Ozone therapy promotes the differentiation of basal keratinocytes via increasing Tp63-mediated transcription of KRT10 to improve psoriasis.

Abstract Psoriasis is a chronic immune‐mediated inflammatory dermatosis. Recently, ozone therapy has been applicated to psoriasis treatment; however, the mechanism by which ozone therapy improves psoriasis remains unclear. The excessive proliferation and the differentiation of basal keratinocytes have been considered critical issues during pathological psoriasis process, in which keratin 6 (KRT6) and KRT10 might be involved. In the present study, KRT6, IL‐17 and IL‐22 protein within psoriasis lesions was decreased, while KRT10 and Tp63 protein in psoriasis lesions was increased by ozone treatment in both patient and IMQ mice psoriatic tissues. In the meantime, ozone treatment down‐regulated KRT6 mRNA and protein expression while up‐regulated KRT10 mRNA and protein expression within IL‐22 treated primary KCs; the cell viability of KCs was suppressed by ozone treatment. Moreover, Tp63 bound to KRT10 promoter region to activate its transcription in basal keratinocytes; the promotive effects of ozone on Tp63 and KRT10 were significantly reversed by Tp63 silence. Both TP63 and KRT10 mRNA expression were significantly increased by ozone treatment in psoriasis lesions; there was a positive correlation between Tp63 and KRT10 expression within tissue samples, suggesting that ozone induces the expression of Tp63 to enhance the expression of KRT10 and the differentiation of keratinocytes, therefore improving the psoriasis. In conclusion, the application of ozonated oil could be an efficient and safe treatment for psoriasis; ozone promotes the differentiation of keratinocytes via increasing Tp63‐mediated transcription of KRT10, therefore improving psoriasis.


| INTRODUC TI ON
Psoriasis is a common recrudescent chronic immune-mediated inflammatory dermatosis, which is characterized by hyperplasia of epidermis, angiogenesis, and soakage of inflammation cells. 1 Psoriasis can lead to physical and psychological trauma to patients, therefore affecting up to 2%-4% of the global population. [2][3][4] A number of treatments that have been used to treat psoriasis include topical preparations containing corticosteroids, retinoid derivatives, synthetic vitamin D3 analogs, tar, or anthralin; systemic drugs, including immunosuppressants and calcineurin inhibitors cyclosporine, tacrolimus, acitretin and isotretinoin; as well as photochemotherapy (PUVA) and UVB irradiation. [5][6][7] However, most psoriasis therapies bring up considerable side effects, including skin atrophy, telangiectasia, purpura, hairy and folliculitis. [8][9][10] Ozone (O 3 ) is a strong oxidizing molecule composed of three oxygen atoms. Ozone has anti-infection, accelerates blood metabolism, anti-inflammatory 11 and immunomodulatory effects, 12,13 and can be used in a wide range of diseases. Recently, the ozone therapy has also been applied to psoriasis treatment. 14, 15 Tan et al 15 prepared ozonated oil by dissolving ozone in vegetable oil and compared the efficacy and safety between ozonated oil and compound flumethasone ointment in the treatment of psoriasis vulgaris. After 4-week treatment, the epidermis was approximately normal and few inflammatory cells infiltration in the dermal papilla were observed in both groups. They concluded that the ozonated oil treatment for stable psoriasis is safe and effective, and its efficacy is equivalent to the effect of glucocorticoid topical preparations. Regarding the molecular mechanism, another group has speculated that O 3 could affect the activity of MMP, 14,16,17 thus exerting the effects not only on the occurrence and development of disorders associated with human skin ageing, [18][19][20] but also on other skin diseases including psoriasis and dermatitis. 14,15,[21][22][23][24][25] However, the precise mechanism by which the ozone therapy improves psoriasis remains unclear.
The basal keratinocytes in the lesions of psoriasis proliferate rapidly, and they do not fully differentiate into the epidermal horny layer to form hyperplasia, while the undifferentiated basal keratinocytes in normal skin undergo a long period of process such as proliferation, differentiation and evolution, and eventually enter the cuticle of the epidermis. Studies have found that ozone may affect basal keratinocyte differentiation. The expression of KRT10 (KRT10) is increased after ozone treatment of skin tissue, while KRT10 is a keratin marker produced in well-differentiated upper basal keratinocytes. 13,26 In addition, KRT10 and other differentiation markers are one of the characteristics of psoriasis. 27 KRT10 expression could be significantly down-regulated within lesions and marginal healthy skin cells of diffuse psoriasis; more keratin 6 (KRT6)-positive and KRT10-negative cells were detected in the inner margin of the lesions. 28 Therefore, we speculate that ozone may directly affect the pathological process of psoriasis through the regulation of the differentiation of basal keratinocytes and that KRT6/10 might be involved.
Reportedly, Tp63 could regulate the ability of keratinocytes to proliferate and differentiate. 29,30 Full-length Tp63 could be remarkably inhibited within lesional tissue than that in clinically normal skin from patients and matched healthy controls. 31,32 More important, Tp63 is involved in the regulation of redox status and is regulated by oxidative stress in epithelial cell, including in keratinocytes. 33 Therefore, we speculate that ozone induces Tp63 activation, promotes KRT10 expression and accelerates basal keratinocyte differentiation, therefore improving the psoriatic lesions.
Herein, the efficiency of ozone in the treatment of psoriasis has been further proved. The effects of ozone on the differentiation of basal keratinocytes, as well as the protein levels of KRT6 and KRT10, were also examined. Primary basal keratinocytes, KCs, were then stimulated with IL-22 to establish a cell model of psoriasis 34,35 and treated with ozone and examined for cellular morphological changes and the protein levels of KRT6 and KRT10. Next, the predicted binding between Tp63 and KRT10 promoter region was validated by ChIP and luciferase reporter assays. The effects of Tp63 silence on Tp63 and KRT10 proteins were examined under ozone treatment. Finally, the mRNA expression of Tp63 and KRT10 was examined in psoriasis lesion tissues with or without ozone treatment; we examined the interaction between the expression of Tp63 and KRT10 within tissues using Pearson's correlation analysis. In summary, we provide experimental basis for a novel mechanism by which the ozone therapy improves the psoriasis via regulating the differentiation of basal keratinocyte.

