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Vol. 44, No. 1 7Biol. Pharm. Bull. 44, 7–17 (2021)

© 2021 The Pharmaceutical Society of Japan

Regular Article

Vitamin K2 Suppresses Proliferation and Inflammatory Cytokine Production in Mitogen-Activated Lymphocytes of Atopic Dermatitis Patients through the Inhibition of Mitogen-Activated Protein KinasesMeiyu Zhang,a,b Taro Miura,c Shunsuke Suzuki,c Masako Chiyotanda,d Sachiko Tanaka,a Kentaro Sugiyama,*,a Hisashi Kawashima,d and Toshihiko Hiranoa

a Department of Clinical Pharmacology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences; 1432–1 Horinouchi, Hachioji, Tokyo 192–0392, Japan: b Experimental Research Center, China Academy of Chinese Medical Sciences; No.16, Nanxiaojie, Dongzhimennei, Beijing 100700, China: c Department of Pediatrics, Tokyo Medical University Hachioji Medical Center; 1163 Tatemachi, Hachioji, Tokyo 193–0998, Japan: and d Department of Pediatrics and Adolescent Medicine, Tokyo Medical University; 6–7–1 Nishishinjuku, Shinjuku-ku, Tokyo 160–0023, Japan.Received January 26, 2020; accepted October 16, 2020

Vitamin K2 is suggested to have a suppressive effect on the peripheral blood mononuclear cells (PBMCs) of pediatric atopic dermatitis patients. We examined the molecular targets of vitamin K2 to suppress pro-liferation and cytokine production in T-cell mitogen-activated PBMCs of atopic dermatitis patients from the viewpoint of mitogen-activated protein kinase signaling molecules. The study population included 16 pediat-ric vitamin K2 patients and 21 healthy subjects. The effect of vitamin K2 on concanavalin A-activated PBMC proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell counting assays. T-helper (Th)1/Th2/Th17 cytokine profiles in plasma and PBMC-culture supernatants were analyzed by a cytometric beads array assay. Mitogen-activated protein kinase signaling molecules in concanavalin A-activated PBMCs were examined by enzyme-linked immunosorbent assay (ELISA) assays. At 10–100 µM, vitamin K2 significantly suppressed the proliferation of mitogen-activated PBMCs derived from atopic dermatitis patients and healthy subjects (p < 0.05). The interleukin (IL)-10 concentrations in plasma and the PBMC culture supernatants of atopic dermatitis patients were significantly higher than those of healthy subjects (p < 0.05). The IL-2 concentrations in the culture supernatants of atopic dermatitis PBMCs were significantly lower than those of healthy PBMCs (p < 0.05). Vitamin K2 significantly inhibited the IL-17A, IL-10, and tumor necrosis factor α (TNF-α) production (p < 0.05), and increased the IL-2 production (p < 0.01) in the culture supernatant of atopic dermatitis PBMCs. At 10–100 µM, vitamin K2 markedly de-creased the of Mek1, extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase, and SAPK/c-Jun N-terminal kinase (JNK) expression in atopic dermatitis PBMCs (p < 0.05). Vitamin K2 is sug-gested to attenuate activated T-cell immunity in atopic dermatitis patients through the inhibition of mitogen-activated protein kinase-Mek1-ERK1/2 and SAPK/JNK signaling pathways.

Key words vitamin K2; atopic dermatitis; mitogen-activated protein kinase signaling pathway; cytokine

INTRODUCTION

Atopic dermatitis, also known as atopic eczema, is a chronic inflammatory skin disease characterized by pruritic, erythematous, and scaly skin lesions localized on the flexural surfaces of the body with a remitting, relapsing course. Gener-ally, the onset of atopic dermatitis (AD) is in early childhood, and epidemiological surveys have shown a steady increase in the prevalence of atopic dermatitis in developing countries.1,2) AD is estimated to occur in up to 20% of children and 3% of adults worldwide.3) Among patients with pediatric AD that persists into adulthood, up to 50% of patients are reported to develop asthma and arthritis.4) Atopic dermatitis is reported to have “atopy” characteristics, including a familial tendency to experience asthma, allergic rhinitis, eczema, allergy to het-erogeneous proteins, elevated total serum immunoglobulin E (IgE) and blood eosinophilia.5) Typical AD shows a specific clinical manifestation of eczema and the above characteristics.

AD has been viewed as a T helper (Th)2-mediated aller-gic disease associated with an increased IgE concentration, increased eosinophils, and mast cell activation; the overex-

pression of Th2 cytokines interleukin (IL)-4, IL-5, and IL-13 are also observed.6) The upregulation of novel T lymphocyte subpopulations (Th9, Th17, and Th22) and their secreted cyto-kines (IL-9, IL-10, IL-17, and IL-22) have also been reported in AD.6) Furthermore, the reduced expression of epidermal differentiation complex and filamentous protein genes (FLG), the reduced secretion of antimicrobial peptides, and the de-struction of the epidermal barrier function often occur.6)

The keratinocytes in atopic dermatitis patients produce pro-inflammatory factors, such as thymic stromal lymphopoietin (TSLP), which further affect the balance of Th cells.7) In ad-dition, the mitogen-activated protein kinase pathways, which include the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (c-JNKs) and p38 mitogen-activated protein kinase,8,9) are also known to be implicated in cell pro-liferation, differentiation, cell death and inflammation. The phosphorylation of mitogen-activated protein kinases causes the production of inflammatory mediators, and promotes an inflammatory response. Thus, the inactivation of mitogen-activated protein kinases subsequently decreases the inflam-matory response, which could be an efficient way to treat

* To whom correspondence should be addressed. e-mail: sugiyama@toyaku.ac.jp

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several autoimmune diseases.10,11) These novel insights have, in turn, led us to develop new therapeutic strategies for atopic dermatitis based on the immunological regulation.

