Advertisement

Successful Treatment of Vitiligo with Cold Atmospheric Plasma‒Activated Hydrogel

  • Siyue Zhai
    Affiliations
    Department of Dermatology, The Second Affiliated Hospital, Xi'an Jiaotong University, Xi’an, China

    Center of Plasma Biomedicine, State Key Laboratory of Electrical Insulation and Power Equipment, Xi’an Jiaotong University, Xi’an, China
    Search for articles by this author
  • Meifeng Xu
    Affiliations
    Department of Dermatology, The Second Affiliated Hospital, Xi'an Jiaotong University, Xi’an, China
    Search for articles by this author
  • Qiaosong Li
    Affiliations
    Center of Plasma Biomedicine, State Key Laboratory of Electrical Insulation and Power Equipment, Xi’an Jiaotong University, Xi’an, China

    School of Electrical Engineering, Xi’an Jiaotong University, Xi’an, China
    Search for articles by this author
  • Kun Guo
    Affiliations
    Department of Dermatology, The Second Affiliated Hospital, Xi'an Jiaotong University, Xi’an, China
    Search for articles by this author
  • Hailan Chen
    Affiliations
    Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, Virginia, USA
    Search for articles by this author
  • Michael G. Kong
    Affiliations
    Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, Virginia, USA

    Department of Electrical & Computer Engineering, Batten College of Engineering and Technology, Old Dominion University, Norfolk, Virginia, USA
    Search for articles by this author
  • Yumin Xia
    Correspondence
    Correspondence: Yumin Xia, Department of Dermatology, The Second Affiliated Hospital, Xi’an Jiaotong University, 157 Xiwu Road, Xi’an 710004, China.
    Affiliations
    Department of Dermatology, The Second Affiliated Hospital, Xi'an Jiaotong University, Xi’an, China
    Search for articles by this author
Open ArchivePublished:May 21, 2021DOI:https://doi.org/10.1016/j.jid.2021.04.019
      Vitiligo shows insufficient response to current therapies largely owing to T-lymphocyte dysfunction, abnormal inflammatory activation, and excessive oxidative stress in lesions. Cold atmospheric plasma (CAP) possesses pleiotropic antioxidant and anti-inflammatory properties and may offer an improvement to current treatment options. In this study, the efficacy and safety of CAP were investigated in a mouse model of vitiligo and a randomized and controlled trial of patients with active focal vitiligo. Skin biopsies showed that topical treatment of vitiligo-like lesions on mouse dorsal skin by CAP restored the distribution of melanin. In addition, CAP treatment reduced the infiltration of CD11c+ dendritic cells, CD3+ T cells, and CD8+ T cells; inhibited the release of CXCL10 and cytokine IFN-γ; and enhanced cellular resistance to oxidative stress and excessive immune response by enhancing the expression of the transcription factor NRF2 and attenuating the activity of inducible nitric oxide synthase. In a randomized and controlled trial, CAP treatment achieved partial and complete repigmentation in 80% and 20% of vitiligo lesions, respectively, without hyperpigmentation in surrounding areas or other adverse events during the treatment period and its follow-up period. In conclusion, CAP offers a promising option for the management of vitiligo.

      Abbreviations:

      CAP (cold atmospheric plasma), H2O2 (hydrogen peroxide), iNOS (inducible nitric oxide synthase), NO (nitric oxide)

      Introduction

      Vitiligo is the most frequent cause of depigmentation worldwide, with an estimated prevalence of 1% (
      • Ezzedine K.
      • Eleftheriadou V.
      • Whitton M.
      • van Geel N.
      Vitiligo.
      ). Standard treatment methods for vitiligo include systemic oral drugs combined with topical drugs, phototherapy (narrow-band UVB or 308-nm excimer laser), and surgery. However, vitiligo treatment is challenging owing to the low cure and high recurrence rates (
      • Speeckaert R.
      • van Geel N.
      Vitiligo: an update on pathophysiology and treatment options.
      ). Furthermore, systemic side effects often occur during oral drug administration, and hyperpigmentation in surrounding areas is common with topical treatments. Therefore, it is critical to explore alternative therapies for patients with vitiligo.
      It is widely accepted that the pathogenesis of vitiligo involves oxidative stress pathways, immune inflammation, and T-cell‒mediated destruction of melanocytes (
      • Rashighi M.
      • Harris J.E.
      Vitiligo pathogenesis and emerging treatments.
      ;
      • Speeckaert R.
      • Dugardin J.
      • Lambert J.
      • Lapeere H.
      • Verhaeghe E.
      • Speeckaert M.M.
      • et al.
      Critical appraisal of the oxidative stress pathway in vitiligo: a systematic review and meta-analysis.
      ). The production of excessive ROS due to exogenous and endogenous stimuli and mitochondrial or antioxidant system dysfunction will lead to melanocyte destruction and the initiation of vitiligo (
      • Xie H.
      • Zhou F.
      • Liu L.
      • Zhu G.
      • Li Q.
      • Li C.
      • et al.
      Vitiligo: how do oxidative stress-induced autoantigens trigger autoimmunity?.
      ). CXCL10 is elevated in the skin lesions of both patients with vitiligo and a murine vitiligo model, and vitiligo-like mice lacking CXCL10 receptors or treated with CXCL10-neutralizing antibody display minimal depigmentation (
      • Rashighi M.
      • Agarwal P.
      • Richmond J.M.
      • Harris T.H.
      • Dresser K.
      • Su M.W.
      • et al.
      CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo.
      ). Furthermore, CXCL10 receptors are expressed on pathogenic T cells that infiltrate vitiliginous lesions. In addition, CD8+ T cells, which serve as the critical immune cells of vitiligo autoimmunity, are both necessary and sufficient for melanocyte destruction in vitiligo (
      • Rodrigues M.
      • Ezzedine K.
      • Hamzavi I.
      • Pandya A.G.
      • Harris J.E.
      Vitiligo Working Group
      New discoveries in the pathogenesis and classification of vitiligo.
      ). Proteins involved in melanin synthesis, such as Melan-A/MART-1 and tyrosinase, can be recognized by Langerhans cells or dendritic cells as antigens and then are presented to cytotoxic T lymphocytes, which target melanocytes with the assistance of major histocompatibility complex class I molecules (
      • Ezzedine K.
      • Eleftheriadou V.
      • Whitton M.
      • van Geel N.
      Vitiligo.
      ;
      • Xie H.
      • Zhou F.
      • Liu L.
      • Zhu G.
      • Li Q.
      • Li C.
      • et al.
      Vitiligo: how do oxidative stress-induced autoantigens trigger autoimmunity?.
      ). HIF-1α increases proinflammatory cytokine levels and plays a crucial role in immune cell functions. In vitiligo, HIF-1α induces proinflammatory cytokines in CD8+ T cells and strengthens their cytotoxicity by secreting cytotoxic granules to melanocytes (
      • Deng Q.
      • Wei J.
      • Zou P.
      • Xiao Y.
      • Zeng Z.
      • Shi Y.
      • et al.
      Transcriptome analysis and emerging driver identification of CD8+ T cells in patients with vitiligo.
      ). Therefore, oxidative stress and T-lymphocyte infiltration are critical events in the pathogenesis of vitiligo.
      Cold atmospheric plasma (CAP) is an electrical discharge of plasma generated in the open air at room temperature (
      • Kong M.G.
      • Kroesen G.
      • Morfill G.
      • Nosenko T.
      • Shimizu T.
      • van Dijk J.
      • et al.
      Plasma medicine: an introductory review.
      ). With its generation of transient and low-level ROS species, ions, and other effectors, CAP has been shown to be safe and therapeutically beneficial for a range of human disorders, including chronic wounds, actinic keratosis, and skin cancers (
      • Friedman P.C.
      • Miller V.
      • Fridman G.
      • Lin A.
      • Fridman A.
      Successful treatment of actinic keratoses using nonthermal atmospheric pressure plasma: a case series.
      ;
      • Isbary G.
      • Heinlin J.
      • Shimizu T.
      • Zimmermann J.L.
      • Morfill G.
      • Schmidt H.U.
      • et al.
      Successful and safe use of 2 min cold atmospheric argon plasma in chronic wounds: results of a randomized controlled trial.
      ;
      • Metelmann H.R.
      • Seebauer C.
      • Miller V.
      • Fridman A.
      • Bauer G.
      • Graves D.B.
      • et al.
      Clinical experience with cold plasma in the treatment of locally advanced head and neck cancer.
      ). Whereas oxidative stress by excessive ROS is well-known, physiological ROS counterintuitively promotes stress responses and adaptation (
      • Sies H.
      • Jones D.P.
      Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.
      ) and regulates T-cell function (
      • Kesarwani P.
      • Murali A.K.
      • Al-Khami A.A.
      • Mehrotra S.
      Redox regulation of T-cell function: from molecular mechanisms to significance in human health and disease.
      ). The low-level transient ROS produced during CAP treatments can suppress oxidative stress by promoting antioxidant mechanisms, downregulating proinflammatory genes, and inhibiting excess T-cell activation (
      • Horiba M.
      • Kamiya T.
      • Hara H.
      • Adachi T.
      Cytoprotective effects of mild plasma-activated medium against oxidative stress in human skin fibroblasts.
      ;
      • Lee Y.S.
      • Lee M.H.
      • Kim H.J.
      • Won H.R.
      • Kim C.H.
      Non-thermal atmospheric plasma ameliorates imiquimod-induced psoriasis-like skin inflammation in mice through inhibition of immune responses and up-regulation of PD-L1 expression.
      ,
      • Lee O.J.
      • Ju H.W.
      • Khang G.
      • Sun P.P.
      • Rivera J.
      • Cho J.H.
      • et al.
      An experimental burn wound-healing study of non-thermal atmospheric pressure microplasma jet arrays.
      ). Furthermore, CAP treatment reduced lymphocyte function-associated antigen 1 and intercellular adhesion molecule 1 in psoriasiform mouse skin (
      • Lee Y.S.
      • Lee M.H.
      • Kim H.J.
      • Won H.R.
      • Kim C.H.
      Non-thermal atmospheric plasma ameliorates imiquimod-induced psoriasis-like skin inflammation in mice through inhibition of immune responses and up-regulation of PD-L1 expression.
      ); both are also highly expressed in vitiliginous lesions (
      • Abdallah M.
      • Abdel-Naser M.B.
      • Moussa M.H.
      • Assaf C.
      • Orfanos C.E.
      Sequential immunohistochemical study of depigmenting and repigmenting minigrafts in vitiligo.
      ). Thus, we hypothesized that CAP would strengthen antioxidative responses and inhibit immune reactions in vitiligo. This study was designed to investigate the therapeutic efficacy and side effects of CAP treatments in a vitiligo-like mouse model and in patients with active focal vitiligo as well as the relevant mechanism underlying such functions.

