1-Iodohexadecane Alleviates 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis in Mice: Possible Involvements of the Skin Barrier and Mast Cell SNARE Proteins

Atopic dermatitis (AD) is a chronic inflammatory dermal disease with symptoms that include inflammation, itching, and dry skin. 1-Iodohexadecane is known as a component of Chrysanthemum boreale essential oil that has an inhibitory effect on AD-like lesions. However, its effects on AD-related pathological events have not been investigated. Here, we explored the effects of 1-iodohexadecane on AD lesion-related in vitro and in vivo responses and the mechanism involved using human keratinocytes (HaCaT cells), mast cells (RBL-2H3 cells), and a 2,4-dinitrochlorobenzene (DNCB)-induced mouse model (male BALB/c) of AD. Protein analyses were performed by immunoblotting or immunohistochemistry. In RBL-2H3 cells, 1-iodohexadecane inhibited immunoglobulin E-induced releases of histamine and β-hexosaminidase and the expression of VAMP8 protein (vesicle-associated membrane proteins 8; a soluble N-ethylmaleimide-sensitive factor attachment protein receptor [SNARE] protein). In HaCaT cells, 1-iodohexadecane enhanced filaggrin and loricrin expressions; in DNCB-treated mice, it improved AD-like skin lesions, reduced epidermal thickness, mast cell infiltration, and increased filaggrin and loricrin expressions (skin barrier proteins). In addition, 1-iodohexadecane reduced the β-hexosaminidase level in the serum of DNCB-applied mice. These results suggest that 1-iodohexadecane may ameliorate AD lesion severity by disrupting SNARE protein-linked degranulation and/or by enhancing the expressions of skin barrier-related proteins, and that 1-iodohexadecane has therapeutic potential for the treatment of AD.


Introduction
Atopic dermatitis (AD) is a common inflammatory disease that is prevalent among all ages and has characteristic symptoms such as severe itching, dry skin, erythema, and scaly, thickened skin lesions [1]. Reportedly, AD is caused by immunological abnormalities and/or epidermal barrier dysfunction, which are associated with the complex interplay between genetic, immunological (increased serum immunoglobulin-E (IgE) levels and imbalance between T helper type 1 (Th1) and Th2 cells) responses, and environmental factors such as environmental pollution [2]. Immunological and skin epidermal barrier functional abnormalities are also closely related to the chronicity and relapse of AD lesions [3]. Therefore, controlling these etiologic factors is a crucial aspect of the development of strategies designed to treat, prevent, or manage AD. Two hypotheses have been proposed to explain the pathogenic mechanism responsible for AD; that is, AD results from syntaxin 4, and v-SNARE proteins, such as VAMP7 and VAMP8, control the membrane fusion required for mast cell degranulation [12,32]. To determine whether 1-Iodo affects SNARE proteins, we used Western blotting to examine its effects on the expressions of SNAP23 and syntaxin 4 (its partner protein) and VAMP8 in RBL-2H3 mast cells. Treatment with 1-Iodo (25-100 µg/mL) significantly decreased VAMP8 expression at 75 µg/mL (28.31 ± 1.83% vs. untreated controls) and 100 µg/mL (29.30 ± 1.93% vs. untreated controls) (Figure 1a,b), but 1-Iodo did not influence the expressions of t-SNAREs (SNAP23 and syntaxin 4) in RBL-2H3 mast cells (Figure 1a,c,d).

Effects of 1-Iodohexadecane on the Expressions of SNARE Proteins in Mast Cells
SNARE proteins are membrane fusion-regulatory molecules associated with the regulation of mast cell degranulation, which results in the release of inflammatory mediators of AD such as histamine and cytokines [10][11][12][13]. t-SNARE proteins, such as SNAP23 and syntaxin 4, and v-SNARE proteins, such as VAMP7 and VAMP8, control the membrane fusion required for mast cell degranulation [12,32]. To determine whether 1-Iodo affects SNARE proteins, we used Western blotting to examine its effects on the expressions of SNAP23 and syntaxin 4 (its partner protein) and VAMP8 in RBL-2H3 mast cells. Treatment with 1-Iodo (25-100 μg/mL) significantly decreased VAMP8 expression at 75 μg/mL (28.31 ± 1.83% vs. untreated controls) and 100 μg/mL (29.30 ± 1.93% vs. untreated controls) (Figure 1a,b), but 1-Iodo did not influence the expressions of t-SNAREs (SNAP23 and syntaxin 4) in RBL-2H3 mast cells (Figure 1a,c,d). Results are expressed as mean percentages ± SEMs versus untreated cells (n = 3). * p < 0.05 vs. untreated cells.