We previously reported that vitamin Ks effectively suppress the proliferation and production of inflammatory cytokines from human peripheral-blood mononuclear cells (PBMCs) activated by T cell mitogen.12,13) We also examined the effects of VKs on the proliferation and regulatory T cell-frequency in the PBMCs of pediatric atopic dermatitis patients.14) Among the vitamin Ks that we examined in these studies, vitamin K2 is generally used for the treatment of osteoporosis, and we therefore considered that vitamin K2 would be a candidate therapeutic agent for the safe and efficient treatment of atopic dermatitis.

In the present study, we used PBMCs from patients to newly evaluate the molecular mechanism(s) through which vitamin K2 suppresses activated PBMCs in vitro. This PBMC culture system can simulate the in vivo immune network more closely than cultures with separated cells, such as T cells, B cells, or monocytes, since PBMCs include various types of immune cells—many of which may participate in the immune network response in vivo.15) Thus, we evaluated the effects of on the proliferation, cytokine production and expression of mitogen-activated protein kinase signaling molecules in T-cell mitogen-activated PBMCs of pediatric atopic dermatitis patients.

MATERIALS AND METHODS

Reagents Vitamin K2 (menaquinone-4, 136-16641-100mG) was obtained from Wako Pure Chemical Corporation (Osaka, Japan), and dissolved in ethanol at concentrations of 5–5000 µM as working solutions. RPMI 1640 medium (R8758-500 mL), fetal bovine serum (FBS) and 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Concanavalin A (Con A) and dimethyl sulfoxide (DMSO) were obtained from Wako Pure Chemical Corporation. Ficoll-Hypaque was purchased from Nacalai Tesque Co. (Kyoto, Japan). Calcein-AM was obtained from Invitrogen Co. (Eu-

gene, OR, U.S.A.), and propidium iodide (PI) was obtained from Sigma-Aldrich. Mitogen-activated protein kinase signal-ing enzyme-linked immunosorbent assay (ELISA) kits was purchased from CST Inc. (Chiyoda-ku, Tokyo, Japan). The BD™ Cytometric Bead Array (CBA) Human Th1/Th2/Th17 cytokine kit was obtained from BD Biosciences Inc. (San Jose, CA, U.S.A.). All other reagents were of the highest avail-able grade.

Patients and Healthy Subjects The present study in-cluded 10 pediatric atopic dermatitis patients (male, n = 7; female, n = 3; age, 5.4 ± 4.0 years) and 21 healthy subjects (male, n = 11; female, n = 10; age, 28.2 ± 8.9 years). The basic characteristics and laboratory test indicators of the atopic der-matitis patients are shown in Table 1. There was no difference in the sex ratio between the 10 atopic dermatitis patients and the 21 healthy subjects. Furthermore, there was no difference in the sex ratio between the atopic dermatitis patients and the healthy subjects in any of the experiments in this study. These healthy subjects had no history of immunosuppressive medica-tion. Venous blood (20 mL) was taken from each subject and heparinized. The present study was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). The study was approved by the institutional ethics committee for studies in humans in both Tokyo Medical University Hospital (#3295) and the Tokyo University of Pharmacy and Life Sciences (#16-1). Written informed consent was obtained from all parents of the patients and healthy subjects.

Isolation of PBMCs and Plasma The isolation and culture of PBMCs were carried out according to previously described methods.16) Briefly, heparinized blood (5 mL) was loaded onto 4 mL of Ficoll-Hypaque and centrifuged at 2300 rpm for 20 min at room temperature. After centrifuga-tion, plasma located in the upper layer was transferred into the tube. Buffy coat containing PBMCs was aspirated out, and the cells were rinsed three times with RPIM 1640 medium containing 10% FBS, 100000 IU/L penicillin, and 100 mg/L streptomycin. PBMCs were re-suspended in the RPMI 1640 medium to a final cell density of 1 × 106 cells/mL. The plasma was stored at use under air at −80 °C.

