Nicotine treatment shows the therapeutic effect for imiquimod-induced psoriasiform dermatitis in alpha 7- nicotinic acetylcholine receptor-independent manner

Abstract

The neurotransmitter acetylcholine (ACh) stimulates nicotinic ACh receptors (nAChRs) expressed in both excitable and non-excitable cells. Cholinergic signaling in non-excitable cells induces anti-inflammatory responses; the cholinergic anti-inflammatory system inhibits the development of inflammatory diseases such as multiple sclerosis, colitis and rheumatoid arthritis. The subunits of nAChR are expressed in different layers of the epidermis. Cholinergic anti-inflammatory signaling is involved in the development of cutaneous inflammatory diseases. However, the role of nAChR-related signaling remains unclear in psoriasiform dermatitis. In this study, a mouse model of imiquimod-induced psoriasiform dermatitis demonstrated impaired cholinergic signaling. However, the nAChR subunits expression remained almost unchanged. Nicotine, a nonselective nAChR agonist, ameliorated the dermatitis phenotype and abnormal cholinergic signaling. The therapeutic effect of nAChR-signaling was independent of nAChRα7. Taken together, these results suggest that nAChR-mediated cholinergic anti-inflammatory signaling might be a good therapeutic target for treating dermatitis.

Highlights

It is reported that cholinergic anti-inflammatory signaling via nAChRs is important for the cutaneous inflammatory diseases. However, the hypothesis is not established by in vivo experiments. In this study, a nonselective nAChR agonist ameliorated the dermatitis phenotype and abnormal cholinergic signaling. The finding will be useful in the development for cutaneous inflammatory diseases medicine.

Introduction

Nicotinic acetylcholine receptors (nAChRs) play functional roles in the central and peripheral nervous systems. The neurotransmitter acetylcholine (ACh) is an endogenous ligand for nAChRs. Choline acetyltransferase (ChAT) synthesizes ACh in nerve terminals and vesicular acetylcholine transporter (VAChT) mediates the packaging and transport of ACh for exocytic release. In contrast, acetylcholine esterase (AChE) catalyzes the hydrolysis of ACh. These cholinergic systems play important roles in neurological diseases, such as Alzheimer’s disease [1, 2].

nAChRs are composed of homo- and hetero-pentamers. Thus, cholinergic signaling is mediated by distinct combinations of nAChR subunits. In mammals, 16 homologous genes encode the subunits of nAChR. Various combinations of nAChR subunits regulate specific biological functions. Muscle-type nAChRs include nAChR (α1)₂β1δε (adult receptor) or nAChR (α1)₂β1δγ (fetal receptor). Neuronal-type nAChRs are homo- or hetero-pentamers composed of nine α subunits (α2–α10) and three β subunits (β2–4) [3].

In addition to excitable cells, non-excitable cells such as keratinocytes, as well as immune cells such as Th17 cells, neutrophils, and macrophages, express nAChR subunits [4]. Neuronal cell-released ACh activates nAChR-related cholinergic anti-inflammatory signaling in these cells [5,6,7]. Nicotine is a nonselective agonist of nAChRs [8]. Nicotine-induced nAChR signaling modulates the development of inflammatory diseases such as multiple sclerosis, colitis and rheumatoid arthritis in an nAChRα7-dependent and nAChRα7-independent manner [9,10,11,12,13]. nAChRα7 plays a protective role in inflammatory diseases such as multiple sclerosis and colitis [13, 14]. The activation of nAChRα7 has been reported to mediate the suppression of TNFα from macrophages [15]. In terminally differentiated keratinocytes, nAChRα7 is important for the ACh-mediated regulation of apoptosis [16]. In contrast to other nAChRs, the selective agonist and antagonist of nAChRα7 are commercially available. Thus, understanding the mechanism underlying nAChR signaling will enable the development of therapeutic agents for treating inflammatory diseases.