      Results

      CAP treatments ameliorated vitiligo-like lesions in mice

      A CAP jet generated in flowing helium (
      • Walsh J.L.
      • Kong M.G.
      Contrasting characteristics of linear-field and cross-field atmospheric plasma jets.
      ) and CAP-activated hydrogel prepared with deionized water pretreated with surface air CAP (
      • Chen C.
      • Li F.
      • Chen H.
      • Kong M.G.
      Aqueous reactive species induced by a PCB surface micro-discharge air plasma device: a quantitative study.
      ) were used for direct and indirect CAP treatments (Supplementary Figure S1), respectively. Both CAP treatments effectively reduced depigmentation in mice (Figure 1a). Depigmentation scores, as defined in Supplementary Figure S2, were 57‒72% lower in the CAP jet and CAP-activated hydrogel groups than in the blank and vehicle hydrogel controls (Figure 1b). There was no significant difference between the two CAP treatment groups nor between the two control groups, suggesting that the efficacy of CAP-activated hydrogel was similar to that of the CAP jet and was not due to its hydrogel. Fontana–Masson staining of the biopsied tissues showed that the two CAP groups exhibited more follicular melanocytes in the lesions than the control groups (Figure 1c). Histologic evaluation confirmed the changes in follicular melanocyte distribution (Figure 1d).
      Figure thumbnail gr1
      Figure 1CAP jet and CAP-activated hydrogel ameliorated vitiligo lesions in mice and restored the distribution of the follicular melanocytes. C57BL/6 mice treated with monobenzone cream were used to create the vitiligo model. Mice were then treated with vehicle hydrogel, CAP jet, or CAP-activated hydrogel. (a) Skin lesions were monitored after the 1-month treatment period. (b) The depigmentation score of skin lesions measured in each group. (c) H&E staining and melanin shown in Fontana–Masson–stained sections; arrows indicate representative lesions. (d) Staining score of the follicular melanocytes in each group (n = 6). Representative images are shown; Bar = 25 μm. The data were analyzed by one-way ANOVA or nonparametric tests. ∗P < 0.05. CAP, cold atmospheric plasma.

      CAP treatments reduced inflammatory cell infiltration in vitiligo-like mice

      Vitiligo development is closely related to the aberrant activation of dendritic cells, CD3+ T cells, and CD8+ T cells (
      • Rashighi M.
      • Agarwal P.
      • Richmond J.M.
      • Harris T.H.
      • Dresser K.
      • Su M.W.
      • et al.
      CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo.
      ;
      • Visvanathan S.
      • Baum P.
      • Vinisko R.
      • Schmid R.
      • Flack M.
      • Lalovic B.
      • et al.
      Psoriatic skin molecular and histopathologic profiles after treatment with risankizumab versus ustekinumab.
      ). Immunohistochemistry revealed that CAP jet and CAP-activated hydrogel treatments reduced the infiltration of these cells in skin lesions (Figure 2a). Histologic quantitation showed that in the two CAP treatment groups, cell counts were reduced by 67‒85% for CD11c+ dendritic cells, 68‒82% for CD3+ cells, and 65‒80% for CD8+ cells compared with those in the two controls. There was no significant difference between the blank and CAP jet groups in CD11c+ dendritic cells or in CD3+ cells. There was also no significant difference between the two CAP treatment groups nor between the two control groups (Figure 2b–d).
      Figure thumbnail gr2
      Figure 2CAP jet and CAP-activated hydrogel inhibited the infiltration of immune cells in mice. C57BL/6 mice treated with monobenzone cream were used to create the vitiligo model. Mice were then treated with vehicle hydrogel, CAP jet, or CAP-activated hydrogel. Skin tissues were harvested on day 60. (a) Distribution of CD11c+ dendritic cells, CD3+ T cells, and CD8+ T cells; arrows indicate the representative lesions. (b–d) The staining scores of skin lesions were measured in each of the four groups (n = 6). Representative images are shown; Bar = 25 μm. The data were analyzed by nonparametric test. ∗P < 0.05. CAP, cold atmospheric plasma.

      CAP treatments downregulated inflammatory factor expression in vitiligo-like mice

      The IFN-γ‒CXCL10‒CD8+ T-cell axis (Supplementary Figure S3) is crucial in vitiligo development, and IL-17 promotes vitiligo progression and severity (
      • Singh R.K.
      • Lee K.M.
      • Vujkovic-Cvijin I.
      • Ucmak D.
      • Farahnik B.
      • Abrouk M.
      • et al.
      The role of IL-17 in vitiligo: a review.
      ;
      • Strassner J.P.
      • Harris J.E.
      Understanding mechanisms of autoimmunity through translational research in vitiligo.
      ). Furthermore, HIF-1α exacerbates CD8+ T-cell cytotoxicity by secreting cytotoxic granules in vitiligo (
      • Deng Q.
      • Wei J.
      • Zou P.
      • Xiao Y.
      • Zeng Z.
      • Shi Y.
      • et al.
      Transcriptome analysis and emerging driver identification of CD8+ T cells in patients with vitiligo.
      ). CAP-activated hydrogel reduced the mRNA expression levels of Cxcl10 and Ifnγ, whereas the CAP jet exhibited no such effect (Figure 3a). The advantage of CAP-activated hydrogel over CAP jet was also observed at the protein levels in tissues (Figure 3b). Finally, IL-17 was insignificantly affected by the CAP jet and the CAP-activated hydrogel (Figure 3a and b). Immunohistochemistry showed that the two CAP groups exhibited lower expression levels of CXCL10 and HIF-1α than the control groups (Figure 3c–f). The CAP treatments reduced the protein expression levels of HIF-1α in the skin samples, although its mRNA levels in tissue or protein expression levels in sera were not affected (Supplementary Figure S4).
      Figure thumbnail gr3
      Figure 3CAP jet and CAP-activated hydrogel inhibited the release of inflammatory factors and HIF-1α. C57BL/6 mice treated with monobenzone cream were used to create the vitiligo model. Mice were then treated with vehicle hydrogel, CAP jet, or CAP-activated hydrogel. Skin tissues were harvested after 1 month of treatment. (a) mRNA levels of Cxcl10, Ifnγ, and Il17. (b) Protein concentrations of CXCL10, IFN-γ, and IL-17 in skin tissues. (c) Distribution of CXCL10; arrows indicate representative lesions. (d) CXCL10 scores for skin lesions. (e) Distribution of HIF-1α; arrows indicate representative lesions. (f) HIF-1α scores for skin lesions (n = 5). Representative images are shown; Bar = 25 μm. The data were analyzed by one-way ANOVA or nonparametric test. ∗P < 0.05, compared with the blank group; #P < 0.05, compared with the vehicle hydrogel group. CAP, cold atmospheric plasma.

