Inhibitory effects of nicotine derived from cigarette smoke on thymic stromal lymphopoietin production in epidermal keratinocytes

Cellular Immunology xxx (2016) xxx–xxx

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Research paper

Inhibitory effects of nicotine derived from cigarette smoke on thymic stromal lymphopoietin production in epidermal keratinocytes Jiangxu Dong, Ryosuke Segawa, Natsumi Mizuno, Masahiro Hiratsuka, Noriyasu Hirasawa ⇑ Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan

a r t i c l e

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Article history: Received 30 September 2015 Revised 31 December 2015 Accepted 1 January 2016 Available online xxxx Keywords: TSLP Nicotine CSE Cell signaling AD

a b s t r a c t Thymic stromal lymphopoietin (TSLP) is regarded as the main factor responsible for the pathogenesis of atopic dermatitis (AD). Cigarette smoke is an aggravating factor for allergies, but has been reported to decrease the risk of AD. In the present study, we evaluated the role of nicotine, the main constituent in cigarette smoke extract, and its underlying mechanism of action in the regulation of TSLP expression. We found that nicotine significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced TSLP expression in BALB/c mice and the mouse keratinocyte cell line PAM212. Nicotine inhibition of TSLP production was abolished by pretreatments with a7 nicotinic acetylcholine receptor (a7 nAChR) antagonists, AMP-activated protein kinase (AMPK) inhibitor, and phosphoinositide 3-kinase (PI3K) inhibitors. The same inhibitors abolished inhibition of nuclear factor-jB (NF-jB) activation by nicotine. These results suggest that nicotine inhibits the expression of TSLP by suppressing the activation of NF-jB through the a7 nAChR–PI3K–AMPK signaling pathway. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction The prevalence and severity of atopic dermatitis (AD) have been increasing in recent years, and this condition is caused by allergeninduced T helper 2 (Th2) cellular responses characterized by the production of Th2 cytokines [1,2]. Large amounts of thymic stromal lymphopoietin (TSLP), an interleukin-7 (IL-7)-like cytokine, are produced by epithelial cells in AD lesions. TSLP appears to be a master regulator of Th2-type allergic inflammation by inducing the maturation, activation, and migration of dendritic cells (DCs) (Langerhans cells) and Th2-type lymphocytes [3–7]. The overexpression of TSLP in keratinocytes has been shown to trigger an AD-like skin phenotype in mice, suggesting that it is responsible for the initiation, maintenance, and aggravation of AD [8–10]. Previous studies have demonstrated that the mitogen-activated protein kinase (MAPK) and nuclear factor-jB (NF-jB) pathways play crucial roles in the expression of TSLP [11,12].

Abbreviations: TSLP, thymic stromal lymphopoietin; AD, atopic dermatitis; MAPK, mitogen-activated protein kinase; NF-jB, nuclear factor-jB; ERK, extracellular signal-regulated kinase; nAChR, nicotinic acetylcholine receptor; PI3K, phosphoinositide 3-kinase; AMPK, AMP-activated protein kinase; CSE, cigarette smoke extract; TPA, 12-O-tetradecanoylphorbol-13-acetate; MLA, methyllycaconitine; a-BTX, a-bungarotoxin; MCL, mecamylamine. ⇑ Corresponding author. E-mail address: [email protected] (N. Hirasawa).

Among several indoor pollutants, cigarette smoke has been suggested as a risk factor contributing to the development of AD [13–15]. On the other hand, exposure to cigarette smoke during early adult life as a result of parental smoking or personal smoking habits during adolescence is associated with a lower risk of allergic disease [16–18]. Although the toxicity and allergenicity associated with cigarette smoke may be due to the combined action of a complex mixture of more than 4700 chemical compounds, nicotine is the main, as well as most abundant, active constituent of the pathogenic compounds in cigarette smoke [19]. Increasing evidence indicates that nicotine exerts an anti-inflammatory effect on some inflammatory diseases including ulcerative colitis and obesity [20,21], and inhibits the production of inflammatory cytokines such as interleukins (ILs) [22,23], tumor necrosis factor-a (TNF-a) [24,25] and highmobility group box-1 (HMGB1) [26] in multiple cell types [27]. Nicotine exerts its effects through the activation of heteromeric nicotinic acetylcholine receptors (nAChRs), formed by a combination of a4 and b2 subunits, and/or homomeric nAChRs composed of a7 subunits (a7 nAChRs). These receptors exhibit high nicotine affinity and are located on non-neuronal cells [28–30]. The antiinflammatory effects of nicotine are mediated by the activation of a7 nAChRs expressed in many different tissues and cells, including immune system cells such as macrophages and DCs, airway epithelial cells and endothelial cells [22,24,27,30]. Nicotine inhibits

http://dx.doi.org/10.1016/j.cellimm.2016.01.001 0008-8749/Ó 2016 Elsevier Inc. All rights reserved.

