Antroquinonol Exerts Immunosuppressive Effect on CD8+ T Cell Proliferation and Activation to Resist Depigmentation Induced by H2O2

Antroquinonol was investigated as antioxidant and inhibition of inflammatory responses. Our study was to evaluate its immunosuppressive effect on CD8+ T cells and protective effect on depigmentation. CD8+ T cells were treated with antroquinonol in vitro, and C57BL/6 mice were treated with antroquinonol with or without H2O2 in vivo for 50 consecutive days. We found antroquinonol could inhibit proliferation of CD8+ T cells and suppress the production of cytokines IL-2 and IFN-γ and T cell activation markers CD69 and CD137 in vitro. H2O2 treatment induced depigmentation and reduced hair follicle length, skin thickness, and tyrosinase expression in vivo. Whereas, antroquinonol obviously ameliorated depigmentation of mice skin and resisted the reduction of hair follicle length, skin thickness, and tyrosinase expression induced by H2O2. Antroquinonol decreased CD8+ T cell infiltration in mice skin, inhibited the production of IL-2 and IFN-γ, and decreased the expression of CXCL10 and CXCR3. Summarily, our data shows antroquinonol inhibits CD8+ T cell proliferation in vitro. It also reduces CD8+ T cell infiltration and proinflammatory cytokine secretion and suppresses the thinning of epidermal layer in vivo. Our findings suggest that antroquinonol exerts immunosuppressive effects on CD8+ T cell proliferation and activation to resist depigmentation induced by H2O2.


Introduction
Vitiligo is a common dermatological disorder characterized by the progressive depigmentation caused by a loss of melanocytes in the epidermis. Absence of melanocytes in the skin lesion has been considered as a core event in the pathogenesis of vitiligo [1]. A single dominant pathway appears unable to explain all causes of vitiligo. Obviously, loss of melanocytes in vitiligo seems to occur through a complex interaction of several mechanisms including environmental, biochemical, immunological, and genetic events that act in concert [2]. In vitiligo epidermis, the increased levels of reactive oxygen species (ROS) were observed [3,4]. −89 A/T polymorphisms of catalase in vitiligo patients showed significantly increased lipid peroxidation levels [5]. Increased malondialdehyde and decreased catalase were found in vitiligo patient blood [6]. Higher activity of superoxide dismutase has been demonstrated in both lesional and nonlesional epidermis [7]. Lymphocyte analysis to peripheral blood of patients with vitiligo showed the total levels of T-cells are normal, but the ratio of CD4 + /CD8 + is decreased. The decreased CD4 + / CD8 + ratio of skin-infiltrating T cells and CD8 + T cells from vitiligo skin are observed in progressive disease [8]. Significantly higher number of circulation CD8 + T cells was shown in progressive generalized vitiligo [9]. Decreased CD4 + /CD8 + ratio was shown in active generalized vitiligo patients, which is involved in the pathogenesis of vitiligo [10]. Increased ROS are thought to be involved in onset of vitiligo, and the infiltration of melanocyte-specific cytotoxic CD8 + T cells into the perilesional margin directly result in melanocyte loss [11,12]. One study [13] reported that oxidative stress leads to chemokine production and causes CD8 + T cell skin trafficking and melanocyte destruction in vitiligo. Blockade of oxidative stress can ameliorate melanocyte apoptosis through anti-inflammatory and antiapoptotic processes. CXC chemokine ligand10 (CXCL10) was highly expressed in the skin and serum of patients with vitiligo and is critical to the progression and maintenance of depigmentation in a mouse model of vitiligo. CXCL10-CXCR3 (CXC chemokine receptor 3) axis is critical to both the progression and maintenance of depigmentation in vitiligo mouse models [14,15].
Antrodia camphorate is a mushroom growing on camphor tree in Taiwan forests. It is a traditional Chinese herbal medicine with several pharmacological effects, such as antioxidant and free radical-scavenging activities [16,17] and inhibition of inflammatory responses [18,19]. Antroquinonol is a major active component of Antrodia camphorate and was identified with its anti-inflammatory activity and anticancer potential [20][21][22]. Antroquinonol displayed anticancer potential for human hepatocellular carcinoma cells by adenosine 5 ′ -monophosphate-(AMP-) activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathways [23] and could protect the kidney from immunologic damage via blocking tumor necrosis factor-α (TNF-α) and interleukin-1β-(IL-1β-) mediated inflammatory process [24]. Antroquinonol differentially modulates T cell activity and reduced IL-18 production of murine accelerated severe lupus nephritis [25]. However, it remains to be determined whether antroquinonol is capable of preventing the various depigmentation histopathologic features of C57BL/6 mice treated by hydrogen peroxide (H 2 O 2 ). Immunosuppressive effect of antroquinonol on CD8 + T cells is still unknown.
We hypothesize that antroquinonol might exert immunosuppressive effect on CD8 + T cell proliferation and activation to resist depigmentation induced by H 2 O 2 . To test this, we investigated effects of antroquinonol on depigmentation model induced by H 2 O 2 that mimics vitiligo in vivo.

