DOI: https://doi.org/10.21203/rs.3.rs-1699070/v1
Benvitimod is a newly synthesized non-steroidal small molecule targeting the aryl hydrocarbon receptor(AhR). Clinical studies have demonstrated its efficacy for patients with psoriasis and atopic dermatitis(AD).
To evaluate the therapeutic effects of benvitimod on peripheral blood mononuclear cells (PBMCs) and tumor necrosis factor α (TNFα)/interferon γ (IFNγ) stimulated human keratinocytes (HaCaT).
PBMCs were obtained from 6 patients with AD. PBMCs and HaCaT cells were cultured and the effects of benvitimod on cell viability were evaluated. The expression of inflammatory cytokines and chemokines in PBMCs and HaCaT cells was measured and the nuclear translocation of AhR in HaCaT cells was visualized. Reverse transcription quantitative polymerase chain reaction(RT-PCR) and western-blot(WB) were performed to detect the expression of cytochrome P4501A1(CYP1A1), filaggrin (FLG), involucrin (IVL) and thymic stromal lymphopoietin (TSLP) in HaCaT cells. Phosphorylation of STAT1 was determined by western-blot.
Our research demonstrated that the cell proliferation of PBMCs and HaCaT cells were suppressed by benvitimod in a dose dependent manner. Benvitimod significantly suppressed TNFα/IFNγ(TI)-induced cytokine secretion and upregulated the expression of FLG and IVL in HaCaT cells. In addition, benvitimod induced the nuclear staining of AhR and inhibited the phosphorylation of STAT1 in HaCaT cells.
Benvitimod inhibited inflammatory effects in PBMCs of AD patients and underpinned the barrier-repairing effects by targeting AhR and STAT1 in TI-stimulated HaCaT cells. Benvitimod may be a potential therapeutic drug for inflammatory skin diseases such as AD.
Atopic dermatitis(AD) is an inflammatory skin disease characterized by chronic recurrent dermatitis and pruritus. The pathogenesis of AD remains unknown[1]. It is characterized by defective skin barrier, infiltration of immune cells, elevated serum immunoglobulin (IgE) levels, eosinophilia and the imbalance of T helper (Th1/Th2) cytokine profile[2]. The acute phase of AD is characterized by the stimulation of Th2 lymphocytes with its cytokine pattern including upregulation of IL-4, IL-5, and IL-13 and IgE, while the chronic phase is characterized by the stimulation of Th1 cytokines including interferon gamma (IFNγ) and interleukin-1β[3].
Benvitimod is a non-steroidal topical agent which acts on AhR[4]. It significantly inhibits the production of pro-inflammatory cytokines such as IFNγ, IL-2 and TNFα[5]. Benvitimod cream has been proven to display good efficacy and safety in patients with psoriasis and AD in clinical trials, although its mechanism was not fully understood[6].
Skin barrier maturation is accomplished by sequential and coordinated expression of various skin barrier proteins, such as filaggrin (FLG) and involucrin (IVL). Impairment of barrier function is critical for the development of AD[7]. In accordance with these observations, FLG and IVL expression levels have been reported to be reduced in lesional skin of AD. Furthermore, the stimulated keratinocytes can produce inflammatory cytokines, TARC and TSLP[8]. These cytokines and chemokines can significantly down-regulate skin barrier proteins such as FLG and IVL, which may contribute to skin barrier abnormalities[9]. Thus, skin barrier repair by blocking production of inflammatory cytokines and promoting skin barrier protein suggests a probable treatment strategy for AD[10].
Aryl hydrocarbon receptor(AhR) is a transcription factor expressed in many cells, controlling many physiological processes including cell proliferation differentiation, adhesion, migration and apoptosis. It responds to exogenous and endogenous chemicals by inducing or repressing the expression of several genes such as cytochrome P4501A1 (CYP1A1) with toxic or protective effects in a wide range of species and tissues[11]. AhR has been recognized as modulating expression of various cytokines, such as IL-1β, IL-4, IL-6, IL-10, IL-17, IL-22, TNFα and other chemokines[12]. Moreover, it can play a critical role in the regulation of inflammatory responses and open the possibility for novel therapeutic strategies in chronic inflammatory disorders. Some studies have indicated that AhR expression appears to be aberrantly induced in chronical inflammatory skin disease, such as psoriasis, AD and vitiligo[13]. In recent years, exploring safe and effective topical ligands for AhR is a potential approach to developing new treatments for AD. Topical application of AhR agonist such as coal tar has been used for the treatment of inflammatory skin diseases for quite a long time. Benvitimod, which targets the AhR signaling pathway in human skin, has been recently shown to be an efficacious novel topical treatment for AD[14].