| Cell line and cell transfection
Primary healthy human KCs were obtained from ATCC (ATCC ® PCS-200-011™, ATCC). Cells were cultured in Medium 154 (M154500; Gibco) supplemented with Human Keratinocytes Growth Supplement (S0015, Gibco). All cells were maintained at 37°C in a humidified tissue culture incubator with 5% CO 2 . KCs were stimulated with 100 ng/mL human recombinant cytokines IL-22 (13059-HNAE; Sino Biology) as described previously 36 to mimic the cellular alterations during psoriasis-like dermatitis. For O 3 treatments, ozone gas was dissolved in PBS solution to maintain a final concentration of 0.3 mg/L. Ozonated PBS was added to the medium, the cells were further cultured for 30 minutes, and the medium was changed. The above operations were performed once a day for a total of 3 days.

| Psoriasis-like dermatitis mouse model
Psoriasis-like dermatitis model was established in healthy BALB/C mice. Mice were randomly divided into the blank, imiquimod group (IMQ), IMQ + vehicle group and IMQ + Ozone group. IMQ cream (62.5 mg, 5%) was applied following the methods described previously. 37 For O 3 therapy, lesion skin was smeared with ozonated oil once a day for 1 week. The morphological changes were photographed and evaluated for the total sign score (TSS). 38 The pathology changes were examined using H&E staining. The protein expression level of IL-17, IL-22, Tp63, KRT6 and KRT10 in skin tissues was examined by immunohistochemistry (IHC) staining.

| Immunoblotting assays
Protein samples were separated by SDS-PAGE, transferred from gel to a PVDF membrane. The membrane was incubated with antibodies against KRT6, KRT10 and Tp63 following the methods described previously. 41 After incubating with the primary antibodies mentioned above, the blots were incubated for 1 hour with HRP-conjugated goat anti-mouse or goat anti-rabbit IgG (Beyotime). Protein levels in each lane were normalized to the levels of GAPDH.