Table 1. Characteristics and Laboratory Data Pediatric Patients with Atopic Dermatosis

Case number Age (years) Sex SCORAD Complications Another allergy

historyPeripheral eosinophil

Total serum IgE level (U/mL)

Specific IgE serum (Dermatophagoi-des pteronyssinus)

(UA/mL)

TARK (pg/mL)

Treatment duration (years)

1 10 Male 20 None BA, AR 238 138 0.25 374 ≥ 2 years2 13 Male 15 None BA, AR 280 406 25.4 287 ≥ 5 years3 4 Female 36 None None 1150 438 53.3 1834 ≥ 3 months4 6 Male 22 None FA 437 763 19.1 175 ≥ 1 year5 1 Male 35 None None 300 60 0.1 1086 ≥ 1 year6 2 Male 10 None FA 486 108 0.1 806 ≥ 1 year7 2 Female 22.5 None None 913 617 158 615 ≥ 1 year8 8 Male 10 None FA, AR 561 1090 376 422 ≥ 1 year9 2 Male 17 None FA 304 181 36.1 — ≥ 1 year

10 6 Female 18 None BA, AR, FA 172 834 22.5 389 ≥ 4 months

Mean (S.D.) 5.4 (4.0) 20.6 (9.0) 484.1 (316.5) 463.5 (353.1) 69.1 (24.0) 665.3 (520.7)Median 5 19 370.5 422 24 422.0Minimum 1 10 172 60 0.1 175.0Maximum 13 36 1150 1090 376 1834.0

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MTT Assay Procedures Each of the 186-µL PBMC sus-pensions was prepared as described above and plated on non-treated microplates with 96 flat-bottom wells. Ten microliters of Con A solution was pipetted into each well at a final con-centration of 5.0 µg/mL. Subsequently, 4 µL of ethanol solution containing vitamin K2 was added to give final drug con-centrations of 0.1, 1, 10, and 100 µM, respectively. The same volume of each vehicle solution was pipetted into the control wells. The PBMCs were incubated for 96 h at 37 °C under 5% CO2. After incubation, the MTT assay procedures were per-formed, and the absorbance (OD) at 550 nm was read in each well with a microplate reader Varioskan® Flash was manufac-tured by Thermo Scientific Inc (Waltham, MA, U.S.A.).

Cell Counting Assay Procedures After 96 h of incu-bation in a CellCarrier 96-well plate (PerkinElmer, Inc., Waltham, MA, U.S.A.), the cells were washed using 200 µL PBS, and re-suspended in 100 µL of the staining solution containing fluorescent dyes, Calcein AM and PI, at final concentrations of 2 µM and 100 µg/mL, respectively. The cells were incubated at 37 °C for 30 min, and then fixed with 100 µL of 1% formaldehyde at room temperature for 15 min. After re-washing, the cells were re-suspended in 100 µL of 1 × PBS. For the measurement of cell viability, live and dead cells were stained with Calcein AM and PI dyes, respectively. Subsequently, the cells in each well were scanned and imaged with an Operetta high-content imaging system (PerkinElmer, Inc.) at 10× air objective and the non-confocal UV channel. Nine regions of view (1292 × 1292 µm) corresponding to ap-proximately 65% of the area were recorded for each well. An image analysis and evaluation were then performed using the Harmony software program (version 4.5) with PhenoLOGIC (PerkinElmer, Inc.). The number of living cells was counted by detecting the number of nuclei in 9 regions per well, and the numbers of cells in different groups were calculated from the average in two wells of each group, and reported as the mean (standard deviation (S.D.)). The dose–response curves and the percentage of inhibition by vitamin K2 were deter-mined using the cell count data.

The Th1/Th2/Th17 Cytokine Analysis The concentra-tions of interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-2, IL-4, IL-6, IL-10 and IL-17A in the plasma and the supernatant of the culture medium were measured using a BD™ CBA Human Th1/Th2/Th17 Cytokine Kit fol-lowed by flow cytometry. The assays were performed accord-ing to the manufacturer’s instructions. Briefly, seven capture bead populations with distinct fluorescence intensities coated with specific capture antibodies were mixed (i.e., 50 µL of bead mixture, 50 µL of sample, and 50 µL of phycoerythrin (PE) detection reagent were mixed and vortexed thoroughly). The mixture was incubated for 3 h at room temperature pro-tecting from light in order to form sandwich complexes. The beads were washed with the washing buffer and re-suspended in 300 µL of the buffer. The samples were then analyzed by flow cytometry with a FACSCanto™ II (BD Biosciences, Inc.), using BD FACSDiva™ software program. The BD FACSDiva and FCAP Array™ (V3.0) software programs were used for the analysis. These experiments were carried out according to the manufacturer’s instructions (BD Biosciences, Inc.).

Phosphorylated Mitogen-Activated Protein Kinase As-says After 96 h of incubation, cells were harvested and stored at −80 °C. The expression levels of p-Erk1/2 mitogen-

activated protein kinase, p-p38 mitogen-activated protein kinase, MEK1, p-MEK1/2, SAPK/JNK, or p-SAPK/JNK were measured by a human mitogen-activated protein kinase, multi-target sandwich ELISA kit according to the manu-facturer's instructions. We then also examined the amounts of p-ERK1/2, MEK1, SAPK/JNK, which were measured by human p-p42/44 mitogen-activated protein kinase, total MEK1, and SAPK/JNK sandwich ELISA kits according to the manufacturer’s instructions.