Effects of 1-Iodohexadecane on Skin Barrier Protein Expressions in Human Keratinocytes
FLG and LOR play important functional roles in the barrier properties of skin [22,35], and low levels of these proteins are associated with skin barrier disruption and AD development [22,35]. Tumor necrosis factor-α (TNF-α) is known to impair the skin barrier by inhibiting FLG and LOR expression in keratinocytes [36]. Thus, to determine the influence of 1-Iodo on FLG and LOR in keratinocytes, we assessed their levels in tumor necrosis factor-α (TNF-α)-stimulated HaCaT cells in the presence or absence of 1-Iodo by immunoblotting. As shown in Figure 3, TNF-α reduced the expressions of FLG (46.51 ± 27.34% vs. untreated controls) and LOR (34.65 ± 16.03% vs. untreated controls). Treatment with 1-Iodo (25-100 μg/mL) significantly prevented TNF-α induced reduction in FLG at concentrations of 25 to 100 μg/mL (Figure 3a (c) Cell viability in RBL-2H3 cells exposed to 1-Iodo. Cells were treated with 1-Iodo (0.1-100 µg/mL) for 48 h. Cell viabilities were measured using an EZ-CyTox kit. The response levels are expressed as percentages of levels in untreated cells (n = 5). Results are expressed as means ± SEMs. * p < 0.05 vs. untreated cells.

Effects of 1-Iodohexadecane on Skin Barrier Protein Expressions in Human Keratinocytes
FLG and LOR play important functional roles in the barrier properties of skin [22,35], and low levels of these proteins are associated with skin barrier disruption and AD development [22,35]. Tumor necrosis factor-α (TNF-α) is known to impair the skin barrier by inhibiting FLG and LOR expression in keratinocytes [36]. Thus, to determine the influence of 1-Iodo on FLG and LOR in keratinocytes, we assessed their levels in tumor necrosis factor-α (TNF-α)-stimulated HaCaT cells in the presence or absence of 1-Iodo by immunoblotting. As shown in Figure 3, TNF-α reduced the expressions of FLG (46.51 ± 27.34% vs. untreated controls) and LOR (34.65 ± 16.03% vs. untreated controls). Treatment with 1-Iodo (25-100 µg/mL) significantly prevented TNF-α induced reduction in FLG at concentrations of 25 to 100 µg/mL (Figure 3a,b) and LOR protein levels at concentrations of 25 to 75 µg/mL (Figure 3a,c). These effects were highest at a 1-Iodo concentration of 75 µg/mL (FLG expression, 189.57 ± 3.97% of untreated controls [ Figure 3b]; LOR expression, 217.62 ± 16.95% of untreated controls [ Figure 3c]). Furthermore, 1-Iodo did not affect HaCaT cell viability at concentrations up to 100 µg/mL (Figure 3d).

Effect of 1-Iodohexadecane on Skin Lesions in the DNCB-Induced Murine Model
To determine the in vivo effects of 1-Iodo, we investigated its effect on the severity of DNCB-induced skin lesions using SCORAD scores. As shown in Figure 4, DNCB reduced SCORAD scores, and these scores were markedly improved in the 50 and 100 µg/mL 1-Iodo groups and in the DEX group.

Effect of 1-Iodohexadecane on Skin Lesions in the DNCB-Induced Murine Model
To determine the in vivo effects of 1-Iodo, we investigated its effect on the severity of DNCB-induced skin lesions using SCORAD scores. As shown in Figure 4, DNCB reduced SCORAD scores, and these scores were markedly improved in the 50 and 100 μg/mL 1-Iodo groups and in the DEX group.