| PCR-based analysis
Total RNA was extracted from cultured cells using Trizol reagent (Invitrogen). The expression of mRNA was measured using a SYBR Green qPCR assay (Takara). The expression of GAPDH served as an endogenous control. The 2 −ΔCT method was applied for data processing.

| Immunofluorescence
Treated target cells were plated on a glass slide at a density of 1 × 10 4 cells/mL, fixed by 4% precooled formaldehyde, washed by PBS containing 0.1%Triton X-100 for three times and then blocked with 5% BSA at room temperature for 2 hours. The slides were incubated with anti-KRT6 or anti-KRT10 antibodies for 1 hour at 37°C and with Cy5 or FITC labelled secondary antibodies (Beyotime) for 1 hour at room temperature. The nucleus was staining by DAPI (Beyotime) for 5 minutes at room temperature.
The slides were observed by inverted fluorescence microscope (Olympus).

| Chromatin immunoprecipitation (ChIP)
To validate the binding between Tp63 and KRT10 promoter region, ChIP assays were performed using anti-Tp63 (Cat.687203, Biolegend) following the methods described previously. 42 RNA polymerase II was used as a positive control antibody and non-immune IgG was used as a negative control demonstrate the efficacy of the kit reagents (Epigentek Group Inc, P-2025-48). The immunoprecipitated DNA was eluted and examined for fold-enrichment according to the methods described previously. 42

| Statistical analysis
Results from at least three independent experiments were processed using GraphPad and then expressed as means ± SD. Data were statistically analysed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test or independent sample t test. A P-value of <.05 was considered as statistically different.

| Clinical ozone (O 3 ) therapy improves psoriatic lesions in vivo
The appearance of the lesions in patients before and after receiving the ozone therapy is shown in Figure 1A,B to demonstrate the efficiency of the ozone therapy. Before the ozone treatment, there were obvious erythema, plaque, infiltration and scales on the psoriasis lesion area. After 4 weeks of treatment, the mentioned erythema, plaque, infiltration and scales were all improved, and some pigmentation remained. As further revealed by PASI scores, the PASI scores of patients after ozone therapy were significantly reduced, compared to that before ozone therapy ( Figure 1C). Histopathological

| The ozone therapy modulates the protein levels of KRT6/10 in psoriasis lesions in vivo
As we have mentioned, the reduction of KRT10 and other differentiation markers is one of the characteristics of psoriasis. 27 To investigate whether the ozone therapy improves psoriasis through KRT6/10, the levels of KRT6 and KRT10 in normal skin, psoriasis lesions with or without the ozone therapy were examined by immunohistochemical (IHC) staining. As shown in Figure 2A Figure 2C); moreover, the ratio of KRT6/KRT10 was significantly increased in psoriasis lesion while reduced after ozone therapy ( Figure 2C), indicating that KRT6/10 might be involved in the effects of ozone on psoriasis lesions.

| The ozone therapy improves IMQ-induced psoriasis-like dermatitis in vivo
To further confirm that KRT6 and KRT10 are involved in the effects of the ozone therapy on psoriasis, we established psoriasis-like dermatitis model in mice by applying IMQ cream. Mice were subjected to blank, IMQ, IMQ + Vehicle, or IMQ + ozone treatment and examined for the appearance of the dorsal skin; the dorsal skin of mice from IMQ and IMQ + Vehicle groups presented as a typical psoriasislike dermatitis, whereas the ozone therapy remarkably improved the psoriasis-like appearance ( Figure 3A). Consistently, the TSS scores in IMQ and IMQ + Vehicle groups remarkably increased whereas the ozone therapy significantly reduced the TSS scores ( Figure 3A). H&E staining showed the typical histopathological features of psoriasislike dermatitis including parakeratosis and Munro's micro-abscesses, obvious acanthosis with rete ridges extended and large inflammatory cells infiltrated in both IMQ and IMQ + Vehicle groups; the ozone therapy partially reversed these pathological changes ( Figure 3B).
IHC staining showed that psoriasis-related cytokines IL-17 and IL-22 were increased in IMQ and IMQ + Vehicle groups compared to those in control group while ozone therapy decreases their expression ( Figure 3C). In Figure 3D