Statistical Analysis All values were presented as the mean ± standard deviation (S.D.) A one-way ANOVA with a post-hoc Dunnett’s T3 test and Bonferroni test was used to assess the difference in cell proliferation between PBMCs treated with serial concentrations of VK2 and control PBMCs. Differences in the amounts of Th1/Th2/Th17 cytokines pro-duced in the culture supernatant (CS) between PBMCs treated with vitamin K2 and control PBMCs were also analyzed in the same manner as described above. Wilcoxon’s signed-rank test was applied to evaluate the differences in the expression levels of phosphorylated mitogen-activated protein kinases between PBMCs treated with and without vitamin K2. When necessary, the difference in the mean concentration of cyto-kines between plasma and CS of PBMCs was analyzed by Student’s t-test. The difference in sex ratio between atopic dermatitis patients and healthy subjects was analyzed by an χ2 test. A statistical ANOVA was performed using the SPSS 26 software package (IBM Inc. Chicago, IL, U.S.A.), Excel 2016 (Microsoft) and GraphPad PRISM 7 (GraphPad Software Inc., San Diego, CA, U.S.A.). p Values of <0.05 were considered to indicate statistical significance.

RESULTS

The Anti-proliferative Potency of Vitamin K2 in PBMCs of Healthy Subjects and Atopic Dermatitis Patients, as As-sessed by an MTT Assay and Calcein AM/PI Stained-Cell Counting Assay We first examined the pharmacological ef-ficacy of vitamin K2 on the proliferation of Con A-activated PBMCs of healthy subjects and atopic dermatitis patients with an MTT assay (Fig. 1A). To further confirm the efficacy of vitamin K2 on Con A-activated PBMC proliferation, we also performed a cell counting assay with dual-staining of treated cells by Calcein AM/PI staining, which can co-labels viable and non-viable cells (Figs. 1B, C). Green calcein-positive cells were considered to be alive, while red PI-positive cells were counted as dead cells.

Vitamin K2 inhibited the proliferation of PBMCs of healthy subjects in a dose-dependent manner with a significant effect at 100 µM (p < 0.001), as assessed by an MTT assay (Fig. 1A), and at 10 and 100 µM (p < 0.05 and p < 0.01), as assessed by a cell counting assay (Figs. 1B, C). The IC50 values of vitamin K2, as estimated by these assays, are shown in Table 2. The mean (S.D.) and median IC50 values of vitamin K2 in PBMCs of healthy subjects, as assessed by the MTT assay, were 8.1 (10.0) and 4.6 µM, respectively; the IC50 values varied among the subjects (n = 13), ranging from 0.3 to 30.8 µM. On the other hand, the mean (S.D.) and median IC50 values of vitamin K2 in PBMCs of healthy subjects, as estimated by the cell counting assay, were 8.7 (11.1) and 4.0 µM, respectively; the IC50 values showed variation among the subjects (n = 9), rang-ing from 1.6 to 36.7 µM.

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Fig. 1. The Effects of Vitamin K2 on the Proliferation of Con A-Activated PBMCs Derived from Healthy Subjects and Atopic Dermatitis Patients(A) PBMCs derived from healthy subjects (n = 13) and atopic dermatitis patients (n = 10) were cultured with Con A in the presence or absence of vitamin K2, and the cell

proliferation was estimated by an MTT assay. The data are presented as the mean ± standard deviation (S.D.), and the differences in the PBMC proliferation (%) in each subject group between cells treated with and without serial concentrations of vitamin K2 were analyzed by a one-way ANOVA with a post-hoc Dunnett’s T3 test (equal variance not assumed) and Bonferroni (equal variances assumed) tests. ** p < 0.01, *** p < 0.001, in comparison to ConA(+)Lym(+) group (see Materials and Methods). No significant difference was observed in the PBMC proliferation (%) between the two subject groups. (B) Fluorescence images of PBMCs derived from healthy subjects and atopic dermatitis patients treated with serial concentrations of vitamin K2, as detected by co-labeling with Calcein-AM/PI (magnification 100×). The ConA(-)Lym(+) group shows PBMCs cultured in the absence of Con A and vitamin K2. All of the other cells were cultured with Con A in the presence or absence (ConA(+)Lym(+) group) of serial concentrations of vitamin K2. (C) The comparison of the inhibitory effects of vitamin K2 on the cell viability in PBMCs of healthy subjects and atopic dermatitis patients, as measured by a cell counting analysis. Wilcoxon’s signed-rank test was used to analyze the difference in the number of living cells treated with serial concentra-tions of vitamin K2 between healthy subjects (n = 9) and atopic dermatitis patients (n = 10). The data are presented as the mean ± S.D., and the differences in the number of living cells in the PBMCs of each subject group between cells treated with and without serial concentrations of vitamin K2 were analyzed by Dunnett’s T3 test. * p < 0.05, ** p < 0.01 in healthy PBMCs, and #p < 0.05 and ##p < 0.01 in atopic dermatitis PBMCs, in comparison to each group of ConA(+)Lym(+). The number of living cells in the two subject groups did not differ to a statistically significant extent. (Color figure can be accessed in the online version.)

