Effect of cinnamamides on atopic dermatitis through regulation of IL-4 in CD4+ cells

Abstract This study aimed to evaluate the effects of cinnamamides on atopic dermatitis (AD) and the mechanisms underlying these effects. To this end, the actions of two cinnamamides, (E)-3-(4-hydroxyphenyl)-N-phenylethyl acrylamide (NCT) and N-trans-coumaroyltyramine (NCPA), were determined on AD by orally administering them to mice. Oral administration of the cinnamamides ameliorated the increase in epidermal and dermal thickness as well as mast cell infiltration. Cinnamamides suppressed serum immunoglobulin (Ig) levels and expression of T-helper (Th)1/Th2 cytokines. Moreover, cinnamamides suppressed interleukin (IL)-4, which plays a crucial role in preparing naïve clusters of differentiation (CD)4+ T cells, and decreased the cervical lymph node size and weight. Interestingly, in almost all cases, NCPA exhibited higher anti-AD activity compared to NCT. These results strongly indicate that NCPA may have potential as an anti-AD agent, and further mechanistic comparative studies of NCT and NCPA are required to determine the cause of differences in biological activity.


Introduction
Atopic dermatitis (AD) is a complex incurable inflammatory skin disease associated with pruritus, dryness, erythema, weeping, and eczematous skin lesions 1 . AD usually presents during early infancy and childhood; however, it can start or may continue into middle age. AD is a multifaceted condition with precipitating factors including an abnormal immune system, genetic disorders, and environmental factors [2][3][4][5] . Distributed skin barrier functions lead to transepidermal water loss and T-helper 1 (Th1)-and Th2-mediated immunological responses. Th2 cell-mediated changes in interleukin-4 (IL-4), IL-5, and IL-13 expressions have been reported in the acute phase of AD, whereas in the chromic stages, Th1 cell-mediated AD lesions were found 6,7 . In addition, tumour necrosis factor (TNF)-a and interferon (IFN)-c are involved in acute and chronic AD 8,9 . Moisturisers and topical steroids are used for AD treatment to maintain the moisture level of the skin and reduce inflammation, respectively. However, long-term use of topical steroids or short, repeated courses of stronger steroids are associated with adverse events 10 . Therefore, numerous research studies have recently focused on developing natural anti-inflammatory agents with low incidence of side effects 11 .
Biochemical properties of polyphenolic secondary plant metabolites such as esters of cinnamic acids (e.g. caffeic, ferulic, and p-, and o-coumaric acids) have attracted considerable attention in medicine 12 . Cinnamic acid derivatives have low toxicity and show anti-allergic, anti-inflammatory, free radical scavenging, antitumor, and antimicrobial activities [13][14][15] . Cinnamamides are Nsubstituted amides of cinnamic acids and, as a scaffold of interest to medicinal chemistry, have been incorporated into synthetic compounds with antimicrobial 16 , neuroprotective 17 , and antiinflammatory properties 18 .
This study evaluated and compared the inhibitory effect of synthesised cinnamamides, (2E)-3-(4-hydroxyphenyl)-N-(2-phenylethyl) acrylamide (NCPA) and N-p-trans-coumaroyltyramine (NCT) on AD induced by Dermatophagoides farinae extract (DFE, house dust mite extract) and 2,4-dinitrochlorobenzene (DNCB) in BALB/c mice. Specifically, it investigated the effect of the compounds on epidermal and dermal ear thickness, mast cell infiltration, serum immunoglobulin (Ig) levels, and mRNA expression of Th1/Th2 cytokines in ear skin tissue. In addition, the expression of IL-4 in clusters of differentiation (CD)4 þ T cells, which play central roles in the function of the immune system 19 , in draining lymph nodes (dLN), and the size and weight of cervical lymph nodes were determined.

Materials and cell culture
The TRIzol reagent for RNA extraction was obtained from Invitrogen (Carlsbad, CA, USA), and the primary antibodies and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Jurkat cells (human T lymphocytes) were obtained from the ATCC (Manassas, VA, USA) and cultured in RPMI-1640 Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37 C in a 5% CO 2 atmosphere. All other reagents were of the highest grade commercially available at the time of the study.
General procedure and characterisation of amide derivatives p-Coumaric acid (1.0 g, 61.09 mmol, 1.0 equiv.) was dissolved in dimethylformamide (20 mL) and stirred at 0 C for 5 min. Triethylamine (0.85 mL, 6.09 mmol, 1.0 equiv.) was added drop-wise and stirred for 5 min before the corresponding amines (6.09 mmol, 1.0 equiv.) were added, followed by a solution of benzotriazol-1yloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) in dichloromethane (CH 2 Cl 2 , 20 mL). The mixture was stirred at 0 C for 30 min and then at room temperature overnight. The reaction mixture was evaporated under reduced pressure; ethyl acetate (30 mL) and water (H 2 O, 20 mL) were added, and the organic layer was washed with 1 N hydrochloric acid (HCl, aq., 20 mL) and sodium bicarbonate (NaHCO 3 , aq., saturated, 20 mL). The organic layer was separated and dried over anhydrous magnesium sulfate (MgSO 4 ). The solvent was evaporated under reduced pressure, and the residue was further purified using an openbed silica gel column (4 Â 30 cm, ethyl acetate:n-hexane:methanol, 6:2:1, v/v/v). Fractions containing the product were combined and evaporated under reduced pressure to obtain the corresponding amides.