Please cite this article in press as: J. Dong et al., Inhibitory effects of nicotine derived from cigarette smoke on thymic stromal lymphopoietin production in epidermal keratinocytes, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.01.001

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signaling by NF-jB, a pro-inflammatory transcription factor [24–26], or induces the expression of tristetraprolin (TTP) via the Janus kinase2-signal transducer and activator of transcription 3 (JAK2–STAT3) pathway, thereby increasing the decay of messenger RNA (mRNA) [31,32]. In addition, the phosphoinositide 3-kinase (PI3K) and AMP-activated protein kinase (AMPK) pathways may converge and cooperate to relay signals that intervene in the activation of nAChRs and NF-jB, and thereby interfere with the production of various cytokines [27,33,34]. However, epidermal keratinocytes are the most prevalent cell type in the skin, and the effects of nicotine on TSLP production and the mechanisms underlying its regulation have not yet been elucidated. Therefore, we investigated the role of nicotine in epidermal TSLP expression. This is based on the hypothesis that nicotine adsorption on the skin surface through natural moisture or perspiration plays a direct role in modifying the Th1/Th2 balance in skin lesions [35]. We also examined the effects of cigarette smoke extract (CSE) and nicotine on the regulation of TSLP in the mouse keratinocyte cell line PAM212 derived from BALB/c mice. Our results showed that treatment of keratinocytes with CSE and nicotine did not induce the expression of TSLP, but did inhibit TPAinduced TSLP expression.

2.4. Treatment of cells PAM212 cells (1
105 cell/mL) in MEMa medium were seeded in 6-well culture plates for the preparation of cell lysates, 12-well culture plates for RNA extraction, and 24-well culture plates for cell culture supernatants. Cell cultures at 70–80% confluency were stimulated with 30 nM TPA for an appropriate period, with or without pre-incubation with nicotine (2–200 lM) for 1 h. In some experiments, cells were pretreated 30 min before the addition of nicotine with inhibitors such as LY294002 and wortmannin for PI3K, compound C for AMPK, and a-BTX, MLA, and MCL for nAChRs. The viability of PAM212 cells was assessed using the MTT cell proliferation assay, and shown to be greater than 90% regardless of the 24 h TPA treatment applied. 2.5. Enzyme-linked immunosorbent assay (ELISA) of TSLP At the times indicated, supernatants of the culture media were collected and stored at 30 °C. TSLP levels were assayed with the DuoSetÒ ELISA Development System (R&D Systems, Minneapolis, MN, USA) according to the manufacturer instructions. 2.6. Western blotting

2. Materials and methods 2.1. Materials 12-O-Tetradecanoylphorbol-13-acetate (TPA), nicotine, and mecamylamine (MCL) were purchased from Wako Pure Chemical Ind. (Osaka, Japan). a-Bungarotoxin (a-BTX) and methyllycaconitine (MLA) were obtained from Abcam Biochemicals (Cambridge, MA, USA), and TNF-a was acquired from PeproTech, Inc. (Rocky Hill, NJ, USA). LY294002 and wortmannin were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Compound C (dorsomorphin) was acquired from Sigma–Aldrich (St. Louis, MO, USA).

2.2. Preparation of CSE An extemporaneous preparation of CSE was created using commercially available cigarettes (Hi-lite, Japan Tobacco Inc., nominal nicotine 1.6 mg). Briefly, 50 mL of smoke was drawn slowly through a connector into a syringe containing either 5 mL of Minimum Essential Medium alpha (MEMa) culture medium (Life Technologies, Grand Island, NY, USA) or 2 mL of deionized (DI) water and vortexed for 1 min until fully dissolved. The resulting solutions were defined as 100% and 250% CSE, respectively. CSE was then passed through a 0.22-lm Millipore filter (Millipore Corp., Bedford, MA, USA) for immediate use. The concentration of nicotine in the 100% CSE solution was estimated to be approximately 2.0 mM based upon its absorbance at 260 nm.