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We also examined the effects of vitamin K2 on the Con A-induced proliferation of PBMCs obtained from atopic dermatitis patients using an MTT assay (Fig. 1A) and a cell counting assay (Figs. 1B, C) (Table 2). Vitamin K2 inhibited the mitogen-activated proliferation of PBMCs of AD patients at 10 and 100 µM (p < 0.01 and p < 0.001) in a dose-dependent manner, as estimated by both the MTT and cell counting as-says. The IC50 values of vitamin K2 are shown in Table 2. The mean (S.D.) and median IC50 values of vitamin K2 in PBMCs of atopic dermatitis patients, as estimated by the MTT assay, were 4.4 (2.2) and 4.1 µM, respectively; the IC50 values showed variation among the subjects (n = 10), ranging from 1.6 to 8.8 µM. The mean (S.D.) IC50 values of vitamin K2 in PBMCs of atopic dermatitis patients, as estimated by the cell counting assay, were 3.8 (2.9) and 2.7 µM, respectively; the IC50 values showed variation among the subjects (n = 10), ranging from 0.8 to 10.4 µM.

Figure 1B shows fluorescence images of vitamin K2-treated PBMCs of healthy subjects (n = 9) and AD patients (n = 10). The number of viable cells in the PBMCs of healthy subjects was significantly reduced in a dose-dependent manner by vitamin K2 at concentrations of 10 to 100 μΜ (p < 0.05 and p < 0.01). Similarly, the number of viable cells in the PBMCs of AD patients was significantly reduced by vitamin K2 at concentrations of 10 to 100 μΜ (p < 0.01). As shown in Fig. 1C, the number of living cells was also reduced by vitamin K2 at concentrations of 10 to 100 μΜ (p < 0.05 and p < 0.01) in both healthy subjects and atopic dermatitis patients. Thus, vitamin K2 efficiently inhibited the mitogen-activated prolif-eration of PBMCs of both healthy subjects and vitamin K2 patients.

The Cytokine Concentrations in Plasma and Culture Su-pernatant of PBMCs without Con A Activation in Healthy Subjects and Patients The IL-10 concentrations in plasma and culture supernatant (CS) of PBMCs of atopic dermatitis patients were significantly higher in comparison to those in the plasma and CS of PBMCs of healthy subjects (p < 0.05 and p < 0.01) (Fig. 2). In contrast, the IL-2 concentrations in the CS of PBMCs of atopic dermatitis patients were signifi-cantly lower in comparison to healthy subjects (p < 0.05) (Fig. 2).

The IL-17A and TNF-α concentrations in plasma and CS of PBMCs of atopic dermatitis patients tended to be higher in comparison to those in PBMCs of healthy subjects. The IL-6 and IFN-γ concentrations in CS of PBMCs of atopic dermatitis patients also tended to be higher in comparison to PBMCs of healthy subjects. The IL-4 concentrations in plasma and CS of PBMCs did not differ between atopic dermatitis patients and healthy subjects. The differences in the IL-17A, TNF-α, IL-6,

IFN-γ, and IL-4 concentrations between atopic dermatitis patients and healthy subjects were not statistically significant.

The Effects of Vitamin K2 on Th1/Th2/Th17 Cytokine Production from Mitogen-Activated PBMCs of Healthy Subjects and Atopic Dermatitis Patients The effects of vitamin K2 on the production of Th1/Th2/Th17 cytokines in the CS of Con A-activated PBMCs of healthy subjects and atopic dermatitis patients were examined (Fig. 3). In the CS of Con A-activated PBMCs, the concentrations of most cytokines were obviously increased in comparison to the concentrations in the CS of non-activated PBMCs (Figs. 2, 3). The IFN-γ con-centration in the CS of Con A-activated PBMCs, for instance, increased almost 1000-fold in comparison to the concentration in the CS of non-activated PBMCs. The concentrations of six other cytokines in the CS also increased approximately 10- to 20-fold after activation (see Figs. 2 and 3).

At a concentration of 100 µM, vitamin K2 significantly in-hibited the production of IFN-γ, TNF-α, and IL-10 (p < 0.05 and p < 0.01), while vitamin K2 increased the production of IL-2 and IL-4 by the activated PBMCs of healthy subjects (p < 0.01). Similarly, at a concentration of 10-100 µM, vitamin K2 significantly decreased the concentrations of IL-17A and IL-10 (p < 0.05 and p < 0.01), and 100 µM vitamin K2 signifi-cantly inhibited the production of TNF-α (p < 0.05) by acti-vated PBMCs of atopic dermatitis patients. At a concentration of 100 µM, vitamin K2 increased the production of IL-2 by the activated PBMCs of atopic dermatitis patients (p < 0.01). Vitamin K2 decreased the concentration of IFN-γ and in-creased concentration of IL-4 in the CS of Con A-activated PBMCs of healthy subjects (p < 0.01). This tendency was also observed in the effects of vitamin K2 on the PBMCs of atopic dermatitis patients; however, the effects were not statistically significant.

The difference in the TNF-α concentrations in the CS of PBMCs cultured in the presence of serial concentrations of vitamin K2 between healthy subjects and atopic dermatitis patients was statistically significant (p < 0.05). The difference in the IFN-γ concentrations in CS of PBMCs cultured in the presence of 10 and 100 µM of vitamin K2 between healthy subjects and atopic dermatitis patients was also statistically significant (p < 0.05).