Effect of 1-Iodohexadecane on Skin Barrier Proteins in the DNCB-Induced Murine Model
Immunostaining was used to determine whether 1-Iodo affected LOR and FLG levels in lesioned skin tissues. LOR expression in skin was lower in the DNCB control group than in normal controls (59.21 ± 7.53% of normal control levels) but at 99.71 ± 1.02% and 138.70 ± 13.59% of normal control levels in the 50 and 100 µg/mL 1-Iodo groups, respectively (Figure 6a,b). FLG protein levels were lower in the DNCB control group than in normal controls (82.40 ± 0.01% vs. normal controls) but were at 142.50 ± 13.89% of the normal control level in the 100 µg/mL 1-Iodo group. However, 50 µg/mL of 1-Iodo had no effect (Figure 6a,c). In addition, to determine whether 1-Iodo influences mast cell degranulation markers in vivo, we assessed histamine and β-hexosaminidase levels in the serum of DNCB-induced mice model treated with 1-Iodo. DNCB elevated histamine levels (205.36 ± 5.48% in lesioned skin tissues. LOR expression in skin was lower in the DNCB control group than in normal controls (59.21 ± 7.53% of normal control levels) but at 99.71 ± 1.02% and 138.70 ± 13.59% of normal control levels in the 50 and 100 µ g/mL 1-Iodo groups, respectively (Figure 6a,b). FLG protein levels were lower in the DNCB control group than in normal controls (82.40 ± 0.01% vs. normal controls) but were at 142.50 ± 13.89% of the normal control level in the 100 µ g/mL 1-Iodo group. However, 50 μg/mL of 1-Iodo had no effect (Figure 6a,c).