Psoriasiform dermatitis is a chronic inflammatory skin disease characterized by erythema (redness), induration (thickness), desquamation (scaling), and itching, which reduce the quality of life. Despite regional differences in the prevalence of psoriasis, the disease affects approximately 2–4% of the global population [17]. Multiple factors such as genetic susceptibility and environmental conditions are involved in the pathogenesis of psoriasis [18, 19]. For elucidating its pathogenesis, some studies used the TLR 7/8 agonist imiquimod (IMQ) and found that it caused psoriasiform dermatitis resembling psoriasis in humans similar to skin thickening, erythema, scaling, acanthosis, and parakeratosis developing in mice and humans [20,21,22]. Keratinocyte is a major component of the epidermis. Abnormal proliferation and differentiation of keratinocytes are observed in psoriasis [23]. Various nAChR subunits are expressed in different layers of the epidermis, and cholinergic anti-inflammatory signaling on the keratinocytes regulates the development of cutaneous inflammatory disease [24, 25]. These findings suggested that the nAChR subunits may be useful therapeutic target for psoriasiform dermatitis. However, its potential as a therapeutic agent using in vivo models remain to be elucidated. Thus, this study aimed to analyze the mechanism of nAChR signaling using nAChR agonists and antagonists in an IMQ-induced psoriasiform dermatitis mouse model.

Materials and Methods

1. Ethics statement

All animal experiments were approved by the Animal Care and Use Committee in Kansai Medical University (KMU, approval numbers: 19-104 and 20-091) and conducted according to the institutional ethical guidelines for animal experiments and safety guidelines for gene manipulation experiments.

2. IMQ-induced psoriasiform dermatitis

Female C57BL/6J mice were obtained from CLEA Japan (Tokyo, Japan). The mice were maintained under specific pathogen-free conditions at the animal facility in KMU. According to a previous study [21], the mouse model of IMQ-induced psoriasiform dermatitis was developed under appropriate conditions at the animal facility in KMU. The mice were subjected to daily application of approx. 18 mg (just a spoonful of cream with micro spatula [Cat No. 6-524-05, AS ONE, Osaka, Japan]) of Beselna Cream (5% IMQ; Mochida Pharmaceutical, Tokyo, Japan) on the ventral surface of both ears under isoflurane anesthesia using the NARCOBIT-E type II (Natsume Seisakusho, Tokyo, Japan).

To examine the effect of an nAChR-nonselective agonist on psoriasiform dermatitis, mice were given daily intraperitoneal injections of nicotine tartrate dihydrate solution (0.2 mg/ml, as a nicotine-free base, Nacalai Tesque, Kyoto, Japan) in 0.9% saline (Otsuka Pharmaceutical, Tokyo, Japan) at a dose of 2 mg/kg [26]. To examine the effect of an nAChRα7-selective agonist on psoriasiform dermatitis, mice were given daily intraperitoneal injections of 0.1 mg/ml of PHA 568487 (PHA; nAChRα7-selective agonist, Tocris, Bristol, UK) in 0.9% saline at a dose of 1 mg/kg [27]. To examine the effect of an nAChRα7-selective antagonist on psoriasiform dermatitis, mice were given daily intraperitoneal injections of 0.1 µg/ml of α-bungarotoxin (BGT; nAChRα7-selective antagonist, Abcam, Cambridge, UK) in 0.9% saline 15 min prior to nicotine treatment (1 µg/kg) [28, 29].

The severity of psoriasiform dermatitis was scored based on the ear thickness. Ear thickness was measured daily at three points per ear using a caliper (PEACOCK G-1A, Ozaki Corp., Tokyo, Japan). The extent of inflammation in the IMQ-induced psoriasiform dermatitis model was assessed based on the expression of inflammatory cytokines in the ear using real-time polymerase chain reaction (PCR). Thickness was calculated as the percentage of thickness on day 0. Mouse ear biopsy samples were subject to hematoxylin and eosin staining as described earlier [21]. To examine gene expression by real-time PCR analyses, the dermatitis ear was harvested at the end of the experiments.

3. Real-time PCR

According to a previous study [21], total RNA was extracted from the ear using Sepasol-RNA I Super (Nacalai Tesque). One microgram of total RNA was reverse-transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer’s recommendations. Real-time PCR was performed on the Rotor-Gene Q HRM System (Qiagen, Hilden, Germany) using 20 ng cDNA for each reaction. PCR was performed using the THUNDERBIRD SYBR qPCR Mix (TOYOBO) and 0.5 µM of each primer at a final volume of 20 µl. For all the analyses, cDNAs were amplified using 50 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 60 sec. The primer sequences are described in Table 1 [21, 30,31,32,33,34,35]. The 2^-ΔΔCt method was used for analysis, and the relative expression level of target genes included was normalized with Gapdh [36].