      CAP treatments promoted resistance to oxidative stress in vitiligo-like mice

      The concentrations of hydrogen peroxide (H2O2) and nitric oxide (NO) were assessed in deionized water activated by the surface CAP, and it was determined that H2O2 and NO were maintained at their levels for 12 hours after CAP activation (Figure 4a and b). The peak H2O2 concentration of approximately 15 μM was within its range for mitigating excessive inflammatory responses (
      • Sies H.
      • Jones D.P.
      Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.
      ) and promoting cytoprotection in psoriasis-like mouse skin by CAP (
      • Lee Y.S.
      • Lee M.H.
      • Kim H.J.
      • Won H.R.
      • Kim C.H.
      Non-thermal atmospheric plasma ameliorates imiquimod-induced psoriasis-like skin inflammation in mice through inhibition of immune responses and up-regulation of PD-L1 expression.
      ). It was also two orders of magnitude below approximately 1 mM of H2O2 found in lesional skin of patients with vitiligo (
      • Schallreuter K.U.
      • Moore J.
      • Wood J.M.
      • Beazley W.D.
      • Gaze D.C.
      • Tobin D.J.
      • et al.
      In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase.
      ). H2O2 and NO levels in CAP-activated hydrogel were expected to be similar, although there was interference in their measurements by the lack of fluidity of the hydrogel. Their possible benefits were evaluated by assessing NRF2 and inducible NO synthase (iNOS), which mitigate and promote oxidative stress, respectively (
      • Glassman S.J.
      Vitiligo, reactive oxygen species and T-cells.
      ;
      • Jian Z.
      • Li K.
      • Song P.
      • Zhu G.
      • Zhu L.
      • Cui T.
      • et al.
      Impaired activation of the Nrf2-ARE signaling path-way undermines H2O2-induced oxidative stress response: a possible mechanism for melanocyte degeneration in vitiligo.
      ). Both mRNA and protein levels of iNOS in skin tissues significantly decreased in both CAP jet and CAP-activated hydrogel groups (Figure 4c–e), whereas its protein expression in sera showed no significant difference (Figure 4f). However, the protein level of NRF2 increased in the tissue samples and peripheral sera in the CAP-activated hydrogel group, whereas the CAP jet only increased the level of NRF2 in tissue samples (Figure 4d–f).
      Figure thumbnail gr4
      Figure 4CAP jet and CAP-activated hydrogel attenuated oxidative stress. C57BL/6 mice treated with monobenzone cream were used to create the vitiligo model. Mice were then treated with vehicle hydrogel, CAP jet, or CAP-activated hydrogel. Skin tissues and peripheral sera were harvested after 1 month of treatment. Formation of (a) H2O2 and (b) NO in deionized water after 6 minutes CAP treatment (n = 3). (c) The mRNA and (d) protein expression levels of iNOS and NRF2 in skin tissues. (e) Blot intensity level of iNOS and NRF2 protein in lesional tissues measured with ImageJ software. (f) The protein levels of iNOS and NRF2 in sera (n = 5). The data were analyzed by one-way ANOVA or nonparametric test. Representative images are shown. ∗P < 0.05, compared with the blank group. #P < 0.05, compared with the vehicle hydrogel group. CAP, cold atmospheric plasma; h, hour; H2O2, hydrogen peroxide; iNOS, inducible nitric oxide synthase; NO, nitric oxide; ns, not significant.

      CAP-activated hydrogel ameliorated vitiligo in patients

      CAP-activated hydrogel was topically administered to patients with active focal vitiligo (Supplementary Table S1). We observed that the vitiligo eruptions became smaller or were repigmented in all cases (Figure 5a). Of 15 CAP-hydrogel‒treated lesions, partial response was observed in 12 cases (80.0%), and complete response was observed in three cases (20.0%). By contrast, no response was observed in the eruptions that received the vehicle hydrogel treatment (Figure 5b) or received no treatment (data not shown). Vitiligo area severity index scores were also calculated, reflecting significant improvement in CAP-hydrogel‒treated lesions (Figure 5c). We observed neither hyperpigmentation of the surrounding areas nor treatment-related adverse events. Furthermore, no reappearance of depigmentation was observed during the trial nor in the follow-up period of 6–8 months. Moreover, white blood cells, red blood cells, platelets, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and serum creatinine were not affected by CAP treatments (Supplementary Table S2).
      Figure thumbnail gr5
      Figure 5CAP-activated hydrogel repigmented the vitiligo-like skin in patients. Lesions were either treated with CAP-activated hydrogel, treatment group; vehicle hydrogel, control group; or left untreated, blank/negative control. (a) Representative images of lesions in patients 6–15 are shown at weeks 0 and 12. Cases 1 and 2 received CAP-hydrogel treatment. (b) Representative images of lesions in patients 16–20 (under Wood’s lamp, right side) are shown. White and black arrows indicate the lesions treated with vehicle hydrogel and CAP-activated hydrogel, respectively. (c) Modified VASI scores in different groups are shown. The data were analyzed by paired Student’s t-tests or the Wilcoxon matched-pairs signed-rank test. CAP, cold atmospheric plasma; VASI, Vitiligo Area Scoring Index.

      CAP-activated hydrogel attenuated histologic changes in patients

      Histologic changes were observed in the CAP-hydrogel‒treated sites (Figure 6a). Melanocytes and melanin particles were absent in H&E-stained sections before treatment, but CAP-activated hydrogel led to their partial recovery. The histologic changes were also mirrored by immunohistochemistry, showing more cells positive for glycoprotein 100, which is an important melanocytic differentiation antigen. Similarly, CAP-activated hydrogel reduced intense infiltration of CD8+ T cells under the epidermis of skin lesions. Quantitation confirmed these changes in glycoprotein 100+ cells and in CD8+ T cells (Figure 6b).
      Figure thumbnail gr6
      Figure 6Histologic changes after CAP-activated hydrogel treatment. (a) Melanocytes and melanin particles were observed in H&E-stained sections. Using immunohistochemistry, gp100+ and CD8+ T cells were observed in the epidermis and underneath the epidermis, respectively. (b) The scores of gp100+ and CD8+ cells were measured in the blank control and CAP-hydrogel groups. Representative images are shown (n = 4); Bar = 10 μm. The data were analyzed by an unpaired Student’s t-test. CAP, cold atmospheric plasma; gp100, glycoprotein 100.