Discussion
Worldwide, AD has a prevalence of 15-20% among children and of 1-3% among adults, and its prevalence continues to increase [39]. In severe cases, AD can profoundly impair the quality of life and mental health of patients and of relatives, etc. [40]. Available treatments for AD include topical and systemic corticosteroids, antihistamines, emollients, and immunosuppressants [3]. However, these treatments have limitations in overcoming AD. Thus, much research has been conducted to devise ways of treating AD effectively [41]. DNCB-induced animal models have often been used for AD treatment and pathogenesis studies, because they exhibit symptoms with histological and immunological features similar to those of human AD [42]. 1-Iodo is a component of Chrysanthemum boreale MAKINO flower essential oil with potential anti-AD activity [28]. Thus, in the present study, we investigated its effects on DNCB-induced AD-like lesions in mice. Similar to the dorsal skin tissues in DNCB-exposed AD-like model mice [27,43], DNCB exposure exacerbated lesion severity, as determined by SCORAD scores, epidermal thickness (hyperplasia), and mast cell infiltration into dorsal skin tissues. These DNCB-induced pathological lesions in mice were improved by topical treatment with 1-Iodo. Therefore, 1-Iodo may have an ameliorative effect on AD-like skin lesions.
Studies have suggested that the regulation of mast cell degranulation by SNARE proteins may be a therapeutic target in AD [13]. Degranulation in mast cells is closely associated with membrane fusion events between vesicles/granules and the plasma membrane and results in the release of allergic inflammatory mediators [9,10]. SNARE proteins, such as v-SNAREs and t-SNAREs, are important players in degranulation-associated membrane fusion in mast cells [9,44]. The t-SNARE proteins SNAP23 and syntaxin 4 are localized in the plasma membrane, and the v-SNARE proteins VAMP7 and VAMP8 are localized on intracellular granules. These two types of SNARE proteins form complexes with each other, and thus, induce membrane fusion between vesicles/granules and the plasma membrane leading to degranulation [9][10][11]. Furthermore, it has been reported that knockdown of SNAP23, syntaxin 4, or VAMP8 reduces mast cell degranulation [9]. In the present study, 1-Iodo reduced the expression of VAMP8 in RBL-2H3 cells but not those of SNAP23 and syntaxin 4. It has been reported that VAMP8 deficiency caused impaired mast cell degranulation and inhibited IgE-stimulated release of β-hexosaminidase and histamine by mast cells [45]. On the other hand, another study showed that VAMP8 deficiency reduced β-hexosaminidase release but not histamine release in mast cells [46]. These two reports indicate VAMP8 may affect mast cell degranulation, though its effects on histamine release effects require clarification. These findings imply 1-Iodo may affect SNARE complex assembly-related degranulation, probably by downregulating VAMP8, and it may suppress the release of degranulation-mediated granule contents by interfering with SNARE complex formation. To the best of our knowledge, these results are the first to indicate 1-Iodo inhibits SNARE protein-associated mast cell degranulation.
Degranulation in mast cells is activated by allergens or IgE and results in the release of inflammatory mediators such as histamine, β-hexosaminidase, and cytokines [7,47]. These inflammatory mediators can induce allergic reaction-linked pathological events, such as skin barrier abnormalities and/or immune dysregulation in AD [10][11][12][13]. These reports indicate that suppressing mast cell degranulation or secretion of histamine and β-hexosaminidase may be a useful strategy to improve the condition of AD. In the present study, 1-Iodo treatment reduced β-hexosaminidase and histamine release, both indicators of mast cell degranulation [32,33], from IgE-exposed RBL-2H3 cells. Moreover, 1-Iodo downregulated VAMP8 expression in RBL-2H3 cells, As mentioned above, VAMP8 play essential role in mast cell degranulation through SNARE complex formation and its deficiency impaired mast cell degranulation and inhibited release of β-hexosaminidase and histamine by mast cells [45]. Our findings support the notion that 1-Iodo suppresses the release of degranulation-mediated granule contents by interfering with SNARE complex formation in mast cells. In an animal model of AD, mast cell degranulation inhibition suppressed β-hexosaminidase, histamine, and cytokine secretion and ameliorated ADlike lesions [13,48]. In the present study, we found that topical treatment with 1-Iodo inhibited mast cell infiltrations into dorsal skin tissues in AD-like model mice, implying that mast cells may be associated with skin lesions ameliorated by 1-Iodo in like model mice. Moreover, 1-Iodo significantly reduced the β-hexosaminidase level and slightly attenuated the histamine level in serum of AD-like model mice. Therefore, it can be assumed that 1-Iodo may help alleviate the condition of AD, probably by inhibiting mast cell degranulation-related responses. However, further study will need to clarify the mechanisms involved in the effects of 1-Iodo on these responses in an animal model of AD.
Epidermal skin barrier dysfunction and inflammation play key roles in the development of AD [49], and epidermal structural proteins such as FLG, LOR, and involucrin are key players in epidermal skin barrier formation [50]. FLG is a major cytoskeleton protein of the cornified envelope, and LOR is a main structural cornified envelope protein that constitutes 70-85% of total protein mass in the cornified layer [19]. Involucrin is an assembly and scaffold protein of the cornified envelope [19]. Reduced expressions of these epidermal barrier proteins are major pathological characteristics in the skin of AD patients [50]. Reduced levels of FLG and LOR expressions are associated with barrier disruption and skin inflammation, weakening of the epidermal barrier function, and are also observed in inflamed skin [51,52]. In the present study, treatment with 1-Iodo prevented TNF-α-induced reduction in the expressions of FLG and LOR in keratinocytes and DNCB-induced reduction in the expressions of FLG and LOR in dorsal skins in our murine model. It has been reported that FLG and LOR expressions are decreased by histamine in keratinocytes and human AD skin and suggested that this may be associated with impaired skin barrier function [53]. As mentioned above, we also observed that 1-Iodo inhibited histamine release by IgE-activated RBL-2H3 mast cells Moreover, 1-Iodo decreased mast cell infiltrations into dorsal skin tissues and reduced, but not significantly, serum level of histamine in AD-like model mice. These results suggest 1-Iodo upregulates FLG and LOR by suppressing histamine release by mast cells, and that 1-Iodo might have a positive effect on the skin barrier function.