2.3. Cell culture The murine keratinocyte cell line PAM212 [36] derived from BALB/c mouse skin was kindly provided by Dr. Yuspa (National Institute of Health, NCI, MD, USA), and cultured in MEMa supplemented with 10% heat-inactivated fetal bovine serum, penicillin G potassium (15 lg/mL), and streptomycin (50 lg/mL) (Meiji Seika, Tokyo, Japan). The preparation was maintained at 37 °C in a humidified 5% CO2/95% air atmosphere. These cells were used within five to 12 passaging cycles, and cell cultures were passaged every 3–4 days.

Western blotting was performed as described previously [37]. The monoclonal antibodies (mAbs) used as the primary antibodies included: (1) phospho-IjB-a (Ser 32) Rabbit mAb (New England BioLabs Inc., Ipswich, MA, USA), (2) IjB-a (Arg 29) Rabbit mAb, (3) phospho-AMPKa (Thr172) (40H9) Rabbit mAb, AMPKa (#2532) Rabbit mAb, (4) phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb, (5) p44/42 MAPK (Erk1/2) Rabbit mAb (Cell Signaling Technology, Inc., Danvers, MA, USA), and (6) NFjB p65 (C-20) Rabbit mAb (Santa Ceruz Biotechnology, Inc., Dallas, TX, USA). 2.7. Preparation of nuclear extracts PAM212 cells stimulated with TPA (30 nM) for 45 min were used to prepare nuclear extracts with a nuclear extraction kit (Active Motif Corp, Carlsbad, CA, USA) according to the manufacturer instructions. The extracts were stored at 80 °C for subsequent analyses. 2.8. Real-time RT-PCR Total RNA was prepared from cultured PAM212 cells at the end of the incubation period using RNAiso plus (Takara Bio Company, Shiga, Japan), and cDNA was synthesized from total RNA by reverse transcription with PrimeScriptTM RT master mix (Takara) according to the manufacturer instructions. Real-time PCR was performed using SYBRÒ premix Ex Taq IITM (Takara) in a Thermal Cycler DiceÒ Real Time System TP800 (Takara). The following primers were used: GAPDH (forward: 50 -TGTGTCCGTCGTGGATCTGA-30 ; reverse: 50 -TT GCTGTTGAAGTCGCAGGAG-30 ), and TSLP (forward: 50 -AGCTTGTCT CCTGAAAATCGAG-30 , reverse: 50 -AGGTTTGATTCAGGCAGATGTT-30 ). Relative quantification was based upon the expression levels of a target gene versus the reference GAPDH gene. Cycle threshold (Ct) values were calculated using the Second Derivative Maximum Method. Immediately after PCR, primer specificity was assessed using a melting curve analysis in the same reaction tube. 2.9. Animals Four-week-old male BALB/c mice were purchased from Japan SLC, Inc. (Shizuoka, Japan), raised in standard polyacrylamide cages, and housed in a specific pathogen free (SPF) animal facility

Please cite this article in press as: J. Dong et al., Inhibitory effects of nicotine derived from cigarette smoke on thymic stromal lymphopoietin production in epidermal keratinocytes, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.01.001

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maintained at 22 ± 2 °C, and under a controlled 12-h light/12-h dark cycle. All guidelines related to the ethical treatment of research animals were followed and approved by the Tohoku University Animal Ethics Committee. TPA (20 lg/mL) and nicotine (5 mM) were dissolved in ethanol and DI water respectively, and 20 lL of each solution was smeared on the auricles of mice. Auricular tissue punches 8 mm in diameter were obtained after 24 h and weighed. Tissue samples were homogenized at 4 °C in 10 volumes (w/v) of ice-cold phosphate-buffered saline using a PrecellysÒ 24 Beads Cell Disrupter (Bertin Technologies, France). The concentrations of TSLP in the homogenate supernatants were determined by ELISA as described above.