In this study, we have found that benvitimod can suppress proliferation and regulate inflammatory response in PBMCs of AD patients. It also regulates the production of cytokines, chemokines and skin barrier proteins in TNFα/IFNγ-induced HaCaT cells. Benvitimod can suppress inflammatory response and induce skin barrier repair by activating AhR and phosphorylation of STAT1 in HaCaT cells.
The tested benvitmod powder was donated by Beijing Wenfeng Tianjipharma Company. The dried extract powder was dissolved in dimethyl sulfoxide (DMSO) and diluted up to 100mM for storage at -20℃. Dulbecco's modified Eagle's medium (DMEM; Gibco, USA) was used to dilute the chemicals to desired concentration. The final concentration of DMSO did not exceed 0.1% (v/v) and did not affect cell viability.
A total of 6 AD patients (3 males and 3 females, range from 25 to 74 years old) were recruited in this study. All patients were at an active stage of the disease, before administration of systemic treatment. The informed consents were obtained from each patient. All AD patients satisfied the criteria of Hanifin&Rajka[15] and Chinese criteria for AD[16]. The study was approved by the ethics committee of Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College.
10 ml of venous blood was withdrawn from all patients and controls under total aseptic technique in blood collection tubes containing K2EDTA (dipotassium ethylenediamine tetra-acetic acid) for serum and PBMCs separation. PBMCs were separated from peripheral blood by Ficoll Hypaque density gradient centrifugation and resuspended in RPMI 1640 medium (Gibco, USA).
The HaCaT cell line was obtained from the Peking University People’s hospital (Chinese Type Culture Collection, Beijing, China). The human keratinocyte cell line and PBMCs were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). In all experiments, cells were cultured under the atmosphere containing 5% CO2 at 37℃. Prior to further association studies, the growth medium was changed to serumfree medium. Cells in the control group were treated with DMEM alone. The HaCaT cells were pretreated with benvitimod (0.1µM, 1µM, 10µM, 100µM) for 1 h and subsequently treated with TNFα (10 ng/ml) and IFNγ (10 ng/ml) and incubated for 24 h at 37˚C. In another set of cultures, the cells were coincubated with 100nM StemRegenin1 (SR1, aryl hydrocarbon receptor antagonist, Solarbio, China) for 1 h at 37˚C in a humidifed 5% CO2 incubator.
Cell viability was assessed by the CCK-8 assay according to the manufacturer’s protocol. HaCaT cells and PBMCs were collected during the logarithmic growth phase and plated in triplicate in 96-well (2 × 104 cells/well, n = 4 per group). After 24 h of inoculation, the culture medium in benvitimod groups was replaced with DMEM containing different concentrations of benvitimod (0.1µM, 1µM, 10µM, 100µM, 1mM). A group of untreated control wells (cells treated with medium) and a group of blank control wells (without cells) were established at the same time. After another 24 h of incubation with benvitimod, 10µl of kit reagent was added to 100µl cell solution and incubated for a further 60 min at 37°C. The optical densities were measured at 450 nm using a quantitative automatic microplate reader. The cell viability rate (%) was calculated using the following formula: (OCexperimental group- ODblank control )/(ODcontrol group- ODblank control)×100%.
PBMCs (2×106 cells/well) were cultured in 6-well plates. The cells were then washed and treated with benvitimod of different concentrations (0.1µM, 1µM, 10µM, 100µM) for 24 hours. A group of untreated control (cells treated with serum-free medium) and a group treated with SR1 (aryl hydrocarbon receptor antagonist, 100nM) were established. The supernatants were harvested and cytokine levels (IL-1β、IL-6、TNFα、IL-4 and IL-22) were analyzed by commercial ELISA kit (eBioscience, Germany) according to the manufacturer’s instructions.