      Discussion

      In this study, we showed that the CAP jet or CAP-activated hydrogel treatment ameliorated skin lesions in vitiligo-like mice and in patients with vitiligo. In the mouse experiments, CAP treatment reduced the infiltration of dendritic cells, CD3+ T cells, and CD8+ T cells as well as the production of CXCL10, IFN-γ, and HIF-1α. Moreover, CAP treatment modulated oxidative stress responses by downregulating iNOS expression and upregulating NRF2 expression. In the clinical trial, CAP-activated hydrogel treatment significantly ameliorated skin lesions in patients with active focal vitiligo. There were no adverse events observed during the clinical trial or during the follow-up periods. Therefore, CAP treatment can effectively and safely attenuate vitiligo.
      As an innovative noninvasive treatment, CAP application in dermatology has been studied widely in vitro and in vivo. It has been directly applied to patient or animal skin to treat wounds, skin cancers, actinic keratosis, and psoriasis (
      • Friedman P.C.
      • Miller V.
      • Fridman G.
      • Lin A.
      • Fridman A.
      Successful treatment of actinic keratoses using nonthermal atmospheric pressure plasma: a case series.
      ;
      • Horiba M.
      • Kamiya T.
      • Hara H.
      • Adachi T.
      Cytoprotective effects of mild plasma-activated medium against oxidative stress in human skin fibroblasts.
      ;
      • Isbary G.
      • Heinlin J.
      • Shimizu T.
      • Zimmermann J.L.
      • Morfill G.
      • Schmidt H.U.
      • et al.
      Successful and safe use of 2 min cold atmospheric argon plasma in chronic wounds: results of a randomized controlled trial.
      ;
      • Lee Y.S.
      • Lee M.H.
      • Kim H.J.
      • Won H.R.
      • Kim C.H.
      Non-thermal atmospheric plasma ameliorates imiquimod-induced psoriasis-like skin inflammation in mice through inhibition of immune responses and up-regulation of PD-L1 expression.
      ,
      • Lee O.J.
      • Ju H.W.
      • Khang G.
      • Sun P.P.
      • Rivera J.
      • Cho J.H.
      • et al.
      An experimental burn wound-healing study of non-thermal atmospheric pressure microplasma jet arrays.
      ;
      • Metelmann H.R.
      • Seebauer C.
      • Miller V.
      • Fridman A.
      • Bauer G.
      • Graves D.B.
      • et al.
      Clinical experience with cold plasma in the treatment of locally advanced head and neck cancer.
      ). Its long-term safety for treating wounds was confirmed with a detailed 1-year risk assessment in mice (
      • Schmidt A.
      • von Woedtke T.V.
      • Stenzel J.
      • Lindner T.
      • Polei S.
      • Vollmar B.
      • et al.
      One year follow-up risk assessment in SKH-1 mice and wounds treated with an argon plasma jet.
      ) and was supported by clinical and preclinical studies (
      • Friedman P.C.
      • Miller V.
      • Fridman G.
      • Lin A.
      • Fridman A.
      Successful treatment of actinic keratoses using nonthermal atmospheric pressure plasma: a case series.
      ;
      • Lee O.J.
      • Ju H.W.
      • Khang G.
      • Sun P.P.
      • Rivera J.
      • Cho J.H.
      • et al.
      An experimental burn wound-healing study of non-thermal atmospheric pressure microplasma jet arrays.
      ;
      • Metelmann H.R.
      • Seebauer C.
      • Miller V.
      • Fridman A.
      • Bauer G.
      • Graves D.B.
      • et al.
      Clinical experience with cold plasma in the treatment of locally advanced head and neck cancer.
      ). In this study, both CAP jet and CAP-activated hydrogel were applied to vitiligo-like lesions on mouse dorsal skin and on patient skin, and both demonstrated good tolerance during the treatment period. Remarkably, these two CAP treatments exhibited a comparable improvement effect on the vitiligo-like lesions. For dermatological diseases that benefit from topical therapies, CAP-activated hydrogel offers the advantages of simpler administration and greater patient acceptance than CAP itself. The subtype of active focal vitiligo was chosen because of inflammatory features at vitiligo onset. The results also showed impressive outcomes of CAP-activated hydrogel treatment in patients with active focal vitiligo.
      In terms of the inflammatory reaction, the IFN-γ‒CXCL10‒CD8+ T-cell axis is crucial in vitiligo development (
      • Strassner J.P.
      • Harris J.E.
      Understanding mechanisms of autoimmunity through translational research in vitiligo.
      ). IFN-γ binds to the IFN-γ receptor on the nuclei of keratinocytes, which then secrete more CXCL10 into the dermis. Then, CXCL10 drives CD8+ T cells from blood vessels into the dermis and combines with CXCR-3 on T cells. The conjugate of CXCL10 with CXCR-3 can destroy melanocytes, leading to depigmentation of the skin (Supplementary Figure S3). Our results showed that the production of IFN-γ and CXCL10 was suppressed in vitiligo-like lesions by CAP-activated hydrogel treatment. Moreover, several types of inflammatory cells, including CD8+ T cells, exhibited less infiltration on such treatment. Obviously, the active species of CAP inhibits the IFN-γ‒CXCL10‒CD8+ T-cell axis in vitiligo. Furthermore, studies documented that HIF-1α is elevated in patients with vitiligo and participates in activating CD8+ T cells (
      • Deng Q.
      • Wei J.
      • Zou P.
      • Xiao Y.
      • Zeng Z.
      • Shi Y.
      • et al.
      Transcriptome analysis and emerging driver identification of CD8+ T cells in patients with vitiligo.
      ). Our results showed that HIF-1α decreases in CAP-treated groups compared with that in the controls, further verifying the inhibitory effect of CAP treatment on the IFN-γ‒CXCL10‒CD8+ T-cell axis.
      iNOS stimulates cells to produce NO, which is the primary free radical substance of reactive nitrogen species in tissue (
      • Xia J.
      • Zeng W.
      • Xia Y.
      • Wang B.
      • Xu D.
      • Liu D.
      • et al.
      Cold atmospheric plasma induces apoptosis of melanoma cells via Sestrin2-mediated nitric oxide synthase signaling.
      ). As the main functional component of oxidative stress, iNOS plays an important role in the development of vitiligo. High epidermal levels of iNOS imply increased epidermal peroxynitrite, which is involved in vitiligo pathogenesis (
      • Glassman S.J.
      Vitiligo, reactive oxygen species and T-cells.
      ). NRF2, a critical transcription factor, can regulate antioxidant genes. Under normal conditions, NRF2 is bound to KEAP1 in the cytosol. Once the tissue undergoes an oxidative stress reaction, NRF2 is rapidly released from KEAP1 to the nucleus. Then, NRF2 combines with antioxidant response elements, and the phase II antioxidant gene is induced (
      • Jian Z.
      • Li K.
      • Song P.
      • Zhu G.
      • Zhu L.
      • Cui T.
      • et al.
      Impaired activation of the Nrf2-ARE signaling path-way undermines H2O2-induced oxidative stress response: a possible mechanism for melanocyte degeneration in vitiligo.
      ). In this study, iNOS was significantly downregulated, whereas NRF2 was upregulated in the CAP jet‒ and CAP-activated hydrogel‒treated skin lesions. These findings not only support the role of iNOS/NRF2 signals in vitiligo development but also verify the therapeutic effect of CAP treatment on vitiliginous skin.
      Current antioxidants often target direct ROS scavenging and are of modest clinical efficacy (
      • Felsten L.M.
      • Alikhan A.
      • Petronic-Rosic V.
      Vitiligo: a comprehensive overview part II: treatment options and approach to treatment.
      ;
      • Speeckaert R.
      • van Geel N.
      Vitiligo: an update on pathophysiology and treatment options.
      ). However, antioxidants may be double edged, rescuing melanocytes from cytotoxic CD8+ T cells by alleviating oxidative stress in skin lesions while damaging the DNA of melanocytes by over-reducing their intracellular ROS. Our results show that vitiligo at an early stage can be ameliorated by hydrogel made from CAP containing low-level ROS, for example, H2O2 at 15 μM. This is approximately two orders of magnitude below the 1‒5 mM concentration of H2O2 that elicits oxidation of key residues of tyrosinase and leads to melanogenesis (
      • Khan R.
      • Satyam A.
      • Gupta S.
      • Sharma V.K.
      • Sharma A.
      Circulatory levels of antioxidants and lipid peroxidation in Indian patients with generalized and localized vitiligo.
      ). Low-level ROS, including H2O2 from CAP, are known to activate the cellular antioxidant system and to suppress inflammation (
      • Lee Y.S.
      • Lee M.H.
      • Kim H.J.
      • Won H.R.
      • Kim C.H.
      Non-thermal atmospheric plasma ameliorates imiquimod-induced psoriasis-like skin inflammation in mice through inhibition of immune responses and up-regulation of PD-L1 expression.
      ). This is further supported by the control of ROS in CAP-activated hydrogel that results in an H2O2 level below 50 μM, which is well-tolerated in living tissue (
      • Niethammer P.
      • Grabher C.
      • Look A.T.
      • Mitchison T.J.
      A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.
      ;
      • Schmidt A.
      • von Woedtke T.V.
      • Stenzel J.
      • Lindner T.
      • Polei S.
      • Vollmar B.
      • et al.
      One year follow-up risk assessment in SKH-1 mice and wounds treated with an argon plasma jet.
      ;
      • Yoo S.K.
      • Starnes T.W.
      • Deng Q.
      • Huttenlocher A.
      Lyn is a redox sensor that mediates leukocyte wound attraction in vivo.
      ). In vitiligo, melanocytes are more sensitive to oxidative damage. iNOS inhibitors exhibit a therapeutic effect on vitiligo-like lesions in mice (
      • Mansourpour H.
      • Ziari K.
      • Motamedi S.K.
      • Poor A.H.
      Therapeutic effects of iNOS inhibition against vitiligo in an animal model.
      ). iNOS promotes the development of vitiligo by synthesizing NO, whereas NRF2 ameliorates vitiligo by inhibiting abnormal oxidative stress responses. Indeed, our results showed that iNOS expression was downregulated in the skin lesions of CAP jet‒ or CAP-activated hydrogel‒treated mice, whereas NRF2 expression was upregulated. Clearly, CAP treatments inhibited iNOS generation but elevated NRF2 activity. Therefore, we hypothesize that CAP attenuates vitiligo by activating the antioxidant system, in addition to inhibiting inflammatory cells.
      A limitation in this study was that the clinical trial was randomized and placebo controlled but not double blinded. Moreover, the number of cases was relatively small, and the follow-up period of 6‒8 months was relatively short. In addition, we only considered patients with active focal vitiligo at an early stage; therefore, the therapeutic effect of CAP-activated hydrogel treatment on other subtypes of vitiligo remains unknown. Additional clarification will require further investigation in a double-blinded and large-scale clinical trial.
      In summary, CAP jet or CAP-activated hydrogel treatment exhibits excellent efficacy in treating vitiligo. The therapeutic effect may involve reducing the infiltration of immune cells, including CD11c+ dendritic cells, CD3+ T cells, and CD8+ T cells; inhibiting the release of inflammatory or transcription factors such as CXCL10, IFN-γ, and HIF-1 α; and activating a stronger antioxidant stress response. Thus, CAP jet and CAP-activated hydrogel treatment are promising therapies for vitiligo with efficacy, safety, and little evidence of adverse effects.