| Ozone promotes the differentiation of keratinocytes in vitro
To further investigate the molecular mechanism of ozone improving psoriasis, we evaluated the cellular effects of ozone on primary  Figure 4B). Similar results were revealed by Immunoblotting ( Figure 4C). Moreover, since KRT6 is considered as an activation marker for KCs, 43,44 next, we investigated the cell viability of KCs in response to ozone therapy. As shown in Figure 4D

| Tp63 is involved in ozone-mediated KC differentiation in vitro
Tp63 was found to bind to the KRT10 promoter region in keratinocytes by the Chip-Atlas (http://chip-atlas.org/) Chromatin Immunoprecipitation Database (http://ddbj.nig.ac.jp/). To investigate whether Tp63 is involved in keratinocyte differentiation in psoriasis, we next examined the protein levels of Tp63 in IMQ-induced psoriasis-like dermatitis mice model and in human normal skin tissues and psoriasis lesions with or without the ozone therapy by IHC staining and Immunoblotting. As shown in Figure 5A,B, the protein levels F I G U R E 1 Clinical ozone (O 3 ) therapy improves psoriatic lesions in vivo. A and B, The appearance of the lesions in patients before and after receiving the ozone therapy. C, PASI scores of 20 patients before and after ozone therapy. D, Histopathological characteristics of normal skin (NC, negative control), psoriasis lesion without the ozone therapy and psoriasis lesion with the ozone therapy were shown by H&E staining. E, IL-22 and IL-17 levels in normal skin, psoriasis lesion without the ozone therapy and psoriasis lesion with the ozone therapy were shown by IHC staining of Tp63 were significantly decreased in IMQ and IMQ + Vehicle groups compared to control group while ozone therapy increased Tp63 expression. As shown in Figure 5C,D, the protein levels of Tp63 were remarkably down-regulated in lesion tissues without ozone therapy, compared to those in normal skin tissues; ozone treatment increased the protein levels of Tp63 in psoriasis lesions. To investigate the molecular mechanism, we treated KCs with or without ozone and then examined Tp63 mRNA and protein expression. As shown in Figure 5E,F, IL-22 treatment remarkably reduced, whereas ozone treatment remarkably enhanced Tp63 mRNA and protein expression within KCs. Next, ChIP assays were performed using anti-Tp63 to validate the predicted binding of Tp63 to KRT10 promoter region. As shown in Figure 5G, the levels of promoter abundance binding to Tp63 antibody were significantly higher, compared to those binding to IgG. We validated the effects of Tp63 on KRT10 transcription activity via performing luciferase reporter assays. As shown in Figure 5H

| Expression and correlation of Tp63 and KRT10 in tissue samples in vivo
To provide further evidence, we examined Tp63 and KRT10 expression in psoriasis lesion tissues with or without ozone treatment. As shown in Figure 6A,B, Tp63 and KRT10 mRNA expression could be remarkably up-regulated upon ozone treatment in psoriasis lesions, compared to those in non-treated psoriasis lesions. The expression of Tp63 and KRT10 was positively correlated in tissue samples as revealed by Pearson's correlation analysis ( Figure 6C). The application of ozone therapy in psoriasis has been reported previously. However, oxygen-ozone (O 2 /O 3 ) therapy administered by autohemotransfusion has been reported to cause the gas embolism, which could directly lead to unexpected death of the patient. 45 In the present study, ozone was dissolved in plant oil and applied to the lesion skin to avoid decomposition of ozone into oxygen at room temperature and the gas embolism. The morphometric and pathological examinations revealed that ozonated oil application improved the psoriasis; the red spots, plaques, infiltration and scales are basically subsided after treatment, further indicating that the ozone therapy is an effective and safe strategy for psoriasis.

