD88/NF-κB pathway in HBE16 airway epithelial cells

Department of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, Chongqing, P.R. China.
Cellular Physiology and Biochemistry (Impact Factor: 2.88). 06/2011; 27(5):605-12. DOI: 10.1159/000329982
Source: PubMed


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.

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    • "These molecules include STAT3, suppressors of cytokine signaling 1 and 3 (SOCS-1, SOCS-3), phosphatidylinositol 3-kinase/threonine protein kinase B (PI3K/Akt), myeloid differentiation primary response gene 88 (MyD88), and interleukin-1 receptor-associated kinase-M (IRAK-M). Interestingly, some of these negative regulators of TLR signaling (STAT3, SOCS-3, PI3K and MyD88s) have also been implicated in the anti-inflammatory effect mediated by α7 nAChRs in immune cells [16], [30]–[32]. However, the participation of others, like IRAK-M, is yet to be evaluated. "
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    ABSTRACT: Nicotine stimulation of α7 nicotinic acetylcholine receptor (α7 nAChR) powerfully inhibits pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated macrophages and in experimental models of endotoxemia. A signaling pathway downstream from the α7 nAChRs, which involves the collaboration of JAK2/STAT3 and NF-κB to interfere with signaling by Toll-like receptors (TLRs), has been implicated in this anti-inflammatory effect of nicotine. Here, we identifiy an alternative mechanism involving interleukin-1 receptor-associated kinase M (IRAK-M), a negative regulator of innate TLR-mediated immune responses. Our data show that nicotine up-regulates IRAK-M expression at the mRNA and protein level in human macrophages, and that this effect is secondary to α7 nAChR activation. By using selective inhibitors of different signaling molecules downstream from the receptor, we provide evidence that activation of STAT3, via either JAK2 and/or PI3K, through a single (JAK2/PI3K/STAT3) or two convergent cascades (JAK2/STAT3 and PI3K/STAT3), is necessary for nicotine-induced IRAK-M expression. Moreover, down-regulation of this expression by small interfering RNAs specific to the IRAK-M gene significantly reverses the anti-inflammatory effect of nicotine on LPS-induced TNF-α production. Interestingly, macrophages pre-exposed to nicotine exhibit higher IRAK-M levels and reduced TNF-α response to an additional LPS challenge, a behavior reminiscent of the 'endotoxin tolerant' phenotype identified in monocytes either pre-exposed to LPS or from immunocompromised septic patients. Since nicotine is a major component of tobacco smoke and increased IRAK-M expression has been considered one of the molecular determinants for the induction of the tolerant phenotype, our findings showing IRAK-M overexpression could partially explain the known influence of smoking on the onset and progression of inflammatory and infectious diseases.
    PLoS ONE 09/2014; 9(9):e108397. DOI:10.1371/journal.pone.0108397 · 3.23 Impact Factor
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    • "Furthermore, activation of α7nAChR has been shown to reduce IL-8 expression by epithelial cells of patients with cystic fibrosis [14] and human colon epithelial cells [15]. In addition, α7nAChR activation reduced TNF-α expression by HBE16 airway epithelial cells [16] and reduced expression of IL-6 and IL-8 from fibroblast-like synoviocytes [17]. "
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    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; 63(7). DOI:10.1007/s00011-014-0725-5 · 2.35 Impact Factor
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    • "The intracellular mechanisms of the cholinergic anti-inflammatory action have been investigated in a number of different systems. In HBE16 airway epithelial cells, it was shown that nicotine reduces TNF-α expression at the transcriptional level by blocking NF-κB activation38. In RAW267.4 cells, it was found that nicotine inhibits LPS-induced NO synthesis by suppressing p42/44 MAPK and S6K1-mediated STAT3 phosphorylation at serine 72739. "
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    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; 23(11). DOI:10.1038/cr.2013.116 · 12.41 Impact Factor
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