Table 1. Primers for quantitative real-time PCR

Gene	Forward	Reverse	Reference
Gapdh	5′-TTCACCACCATGGAGAAGGC-3′	5′-GGCATGGACTGTGGTCATGA-3′	[31]
Il17a	5′-TTTAACTCCCTTGGCGCAAAA-3′	5′-CTTTCCCTCCGCATTGACAC-3′	[21]
Tnf	5′-GCCTCCCTCTCATCAGTTCT-3′	5′-CACTTGGTGGTTTGCTACGA-3′	[30]
Chrna1	5′-GGTGCGGAAGGTTTTTATCG-3′	5′-TCTCTGGATGGTCTTTTCATTGTG-3′	[35]
Chrna2	5′-TGCTGACTCTTCGGTGAAGGA-3′	5′-GCCAGAGGAAGATCCGGTCTA-3′	[35]
Chrna3	5′-GCTGAAAATATGAAAGCACAGAATG-3′	5′-ACCATGGCAACATACTTCCAATC-3′	[35]
Chrna4	5′-GAAGGCGTCCAGTACATTGCA-3′	5′-CCTTCACCGAGAAGTCTGTGTCT-3′	[35]
Chrna5	5′- TGGATCCCAGACATCGTTTTG-3′	5′- CGTACTGGCCCCTTCGAA-3′	[35]
Chrna6	5′- AAACATGAAGAGCCACAATGAAAC-3′	5′- CCACCATAGCCATGTATTTCCA-3′	[35]
Chrna7	5′- CAGCAGCTATATCCCCAATGG-3′	5′- GGCTCTTTGCAGCATTCATAGA-3′	[35]
Chrna9	5′- TGCCAGCCTCAGAAAACGT-3′	5′- GATCAAGGCCATGGTAGCTATGT-3′	[35]
Chrna10	5′- AGCATGCCACCTGCAGAGA-3′	5′- CTGTGGAGAATGTGACCATGGT-3′	[35]
Chrnb1	5′- CCGCCAGATCTTCATTCACA-3′	5′-GGGTTTGGGCCTCTTCAGA-3′	[35]
Chrnb2	5′-CGGACCATATGCGAAGTGAA-3′	5′-TGGCAACGTATTTCCAATCCT-3′	[35]
Chrnb3	5′-GAGGCATGTGAAGAAGGAACACT-3′	5′-TGAGCCACGAATTTCCAGTCT-3′	[35]
Chrnb4	5′-GTCGTCGACCGCCTGTTC-3′	5′-CCCCATGGTGCCCAGAA-3′	[35]
Chrng	5′-GCAGGCAGTATTGGAGAAGC-3′	5′-ACGAGCCATGAGGTTACAGG-3′	[34]
Chrnd	5′-GAATGAGGAACAAAGGCTGATCC-3′	5′-GGTGAGACTTAGGGCGACAT-3′	[33]
Chrne	5′-ACCGCAGCTTTTACCGAGAA-3′	5′-CGACGGATGATGAGCGTGTA-3′	[33]
Ache	5′-CTACACCACGGAGGAGAGGA-3′	5′-CTGGTTCTTCCAGTGCACCA-3′	[32]
Chat	5′-CCTGGATGGTCCAGGCACT-3′	5′-GTCATACCAACGATTCGCTCC-3′	[32]
Vacht	5′-GAGAGTACTTTGCCTGGGAGGA-3′	5′-GGCCACAGTAAGACCTCCCTTG-3′	[32]

4. Statistics

All results are expressed as average ± standard error of mean. Student’s t-test was used to statistically analyze all results. All statistical analyses were performed using Statcel3 software (OMS Publishing, Tokyo, Japan). Results with P<0.05 were considered statistically significant.

Results and Discussion

1. Cholinergic signaling was impaired in psoriasiform dermatitis ear skin samples

To analyze the role of cholinergic anti-inflammatory signaling in psoriasiform dermatitis development, IMQ-induced psoriasiform dermatitis was developed (Fig. 1A and 1B). To elucidate which combinations of nAChR subunits mediate cholinergic signaling in psoriasiform dermatitis, real-time PCR analysis revealed the mRNA levels of the nAChR subunits in ear skin samples (Fig. 1C). It is reported that mRNAs of nAChRα3, nAChRα5, nAChRα7, nAChRα9, nAChRα10, nAChRβ1, nAChRβ2, and nAChRβ4––but not of nAChRα1, nAChRα2, nAChRα4, nAChRα6, and nAChRβ3––are expressed in the different layers of human scalp split skin [37]. In the skin samples from dermatitis mice, nAChRα1 and nAChRα3 mRNA levels were downregulated. However, nAChRα2 mRNA was not detected in intact and dermatitis skin samples. The mRNA expression of other nAChR subunits was comparable in dermatitis and healthy skin samples.