Cell Culture
Rat basophilic leukemia (RBL-2H3) cells were purchased from the Korean Cell Line Bank and human skin keratinocytes (HaCaT cells) from the Daegu Gyeongbuk Institute for Oriental Medicine Industry (Daegu, South Korea). Cells were cultured in DMEM supplemented with 10% FBS and antibiotics (100 U/mL penicillin/100 µg/mL streptomycin, and 200 mM glutamine) at 37 • C in a humidified 5% CO 2 atmosphere.

β-Hexosaminidase and Histamine Release Assays
In vitro analysis: RBL-2H3 cells (2 × 10 4 cells/well) were seeded in a 24-well plate and cultured for 12 h. After washing with PBS, cells were incubated with different concentrations of the test sample (1-Iodo) for 48 h and then sensitized with anti-DNP-IgE (200 ng/mL) for 10 h at 37 • C. After washing with Siraganian buffer (119 mM NaCl, 5 mM KCl, 5.6 mM Glucose, 25 mM PIPES, 0.4 mM MgCl 2 , CaCl 2 1 mM, 0.1% BSA, pH 7.2), cells were incubated with DNP-BSA (10 ng/mL) for 1 h at 37 • C. Media were collected and centrifuged at 10,000× g for 10 min at 4 • C. For β-hexosaminidase release analysis, the supernatants (conditioned media; 50 µL/well) were transferred to a 96-well plate, incubated with 100 µL of the substrate (1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide in 0.05 M citrate buffer, pH 4.5) for 1 h at 37 • C; then, reaction stop solution (0.05 M sodium carbonate buffer (pH 10.0)) was added. Absorbances were measured at 405 nm using an ELISA reader (Synergy 2, Bio-Tek Instruments). For histamine release analysis, supernatants (100 µL/well) were transferred to a 96-well plate and histamine contents were determined using a histamine enzyme assay kit (Cayman Chemical, Ann Arbor, MI, USA). In vivo analysis: Serum was diluted 1:5 in PBS, and then β-hexosaminidase and histamine levels were analyzed as described above for β-hexosaminidase and histamine release analysis in cells. The levels of histamine content and β-hexosaminidase release were expressed as a percentage of the control (anti-DNP-IgE alone group, in vitro; non-treated group, in vivo).

DNCB-Induced Atopic Dermatitis Animal Model
All tests involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85-23, revised 1996) and approved by the Animal Subjects Committee of Konkuk University, Korea (Approval number: KU20083). The atopic dermatitis was induced in shaved dorsal skin using DNCB as previously reported [27,43].
BALB/c mice (male, 5-6 weeks old; Orient Bio, Korea) were divided into five groups: the normal control group (n = 5), a DNCB plus topical olive oil group (n = 5; the DNCB control group), a DNCB plus topical 0.1% dexamethasone (n = 5; the DEX group), a DNCB plus topical 1-Iodo 50 µg/mL (n = 5; the 50 µg/mL 1-Iodo group), and a DNCB plus topical 1-Iodo 100 µg/mL (n = 5; the 100 µg/mL 1-Iodo group). DNCB-induced atopic dermatitis in animals was induced using the following protocol. The dorsal skin was shaved 1 day before DNCB sensitization (day 0). For all groups except the normal control group, on days 1, 3, 5, and 7, 200 µL of DNCB (1%; dissolved in acetone and olive oil mixture (4:1, v/v) was applied to the dorsal skin (the sensitization period). The 1% DNCB-applied dorsal skins were treated with 0.5% DNCB solution daily from day 8 to day 14, to induce dermatitis (the induction period). The dorsal skins of dermatitis-induced mice were treated with 0.2% DNCB plus 100% olive oil (extra pure grade, Duksan, Korea) (the DNCB control group) and with 0.2% DNCB plus 50 µg/mL or 100 µg/mL of 1-Iodo dissolved in 100% olive oil (the 50 and 100 µg/mL 1-Iodo groups) daily from day 15 to day 35. Animals in the DEX group, a positive control group, were also treated with 0.2% DNCB plus 0.1% dexamethasone dissolved in 100% olive oil from day 15 to day 35 in the same manner as treatments were applied in the 50 and 100 µg/mL 1-Iodo groups. In addition, animals in the normal control groups were treated with 100% olive oil daily from day 15 to day 35. On day 35, images of animals were obtained, some animals were sacrificed by CO 2 inhalation, and skin samples were excised. Other animals were anesthetized with isoflurane, blood was collected by a cardiac puncture, and the animals were sacrificed by CO 2 inhalation, followed by excision of skin samples. The collected blood was centrifuged for 10 min to obtain the serum. The serum was stored at −80 • C for experiments.