      Materials and Methods

      CAP and CAP-activated hydrogel

      A surface air CAP generated on the surface of a perforated electrode at 8 kV and 9 kHz (
      • Chen C.
      • Li F.
      • Chen H.
      • Kong M.G.
      Aqueous reactive species induced by a PCB surface micro-discharge air plasma device: a quantitative study.
      ) is shown in Supplementary Figure S1. It was used to activate deionized water that was located 10 mm downstream of the perforated electrode. The activated water was mixed with hydroxyethyl cellulose to form CAP-activated hydrogel. In addition, a CAP jet, also known as a CAP plume, in flowing helium (
      • Walsh J.L.
      • Kong M.G.
      Contrasting characteristics of linear-field and cross-field atmospheric plasma jets.
      ) generated with the same power supply was used to directly treat mouse skin (Supplementary Figure S1).

      A vitiligo-like mouse model

      All animal protocols were approved by the Second Affiliated Hospital Research Ethics Committee of Xi'an Jiaotong University. Female C57BL/6 mice (aged 4 weeks, 13–16 g) were purchased from the Medical Animal Center of Xi’an Jiaotong University in China and bred under specific pathogen‒free conditions. A 2 × 4 cm area was shaved on the dorsal surface of the mouse. The vitiligo-like model was established by daily topical application of 40% monobenzone cream to the dorsal surface (
      • Speeckaert R.
      • Voet S.
      • Hoste E.
      • van Geel N.
      S100B is a potential disease activity marker in nonsegmental vitiligo.
      ). On day 30, there was depigmentation on the dorsal surface, or the back hair changed from black to white (Supplementary Figure S5).

      CAP treatment of vitiligo-like mice

      Mice with vitiligo-like lesions were randomly divided into four groups, with six mice per group: (i) the blank (no treatment), (ii) the sham (vehicle hydrogel), (iii) the CAP jet, and (iv) the CAP-activated hydrogel groups. To maintain the advanced vitiligo status, all mice received monobenzone application every other day during the treatment period. Mice in the sham group or in the CAP-activated hydrogel group were given either vehicle hydrogel or CAP-activated hydrogel once every other day, whereas mice in the CAP jet group received one weekly 1.5-minute treatment of the CAP jet placed 10 mm upstream of the mouse skin. These interventions lasted for 1 month. Subsequently, the skin lesion was scored with a depigmentation scoring method shown in Supplementary Figure S2, and the skin tissues and peripheral sera were harvested for further analysis on the last day of the treatment.

      CAP treatment of patients with vitiligo

      This study was randomized and placebo controlled but was single blinded in patients with active focal vitiligo and followed the Declaration of Helsinki protocols. The registered date of this clinical trial was 15 December 2017 in the Chinese Clinical Trial Registry, with a registration number of ChiCTR-OPB-17013944. It was also approved by the Institutional Review Board of the Second Affiliated Hospital of Xi’an Jiaotong University in China (number 2016105). All participants had provided their written informed consent. Supplementary Table S1 lists the details regarding the study participants, and the participant flow or the relative registration information is detailed in Supplementary Materials and Methods and Supplementary Figure S6. Briefly, 20 patients completed the study. Patients 1–5 with single lesions received no treatment (the blank control, n = 5). Patients 6–15 with single lesions received CAP-hydrogel treatment daily. Patients 16–20 each had two lesions of similar size and location, and one lesion was treated with vehicle hydrogel, and the other was treated with CAP-hydrogel treatment daily (n = 5). In total, 15 lesions (n = 15) were included in the CAP group. These treatments lasted for 2‒28 weeks until at least one lesion partially recovered (Supplementary Table S1). The same protocol was used for the vehicle hydrogel group. For assessment, serum and tissue samples were harvested before treatment (day 0) and after the final treatment. To avoid histologic variance, representative skin lesions were selected for biopsy by a dermatologist blind to the grouping. Lesional areas were scored with a software-based method, which was modified from the Vitiligo Area Scoring Index scoring system (
      • Rothstein B.
      • Joshipura D.
      • Saraiya A.
      • Abdat R.
      • Ashkar H.
      • Turkowski Y.
      • et al.
      Treatment of vitiligo with the topical Janus kinase inhibitor ruxolitinib.
      ). Digital images were measured to determine the depigmentation percentages by Rapid CAD Editor software (Huduntech, Shanghai, China). White blood cells, red blood cells, and platelets were routinely measured in serum samples (Supplementary Materials and Methods and Supplementary Table S2).

      H2O2 and NO assays

      The H2O2 in deionized water before or after the surface CAP treatment was measured with a commercial H2O2 Assay Kit (Beyotime Biotechnology, Shanghai, China). After adding xylenol orange, the violet color of the product in solution was measured at 560 nm by a microplate reader (Thermo Fisher Scientific, Waltham, MA). NO was inferred from nitrite and nitrate measured with a Griess assay kit (Beyotime Biotechnology) as previously described (
      • Chen C.
      • Li F.
      • Chen H.
      • Kong M.G.
      Aqueous reactive species induced by a PCB surface micro-discharge air plasma device: a quantitative study.
      ).

      Histologic evaluation

      Skin tissues were processed for paraffin embedding. Some sections were stained with H&E. A commercial kit for Fontana–Masson staining (Abcam, Cambridge, MA) was used to detect melanocytes. Immunohistochemistry was performed as previously described (
      • Peng L.
      • Li Q.
      • Wang H.
      • Wu J.
      • Li C.
      • Liu Y.
      • et al.
      Fn14 deficiency ameliorates psoriasis-like skin disease in a murine model.
      ). The Fontana–Masson and immunohistochemical staining methods are detailed in the Supplementary Materials and Methods.

      Quantitative real-time reverse transcriptase‒PCR

      TRIzol reagent (Ambion, Carlsbad, CA) was utilized to extract total RNA from mouse skin tissue. A commercial kit (Takara Bio, Kyoto, Japan) was used to generate cDNA. Quantitative real-time reverse transcriptase‒PCR was performed in duplicate with TB Green stain (Takara Bio, Beijing, China) and an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Waltham, MA). The PCR primers used (Sangon Biotech, Shanghai, China) are listed in Supplementary Table S3. The expression level of the target gene was calculated by the 2–ΔΔCt method.

      ELISA

      Tissue lysates and serum samples were analyzed with ELISA. Mouse CXCL10, IFN-γ, IL-17, iNOS, and HIF-1α kits (Elabscience, Wuhan, China) and an NRF2 kit (Xitang Biological Technology, Shanghai, China) were used, and optical density values were read at 450 nm.

      Western blotting

      Proteins were extracted from fresh tissues using radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitor cocktail (HEART Biotech, Xi’an, China). They were calibrated to have the same protein concentration and were then loaded in polyacrylamide gels. The extract transferring, antibody incubation, and signal generation are detailed in the Supplementary Materials and Methods. Band intensities were measured by ImageJ software (National Institute of Health, Bethesda, MD). The relative values of the target proteins (normalized to GAPDH accordingly) represent their expression levels.

      Statistical analysis

      Quantitative data are expressed as the mean ± SEM. GraphPad Prism 6 software (GraphPad Software, San Diego, CA) was used for statistical analysis. See Supplementary Materials and Methods for details. ANOVA was used for the comparisons of >2 groups, followed by Bonferroni’s or Dunn’s multiple comparisons test. Student’s t-test was used to compare two groups, and differences were considered significant at P < 0.05.

      Data availability statement

      No datasets were generated or analyzed during this study.

      ORCIDs

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We are very grateful to Chunying Li (Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, China) for her valuable advice. We also acknowledge the helpful discussions with Bo Guo, Chen Chen, Yingying Dong, and Dehui Xu (Xi’an Jiaotong University, China). This study was partially supported by the National Natural Science Foundation of China (number 51707149), the Innovation Capability Support Plan of Shaanxi Province (number 2019TD-034), the World-Class Universities (Disciplines) and the Characteristic Development Guidance Funds for the Central Universities (number PY3A038), and Batten Endowed Chair Professorship of Old Dominion University, Norfolk, VA. This clinical trial was registered on the Chinese Clinical Trial Registry, and its registration number was ChiCTR-OPB-17013944. The protocol was approved by the Institutional Review Board of the Second Affiliated Hospital of Xi’an Jiaotong University in China (number 2016105). The trials were carried out in accordance with Good Clinical Practice and the Declaration of Helsinki. Written informed consent was obtained from all patients.