Materials and Methods
2.1. Study Subjects. This study was approved by the ethics committee of the third people's hospital of Hangzhou. Twenty healthy control's blood samples (Table 1) whose CD + T cells are outrange of reference were collected randomly from physical examination center of the third people's hospital of Hangzhou. Informed consent was obtained, and this study was approved by local ethics committees.

Animals and Treatment.
Four-week-old female pathogen-free C57BL/6 mice (weighing 18-20 g) were purchased from Changzhou Cavens Experimental Animal Co. Ltd. (Changzhou, Jiangsu, China) and fed in the laboratory animal research center of Zhejiang Chinese medical university. Mice were housed in groups under specific pathogen-free conditions (22 ± 2°C, RH 50-60%, and a 12 h light/dark cycle). Each mouse was individually weighed and randomly assigned to an experimental group. The mice were housed in polycarbonate cages and fed a standard animal diet with water. All mice were treated in strict accordance with the Zhejiang Chinese Medical University Animal Care and Use committee's guidelines for the care and use of laboratory animals. Before treatment, the back skin of all mice was shaved (area: 2 × 2 cm) and a depilatory cream (Veet, London, UK) was applied to areas. This is aimed to promote hair follicle transferred from telogen stage to anagen stage. Mice were grouped into three: One group of mice was smeared with 1 ml of PBS as control. One group of mice was smeared with 1 ml of 5% H 2 O 2 in the experimental skin area for 3 minutes at 3 pm. The third group of mice was administered with antroquinonol at 50 mg/kg per day by intragastric administration at 9 am, and H 2 O 2 was smeared at 3 pm. The mice were treated once per day for continuous 50 days and shaved daily. Three mice were used in one group.

Measurement of Hair Growth, Skin Thickness, and
Pigmentation. The distance from the dermal papilla to the epidermis was measured using straight line as hair follicle (HF) length. The width of the surface of the epidermis to the muscle in the photomicrograph was measured as skin thickness. Irregular shape simulated the depilation area, and repigmentation percentage was estimated. All data were normalized to the controls and analyzed statistically.
2.5. Preparation of CD8 + T Lymphocytes. Peripheral blood mononuclear cells (PBMC) were isolated by density 2.11. Statistical Analysis. SPSS13.0 software (SPSS, Chicago, IL) was employed for statistical analysis. The data are presented as the mean ± SD. One-way analysis of variance (ANOVA) was performed for comparing means across multiple groups. P values less than 0.05 were considered statistically significant.

Effects of Antroquinonol on Proliferation of Human CD8 + T Cells.
To determine the effect of antroquinonol on proliferation of human CD8 + T cells, a CFSE assay was performed quantificationally. CD8 + T cells were treated with antroquinonol (0-40 μM) for 48 h, and the results indicated that antroquinonol exhibited inhibition in CD8 + T cell proliferation. Treatment of antroquinonol at 20 μM showed 35% growth inhibition, and treatment of antroquinonol at 20 and 40 μM indicated similar inhibitory effect on cell proliferation. Compared with control, treatment of antroquinonol at 20 μM for 48 h effectively enhanced the proliferation by 4 times (P = 0 0001). Whereas, similar increase at 20 μM for 48 h and 96 h was observed (data not shown). Taken together, the results suggested that treatment of antroquinonol at 20 μM for 48 h was used for following experiments (Figure 1).

Antroquinonol Reduced Production of Cytokines in
Human CD8 + T Cells. To investigate the effect of antroquinonol on the production of cytokines associated with CD8 + T cells, levels of IL-2 and IFN-γ were analyzed by ELISA ( Figure 2). The amounts of IL-2 (26.43 ± 4.63 pg/ml) and IFN-γ (38.87 ± 0.88 pg/ml) in the antroquinonol-treated CD8 + T cells were significantly lower compared with those in the control group IL-2 (63.98 ± 2.98 pg/ml) (P = 0 0002, Figure 2(a)) and IFN-γ (61.52 ± 0.96 pg/ml) (P = 0 0004, Figure 2(b)). Additionally, as activator of CD8 + T cells, CD69 and CD137 play an important role in CD8 + T cell activation. Therefore, we also examined the levels of CD69 and CD137. The results demonstrated that the concentration of CD69 (14.87 ± 0.67) and CD137 (11.83 ± 0.78) was less in the CD8 + T cells treated with antroquinonol than that in the control CD69 (31.16 3.3. Mice Observation. The pigmentation and hair growth of mice treated with antroquinonol were evaluated. In the antroquinonol/H 2 O 2 group, pigment islands were observed in about 70% of the experimental area and black hair grew from the pigment islands. In the control group, pigment islands were observed in about 57% of the experimental area and black hair grew from the pigment islands. Whereas, a little of pigment islands in the experimental area of the H 2 O 2 group were shown and few black hair grew from the pigment islands ( Figure 3). This indicated that H 2 O 2 could induce depigmentation, whereas antroquinonol could inhibit the induction of H 2 O 2 in depigmentation.