Fig. 1.

The expression of nAChR subunits and ACh-related molecules in psoriasiform dermatitis skin samples. Mice were treated daily with (IMQ) or without (CTL) IMQ cream for 7 days. The relative mRNA expression levels in the ear on day 7 were determined by real-time PCR. (A) Ear thickness was measured daily using a caliper. Ear swelling is shown as the percentage of ear thickness compared with the thickness on day 0 (CTL, IMQ; n=5 each). (B–D) The relative mRNA expression of TNFα (B), nAChR subunits (C), and ACh-related genes (D) (CTL, IMQ; n=10 each). Average and SEM are shown. *P<0.05, **P<0.01, and ***P<0.001. Student’s t-test. ND, not detected.

Neuronal-type nAChRs are homo- or hetero-pentamers composed of nine α subunits (α2–α10) and three β subunits (β2–4). α4β2- and α6β2-containing nAChR subunits have the highest affinity for nicotine [38]. nAChR subunits such as α3β2 and α3β4 subtype with or without α5, α7 and α9 are expressed in different layers of the epidermis [24, 25]. nAChRα3 is downregulated in dermatitis skin but the polymorphisms in the CHRNA5/CHRNA3/CHRNB4 cluster and CHRNA4 are not associated with an increased risk of psoriasis [39]. Furthermore, variants in the CHRNB3–CHRNA6 region are not important for the development of psoriasis [40]. nAChRβ2 is a constituent of various nAChRs along with nAChRα2, nAChRα3, nAChRα4, nAChRα6, and nAChRβ3 [41]. Thus, nAChRβ2 may not be important for the development of psoriasis as the polymorphisms of subunit pair genes are not associated with the risk of psoriasis, and the other subunit pair nAChRα2 is not detected in human [37] and mouse skin samples (Fig. 1C). Taken together, nAChR complex consisting of these nAChR subunits may not be involved in the development of psoriasiform dermatitis.

ACh induces anti-inflammatory cholinergic signaling via nAChRs. ACh is secreted differentially in the different layers of the epidermis [42]. ACh synthetase ChAT and ACh transporter VAChT mRNA expression were comparable in healthy and dermatitis skin (Fig. 1D). These results suggest that ACh synthetic pathway is intact in dermatitis skin. However, the ACh-degrading enzyme AChE mRNA was higher in the dermatitis skin samples than in the healthy skin samples (Fig. 1D). Thus, degradation of ACh was enhanced in psoriasiform dermatitis skin samples.

2. Activation of nAChRs improved the dermatitis phenotype and cholinergic signaling

To elucidate the therapeutic effect of nAChR signaling, the dermatitis mice were treated with nicotine. Nicotine-treated mice exhibited mild dermatitis compared to the control dermatitis mice (Fig. 2A). Keratinocytes in the epidermis undergo proliferation and differentiation to form different layers of the skin, thus keratinocyte constitute the major proportion of the epidermis [43, 44]. Apoptosis of keratinocytes is important for epidermal remodeling. The psoriatic dermis exhibits increased keratinocyte proliferation, thereby causing skin hypertrophy [45]. Thus, keratinocyte apoptosis helps to maintain a healthy skin [46]. TNFα is primarily expressed by the keratinocytes in mice with dermatitis; it enhances the expression of adhesion molecules and induces the hyperproliferation of keratinocytes [47, 48]. And also TNFα is expressed by macrophages in dermatitis; which is dominant immune population [49]. Nicotine administration decreased TNFα mRNA levels (Fig. 2B).

Fig. 2.

nAChR activation ameliorated the IMQ-induced psoriasiform dermatitis. Nicotine (NIC) or saline as a control (CTL) was injected daily, along with the application of IMQ cream, from day 0 (CTL, NIC; n=6 each). (A) Ear thickness was measured daily using a caliper (left). Ear swelling is shown as the percentage of ear thickness compared with the thickness on day 0. (right) H&E sections of mouse ears on 5 after the start of IMQ cream application. A representative sections is shown. Scale bar: 100 µm. (B, C) The relative mRNA expression of TNFα (B) and ACh-related genes (C) in the ear on day 5 after the start of IMQ cream application was determined by real-time PCR. Average and SEM are shown. *P<0.05, *P<0.01, and *P<0.001. Student’s t-test.