3.2. a7 nAChR antagonists attenuated the nicotinic inhibition of TPA-induced TSLP production

2.10. Statistical analysis

3.3. Nicotine inhibited TPA-induced TSLP production in vivo via a7 nAChRs

The statistical significance of the results was determined using Dunnett’s test or the Student–Newman–Keuls test for multiple comparisons. 3. Results 3.1. TPA-induced TSLP production in keratinocytes was inhibited by pre-exposure to CSE and nicotine In order to determine the effects of CSE on TSLP production under physiological and pathological conditions, PAM212 cells were stimulated for 24 h with 10% CSE alone, or in the presence of 30 nM TPA. As shown in Fig. 1a, CSE did not affect TSLP production, but it did significantly reduce TPA-induced TSLP production (p < 0.01). Since the estimated nicotine concentration in 10% CSE was approximately 200 lM based on UV absorbance at 260 nm (data not shown), the effects of nicotine at concentrations lower than 200 lM were also examined. The results demonstrate that nicotine also reduced the TPA-induced expression of TSLP in a dose-dependent manner (Fig. 1b). A similar dose-dependent inhibition of TSLP production induced by the endogenic stimulant TNF-a (30 ng/mL) was also exhibited (Fig. 1c). Cell viability was not affected by any stimulation (data not shown). Nicotine significantly reduced TPA-induced increases in TSLP mRNA levels (p < 0.01) (Fig. 1d), indicating that nicotine inhibited the production of TSLP at the transcription level.

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In order to clarify the subtype of the nAChR responsible for the inhibition of TSLP production in PAM212 cells, the effects of three types of antagonists were examined. As shown in Fig. 2, pretreatment with MLA and a-BTX significantly reversed nicotineinduced reductions in TPA-induced TSLP production by 66.3% and 84.7%, respectively. In contrast, pretreatment with the nonselective antagonist MCL, which acts on the combination of a and b subunits of nAChR, was completely ineffective. These results suggest the involvement of a7 nAChRs in the inhibitory effects of nicotine.

We previously reported that the topical application of TPA on mice auricles induced significant production of TSLP at 24 h [38]. Using this murine model, we determined whether CSE and nicotine had inhibitory effects on TSLP production in vivo. We painted CSE or nicotine on the surface of the auricles of BALB/c mice once a day for 7 days, and TPA was then applied to the same region. TSLP levels were measured in the homogenates of whole-mount auricles excised 24 h after TPA treatment. As shown in Fig. 3a, TPA induced a marked increase in TSLP levels, and this was significantly reduced by the treatment with either CSE or nicotine. Long-term and single exposures to nicotine also attenuated TPA-induced increases in TSLP levels, and these were blocked by peritoneal injection of MLA prior to nicotine treatment (Fig. 3b). These results suggest that exposure to nicotine had inhibitory in vivo effects on TSLP production primarily through a7 nAChRs. 3.4. PI3-kinase and AMP-activated protein kinase signaling cascades participate in the inhibitory effects of nicotine on TPA-induced TSLP production In an attempt to elucidate the downstream effects of a7 nAChRs activation, we focused on the PI3K and AMPK signaling cascades. The PI3K inhibitors, wortmannin and LY294002, and AMPK inhibitor, compound C, did not affect TPA-induced TSLP production. However, the inhibitory effects of nicotine on TSLP production

Fig. 1. Effects of CSE and nicotine on TSLP expression. PAM212 cells were treated with CSE (10%) (a) or nicotine at the indicated concentrations (b–d) for 1 h before stimulation with TPA (30 nM) (a, b, and d) or TNF-a (30 ng/mL) (c). TSLP levels in the culture supernatant collected after 24 h (a and b) or 48 h (c), and mRNA after 4 h (d) were determined. Data are shown as the mean ± S.E.M. of four samples. Significance: *p < 0.05, **p < 0.01 vs. the corresponding stimulation group.

Please cite this article in press as: J. Dong et al., Inhibitory effects of nicotine derived from cigarette smoke on thymic stromal lymphopoietin production in epidermal keratinocytes, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.01.001

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Fig. 2. Involvement of a7 nAChR in inhibitory effects of nicotine on TPA-induced TSLP production. PAM212 cells were treated with nicotine (NT, 200 lM) for 1 h in the presence or absence of methyllycaconitine (MLA, 2 lM) (a), a-bungarotoxin (a-BTX, 10 lM) (b), and mecamylamine (MCL, 50 lM) (c), and were then stimulated with TPA (30 nM) for 24 h. TSLP levels in the culture supernatant were determined. Data are shown as the mean ± S.E.M. of four samples. Significance: **p < 0.01 vs. TPA alone, ## p < 0.01 vs. TPA + NT group.