      Author Contributions

      Conceptualization: MGK, YX; Formal Analysis: SZ; Funding Acquisition: KG, MGK, YX; Investigation: SZ, QL; Methodology: MGK, SZ, MX; Resources: MGK; Supervision: MGK, YX; Writing - Original Draft Preparation: SZ, HC

      Supplementary Materials and Methods

      Clinical study design and patients

      This study was performed at the Department of Dermatology in the Second Affiliated Hospital of Xi’an Jiaotong University (Xi’an, China) from 1 May 2017 to 31 May 2018 and was followed up until 31 December 2018. The registered date of this clinical trial was 15 December 2017 in the Chinese Clinical Trial Registry, with a registration number of ChiCTR-OPB-17013944. It was also approved by the Institutional Review Board of the Second Affiliated Hospital of Xi’an Jiaotong University in China (number 2016105). All patients were diagnosed with active focal vitiligo (
      • Ezzedine K.
      • Eleftheriadou V.
      • Whitton M.
      • van Geel N.
      Vitiligo.
      ), and they also met our inclusion criteria before their participation in this study. The inclusion criteria were as follows: aged 12 years and above, with a duration of less than 3 months, without other systemic diseases or skin diseases, and receiving no treatment. The exclusion criteria were as follows: having other systemic diseases or skin diseases, receiving other treatment, sensitivity to hydroxyethyl cellulose, or withdrawal from the clinical trial because of personal reasons.

      Cold atmospheric plasma and cold atmospheric plasma–activated hydrogel

      A power supply (CTP-2000K, Suman Electronics, Nanjing, China) was connected to two parallel electrodes sandwiching a ceramic slab to strike a surface discharge plasma in air on the sample-facing electrode (
      • Chen C.
      • Li F.
      • Chen H.
      • Kong M.G.
      Aqueous reactive species induced by a PCB surface micro-discharge air plasma device: a quantitative study.
      ), with an averaged electric power density of 54 mW/cm2 at a peak applied voltage of 8 kV and an excitation frequency of 9 kHz. As shown in Supplementary Figure S1, the electrical measurement was performed with a high-voltage probe (P6015A, Tektronix, Shanghai, China) and a digital storage oscilloscope (DSO-X 2014A, Agilent Technologies, Palo Alto, CA).
      A petri dish containing 5 ml of deionized water was treated for 6 minutes using surface air cold atmospheric plasma (CAP) placed 10 mm above the water surface (Supplementary Figure S1). CAP-activated hydrogel was prepared by mixing CAP-treated water with hydroxyethyl cellulose (30 mg/ml H2O) and triethanolamine (20 mg/ml H2O) for 1 minute at room temperature using a common gel preparation technique (
      • Kumar L.
      • Verma R.
      In vitro evaluation of topical gel prepared using natural polymers.
      ). CAP-activated hydrogel was freshly prepared before it was administered to the patients. Hydrogel prepared with untreated deionized water was used as vehicle hydrogel. In addition, a CAP jet (
      • Walsh J.L.
      • Kong M.G.
      Contrasting characteristics of linear-field and cross-field atmospheric plasma jets.
      ), also known as a CAP plume, was used for direct treatment of the mouse skin. The working gas of the CAP jet was helium flowing at 5 standard liters per minute.

      Depigmentation scoring in mice

      Scoring of vitiligo lesions in mice was performed by two investigators who were blinded to the treatments. On the basis of the scoring method given by
      • Rashighi M.
      • Agarwal P.
      • Richmond J.M.
      • Harris T.H.
      • Dresser K.
      • Su M.W.
      • et al.
      CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo.
      for mouse vitiligo lesions, we designed the scoring method used for this experiment. Shaved dorsal skin of the mouse was divided into four quadrants, and each quadrant was further divided equally into four parts labeled as Q1-1, Q1-2...Q4-4. In a quadrant, no white hair in any part of the quadrant was designated 0 points, in the 0 to ¼ mark as 1, in the ¼ to ½ mark as 2, in the ½ to 1 mark as 3, and in the entire quadrant as 4. In each section of the quadrant, the higher limit was excluded for score assignment. Then, the scores for all four quadrants were added to obtain the score for each mouse (Supplementary Figure S2).

      Fontana–Masson staining

      Fontana– Masson staining was performed as previously described (
      • Hu M.
      • Chen C.
      • Liu J.
      • Cai L.
      • Shao J.
      • Chen Z.
      • et al.
      The melanogenic effects and underlying mechanism of paeoniflorin in human melanocytes and vitiligo mice.
      ). After deparaffinization and rehydration, the slides were incubated in hot ammonia silver solution for 30 minutes till the slides became brown. The slides were then placed in 0.2% gold chloride solution at room temperature for 30 seconds, in 5% sodium thiosulfate solution for 90 seconds, and in nuclear fast red solution for 5 minutes. Finally, fresh absolute alcohol was used for dehydration, and neutral balsam was used for mounting.

      Immunohistochemistry

      Antigen retrieval was performed by boiling deparaffinized sections. After peroxidase blockade, rabbit anti–human CD8 (1:500 Abcam, Cambridge, MA), rabbit anti–human glycoprotein100 IgG (1:500 Abcam), rabbit anti–mouse CD8 (1:2,000, Abcam), rabbit anti–mouse CD3 (1:150, Abcam), rabbit anti–mouse CD11C (1:350, Cell Signaling Technology, Danvers, MA), rabbit anti–mouse CXCL10 (1:2,000, GeneTex, Irvine, CA), and rabbit anti–mouse HIF-1α IgG (1:500, Abcam) were used as the primary antibody. Polymer horseradish peroxidase–labeled goat anti–rabbit IgG (DAKO, Glostrup, Denmark) was used as the secondary antibody (2 μg/ml). The brown-yellow color was developed using 3,3′-diaminobenzine-chromogen substrate (DAKO). ImageJ software was used to score all staining. Finally, human skin pigmentation was evaluated with a Wood’s lamp and photography.

      Western blotting

      The protein extracts were separated on electrophoresis gels and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). Membranes were incubated with rabbit IgG targeting mouse inducible nitric oxide synthase (1:1,000, Abcam), NRF2 (1:1,000, Cell Signaling Technology), or HIF-1α (1:1,000, Abcam). Horseradish peroxidase–labeled goat anti–rabbit IgG (1:2,000, Zhuangzhi Biotechnology, Shaanxi, China) were applied to the membranes. Signal generation was performed through the use of a commercial enhanced chemiluminescence kit (Millipore).

      Serum and blood cell assays

      White blood cells, red blood cells, and platelets were routinely measured in serum samples (Supplementary Table S2). Alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and serum creatinine were determined using Beckman Synchron CX5 Clinical Analyzer (Beckman Instruments, Fullerton, CA).