The Effects of Vitamin K2 on Mitogen-Activated Pro-tein Kinase Signaling Molecules in the PBMCs of Healthy Subjects and Atopic Dermatitis Patients To understand the molecular mechanism underlying the inhibitory effects of vitamin K2 on T-cell proliferation and cytokine production, we examined the effects of vitamin K2 on the phosphoryla-tion of mitogen-activated protein kinase signaling molecules in the Con A-activated PBMCs of healthy subjects and atopic

Table 2. The Comparison of the IC50 Values of VK2 on the Proliferation of Con A-Activated PBMCs from Healthy and AD Subjects, as Assessed by the MTT and Cell-Counting Assays

Subjects Assessed by MTT and cell-counting assays (number of subjects)

VK2 IC50 values (µM)

Mean (S.D.) Median Minimum Maximum

Healthy MTT (n = 13) 8.1 (10.0) 4.6 0.3 30.8

Cell counting (n = 9) 8.7 (11.1) 4.0 1.6 36.7

AD patients MTT (n = 10) 4.4 (2.2) 4.1 1.6 8.8

Cell counting (n = 10) 3.8 (2.9) 2.7 0.8 10.4

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dermatitis patients using a mitogen-activated protein kinase, multi-target sandwich ELISA assay (Fig. 4). At a concentra-tion of 20 µM, vitamin K2 significantly inhibited the expres-sion levels of MEK1, SAPK/JNK, and Erk1/2 (p < 0.05)—but did not significantly inhibit the expression of phospho-p38 mi-togen-activated protein kinase, phospho-MEK1, or phospho-SAPK/JNK—in both healthy PBMCs and atopic dermatitis PBMCs.

The concentration-dependent effects of vitamin K2 on the amounts of MEK1, SAPK/JNK, and phospho-ERK1/2 mito-gen-activated protein kinase, (p < 0.05) in Con A-activated PBMCs were also examined, as shown in Fig. 5. These mark-ers did not differ to a statistically significant extent between healthy PBMCs and atopic dermatitis PBMCs. At concentra-tions of 10–100 µM, vitamin K2 significantly inhibited the

amounts of MEK1, SAPK/JNK, and phospho-Erk1/2 mitogen-activated protein kinase (p < 0.05) in a concentration-depen-dent manner in both healthy PBMCs and atopic dermatitis PBMCs.

DISCUSSION

Vitamin K2 has been used in the treatment of osteopo-rosis, vascular calcification, and bone catabolism in cancer metastasis and arthritis.17–19) We have previously reported that vitamin Ks have effectively suppress the proliferation of T cell mitogen-activated PBMCs of pediatric atopic dermatitis patients.14) The present study was carried out to investigate the implications of the regulation of mitogen-activated protein kinase, a signaling-molecule, on the suppressive efficacy of

Fig. 2. Th1/Th2/Th17 Cytokine Concentrations in the Plasma and Culture Supernatant (CS) of PBMCs of Healthy Subjects (Open Bars) and Atopic Dermatitis Patients (Shaded Bars)

Differences in the amounts of Th1/Th2/Th17 cytokines in the plasma and culture supernatant of PBMCs (CS) between healthy subjects (n = 13) and AD patients (n = 10) were analyzed by Wilcoxon’s signed-rank test. In the case of IL-4, a significant difference was observed between the two subject groups. * p < 0.05 and ##p < 0.01.

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vitamin K2 on proliferation and cytokine production in T cell mitogen-activated PBMCs of healthy subjects and atopic der-matitis patients.

The results showed that vitamin K2 at 10–100 µM sig-nificantly suppressed the T-cell proliferative response and modulated Th1/Th2/Th17 cytokine production in T cell mitogen-activated PBMCs derived from healthy subjects and pediatric atopic dermatitis patients, which are consistent with our previous findings.13,14) In our previous study, we examined the suppressive effects of vitamin Ks by MTT assays.14) In the present study in addition to the MTT assay, we confirmed the vitamin K2 efficacy by Calcein AM/PI double staining fol-lowed by cell counting.

At higher concentrations, vitamin K2 significantly de-

creased the levels of TNF-α, IL-10, and IL-17A, but markedly increased the level of IL-2 in culture supernatant of atopic dermatitis PBMCs that were activated by T-cell mitogen. Thus, our present data, together with the data of our previous study,14) suggested that the mechanism underlying the thera-peutic efficacy of vitamin K2 in atopic dermatitis involves not only the inhibition of the excess proliferative response of T lymphocytes but also the modulation of the balance of pro-inflammatory and anti-inflammatory cytokines. We previously reported that the percentage of peripheral CD4+ cells was sig-nificantly decreased by treatment with 10 and 100 µM vitamin K2, and that 100 µM vitamin K2 also reduced the percentage of CD4+CD25+ lymphocytes.14) These observations may sup-port our present findings.

Fig. 3. The Effects of Vitamin K2 on the Th1/Th2/Th17 Cytokine Production in the Culture Supernatant of T-Cell Mitogen-Activated PBMCs of Healthy Subjects (Open Bars) and Atopic Dermatitis Patients (Shaded Bars)

PBMCs were activated with Con A for 96 h in the absence (0 µM) or presence (0.1–100 µM) of vitamin K2. The concentrations of IL-17A, IFN-γ, TNF-α, IL-10, IL-6, IL-4, and IL-2 in the culture supernatant were measured by beads-array procedures, followed by flow cytometry. The data are presented as the mean ± S.D., and the dif-ferences in the cytokine concentrations between PBMCs cultured with serial concentrations of vitamin K2 and those cultured without vitamin K2 (ConA(+)Lym(+) group) were analyzed by a one-way ANOVA with a post-hoc Dunnett’s T3 test (equal variances not assumed) and Bonferroni (equal variances assumed) tests. * p < 0.05 and ** p < 0.01 in healthy PBMCs; #p < 0.05 and ##p < 0.01 in atopic dermatitis PBMCs; ◊ p < 0.05 and ◊◊ p < 0.01 between healthy PBMCs and atopic dermatitis PBMCs.