with a-BTX, compound C, and wortmannin (Fig. 4e). These results suggest that PI3K and AMPK signaling pathways contributed to nicotine-induced reductions in TSLP transcription. 3.5. Nicotine-induced PI3K activation was required for AMPK activation The phosphorylation of AMPK was accelerated 10 min following the initiation of nicotine stimulation, and it was maintained for a period of 1 h or longer (Fig. 5a). Furthermore, the observed increase in AMPK phosphorylation by nicotine was significantly abolished with a-BTX, wortmannin, and compound C pretreatments (Fig. 5b). These results suggest that the nicotine-induced phosphorylation of AMPK was related to the activation of PI3K via the a7 nAChRs, and was responsible for the inhibitory effects of nicotine on TPAinduced TSLP production. 3.6. Nicotine inhibition of TPA-induced IjBa phosphorylation and nuclear translocation of p65

Fig. 3. Effects of CSE and nicotine on TPA-induced TSLP production in vivo. (a) Twenty microliters of CSE (250%) or nicotine (NT, 5 mM) was painted on the auricles of BALB/c mice once per day for 7 days, and 20 lL of TPA (20 lg/mL) was then smeared on the same region. (b) MLA (5 mg/kg) was intraperitoneally administered 10 min prior to the treatment with nicotine (5 mM, once), and mice were then stimulated with TPA in the same manner. TSLP levels in the homogenates of auricles excised at 24 h were determined. Data are shown as the mean ± S.E.M. of 10 samples. Significance: **p < 0.01 vs. TPA alone, ##p < 0.01 vs. the TPA + NT group.

were completely abolished by wortmannin and LY294002 (Fig. 4a and b) as well as compound C (Fig. 4c). The involvement of AMPK was confirmed by using the AMPK activator metformin (Metf). At a concentration of 1000 lM, Metf significantly reduced TPA-induced TSLP production (Fig. 4d). The nicotine-induced reduction in TSLP mRNA levels was also reversed by pretreatment

In order to understand the mechanism by which nicotine inhibits the TPA-induced TSLP secretion in PAM212 cells, we investigated the effects of nicotine on IjBa phosphorylation and p65 nuclear accumulation as indices of NF-jB activation. We also examined the phosphorylation of ERK in response to TPA stimulation. TPA treatment resulted in IjBa phosphorylation at 15 min, and the nuclear translocation of p65 at 45 min. These responses gradually diminished at later time points (data not shown). The TPA-induced nuclear translocation of p65 (Fig. 6a) and phosphorylation of IjBa (Fig. 6b) were suppressed by nicotine, but this effect was reversed by pretreatment with wortmannin, compound C, and a-BTX. In contrast, TPA-induced phosphorylation of ERK was not inhibited by nicotine (Fig. 6c). These results suggest that nicotine/a7nAChR signaling weakened the NF-jB signaling pathway rather than the MAPK signaling pathway, with corresponding reductions in the production of TSLP in PAM212 cells. 4. Discussion Cigarette smoking is regarded as a potential risk factor for AD. However, previous findings from individual epidemiological

Please cite this article in press as: J. Dong et al., Inhibitory effects of nicotine derived from cigarette smoke on thymic stromal lymphopoietin production in epidermal keratinocytes, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.01.001

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Fig. 4. Involvement of PI3K and AMPK in inhibitory effects of nicotine on TPA-induced TSLP expression in PAM212 cells. PAM212 cells were treated with nicotine (NT, 200 lM) in the presence or absence of the inhibitors wortmannin (Wort, 200 nM) (a and e), LY294002 (LY, 1 lM) (b), and compound C (Com. C, 1 lM) (c and e), or abungarotoxin (a-BTX, 10 lM) (e). (d) Cells were treated with metformin (Metf, 10–1000 lM) for 1 h, and then stimulated with TPA (30 nM). Cells were then incubated for 24 h for the determination of TSLP protein levels (a–d) or 4 h for TSLP mRNA levels (e). Data are shown as the mean ± S.E.M. of four samples. Significance: **p < 0.01 vs. TPA alone, ##p < 0.01 vs. the TPA + NT group.