HaCaT cells (1×106 cells/well) were cultured in 6-well plates. After reaching a confluent state, the cells were washed and treated with benvitimod of different concentrations(1µM, 10µM, 100µM) in 2ml medium that contained tumor necrosis factor-α(TNFα) and interferon-γ (IFNγ) (each 10 ng/mL; R&D Systems, USA) for 24h. A group of untreated control well (cells cultured in serum-free medium), a group of treated with only TI stimulants (TNFα and IFNγ, each 10 ng/mL) and a group of treated with 100nM SR1 were established at the same time. The supernatants were harvested and IL-4, IL-10, IL-22 and TARC levels were analyzed by commercial ELISA kit (eBioscience, Germany) according to the manufacturer’s instructions.
HaCaT cells in 6-well plates were treated with TI (10ng/ml) and benvitimod of different concentrations (0.1µM, 1µM, 10µM) for 24 hours. A group of untreated control wells and a group of treated with 100nM SR1 were also established. Total RNA was isolated from the treated cells using RNA blood mini kit (Qiagen, Germany) according to the manufacturer’s instructions. The concentration and purity of RNA were measured at 260nm and 280nm using NanoDrop2000 Spectrophotometer (Thermo Scientific, USA). A260: A280 ratio greater than 1.8 and lower than 2.1 indicated highly pure RNA. The integrality of RNA was observed by agarose gel electrophoresis. RNA was then reverse transcribed with the Revert Aid First Strand cDNA Synthesis Kit (Reverse Transcription Kit, Tiangen, China). The reverse transcription mixture was incubated for 15 min at 42°C followed by 5 min at 95°C. The mRNA levels of AhR, CYP1A1, FLG, IVL and TSLP were tested by real-time quantitative polymerase chain reaction (RT-PCR). RT-PCR was performed using the SYBR Green Dye method which was carried out using cDNAs supplemented with SYBR Green supermix (Bio-rad, Hercules, CA, USA). The PCR amplification used gene-specific primers for β-actin (forward, 5’-TGGCACCCAGCACAATGAA-3’; reverse, 5’-CTAAGTCATAGTCCGCCTAGAAGCA-3’), AhR(forward, 5’-ATACCGAAGACCGAGCTGAAT-3’; reverse, 5’-CCAGCAGACACCTTAGACGACT-3’), CYP1A1(forward, 5’-GTCATCTGTGCCATTTGCTTTG-3’; reverse, 5’-CAACCACCTCCCCGAAATTATT-3’), FLG(forward, 5’- TGAAGCCTATGACACCACTGA-3’; reverse, 5’- TCCCCTACGCTTTCTTGTCCT-3’), FLG(forward, 5’- TGAAGCCTATGACACCACTGA-3’; reverse, 5’- TCCCCTACGCTTTCTTGTCCT-3’), IVL(forward, 5’- ACAAGGGAAGAGAGAGCCACTG-3’; reverse, 5’- TGTAGAGGGACAGAGTCAAGTTCA-3’), TARC(forward, 5’- ACTGCTCCAGGGATGCCATCGTTTT-3’; reverse, 5’- ACAAGGGGATGGGATCTCCCTCACTG-3’), TSLP(forward, 5’- CCCAGGCTATTCGGAAACTCAG-3’; reverse, 5’- CGCCACAATCCTTGTAATTGTG-3’). The PCR protocol consisted of a cycle at 95°C for 60 seconds followed by 40 cycles consisting of 15 s at 95°C, 15 s at 60°C and 15 s at 72°C. β-actin was used as an endogenous reference. The average Ct was calculated for the target genes and internal control (β-actin). ΔCt (Cttarget - Ctβ−actin) values were determined. The expression levels of target genes in AD patients was determined relative to controls as 2−ΔΔCt, where ΔΔCt = ΔCt (patient or control) – ΔCt (average for controls). The results were converted into relative expression in this article.