      Statistical analysis

      Quantitative data are expressed as mean ± SEM. GraphPad Prism 6 software (GraphPad Software, San Diego, CA) was used for statistical analysis. One-way ANOVA was used for comparing more than two groups, and this was followed by Bonferroni’s or Dunn’s multiple comparisons test. When the data were in accordance with normal distribution and homoscedasticity, we used the one-way ANOVA test and then Bonferroni’s multiple comparisons test (e.g., IFN-γ in Figure 3a, CXCL10 and IFN-γ in Figure 3b, HIF-1α in Figure 3f, NRF2 in Figure 4e, and inducible nitric oxide synthase in Figure 4f). When the data did not accord with the normal distribution (e.g. CXCL10 and IL-17 in Figure 3a, IL-17 in Figure 3b, CXCL10 in Figure 3d, inducible nitric oxide synthase and NRF2 in Figure 4c, inducible nitric oxide synthase in Figure 4e, and NRF2 in Figure 4f), we used the nonparametric test and then Dunn’s multiple comparisons test. Comparing two independent samples, we used the paired Student’s t-test (blank and vehicle hydrogel groups in Figure 5c) or unpaired Student’s t-test (Figure 6b) if the data were in accordance with both normal distribution and homoscedasticity. When the data were not in accordance with the normal distribution, we used the Wilcoxon matched-pairs signed-rank test (CAP-hydrogel group in Figure 5c). Differences were considered significant at P < 0.05.
      Figure thumbnail fx1
      Supplementary Figure S1CAP and CAP-activated hydrogel. (a) The CAP-generating apparatus with its power supply and voltage monitoring devices. (b) A surface air CAP with its end view in open air. (c) Water was activated by the surface air CAP in a dish approximately 10 mm beneath the electrode. The activated water was then mixed with hydroxyethyl cellulose to form CAP-activated hydrogel. (d) A CAP jet, also known as a CAP plume, in flowing helium at 5 standard liters per minute was used to (e) treat a mouse placed 10 mm downstream of the jet nozzle. CAP, cold atmospheric plasma.
      Figure thumbnail fx2
      Supplementary Figure S2Treatment and depigmentation assessment in vitiligo mice. (a) Disease induction and treatment of the mice in each group. The monobenzone cream application and the treatment days were staggered. (b) The back skin of the mouse was divided into four quadrants, and each quadrant was divided equally into four parts. One quadrant was assigned with a maximum of 4 points. When white hair appeared in one part, we calculated it as 1 point; the number at the top of each image in (b) is the mouse’s score.
      Figure thumbnail fx3
      Supplementary Figure S3The IFN-γ–CXCL10–CD8+ T-cell axis in the development of vitiligo. IFN-γ binds to the IFN-γR. Keratinocytes secrete CXCL10 into the dermis. CXCL10 drives CD8+ T cells from blood vessels to the dermis. CXCL10 combines with CXCR-3 to attack melanocytes. The melanocytes are destroyed, and skin color turns white. IFN-γR, IFN-γ receptor.
      Figure thumbnail fx4
      Supplementary Figure S4HIF-1α activity in skin tissues and peripheral areas of vitiliginous lesions. Skin tissues and peripheral sera were harvested on the last day of the 1-month treatment period. (a) The protein expression levels in sera samples. (b) The mRNA expression levels in skin tissue. (c, d) The protein expression levels in skin tissue. n = 5. The data were analyzed by one-way ANOVA test. ∗P < 0.05, compared with the blank group; #P < 0.05, compared with the vehicle hydrogel group. CAP, cold atmospheric plasma; ns, not significant.
      Figure thumbnail fx5
      Supplementary Figure S5The vitiligo-like mouse model. Shaved dorsal surface of the mouse was treated with 50 mg of 40% monobenzone cream twice daily for 30 days. (a) Dorsal images before and after the vitiligo model was induced in mice. (b) Distribution of the follicular melanocytes and immune cells in skin lesions. Representative images are shown. Bar = 25 μm.
      Figure thumbnail fx6
      Supplementary Figure S6Consort flow diagram. At the beginning of this clinical study, 36 patients were accepted; in total, 8 patients did not meet the inclusion criteria, and 5 patients declined to participate in the clinical study. In total, 23 patients participated in this study, 20 of whom completed the study.
      Supplementary Table S1Demographic Characteristics of Patients with Vitiligo
      Case No.No. of lesionGroupSexAge (y)Time 1
      History of disease before CAP-activated hydrogel treatment.
      (wk)
      Time 2
      Duration of CAP-activated hydrogel treatment regimen.
      (wk)
      11BlankFemale461
      21BlankMale362
      31BlankFemale674
      41BlankMale543
      51BlankMale454
      61CAP-activated hydrogelMale26424
      71CAP-activated hydrogelFemale65828
      8
      Skin tissue was obtained for immunohistochemistry.
      1CAP-activated hydrogelMale29220
      9
      Skin tissue was obtained for immunohistochemistry.
      1CAP-activated hydrogelFemale65320
      101CAP-activated hydrogelFemale6844
      111CAP-activated hydrogelMale1218
      12
      Skin tissue was obtained for immunohistochemistry.
      1CAP-activated hydrogelMale43220
      131CAP-activated hydrogelMale22316
      141CAP-activated hydrogelMale4514
      15
      Skin tissue was obtained for immunohistochemistry.
      1CAP-activated hydrogelFemale3218
      16
      Patients with two similar vitiligo lesions.
      2Vehicle hydrogel and CAP-activated hydrogelMale3414
      17
      Patients with two similar vitiligo lesions.
      2Vehicle hydrogel and CAP-activated hydrogelMale2022
      18
      Patients with two similar vitiligo lesions.
      2Vehicle hydrogel and CAP-activated hydrogelMale67216
      19
      Patients with two similar vitiligo lesions.
      2Vehicle hydrogel and CAP-activated hydrogelMale17316
      20
      Patients with two similar vitiligo lesions.
      2Vehicle hydrogel and CAP-activated hydrogelMale2448
      Abbreviation: CAP, cold atmospheric plasma; No., number.
      1 History of disease before CAP-activated hydrogel treatment.
      2 Duration of CAP-activated hydrogel treatment regimen.
      3 Skin tissue was obtained for immunohistochemistry.
      4 Patients with two similar vitiligo lesions.
      Supplementary Table S2Results of Serum and Blood Cell Assays
      Case No.WBC
      Normal ranges: (4.0–10.0) × 109/l for adult; (5.0–12.0) × 109/l for children.
      RBC
      Normal ranges: (4.00–5.50) × 1012/l for adult male; (3.5–5.0) × 1012/l for adult female; (4.2–5.2) × 1012/l for children.
      PLT
      Normal range: (100–300) × 109/l.
      ALT
      Normal range: (7–40) U/l.
      AST
      Normal range: (13–35) U/l.
      BUN
      Normal range: (2.90–7.50) mmol/l.
      SCr
      Normal ranges: (57–111) μmol/l for adult male; (47–81) μmol/l for adult female; (25–69) μmol/l for children.
      6N (6.03)N (4.15)N (153)N (26)N (20)N (3.74)N (65.43)
      7N (6.26)N (4.62)N (243)N (32)N (29)N (4.76)N (68.91)
      8N (7.97)N (4.71)N (168)N (15)N (23)N (5.32)N (75.32)
      9N (6.83)N (4.06)N (237)N (18)N (20)N (4.13)N (80.21)
      10N (6.31)N (4.03)N (209)N (25)N (21)N (3.75)N (55.73)
      11N (7.22)N (4.22)N (267)N (22)N (30)N (3.87)N (58.59)
      12N (8.33)N (4.57)N (187)N (22)N (27)N (6.12)N (65.31)
      13N (5.27)N (4.53)N (169)N (17)N (23)N (3.57)N (83.26)
      14N (7.18)N (5.10)N (235)N (25)N (28)N (7.21)N (74.26)
      15N (7.83)N (4.77)N (174)N (17)N (29)N (6.46)N (62.15)
      16N (6.99)N (5.12)N (218)N (10)N (14)N (4.58)N (69.43)
      17N (7.02)N (5.00)N (159)N (21)N (25)N (5.92)N (79.32)
      18N (6.55)N (5.10)N (257)N (24)N (26)N (7.14)N (81.27)
      19N (7.68)N (4.81)N (278)N (23)N (31)N (5.32)N (64.24)
      20N (8.09)N (4.94)N (203)N (28)N (26)N (6.42)N (71.89)
      Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; N, normal; No., number; PLT, platelet; RBC, red blood cell; SCr, serum creatinine; WBC, white blood cell.
      1 Normal ranges: (4.0–10.0) × 109/l for adult; (5.0–12.0) × 109/l for children.
      2 Normal ranges: (4.00–5.50) × 1012/l for adult male; (3.5–5.0) × 1012/l for adult female; (4.2–5.2) × 1012/l for children.
      3 Normal range: (100–300) × 109/l.
      4 Normal range: (7–40) U/l.
      5 Normal range: (13–35) U/l.
      6 Normal range: (2.90–7.50) mmol/l.
      7 Normal ranges: (57–111) μmol/l for adult male; (47–81) μmol/l for adult female; (25–69) μmol/l for children.
      Supplementary Table S3Primer Sets Used for Sequencing Expression Genes
      Target GenePrimer (5′-3′)
      Mouse GapdhF: 5′-CTCATGACCACAGTCCATGC-3′

      R: 5′-ACACATTGGGGGTAGGAACA-3′
      Mouse IfnγF: 5′-CTTGAAAGACAATCAGGCCATC-3′

      R: 5′-CTTGGCAATACTCATGAATGCA-3′
      Mouse Cxcl10F: 5′-AGGGGAGTGATGGAGAGAGG-3′

      R: 5′-TGAAAGCGTTTAGCCAAAAAAGG-3′
      Mouse Il17F: 5′-CTCAGACTACCTCAACCGTTCC-3′

      R: 5′-ATGTGGTGGTCCAGCTTTCC-3′
      Mouse iNosF: 5′-CACCAAGCTGAACTTGAGCG-3′

      R: 5′-CGTGGCTTTGGGCTCCTC-3′
      Mouse Nrf2F: 5′-CAGCCATGACTGATTTAAGCAG-3′

      R: 5′-CAGCTGCTTGTTTTCGGTATTA-3′
      Mouse Hif1aF: 5′-GAATGAAGTGCACCCTAACAAG-3′

      R: 5′-GAGGAATGGGTTCACAAATCAG-3′
      Abbreviations: F, forward; R, reverse.