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In the present study, we also investigated the possible molecular mechanism(s) underlying the suppression of acti-vated T lymphocytes by vitamin K2 from the viewpoint of mitogen-activated protein kinase regulation. The data of the present study firstly showed that vitamin K2 significantly decreased the expression of Mek1, ERK1/2, and SAPK/JNK proteins, in a dose-dependent manner, in the T-cell mitogen-activated PBMCs of healthy subjects and atopic dermatitis patients, while vitamin K2 did not inhibit the expression of

p38 mitogen-activated protein kinase, protein. This result indi-cated that p38 mitogen-activated protein is not involved in the vitamin K2-related inhibition of proliferation and the inflam-matory responses in T cell mitogen-activated atopic dermatitis PBMCs, whereas the Mek1-ERK1/2 and SAPK/JNK signaling pathways are critical to these processes. SAPK/JNK plays im-portant roles in both innate and adaptive immune responses. JNK1 was shown to be required for the IL-17A-mediated induction of inflammatory cytokines and antimicrobial pep-

Fig. 5. The Effects of Vitamin K2 on the Expression of Target Proteins of MEK1, SAPK/JNK, and ERK1/2 Mitogen-Activated Protein Kinase in T Cell Mitogen-Activated PBMCs of Healthy Subjects (Open Bars) and Atopic Dermatitis Patients (Shaded Bars)

PBMCs were activated with Con A for 96 h in the absence or presence of serial concentrations (0.1–100 µM) of vitamin K2. Wilcoxon’s signed-rank test was used to ana-lyze the differences in the expression of three target proteins between PBMCs treated with and without vitamin K2. * p < 0.05 and ** p < 0.01 for healthy PBMCs; #p < 0.05 and ##p < 0.01 for atopic dermatitis PBMCs. The expression levels of these proteins did not differ between the two groups.

Fig. 4. The Effects of Vitamin K2 on Mitogen-Activated Protein Kinase Signaling Molecules in T-Cell Mitogen-Activated PBMCs of Healthy Sub-jects (Open Bars) and Atopic Dermatitis Patients (Shaded Bars)

PBMCs were stimulated with Con A for 96 h in the absence (0 µM) or presence (20 µM) of vitamin K2. A Wilcoxon signed ranks test was used to analyze the differences in the expression of mitogen-activated protein kinase pathway multi-target proteins between PBMCs cultured with and without vitamin K2. * p < 0.05 for healthy PBMCs, and #p < 0.05 for atopic dermatitis PBMCs.

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tides in both lung inflammation and the host defense.20) The anti-inflammatory effect of vitamin K2 was suggested to be exerted mainly via the inhibition of the SAPK/JNK signaling pathway in atopic dermatitis, in which the downregulation of IL-10 production and simultaneous upregulation of IL-2 pro-duction are observed in peripheral T cells.

Intracellular vitamin K receptor is suggested to medi-ate these immunopharmacological actions of vitamin K2 in PBMCs. Thus, it might be important to compare the vitamin K receptor expression and/or function in PBMCs between atopic dermatitis patients and healthy subjects. However, we could not examine the vitamin K receptor expression in the present study, mainly because the PBMC samples obtained from the infant atopic dermatitis patients were insufficient for these experiments. Thus, the above points should be investi-gated in a future study.

It has been reported that mitogen-activated protein kinases are involved in several immune responses in mammalian species.21) ERKs are known to be activated by mitogens and many kinds of growth factors, and are required for the regula-tion of cell proliferation and Th2 cell differentiation.18) JNK1 and JNK2 are essential for naïve CD4+ T cell differentiation and cytokine production,19) while p38 acts as a molecular switch changing the balance between cytokines, including IL-2, IL-10, IL-17A, IL-18, and IFN-γ.22–24) Topical p38 mito-gen-activated protein kinase inhibition was demonstrated to attenuate severe inflammatory responses in the dermis.25) The p38/ERK mitogen-activated protein kinase signaling pathways are also suggested to be involved in the regulation of filaggrin and involucrin by IL-17.26)