Animals
Eight-week-old female BALB/c mice were purchased from Samtako (Osan, Republic of Korea) and housed under specific-pathogenfree conditions. All the experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University, Korea (Protocol no. KU14012).

Induction of AD lesions in the ear
AD was induced in the mice by repeated local exposure of the ears to DFE and DNCB, as previously described 20 . For the induction of AD, the mice were divided into six groups (control, ADonly, NCT-only, NCPA-only, AD þ NCT, and AD þ NCPA). Structures of NCT and NCPA and experimental design are shown in Figure 1(a,b), respectively. To induce AD, the surfaces of both earlobes were stripped five times using surgical tape (Nichiban, Tokyo, Japan), and 20 mL 1% DNCB was applied to each ear, followed 4 days later by 20 mL of DFE (10 mg/mL). DFE and DNCB treatments were administered alternately once per week for 4 weeks. The animals in NCT-only, NCPA-only, AD þ NCT, and AD þ NCPA groups were orally administrated NCT or NCPA (50 mg/kg/day) throughout the 4-week AD induction period.
The ear thickness was measured 24 h after DNCB or DFE applications using a dial thickness gauge (ID-C1012XBS, Mitutoyo Co., Kawasaki, Japan). On days 14 and 28, blood samples were collected by orbital puncture; plasma samples were prepared, and stored at -70 C for further analysis. After blood collection on day 28, mice ears were removed and histopathologically analysed. Serum IgE and IgG2a levels were measured on days 14 and 28 after the first induction using an IgE enzyme-linked immunoassay kit (Bethyl Laboratories, Inc., Montgomery, TX, USA) according to the manufacturer's instructions.

Histological observations
The excised mouse ears were fixed in 4% paraformaldehyde for 16 h and embedded in paraffin, and thin (6 mm) sections were cut and stained with hematoxylin and eosin (H&E). The thicknesses of the epidermis and dermis were measured under a microscope. For determining mast cell infiltration, skin sections were stained with toluidine blue, and the mast cells were counted in five randomly chosen fields of view.
Jurkat cells were stimulated with phorbol myristate acetate (PMA)/calcium ionophore A23187 for 24 h. The supernatants were collected, and IL-2 mRNA levels were determined.

Analysis of mRNA expression
For the reverse transcription-polymerase chain reaction (RT-PCR), the total cellular RNA was isolated from the ear tissue and the CD4 þ T cell in draining lymph nodes (dLNs) and non-dLNs using TRIzol according to the manufacturer's protocol [20]. The firststrand complementary DNA (cDNA) was synthesised using Superscript II reverse transcriptase (Invitrogen). The conditions for RT-PCR were similar to those employed in previously described studies 21 . where C t is the threshold cycle value. Expression levels of the analysed genes were normalised to GAPDH for each sample and presented as relative mRNA levels.

Statistical analysis
Statistical analysis was carried out using SAS 9.1.3 software (SAS Institute, Cary, NC, USA). Differences between groups were analysed using a one-way analysis of variance (ANOVA) followed by Dunnett's multiple range test. Data are expressed as means ± standard deviations (SDs) of comparative fold differences and are representative of three independent experiments. The threshold for significance was set at p < 0.05.

Results and discussion
Effect of NCT and NCPA on ear thickness, histopathological observations, and serum Ig levels Ear thickness and AD lesions significantly increased following the application of DFE and DNCB. Oral administration of NCT and NCPA suppressed the increase in dermal and epidermal thickness (Figures 1(c,d) and 2(a,b)). In addition, NCT and NCPA-treated groups showed a significant reduction in the number of infiltrated mast cells compared with the AD-only group (Figure 2(c,d)). Moreover, AD mice exhibited higher IgE and IgG2a levels compared to control. On the contrary, NCT and NCPA-treated mice showed significantly reduced total and DFE-specific IgE and IgG2a levels compared with the AD-only group (Figure 2(e)).

Effect of NCT and NCPA on inflammatory stress in stimulated T cells
Though the molecular basis of the process remains unclear, T-cell infiltration of the skin in AD has been well documented 22 . Therefore, the effects of cinnamamides on T cells were examined using Jurkat cells. Pretreatment with NCT and NCPA reduced IL-2 mRNA levels of Jurkat cells (Figure 3).

Effect of NCT and NCPA on the expression of various pathogenic cytokines in vivo
The mRNA levels of the evaluated cytokines were upregulated in ear tissues of AD mice. Treatment with NCPA or NCT reduced the expression of Th1-related cytokines, IFN-c and TNF-a, and Th2-related cytokines, IL-4, IL-5, and IL-13, in mouse ear tissues (Figure 4(a)). NCPA was significantly more effective than NCT in reducing mRNA expression (p < 0.05). In addition, as shown in Figure 4(b), NCT and NCPA suppressed IL-4 mRNA expression in CD4 þ T cells in dLNs.