HaCaT cells (1×106 cells/well) were cultured in 6-well plates. After washing with PBS, they were fixed with acetone for 10 min. After 24 h of inoculation, the culture medium in benvitimod groups was replaced with DMEM containing different concentrations of benvitimod (1µM, 10µM, 20µM). A group of untreated control wells (cells treated with medium) was established at the same time. After washing in PBS with 0.5 mg/ml Tween 20 (PBST), the fixed HaCaT cells were treated with 100 mg/mL bovine serum albumin (BSA) in PBST for 1 hour. Samples were then incubated with anti-AhR rabbit IgG (1:100 dilution; Abcam, Cambridge, MA, USA) overnight at 4 ℃. Next, samples were washed with PBST for three times and incubated with Alexa Fluor 546-conjugated anti-rabbit secondary antibody(Beyotime Biotechnology, China) for 1 hour at room temperature. Samples were then washed with PBST and mounted with SlowFade Gold Antifade Mountant (Beyotime Biotechnology, China) after nuclear staining with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, China) for 10 minutes. All samples were analyzed using an fluorescence microscope (Keyence, Osaka, Japan).
HaCat cells (1×106 cells/well) were collected by centrifugation, washed twice with phosphate buffered saline, and suspended in extraction lysis buffer (Solarbio, China) containing protease inhibitors (Solarbio, China). The protein concentration was determined using a protein assay reagent (Solarbio, China) according to the manufacturer’s instructions. Equal amounts of nuclear extract (30µg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was incubated with blocking solution (5% bovine serum albumin), followed by overnight incubation at 4°C with the appropriate primary antibody. The following primary antibodies and dilutions were used: mouse anti human GAPDH antibody (1:1000 dilution; Beyotime Biotechnology, China), rabbit anti-human FLG antibody (1:1000 dilution; Abcam, Cambridge, MA, USA), rabbit anti-human IVL antibody (1:1000 dilution; Abcam, Cambridge, MA, USA), rabbit anti-human TSLP antibody (1:1000 dilution; Abcam, Cambridge, MA, USA), anti-signal transducer and activator of transcription (STAT) 1, and anti-phospho-STAT1 antibody (1:1000 dilution; Cell Signaling Technology, Danvers, USA). The membranes were washed three times with Tris-buffered saline (TBS, 20 mM Tris, 0.2 M NaCl, pH 7.5) containing 0.05% Tween 20 (TBST), and then incubated with a 1:2000 dilution of a horseradish peroxidase-conjugated secondary antibody (Beyotime Biotechnology, China) for 1 hour at room temperature. The membranes were again washed three times with TBST, and then developed using an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, USA). Image capture was performed using ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA). To investigate multiple protein targets under the same treatment condition, the blot was stripped and reused. Equal loading of samples was confirmed by GAPDH levels in the whole-cell lysates. The integrated optical density for the protein bands was then analyzed using ImageJ software (version 1.46r; NIH, USA), and the values were normalized to GAPDH. The relative absorbance ratios of target protein to GAPDH were defined as the respective relative values of target proteins.
All the data in this study were obtained from at least four independent experiments and are presented here as the mean ± SEM, with n = 5. A two-tailed unpaired Student’s t test was used to analyze data between two groups. One-way analysis of variance was used to detect significant differences between the control and treatment groups. Dunnett’s test was used for multiple comparisons. Chi-square, Mann-Whitney U tests and Kruskal-Wallis test were used for non-normally distributed variables. These analyses were performed using GraphPad Prism (GraphPad Software 5.0, USA). Differences between groups were considered significant at P < 0.05.
The effects of benvitimod on the proliferation of HaCaT cells and PBMCs were analyzed by CCK-8 assay (Fig. 2). Benvitimod inhibited cell proliferation of PBMCs and HaCaT cells in a dose-dependent manner. The IC50 of PBMCs and HaCaT cells were 6.21µM and 48.54µM, respectively.
The effects of benvitimod on cytokine production of PBMCs from AD patients were shown in Fig. 3. Stimulation with benvitimod significantly induced the release of IL-1β, IL-6 and TNFα and decreased the secretion of IL-4 and IL-22 in PBMCs. These effects were significantly inhibited by SR1 (aryl hydrocarbon receptor antagonist, 100nM).