Antroquinonol Resists Inhibition of Hair Growth and Skin Thickness Induced by H 2 O 2 .
To investigate the role of antroquinonol on the growth of hair and skin, we performed H&E staining to visualize hair follicle length and skin thickness (Figure 4(a)). On the 50th day after depilation, the hair follicle length of the mice in the control group (P = 0 0001) and the antroquinonol/H 2 O 2 group (P = 0 0001) was significantly larger compared to the mice in the H 2 O 2 group (Figure 4(b)). Similarly, skin thickness in the control group (P = 0 005) and the antroquinonol/ H 2 O 2 group (P = 0 0004) was significantly higher than that in the H 2 O 2 group (Figure 4(c)). Collectively, antroquinonol could resist inhibition of hair growth and skin thickness induced by H 2 O 2 .

Antroquinonol Induced Expression of Tyrosinase.
Tyrosinase is the key enzyme of melanogenesis. We detected its expression in the skin with immunohistochemistry ( Figure 5) 3.6. Antroquinonol Could Inhibit Infiltration of Mouse CD8 + T Cells. In order to investigate whether antroquinonol exert immunosuppressive effect on CD8 + T cells, immunofluorescence assay was performed to detect the infiltration of CD8 + T cells. As shown in Figure 6, amount of CD8 + T cells were observed in the experimental area in the H 2 O 2 group. A few of CD8 + T cells were shown in the skin in

Antroquinonol Reduced Production of IL-2 and IFN-γ.
Production of cytokine IL-2 and IFN-γ was determined with ELISA ( Figure 7). Among the three groups, the lowest level of IL