| D ISCUSS I ON
Psoriasis is a commonly seen, chronic and inflammatory dermatosis, which is characterized by immune cell infiltration in the dermis and epidermis and excessive proliferation and abnormal differentiation of keratinocytes. 46  F I G U R E 5 Tp63 is involved in ozone-mediated KC cell differentiation in vitro. A and B, the protein levels of Tp63 were examined in blank, IMQ, IMQ + Vehicle and IMQ + Ozone groups by IHC staining (A) and immunoblotting (B). C and D, The protein levels of Tp63 were examined in normal skin samples and psoriasis lesions with or without the ozone therapy by IHC staining (C) and immunoblotting (D). E and F, KCs were stimulated with IL-22 and examined for the mRNA expression (F) and protein levels of Tp63 (E) with or without ozone treatment. G, ChIP assays were performed in 293T cells with anti-Tp63 antibody to validate the binding of Tp63 to KRT10 promoter region. H, Luciferase reporter assays were performed in 293T cells to validate the effects of Tp63 on KRT10 transcription activity. I and J, KCs were transfected with si-Tp63 in the presence or absence of ozone treatment and examined for the mRNA expression and protein levels of Tp63 and KRT10 by real-time PCR (I) and immunoblotting (J). *P < .05, **P < .01, compared to the control group; # P < .05, ## P < .01, compared to the non-treated group F I G U R E 6 Expression and correlation of Tp63 and KRT10 in tissue samples in vivo. A and B, The expression of Tp63 and KRT10 in psoriasis lesion tissues with or without ozone treatment were determined by real-time PCR. C, The correlation of Tp63 and KRT10 expression in tissue samples was analysed by Pearson's correlation analysis expression, whereas ozone treatment on primary KCs significantly decreased KRT6 mRNA expression and protein levels while increased KRT10 mRNA expression and protein levels. More importantly, the cell viability of KCs could be significantly suppressed by ozone treatment, indicating that the ozone therapy might improve psoriasis through suppressing the excessive proliferation while promoting the differentiation of the basal keratinocytes.
As we have mentioned, KRT10 expression is significantly down-regulated in psoriasis lesions while rescued by ozone treatment. To further understand the mechanism of KRT10 deregulation in the pathogenesis of psoriasis, we analysed online data in the chromatin co-immunoprecipitation database (http://ddbj.nig. ac.jp/) via the Chip-Atlas (http://chip-atlas.org/) and found that Tp63 could activate the transcription of KRT10 via targeting its promoter region. TAp63 isoforms have been considered to provide a contribution to epidermal differentiation. 53 In the process of keratinocyte differentiation of p53-mutant HaCaT cells, TAp63α can be related to the ability of growth differentiation factor 15 (GDF15) to up-regulate and to secrete. TAp63 activates the transcription of GDF15 via targeting its promoter region; the down-regulation of GDF15 could promote cell proliferation and reduce the expression of KRT10 and other differentiation markers upon the stimulation of differentiation. 54 Herein, the predicted binding of Tp63 to KRT10 promoter region was validated. Ozone treatment significantly induced TP63 mRNA expression and protein levels, therefore increasing KRT10 protein levels. After TP63 silence, ozone-induced increases in Tp63 and KRT10 protein levels were significantly reversed. These data indicate that ozone treatment promotes the differentiation of basal keratinocytes via Tp63/KRT10, finally improving psoriasis.
As a further confirmation, Tp63 mRNA and KRT10 mRNA expression were significantly up-regulated in ozone-treated psoriasis lesions; Tp63 and KRT10 mRNA expression in tissue samples was positively correlated. In conclusion, the application of ozonated oil could be an efficient and safe treatment for psoriasis; ozone promotes the differentiation of basal keratinocytes via increasing Tp63mediated transcription of KRT10, therefore improving psoriasis.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests. Informed consent to participate in the study has been obtained from participants.

DATA AVA I L A B I L I T Y S TAT E M E N T
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