Fig. 5. Nicotine-induced phosphorylation of AMPK and inhibition by a7 nAChR and PI3K inhibitors. PAM212 cells were treated with nicotine (NT, 200 lM) for the indicated time (a), or for 10 min in the presence or absence of compound C (Com. C, 1 lM), wortmannin (Wort, 200 nM), or a-bungarotoxin (a-BTX, 10 lM) (b). The phosphorylation of AMPK was determined by western blotting.

studies remain controversial [13–18,39,40]. TSLP is a cytokine that promotes Th2 cytokine-dominant immune responses, and has emerged as a central player in the occurrence and development of AD [3,5,6,8,9]. Although CSE has been shown to induce TSLP expression in the lungs and airways [41,42], its effects on skinderived TSLP currently remain unknown. Therefore, we investigated the effects of CSE and nicotine on TSLP production in epidermal keratinocytes.

Regardless of the time span or frequency (up to 1 week), no significant differences in TSLP production were observed in vitro or in vivo when cells were exposed to CSE. In contrast, CSE downregulated the expression of TSLP under inflammatory conditions, and this may be an effect attributable to nicotine, the main active component of CSE. Particles smaller than 2.5 lm (PM2.5) are the major inflammatory constituents of cigarette smoke. These particles easily enter deep into the airways and activate alveolar macrophages, thereby potentially inducing TSLP production in epithelial cells. However, the skin is composed of multiple layers of dead stratified squamous cells that form a compact mechanical barrier that provides more substantial resistance to many environmental factors. Therefore, painting CSE on normal skin in our experiment did not induce TSLP production. This may be due to the influence of the skin barrier, and its ability to prevent the penetration of particles, but not nicotine. Recent studies have shown that nicotine suppressed the production of inflammatory cytokines in nonneuronal cells, such as macrophages, DCs and endothelial cells [22,23,25,27,30,31], mainly via a7 nAChR and/or a4b2 nAChR [29,31] inhibition. In the present study, the a7 nAChR selective antagonists MLA and a-BTX blocked the reduction in TSLP generated by nicotine in PAM212 cells and BALB/c mice. This effect was not seen with the antagonist MCL because its blocking action may require the presence of b subunits. These results suggested that the a7 nAChRs contributes to the mechanism underlying the inhibition of TSLP production. PI3K is activated via the a7 nAChR, and it functions as a negative regulator of the production of inflammatory cytokines such

Please cite this article in press as: J. Dong et al., Inhibitory effects of nicotine derived from cigarette smoke on thymic stromal lymphopoietin production in epidermal keratinocytes, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.01.001

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Fig. 6. Effects of nicotine on TPA-induced activation of NF-jB and ERK. PAM212 cells were treated with nicotine (NT, 200 lM), then the stimulated with TPA (30 nM) for 45 min (a) or 10 min (b and c). Cells were treated with the proteasome inhibitor MG132 (10 lM) in the presence or absence of wortmannin (Wort, 200 nM), compound C (Com. C, 1 lM), or a-bungarotoxin (a-BTX, 10 lM) (b). A nuclear fraction and cytosol fraction (a) or total cell lysate (b and c) was prepared. p65 and phosphorylated IjBa and ERK levels were determined by western blotting.

as TNF-a in macrophages [33,43]. The results obtained with PI3K inhibitors suggest that the activation of PI3K associated a7 nAChRs were required to elicit the inhibitory effects of nicotine on TPAinduced TSLP production. Furthermore, previous studies showed that the ability of nicotine to inhibit TNF-a production in lipopolysaccharide (LPS)activated macrophages was generally restricted to either the JAK2–STAT3 signaling pathway [31,32] or the AMPK signaling pathway [33,34] by means of the a7 nAChR. The involvement of the JAK2–STAT3 pathway in TSLP inhibition was rejected because NSC74859 (10–100 lM), a selective inhibitor of STAT3 DNAbinding activity, had no effect on actions of nicotine (data not shown). Although the inhibitory effects of nicotine mediated by STAT3 signaling are due to the expression of TTP, a protein that binds to adenylate/uridylate-rich elements of mRNA and promotes their degradation in a number of inflammatory mediators such as TNF-a, IL-6, and COX-2 [32], no specific recognition sequence was identified in TSLP mRNA. In contrast, the involvement of AMPK was indicated in these experiments. Specifically, the AMPK inhibitor compound C was found to be an effective blocker of the inhibitory effects of nicotine on TSLP production. Metf is an AMPK activator, and was found to exert the same effects as nicotine. Additionally, western blot analysis showed that nicotine induced the phosphorylation of AMPK, whereas pretreatment with inhibitors for a7 nAChRs, PI3K, and AMPK blocked this process. These