The effects of benvitimod on chemokines and cytokines production of HaCaT cells were shown in Fig. 4. HaCaT cells treated with TI secreted significantly more TARC and IL-10. By contrast, benvitimod significantly suppressed the production of TARC and induce the release of IL-10. However, benvitimod had no significant effect on IL-1β and IL-6 production (data not shown).
The effects of benvitimod on mRNA expression of pro-inflammatory chemokines and epidermal barrier genes in HaCaT cells were shown in Fig. 5. Although no significant effect on the expression of AhR was found, CYP1A1 gene expression was significantly upregulated in HaCaT cells exposed to benvitimod for 24 hours. We also determined mRNA expression of FLG, IVL, TARC and TSLP by RT-PCR. We found that benvitimod induced upregulation of barrier protein genes including FLG and IVL. Benvitimod also inhibits TNFα/IFNγ-induced upregulation of cellular TARC and TSLP expression in HaCaT cells. The effect of benvitimod on mRNA expression of TSLP was significantly inhibited by SR1.
The AhR-activating capacity of benvitimod in HaCaT cells were shown in Fig. 6. Ligation of AhR was known to induce its cytoplasmic-to-nuclear translocation. AhR was mainly located in the cytoplasm in untreated HaCaT cells. However, nuclear-predominent staining of AhR was enhanced in HaCaT cells treated with benvitimod than control group in a dose-dependent manner (Fig. 6A). The number of HaCaT cells with nuclear-predominant staining of AhR was significantly increased by benvitimod (20µM) treatment (Fig. 6B).
The effects of benvitimod on FLG, IVL and TSLP protein production in TNFα/IFNγ-stimulated HaCaT cells were shown in Fig. 7. HaCaT cells were treated with various concentrations of benvitimod followed by stimulation with TI (10 ng/mL) for 24 hours. We found that benvitimod blocked the down-regulation of FLG and IVL in TI-stimulated HaCaT cells. TI-treated cells exhibited a significant increase in TSLP production and benvitimod significantly inhibited TI-stimulated TSLP production. We also found that the effects of benvitimod on IVL and TSLP were significantly inhibited by SR1.
The effects of benvitimod on STAT1 phosphorylation in TI-stimulated HaCaT cells were shown in Fig. 8. We found that the phosphorylation of STAT1 was significantly increased in TNFα/IFNγ-stimulated HaCaT cells. Furthermore, benvitimod significantly inhibited TI-induced STAT1 phosphorylation in HaCaT cells in a dose dependent manner.
AD is a common inflammatory skin disease characterized by pruritic and eczematous skin lesions. Skin barrier dysfunction and chronic inflammation were considered to be two important factors in pathogenesis of AD[17]. The treatment of AD is mainly focused on symptom relief, predominantly targeting dry skin, itch, and inflammation with emollients and corticosteroids[1]. Most of the current therapies for AD target the immune system (such as corticosteroids and calcineurin inhibitors), whereas emollients are used to improve skin barrier function.
Benvitimod is a non-steroidal topical agent that acts on the AhR signaling pathway. It has performed clinical efficacy for patients with AD (phase II) and psoriasis (phase Ⅲ)[18]. It has been proved to inhibit the production of pro-inflammatory cytokines and migration of T cells. It has also been demonstrated that benvitimod is a direct binding partner and agonist ligand of AhR. It has inherent antioxidant properties through nuclear factor-erythroid 2-related factor-2 pathway activation[19].
Lymphocytes play a central role in the pathogenesis of AD by producing high levels of cytokines and chemokines including IL-4, IL-5, IL-22 and TARC[20]. It has been reported that AhR activation can regulate differentiation and cytokine production in some T-cell subsets[21]. AhR agonist such as polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can also modulate production of various cytokines, including IL-1β, IL-6, IL-10, IL-17, IL-22, TNFα and CCL17 (TARC). Negishi et al[22]. reported that TCDD and other AhR ligands can induce secretion of IL-6, IL-1β and TNFα in mammary MCF-7 cells and dendritic cells. The mechanism seems to involve a suppression of the promoter, leading to a synergistic upregulation in the presence of inflammatory signals[23]. In our study, we found that benvitimod inhibited the proliferation of PBMCs and HaCaT cells. Benvitimod also promoted the production of IL-1β, IL-6, TNFα and inhibited IL-4 and IL-22 production in PBMCs from AD patients. However, the exact mechanism by which AhR ligands induce cytokine expression remains to be determined.