Discussion
Vitiligo is a common dermatological disorder of the epidermis characterized by the acquired loss of melanocytes and melanin. The interplay between oxidative stress and the immune system plays significant roles in the pathogenesis of vitiligo. Increased evidence supported that oxidative stress plays a critical role in the autoimmune initiation in vitiligo [2,26]. Higher level of H 2 O 2 was demonstrated in vitiligo epidermis than that in healthy controls [4].
Here, we induce depigmentation with H 2 O 2 in mouse to simulate vitiligo. 5% H 2 O 2 was applied to smear topically in the skin of mice for inducing depigmentation [27]. depigmentation. In further, H&E staining was applied to investigate the hair follicle length and skin thickness in the experimental area. In the H 2 O 2 group, hair follicle length and skin thickness were significantly lower than those in the control group. In mice, melanocytes grow in hair follicles which provide lieu to melanocyte survival and subsequent melanogenesis. Inhibition of hair follicle growth suppresses biological activity of melanocyte. Tyrosinase has a key role in pigmentation process, and which could be impacted by a range of materials on its activity. Tyrosinase activity in vitiligo patients' lesional skins was lower than that in vitiligo patients' nonlesional skins [28]. In this study, tyrosinase expression is dramatically decreased in the mice treated with H 2 O 2 , which is similar to that in vitiligo patients' lesional skins. Together, it indicates that mice treated with H 2 O 2 could simulate vitiligo patients. Therefore, we used this model to detect antroquinonol effect on the vitiligo.
Several biological activities of natural food-derived components were reported for their promising anti-inflammatory, antioxidant, and antiapoptotic modulatory potential [29][30][31]. Flavonoids present in fruits, vegetables, and herbs exert a positive health effect in neurodegenerative disorders and cancer, owing to their free radical-scavenging activities [32]. Antioxidants, oral vitamins, and supplements have also gained increased interest in the treatment of vitiligo for their antioxidant properties. Ginkgo biloba, resveratrol, and zinc have all been studied either as monotherapies or   Figure 3: Evaluation of mice treated with H 2 O 2 and/or antroquinonol. Mice with different treatment for consecutive 50 days were observed. Pigment islands were observed in about 57% of the experimental area, and black hair grew from the pigment islands in the control group. Pigment islands were observed in about 70% of the experimental area, and black hair grew from the pigment islands in the antroquinonol/ H 2 O 2 group. Whereas, a little of pigment islands in the experimental area of the H 2 O 2 group was shown, and few black hair grew from the pigment islands. The values are presented as mean ± SD. (n = 3). P < 0 05 means statistical difference.
Antrodia camphorata, a parasitic fungus on rotting trees of Cinnamomum kanehirai Hay in Taiwan [20], which is used as a folk medicine and has been shown to have several pharmacologic effects, including antioxidant and free radical-scavenging activities [16], inhibition of the inflammatory response [19], and antitumor cytotoxicity activity [38]. Antroquinonol, a major active component of Antrodia camphorata, has been shown to inhibit T cell activation/proliferation and production of ROS and suppress NF-κB activation and NF-κB-dependent inflammation and activation of Nrf2 [25,39]. In this study, we provide the first demonstration that antroquinonol can inhibit the CD8 + T cell infiltration and reduced tyrosinase induced by H 2 O 2 .
Firstly, we investigate the function of antroquinonol on human CD8 + T cells in vitro. About 20 μM of antroquinonol was incubated in human CD8 + T cells for 48 h. The results showed antroquinonol could inhibit CD8 + T cell proliferation and activation of CD8 + T cells by suppressing production of CD69, CD137, IL-2, and IFN-γ. And then in vivo investigation was performed. The results indicated that antroquinonol could suppress the proliferation and production of cytokines of CD8 + T cells. Moreover, effect of antroquinonol on CD8 + T cells in mice treated with H 2 O 2 was detected. In the antroquinonol/H 2 O 2 group, pigment islands were observed in 80% of the experimental area and black hair grew from the pigment islands. In the control group, pigment islands were observed in 50% of the experimental area and black hair grew from the pigment islands. Whereas, in the H 2 O 2 group, a little of pigment islands in the experimental area was shown and few black hair grew from the pigment islands. This indicated that H 2 O 2 could induce depigmentation, whereas antroquinonol could inhibit the induction of H 2 O 2 in depigmentation. In further, H&E staining was applied to investigate the hair follicle length and skin thickness in the experimental area. In the H 2 O 2 group, hair follicle length and skin thickness were significantly lower than those in the antroquinonol/H 2 O 2 group and the control group. There was no significant difference of hair follicle length between the antroquinonol/H 2 O 2 group and the control group. Skin thickness in the antroquinonol/H 2 O 2 group was higher than that in the control group. Expression of tyrosinase was examined in all groups. In the H 2 O 2 group, a little of tyrosinase was observed in the hair follicle. Contrast to the H 2 O 2 group, increased expression of tyrosinase was detected in the control group and the antroquinonol/H 2 O 2 group. These results showed that antroquinonol could promote hair follicle growth, expression of tyrosinase, and repigmentation. It indicates that antroquinonol could be a potential candidate for interference in depigmentation.
In vitiligo, CD8 + T cells are involved in autoimmune responses, resulting in depigmentation of the skin [40]. Cytokines released by lymphocytes, including IL-1, IFN-γ or TNF-α, can initiate apoptosis of both melanocytes and keratinocytes [41,42]. IFN-γ, as one important cytokine associated with the Th1 immune response, induced protein CXCL10 to express in various cell types, such as lymphocytes, fibroblasts, neutrophils, and other epithelial cells. Some studies have proposed that IFN-γ-induced CXCL10-CXCR3 chemokine pathway plays a vital role in CD8 + T cell skin infiltration [14,15,41,43]. CXCL10 binds to its specific receptor CXCR3 to recruit and activate T cells for regulating immune responses. Increased expression of CXCL10 and CXCR3 was shown in various autoimmune diseases, and they play fundamental parts in leukocyte homing into the inflamed tissues to accelerate the process of tissue damage [44,45]. Highly induced CXCL10 and CXCR3 were found in vitiligo patients [14]. Here, cytokines IL-2 and IFN-γ were examined with ELISA. H 2 O 2 significantly enhanced the level of IL-2 and IFN-γ in mice, and antroquinonol could inhibit the production of IL-2 and IFN-γ. In further, we investigated the IFN-γ-induced expression of CXCL10 and CXCR3. In consistent, highly increased expression of CXCL10 and CXCR3 was found in the mice treated with H 2 O 2 . A little increase expression of CXCL10 and CXCR3 was detected in the mice treated with antroquinonol/H 2 O 2 .

Conclusions
According to our findings in this study, it is suggested that antroquinonol has a potential therapeutic effect on depigmentation. Antroquinonol significantly attenuated histopathologic changes in the mice skins and inhibited the infiltration of CD8 + T cells and expression of chemokines CXCL10 and CXCR3. In addition, antroquinonol could decrease the production of cytokines IL-2 and IFN-γ obviously and promote tyrosinase expression. These results suggest that antroquinonol might be a treatment of choice for preventing depigmentation.

Conflicts of Interest
The authors have no conflicts of interest to declare.