      References

        • Abdallah M.
        • Abdel-Naser M.B.
        • Moussa M.H.
        • Assaf C.
        • Orfanos C.E.
        Sequential immunohistochemical study of depigmenting and repigmenting minigrafts in vitiligo.
        Eur J Dermatol. 2003; 13: 548-552
        • Chen C.
        • Li F.
        • Chen H.
        • Kong M.G.
        Aqueous reactive species induced by a PCB surface micro-discharge air plasma device: a quantitative study.
        J Phys D Appl Phys. 2017; 50: 445208
        • Deng Q.
        • Wei J.
        • Zou P.
        • Xiao Y.
        • Zeng Z.
        • Shi Y.
        • et al.
        Transcriptome analysis and emerging driver identification of CD8+ T cells in patients with vitiligo.
        Oxid Med Cell Longev. 2019; 2019: 2503924
        • Ezzedine K.
        • Eleftheriadou V.
        • Whitton M.
        • van Geel N.
        Vitiligo.
        Lancet. 2015; 386: 74-84
        • Felsten L.M.
        • Alikhan A.
        • Petronic-Rosic V.
        Vitiligo: a comprehensive overview part II: treatment options and approach to treatment.
        J Am Acad Dermatol. 2011; 65: 493-514
        • Friedman P.C.
        • Miller V.
        • Fridman G.
        • Lin A.
        • Fridman A.
        Successful treatment of actinic keratoses using nonthermal atmospheric pressure plasma: a case series.
        J Am Acad Dermatol. 2017; 76: 349-350
        • Glassman S.J.
        Vitiligo, reactive oxygen species and T-cells.
        Clin Sci (Lond). 2011; 120: 99-120
        • Horiba M.
        • Kamiya T.
        • Hara H.
        • Adachi T.
        Cytoprotective effects of mild plasma-activated medium against oxidative stress in human skin fibroblasts.
        Sci Rep. 2017; 7: 42208
        • Isbary G.
        • Heinlin J.
        • Shimizu T.
        • Zimmermann J.L.
        • Morfill G.
        • Schmidt H.U.
        • et al.
        Successful and safe use of 2 min cold atmospheric argon plasma in chronic wounds: results of a randomized controlled trial.
        Br J Dermatol. 2012; 167: 404-410
        • Jian Z.
        • Li K.
        • Song P.
        • Zhu G.
        • Zhu L.
        • Cui T.
        • et al.
        Impaired activation of the Nrf2-ARE signaling path-way undermines H2O2-induced oxidative stress response: a possible mechanism for melanocyte degeneration in vitiligo.
        J Invest Dermatol. 2014; 134: 2221-2230
        • Kesarwani P.
        • Murali A.K.
        • Al-Khami A.A.
        • Mehrotra S.
        Redox regulation of T-cell function: from molecular mechanisms to significance in human health and disease.
        Antioxid Redox Signal. 2013; 18: 1497-1534
        • Khan R.
        • Satyam A.
        • Gupta S.
        • Sharma V.K.
        • Sharma A.
        Circulatory levels of antioxidants and lipid peroxidation in Indian patients with generalized and localized vitiligo.
        Arch Dermatol Res. 2009; 301: 731-737
        • Kong M.G.
        • Kroesen G.
        • Morfill G.
        • Nosenko T.
        • Shimizu T.
        • van Dijk J.
        • et al.
        Plasma medicine: an introductory review.
        New J Phys. 2009; 11: 115012
        • Lee O.J.
        • Ju H.W.
        • Khang G.
        • Sun P.P.
        • Rivera J.
        • Cho J.H.
        • et al.
        An experimental burn wound-healing study of non-thermal atmospheric pressure microplasma jet arrays.
        J Tissue Eng Regen Med. 2016; 10: 348-357
        • Lee Y.S.
        • Lee M.H.
        • Kim H.J.
        • Won H.R.
        • Kim C.H.
        Non-thermal atmospheric plasma ameliorates imiquimod-induced psoriasis-like skin inflammation in mice through inhibition of immune responses and up-regulation of PD-L1 expression.
        Sci Rep. 2017; 7: 15564
        • Mansourpour H.
        • Ziari K.
        • Motamedi S.K.
        • Poor A.H.
        Therapeutic effects of iNOS inhibition against vitiligo in an animal model.
        Eur J Transl Myol. 2019; 29: 8383
        • Metelmann H.R.
        • Seebauer C.
        • Miller V.
        • Fridman A.
        • Bauer G.
        • Graves D.B.
        • et al.
        Clinical experience with cold plasma in the treatment of locally advanced head and neck cancer.
        Clin Plasma Med. 2018; 9: 6-13
        • Niethammer P.
        • Grabher C.
        • Look A.T.
        • Mitchison T.J.
        A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.
        Nature. 2009; 459: 996-999
        • Peng L.
        • Li Q.
        • Wang H.
        • Wu J.
        • Li C.
        • Liu Y.
        • et al.
        Fn14 deficiency ameliorates psoriasis-like skin disease in a murine model.
        Cell Death Dis. 2018; 9: 801
        • Rashighi M.
        • Agarwal P.
        • Richmond J.M.
        • Harris T.H.
        • Dresser K.
        • Su M.W.
        • et al.
        CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo.
        Sci Transl Med. 2014; 6: 223ra23
        • Rashighi M.
        • Harris J.E.
        Vitiligo pathogenesis and emerging treatments.
        Dermatol Clin. 2017; 35: 257-265
        • Rodrigues M.
        • Ezzedine K.
        • Hamzavi I.
        • Pandya A.G.
        • Harris J.E.
        • Vitiligo Working Group
        New discoveries in the pathogenesis and classification of vitiligo.
        J Am Acad Dermatol. 2017; 77: 1-13
        • Rothstein B.
        • Joshipura D.
        • Saraiya A.
        • Abdat R.
        • Ashkar H.
        • Turkowski Y.
        • et al.
        Treatment of vitiligo with the topical Janus kinase inhibitor ruxolitinib.
        J Am Acad Dermatol. 2017; 76: 1054-1060.e1
        • Schallreuter K.U.
        • Moore J.
        • Wood J.M.
        • Beazley W.D.
        • Gaze D.C.
        • Tobin D.J.
        • et al.
        In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase.
        J Investig Dermatol Symp Proc. 1999; 4: 91-96
        • Schmidt A.
        • von Woedtke T.V.
        • Stenzel J.
        • Lindner T.
        • Polei S.
        • Vollmar B.
        • et al.
        One year follow-up risk assessment in SKH-1 mice and wounds treated with an argon plasma jet.
        Int J Mol Sci. 2017; 18: 868
        • Sies H.
        • Jones D.P.
        Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.
        Nat Rev Mol Cell Biol. 2020; 21: 363-383
        • Singh R.K.
        • Lee K.M.
        • Vujkovic-Cvijin I.
        • Ucmak D.
        • Farahnik B.
        • Abrouk M.
        • et al.
        The role of IL-17 in vitiligo: a review.
        Autoimmun Rev. 2016; 15: 397-404
        • Speeckaert R.
        • Dugardin J.
        • Lambert J.
        • Lapeere H.
        • Verhaeghe E.
        • Speeckaert M.M.
        • et al.
        Critical appraisal of the oxidative stress pathway in vitiligo: a systematic review and meta-analysis.
        J Eur Acad Dermatol Venereol. 2018; 32: 1089-1098
        • Speeckaert R.
        • van Geel N.
        Vitiligo: an update on pathophysiology and treatment options.
        Am J Clin Dermatol. 2017; 18: 733-744
        • Speeckaert R.
        • Voet S.
        • Hoste E.
        • van Geel N.
        S100B is a potential disease activity marker in nonsegmental vitiligo.
        J Invest Dermatol. 2017; 137: 1445-1453
        • Strassner J.P.
        • Harris J.E.
        Understanding mechanisms of autoimmunity through translational research in vitiligo.
        Curr Opin Immunol. 2016; 43: 81-88
        • Visvanathan S.
        • Baum P.
        • Vinisko R.
        • Schmid R.
        • Flack M.
        • Lalovic B.
        • et al.
        Psoriatic skin molecular and histopathologic profiles after treatment with risankizumab versus ustekinumab.
        J Allergy Clin Immunol. 2019; 143: 2158-2169
        • Walsh J.L.
        • Kong M.G.
        Contrasting characteristics of linear-field and cross-field atmospheric plasma jets.
        Appl Phys Lett. 2008; 93: 111501
        • Xia J.
        • Zeng W.
        • Xia Y.
        • Wang B.
        • Xu D.
        • Liu D.
        • et al.
        Cold atmospheric plasma induces apoptosis of melanoma cells via Sestrin2-mediated nitric oxide synthase signaling.
        J Biophotonics. 2019; 12e201800046
        • Xie H.
        • Zhou F.
        • Liu L.
        • Zhu G.
        • Li Q.
        • Li C.
        • et al.
        Vitiligo: how do oxidative stress-induced autoantigens trigger autoimmunity?.
        J Dermatol Sci. 2016; 81: 3-9
        • Yoo S.K.
        • Starnes T.W.
        • Deng Q.
        • Huttenlocher A.
        Lyn is a redox sensor that mediates leukocyte wound attraction in vivo.
        Nature. 2011; 480: 109-112