Muscle-type nAChR consists of a combination of nAChR (α1)₂β1δε or nAChR (α1)₂β1δγ [3]. Although the mRNA levels of nAChRα1 decreased in psoriasiform dermatitis skin samples, the mRNA levels of other components of muscle-type nAChR were similar in dermatitis and healthy samples (Fig. 1D). It is reported that nicotine induces suppression of TNFα production from macrophage via nAChRα7, but not nAChRα1 and nAChRα10 [50]. Thus, muscle-type nAChRs and nAChRα10 subunits may not be involved in nicotine-induced nAChR signaling in the case of psoriasiform dermatitis.

Furthermore, ChAT and VAChT mRNA levels increased upon nicotine treatment. In contrast, the mRNA levels of AChE were comparable in the control and nicotine-treated dermatitis mice (Fig. 2C). These results indicated that the activation of nAChR signaling activated cholinergic anti-inflammatory signaling pathways.

3. The role of nAChRα7 in psoriasiform dermatitis

To evaluate the therapeutic potential of nAChRα7 for dermatitis, the mice were treated with PHA and BGT, the selective agonist and antagonist of nAChRα7, respectively. The administration of PHA in dermatitis mice did not affect the severity or the mRNA levels of inflammatory cytokine and ACh-related genes (Fig. 3A–C). Similarly, BGT treatment of mice with dermatitis did not affect the severity or the mRNA levels of inflammatory cytokine- and ACh-related genes (Fig. 3D–F). Collectively, these results show that nAChRα7 is not a suitable target for eliciting the therapeutic potential of nAChRs to alleviate psoriasiform dermatitis.

Fig. 3.

The therapeutic effect of nAChRs signaling on psoriasiform dermatitis was independent of nAChRα7. (A–C) PHA 568487 (PHA) or saline as a control (CTL) was injected daily into mice, along with the application of IMQ cream, from day 0 (CTL, PHA; n=5 each). (A) Ear thickness was measured daily using a caliper. Ear swelling is shown as the percentage of ear thickness compared with the thickness on day 0. (B, C) The relative mRNA expression of TNFα (B) and ACh-related genes (C) in the ear on day 5 after the start of IMQ cream application was determined by real-time PCR. (D–F) α-Bungarotoxin (BGT) or saline as a control (CTL) was injected daily into mice 15 min prior to the nicotine treatment, along with the application of IMQ cream, from day 0 (CTL, BGT; n=3 each). (E) Ear thickness was measured daily using a caliper. Ear swelling is shown as percentage of ear thickness compared with the thickness on day 0. (E, F) The relative mRNA expression of TNFα (E) and ACh-related genes (F) in the ear on day 5 after the start of IMQ cream application was determined by real-time PCR (CTL, BGT; n=3 each). Average and SEM are shown. Student’s t-test.

The nAChRα9 subunit forms functional receptors, i.e., homo- and hetero-pentamers, with nAChRα10 [51, 52]. nAChRα9 is widely expressed in the epidermis and is essential for epidermal differentiation [37]. nAChRα9-mediated cholinergic signaling increases the adhesion and differentiation of keratinocytes [24, 53, 54]. Thus, nAChRα9 might be crucial for the development of psoriasiform dermatitis. However, selective agonist and antagonist for homo-nAChRα9 are not developed at this time.

Taken together, nAChR activation induced by nicotine ameliorated the phenotypes associated with impaired cholinergic signaling and decreased the skin thickness in psoriasiform dermatitis mice. Furthermore, nicotine administration is known to improve inflammatory responses in the central nervous system [13] and arthritis [55], and to improve cognitive function in Alzheimer’s disease [1]. Although the therapeutic roles of nAChR subunits remain to be understood due to the lack of selective agonists and antagonists, nAChR signaling might be a potential therapeutic target for inflammatory diseases such as psoriasiform dermatitis. Thus, the development of selective agonists and antagonists for nAChR subunits is necessary for understandings the molecular mechanism of nAChR subunits and developing novel therapeutic agents for inflammatory diseases.

Conflict of Interest

The author have declared that no conflict of interest exists.

Acknowledgements

This work was supported by SRF Foundation (No. 2019Y005), Koyanagi Foundation (No. 19060062), MEXT-supported program for the Private University Research Branding Project (type B) as a world-leading research university on intractable immune and allergic diseases, and MEXT-supported program for the Strategic Research Foundation at Private Universities.

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