Effect of NCT and NCPA on the morphology of the cervical lymph nodes cytokines in vivo
As AD often progresses as a systemic disease 23 , whether oral administration of NCT and NCPA affected systemic immune responses was examined. AD mice had larger and heavier cervical lymph nodes compared to the untreated control group. Oral administration of NCT and NCPA to AD mice significantly lowered the cervical lymph nodes weight and size compared to the ADonly group (Figure 5(a,b)).  Cinnamamides have a wide range of therapeutic effects, acting as immunomodulatory, anti-allergic, antitumor, and anti-infective agents 13 . In this study, two cinnamamides, NCT, and NCPA, were synthesised, and their therapeutic effects on AD, which was induced in BALB/c mice by alternately applying DFE and DNCB on both earlobes for 4 weeks, were comparatively evaluated. Oral administration of NCT and NCPA significantly suppressed ear thickness and AD lesions compared with the AD-only group, with NCPA showing a significantly higher activity.
Mast cells play a vital role in inflammation and, upon activation, release potent inflammatory mediators, including histamine and cytokines. AD-induced mouse ears were histologically analysed to confirm mast cell infiltration. Excised ear tissues from each group were stained with toluidine blue and microscopically examined. NCT-and NCPA-treated groups showed an inhibitory effect on the number of infiltrated mast cells compared with the AD-only group. In addition, NCPA treatment was more effective than NCT.
An increase in IgE levels is indicative of AD progression 24 , as AD is an immune disorder accompanied by increased serum IgE levels 25,26 . In addition, as chronic AD is frequently associated with high levels of IgG antibodies 27 , serum IgG2a levels were measured. IgE (total and DFE-specific) and IgG2a were measured for each treatment group. As expected, alternately applying DFE and DNCB increased IgE and IgG2a levels compared to control. However, NCT and NCPA significantly reduced total and DFEspecific IgE and IgG2a levels compared with AD-only group mice. Interestingly, NCPA was also more effective than NCT.
Th1/Th2 cell-mediated lesions are critical to AD progression, making the suppression of T-cell activity the optimal therapeutic approach for AD. In this study, treatment with NCT and NCPA reduced IL-2 mRNA levels in activated T cells.
In order to examine whether NCT and NCPA exerted their effects via the Th1 or Th2 response and elucidate the underlying mechanisms, the mRNA expression levels of AD-related cytokines in ear tissues were measured using real-time PCR. NCPA and NCT suppressed the expression of Th1-and Th2-related cytokines in mouse ear tissues. Similar with previous results, NCPA was significantly more effective compared to NCT (p < 0.05).
In addition, Matsui et al. reported that cinnamamides inhibited histamine release and TNF-a production in stimulated rat basophilic leukaemia cells (RBL-2H3) 26 . Acute AD lesions are characterised by an inflammatory cascade caused by the release of soluble factors including Th2-derived cytokines IL-4, IL-5, and IL-13 and increased serum Ig. As IL-4 plays an important role in preparing naïve CD4 þ T cells for conversion into Th2 cells 28 ; thus, the present study investigated whether NCT and NCPA regulate this process in vitro, by examining mRNA expression of IL-4 in CD4 þ T cells in dLNs. In this study, NCT and NCPA reduced IL-4 mRNA expression, with NCPA displaying higher activity. Predominantly Th2-mediated response occurs in the acute phase of AD, whereas chronic AD is characterised by a predominantly Th1-mediated response. Our results suggest that NCT and NCPA inhibit the expression of Th1 and Th2 cytokines, indicating the potential therapeutic use of these cinnamamides in acute and chronic stages of AD.
From the present results, NCPA exhibited a higher effect compared to NCT. NCPA is an amphiphilic compound containing hydrophilic 4-hydroxyl amide groups and a hydrophobic phenyl group. Therefore, we infer that the amphiphilicity contributes to the observed results. Some amphiphilc compounds directly permeate cell membranes 29 . Therefore, we hypothesise that NCPA is effectively distributed between the lipophilic and hydrophilic compartments in AD.

Conclusions
The present study examined the anti-AD potential of the synthesised cinnamamides NCT and NCPA following oral administration, using a mouse model of AD. Oral administration of NCT and NCPA each significantly decreased the severity of AD including histopathological features, production of Ig, and expression of Th1/Th2 cytokines in DFE/DNCB-induced BALB/c mice. Taken together, our results demonstrate that NCT and NCPA effectively suppress the AD-like phenotype by regulating the inflammatory response without inducing any significant effects on the normal immune responses. Furthermore, NCPA exhibited higher anti-AD activity compared to NCT.
Therefore, we propose NCPA as a promising candidate for development of alternative AD treatments. We suggest further mechanistic comparative studies of NCT and NCPA to establish the causes of the differences in biological activity of these compounds.