The skin phenotype of new-onset pediatric atopic dermatitis is known to be substantially different from that of adult atopic dermatitis. Early-onset pediatric atopic dermatitis was charac-terized by Th2/Th17/Th22-centered inflammation and liquid alterations, and the majority of IL-17/TNF-α-regulated genes were significantly upregulated in lesional pediatric atopic dermatitis.27) On the continuous activation of the Th2 (IL-13, IL-31 and CCL17) and T22 (IL-22 and S100As) axes, Th1 skewing (IFN-γ and CXCL10), and the induction of cytokines and antimicrobial peptides of the Th17 type (IL-17A, IL-19, CCL20, LL37 and peptidase inhibitor 3), Th9 (IL-9), IL-33 and innate markers (IL-1β, IL-8 and IFN-α1) were greater in children with atopic dermatitis.28) The non-lesional skin in these patients is also abnormal, showing epidermal prolifera-tion and higher levels of IL-17A, IL-19, LL37, and epidermal proliferation markers (keratin 16 and S100As). Although ex-cess Th2 activation is recognized in both pediatric cases and adult atopic dermatitis, Th9 and Th17 are highly activated at the time of disease inhibition. The increased IL-19 levels might be associated with Th2 and Th17 activation.28)

In our study, all cases involved pediatric patients with chronic clinical atopic dermatitis accompanied by a moderate-to-severe SCORAD score, an increased peripheral eosinophil count, higher levels of total serum IgE and TARC, and in-creased levels of IL-10 and IL-17 in plasma and culture su-pernatant of PBMCs. These results were consistent with those reported by Lesiak et al., who showed significantly higher serum concentrations of IL-10 and IL-13 in atopic dermatitis patients in comparison to healthy controls.29) The importance of this cytokine in the development and/or maintenance of atopic dermatitis has been recently highlighted by the reported

association between polymorphisms in the IL-10 gene and atopic dermatitis in childhood.30) Thus, as a key regulatory cytokine that limits and ultimately terminates the excessive T-cell responses to prevent chronic inflammation and tissue damage, IL-10 could be used as a peripheral screening bio-marker to detect chronic atopic dermatitis.

IL-17 is a key cytokine for the host defense against infec-tion, as well as in the pathogenesis of autoimmune diseases.31) In addition, in keratinocytes, IL-17 has been described as a master regulator of antimicrobial peptides (AMPs).32) IL-17 was usually described as being highly expressed in acute atop-ic dermatitis,33) while being barely detectable in the chronic phase of the disease.34) However, in the present study, we observed that the IL-17 tended to increase in both the plasma and culture supernatant of PBMCs of atopic dermatitis pa-tients in comparison to healthy subjects. Thus, increased lev-els of Th17 and IL-17—as biomarkers of the higher activation of the inflammatory axes—could be detected in peripheral bi-ological samples of chronic atopic dermatitis. In addition, we also observed that the levels of TNF-α in plasma and IFN-γ in the culture supernatant of PBMCs of atopic dermatitis patients tended to increase in comparison to the levels in healthy sub-jects. However, the IL-2 concentrations in the culture superna-tant of atopic dermatitis PBMCs were significantly lower than those in healthy PBMCs. IL-2 is a key cytokine for the Treg function, and has been implicated in the control of chronic al-lergic contact dermatitis.35) The administration of vitamin K2 may have the potential to improve the decreased IL-2 produc-tion in the PBMCs of atopic dermatitis patients (Fig. 3).

Many reports have suggested that atopic dermatitis is closely related to the activation of vascular calcification, which alters the fibrin clot properties associated with the reduced ef-ficiency of clot lysis.36,37) Vitamin K2 plays an important role in the clotting cascade, and vitamin K2-dependent proteins activate a protective mechanism against the development of vascular calcification.38) Furthermore, in numerous trials with healthy and diseased patient cohorts, vitamin K2 has been proven to have a long-term protective effect against the devel-opment of calcification.39,40) These reports, together with our present findings, led us to believe that vitamin K2 can be a candidate for the treatment of pediatric patients with chronic atopic dermatitis. Our present data suggest that vitamin K2 regulates T-cell mediated immunity through the manipulation of the mitogen-activated protein kinase, -Mek1-ERK1/2 and SAPK/JNK signaling pathways, which results in the inhibition of immune cell proliferation and inflammatory responses.

The present study was associated with some limitations. One limitation is that the healthy subjects included in this study were not age-matched to the atopic dermatitis patients. This age difference might have led to a bias in the data inter-pretation. The relatively small size of the samples was another limitation. These points should be considered in future stud-ies.

CONCLUSION

At concentrations of 10–100 µM, vitamin K2 significantly attenuated the T cell mitogen-activated PBMC proliferation and decreased the production of TNF-α, IL-10, and IL-17A by activated PBMCs. In activated atopic PBMCs, vitamin K2 increased the production of IL-2 by the activated PBMCs of

16 Vol. 44, No. 1 (2021)Biol. Pharm. Bull.

atopic dermatitis patients. Vitamin K2 is suggested to attenu-ate T-cell mediated immunity by inhibiting the proliferation and inflammatory responses via the manipulation of the mi-togen-activated protein kinase-Mek1-ERK1/2 and SAPK/JNK signaling pathways in the PBMCs of pediatric atopic derma-titis patients. Taken together, the results suggest that vitamin K2 could be a candidate agent for the treatment of chronic atopic dermatitis. Higher concentrations of IL-10 and lower concentrations of IL-2 were observed in both the peripheral plasma and culture supernatant of T cell mitogen-activated PBMCs of chronic atopic dermatitis patients in comparison to healthy subjects, suggesting that these inflammatory cytokines could be used as a biomarker for the diagnosis and treatment of chronic atopic dermatitis.

Conflict of Interest The authors declare no conflict of interest.

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