Evaluation of Dermatitis Severity
Dermatitis severity was assessed weekly using SCORAD scores [27,43]. In brief, dermatitis severity was assessed by scoring the severities of edema, erythema/hemorrhage, erosion/excoriation, and dryness, which were awarded scores of 0 to 3 (where a score of 0 indicated severe and a score of 3 indicated no symptom). Dermatitis severity was defined as the sum of these four symptom scores.

Histopathological Analysis
Dorsal skin tissues (3 × 4 cm sizes) prepared from five mice per group were fixed in 4% paraformaldehyde, sectioned at 4 µm, deparaffinized, and stained with hematoxylin and eosin (H&E) to evaluate epidermal hyperplasia (thickness) or stained with toluidine blue to evaluate mast cell infiltration. For immunohistochemistry, staining experiments were conducted using the MicroProbe Manual Staining System (Fisher Scientific, Pittsburgh, PA, USA), some deparaffinized skin sections were immersed in 0.3% H 2 O 2 to abolish endogenous peroxidase activity and incubated with polyclonal mouse anti-FLG (diluted 1:500) and anti-LOR (diluted 3 µg/mL) for 30 min at 38 • C. Skin sections were then treated with a secondary antibody and 3-amino-9-ethyl-carbacole as required by the UltraVision LP Detection System (Thermo Scientific, Waltham, MA, USA) and counterstained with hematoxylin for 30 s. All sections were photographed under a microscope (BX50F; Olympus Optical Co., Ltd.), and the images were analyzed using Image Pro Plus Version 4.5 software (Media Cybernetics Co., Silver Spring, MD, USA).

Statistical Analysis
Data are expressed as the means ± standard errors of the means (SEMs) of the indicated numbers of experiments. The significances of differences between pairs of groups were determined using the Student's t-test, and multiple comparisons were performed by oneway analysis of variance (ANOVA) followed by the Newman-Keuls post hoc test. The analysis was performed using GraphPad Prism (Version 6.0; GraphPad Software, San Diego, CA, USA), and p values of <0.05 were considered significant.

Conclusions
The present study showed 1-Iodo reduced histamine and β-hexosaminidase release and SNARE protein VAMP8 expression in RBL-2H3 mast cells, and it upregulated FLG and LOR expressions reduced in TNF-α-exposed HaCaT keratinocytes. In vivo, 1-Iodo reduced the severities of dorsal skin lesions, skin epidermal thicknesses, and mast cell infiltration in DNCB lesioned skin, attenuated the DNCB-induced level of β-hexosaminidase in serum, and prevented DNCB-induced reduction in the expressions of FLG and LOR. These findings suggest that 1-Iodo interferes with mast cell degranulation linked to VAMP8 protein and upregulates the expressions of the skin barrier-related proteins FLG and LOR, thus ameliorating AD severity. Therefore, it appears that 1-iodohexadecane is a promising functional material for regulating mast cell degranulation and skin barrier abnormalities. Furthermore, our findings indicate it has therapeutic potential for the treatment of AD. Additional studies are needed to clarify the mechanism responsible for the ameliorative effects of 1-iodohexadecane on AD-like symptoms.

Institutional Review Board Statement:
All animal experiments and procedures in this study were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85-23, revised 2011) and were approved by the Animal Subjects Committee following the Institutional Guidelines of Konkuk University, Korea (KU20083, 16 June 2020) and conducted according to institutional guidelines.