AhR can also act as an anti-inflammatory factor. Ito et al[24]. reported that AhR ligands exposure such as TCDD and BaP (benzoapyrene) also significantly suppresses the production of the Th2 cell-derived cytokines IL-4 and IL-5 in murine T splenocytes. Increased Th2 cytokines (such as IL-4 and IL-5), Th22 cytokines(such as IL-22) and TARC have been detected in skin lesions and serum of patients with AD. These cytokines and chemokines can recruit inflammatory cells, facilitating their infiltration of inflammatory skin lesions[25]. In our study, benvitimod was proven to inhibit IL4 and IL22 secretion in PBMCs and TARC in HaCaT cells. These results might relate to its therapeutical effects.
AhR is an essential transcription factor for human epidermal barrier proteins including FLG and IVL. Several of these genes, including FLG and IVL, play important roles in barrier function and they can be modulated by AhR agonists such as coal tar and soybean tar[26]. Tsuji et al[27]. reported that AhR activation could upregulate FLG expression via Ovo-like 1(OVOL1) in IL-4-treated normal human epidermal keratinocytes as well as in vitro AD skin model. Our study demonstrated that benvitimod induced the upregulation of FLG, IVL and CYP1A1 expression. These results indicated that benvitimod is potentially beneficial for the treatment of barrier-disrupted skin conditions via its capacity to upregulate barrier proteins.
Chemokines and their receptors play an important role in the pathogenesis of AD. TARC, which induces chemotaxis in lymphocytes by interacting with the chemokine receptor, is responsible for the initiation of AD. TSLP is an epithelial derived cytokine which is known to have wide-ranging impacts on progression of allergic diseases such as AD and asthma[28]. Keratinocytes stimulated with pro-inflammatory cytokines have been identified as important cellular sources of cytokines and chemokines, including TSLP and TARC, which affect the recruitment and differentiation of leukocytes to atopic lesions[29]. In this study, we found that TNFα/IFNγ induced TARC and TSLP over-expresssion was inhibited by benvitimod. These results suggested that suppression of inflammatory responses by benvitimod may contribute to its therapeutic effects.
STAT1 is an essential component of interferon (IFN)-signaling, which mediates several cellular functions in response to stimulation by cytokines, growth factors, and hormones. The binding of IFN-γ to the receptor induces the phosphorylation of Janus tyrosine kinase 1 (JAK1) and JAK2, which phosphorylate a specific tyrosine residue in the STAT protein. These events ultimately stimulate the production of inflammatory chemokines[30]. Therefore, STAT1 is considered as a therapeutic target for the development of antiinflammatory drugs. In this study, we found that phosphorylated STAT1 (p-STAT1) was induced by TNFα/IFNγ stimulation and benvitimod suppressed the expression of pSTAT1. We also found that SR1, an AhR agonist, significantly blocked benvitimod-induced STAT1 phosphorylation, although the underlying cellular and molecular mechanisms still remain to be elucidated.
In conclusion, our study demonstrated that benvitimod regulates cell proliferation and TNF-α/IFN-γ-stimulated cytokine production in the HaCaT cells and PBMCs of AD patients. Benvitimod can also activate AhR to translocate into the nucleus of keratinocytes, where ARNT binds to it, regulating the transcription of downstream genes such as inflammatory cytokines, epidermal barrier protein and STAT1 phosphorylation in TNF-α/IFN-γ-treated cells (Fig. 9). Our data suggested that benvitimod is an attractive therapeutic drug candidate for treating skin dermatitis.
Acknowledgements
Not applicable.
Funding
The present study was supported by “National Natural Science Foundation of China（82103711）” .
Ethics approval
The study was approved by the ethics committee of Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College.
Conflict of interest
The authors have no conflict of interest to declare.
Consent for publication
All authors reviewed and approved of the final manuscript for publication.