results suggest that the inhibitory effects of nicotine on TSLP production were mediated by the activation of AMPK, which is related to the activation of PI3K via the a7 nAChRs. Since TSLP expression was modulated by nicotine at the transcriptional level, we considered it important to further determine the altered transcription factors for TSLP production. Activation of ERK1/2 and NF-jB were two critical signaling routes indicated in the transcription of TSLP. TNF-a and IL-1b induced TSLP expression was previously reported to be suppressed by an ERK1/2 inhibitor [11], and the binding of NF-jB upstream of the TSLP transcription initiation site was shown to be essential for the induction of TSLP gene expression [12]. We found that TPA induced the translocation of NF-jB p65 into the nucleus after 45 min, and this was partially blocked by nicotine pretreatment. In contrast, although ERK1/2 phosphorylation was also induced by TPA stimulation, no change was observed at the maximum time point (10 min) with the nicotine treatment. IjBa is the main regulator of NF-jB because it keeps the p65 subunit sequestered in the cytoplasm in the resting state, and its degradation is essential for transcriptional activity. The phosphorylation and degradation (data not shown) of IjBa in response to TPA was inhibited by nicotine pretreatment, and this was blocked by pretreatments with inhibitors of a7 nAChRs, AMPK, and PI3K. These results suggest that nicotine activates PI3K and AMPK via the a7 nAChR, resulting in the downregulation of NF-jB activation. Taken together, these results indicate that nicotine impedes the expression of TSLP in the skin. This may in turn attenuate Th2-type immune responses while simultaneously augmenting cellmediated immunity by skewing the differentiation of DCs. On the other hand, there is also increasing evidence that cigarette smoke produces high LPS concentrations in indoor air [44,45], and not only induces Staphylococcus aureus biofilm formation, but also increases its adherence to human cells [46]. Therefore, the inhibition of inflammatory cytokines and antimicrobial peptide [47] by cigarette smoke may contribute to an increased risk of bacterial infection. Furthermore, since the barrier function of the skin is compromised in AD lesions, particles in CSE may elicit inflammation [48]. Thus, the effects of exposure to cigarette smoke may differ depending on the pathological condition of patients. In summary, our results demonstrated that the CSE surrogate for smoking exhibited inhibitory effects on the production of TSLP in response to skin irritation, and that these effects may have been caused by nicotine via the a7 nAChR–PI3K–AMPK pathway and the down-regulation of NF-jB activation. Acknowledgment This study was supported in part by the Smoking Research Foundation. References [1] R.M. Locksley, Asthma and allergic inflammation, Cell 140 (2010) 777–783. [2] J.M. Spergel, A.S. Paller, Atopic dermatitis and the atopic march, J. Allergy Clin. Immunol. 112 (2003) S118–S127. [3] T. Ito, Y.H. Wang, O. Duramad, T. Hori, G.J. Delespesse, N. Watanabe, F.X. Qin, Z. Yao, W. Cao, Y.J. Liu, TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand, J. Exp. Med. 202 (2005) 1213– 1223. [4] J. Yoo, M. Omori, D. Gyarmati, B. Zhou, T. Aye, A. Brewer, M.R. Comeau, D.J. Campbell, S.F. Ziegler, Spontaneous atopic dermatitis in mice expressing an inducible thymic stromal lymphopoietin transgene specifically in the skin, J. Exp. Med. 202 (2005) 541–549. [5] Y.J. Liu, Thymic stromal lymphopoietin: master switch for allergic inflammation, J. Exp. Med. 203 (2006) 269–273. [6] S. Nakajima, B.Z. Igyarto, T. Honda, G. Egawa, A. Otsuka, M. Hara-Chikuma, N. Watanabe, S.F. Ziegler, M. Tomura, K. Inaba, Y. Miyachi, D.H. Kaplan, K. Kabashima, Langerhans cells are critical in epicutaneous sensitization with protein antigen via thymic stromal lymphopoietin receptor signaling, J. Allergy Clin. Immunol. 129 (2012). 1048–1055 e1046.

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Please cite this article in press as: J. Dong et al., Inhibitory effects of nicotine derived from cigarette smoke on thymic stromal lymphopoietin production in epidermal keratinocytes, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.01.001