Pseudolaric acid B attenuates atopic dermatitis-like skin lesions by inhibiting interleukin-17-induced inflammation

Pseudolaric acid B (PB), isolated from the extract of the root bark of Pseudolarix kaempferi Gordon, has been used as a traditional remedy for the treatment of skin diseases. However, the information of PB on atopic dermatitis (AD) remains largely unknown. In the present study, oral administration with PB improved the severity scores of AD-like skin lesions dose-dependently in NC/Nga mice through reducing serum IgE, pro-inflammatory cytokines, and the infiltration of inflammatory cells. In addition, PB significantly attenuated the levels of IL-17 and IL-22, and the proportion of Th17 cells in NC/Nga mice, as well as decreased IL-17-induced inflammation in RAW264.7 cells. Moreover, PB inhibited the phosphorylation of IκBα and miR-155 expression both in NC/Nga mice and in IL-17-stimulated RAW264.7 cells, which could be reversed by GW9662, a specific antagonist for PPARγ. The incorporation of GW9662 reversed the inhibitory effect of PB on the RORγ-mediated activation of the Il17 promoter. Transfection with PPARγ luciferase reporter gene further demonstrated the enhancement of PB on PPARγ transactivation. These findings indicate that PB could ameliorate AD-like skin lesions by inhibiting IL-17-induced inflammation in a PPARγ-dependent manner, which would provide experimental evidence of PB for the therapeutic potential on AD and other inflammatory skin diseases.


PB contributes to the decreased number of Th17 cells. Several reports have demonstrated a marked
increase in the IL-17 + CD4 + T-cell population (Th17) derived from AD patients compared with healthy controls, and the highest percentage of IL-17-producing CD4 + T cells was found in severe AD, suggesting Th17 is the major cellular source of IL-17 in AD 14,15 . Therefore, we examined the contribution of PB to the proportion of Th17 cells in the draining lymph nodes of NC/Nga mice. Consistent with previous findings on IL-17, vehicle group had significantly increased amounts of Th17 cells in AD mice, while treatment with PB decreased the percentage of Th17 cells dose-dependently (Fig. 5).

PB attenuates IL-17-induced inflammatory response.
Macrophages are the source of many cytokines that play fundamental roles in pathogenesis of AD. Activated macrophages have a high capacity to produce inflammatory cytokines such as IL-1β and TNF-α 16 . Previously, we have proved that PB could reduce the mRNA expression of IL-1β and TNF-α in RAW 267.4 murine macrophage cells treated with LPS 17 . However, the action of PB on IL-17-induced inflammatory response remains to be demonstrated. Therefore, RAW264.7 cells were firstly incubated with increasing concentrations of PB for 24 h and subjected to the CCK-8 assay to assure whether PB would affect cell viability. As shown in Fig. 6A, PB decreased cell viability to 80% at a concentration of 0.5 μmol/l, higher concentrations of 1.0, 2.0 and 4.0 μmol/l decreased cell viability from 70% to 45%. Therefore, a non-cytotoxic concentration of PB (0.5 μmol/l) was chosen for further experiments. The results showed that PB at 0.5 μmol/l significantly reduced the expression of IL-17RA, IL-1β and TNF-α in RAW 264.7 cells stimulated with IL-17 (Fig. 6B~D).

PB up-regulates Peroxisome proliferator-activated receptor γ (PPARγ) expression and activation.
Considering that PPARγ is a negative regulator of IL-17 and could improve the clinical symptoms of AD 18 , we investigated the role of PPARγ using NC/Nga mice and IL-17-stimulated RAW264.7 cells to the best of our knowledge on PB. Figure 7A indicated that PB could indeed promote PPARγ expression in AD mice. Blocking PPARγ with GW9662, a specific antagonist for PPARγ, remarkably abolished the anti-inflammatory effects of PB in IL-17-induced RAW 264.7 cells (Fig. 6B~D). Moreover, PB could obviously enhance the transcriptional activity of PPARγ, which was also reversed by GW9662 completely (Fig. 7B).
PB regulates RORγ-mediated Il17 promoter activation. Retinoid-related orphan receptor gamma t (RORγt) has been reported to directly regulate the transcription of IL-17 and play a critical role in the differentiation of Th17 19 . Further, Th17 cells are controlled by their master transcriptional factor RORγt, which has been implicated as culprits during the immune response and inflammation of AD 20 . On the basis of the above data, PB could reduce IL-17 level and Th17 cells proportion remarkably. To further examine whether the effect of PB on IL-17 would be due to its regulation on transcriptional activation, we transfected Jurkat cells with RORγ expression plasmid under the control of the Il17 promoter. The results showed that blocking PPARγ using GW9662 also reversed the inhibitory effect of PB on RORγ-mediated activation of Il17 promoter, indicating that the inhibition of PB on Il17 promoter activity might be PPARγ-dependent (Fig. 8A). Moreover, PB administration could also Data are representative of two independent experiments and presented as mean ± SD of n = 8 mice per group. *p < 0.05, **p < 0.01. Vehicle, intact mice with saline treatment; AD, DNFB-sensitized and challenged mice; PB, pseudolaric acid B; PD, prednisolone. lead to a significant decrease for the expression of RORγt mRNA in the draining lymph nodes of NC/Nga mice (Fig. 8B).

PB represses miR-155 expression.
Based on the established anti-AD effects of PB and the important role of miR-155 during the development of AD through enhancing inflammatory response 21 , we thus proposed that PB might produce its beneficial action via affecting miR-155 expression. Figure 9A showed the expression of miR-155 was enhanced in the lesional skin from AD mice, which could be repressed by PB dose-dependently. In addition, miR-155 has a seed region at the 3′UTR of PPARγ gene, leading to regulate the PPRE signaling of PPARγ 22 . Our data confirmed that PB markedly decreased miR-155 expression in IL-17-sitmulated RAW 264.7 cells, as it was reversed by GW9662 (Fig. 9B).
PB suppresses the NF-kB pathway. NF-kB is a transcription factor that modulates the expression of many genes associated with inflammatory processes 23 . Li et al. reported that PB significantly inhibited the nuclear translocation of NF-kB p65 and the phosphorylation of IkBα in active T lymphocytes 24 . To further understand the roles of PB on NF-κB signaling during the development of AD, we measured the phosphorylation of IκBα and found that PB could markedly inhibit the activation of phospho-IκBα both in AD mice and in IL-17-stimulated RAW264.7 cells. The incorporation of GW9662 also reversed the inhibitory effect of PB on phospho-IκBα (Fig. 10).

Discussion
AD is one of the most common chronic or chronically relapsing inflammatory skin diseases with a high prevalence, which could be considered to consist of acute and chronic phases. In the acute phase, infiltrated helper T cells (Th) and the levels of associated cytokines are increased, accompanied with IgE hyper-production by releasing histamine and several pro-inflammatory cytokines that cause pruritus in inflammatory skin lesions 25 . During the late-phase reactions, mast cells favor the recruitment of eosinophils and neutrophils. The complex and unexplored etiology of AD has made the development of therapeutics difficult. For the past few decades, anti-histamine agents, immunosuppressive agents, moisture care therapy, corticosteriods, or localized immunoregulatory agents have been used for treating AD 26 , and the whole body corticosteriods treatment is often chosen for severe cases of AD. However, long-term use of oral and topical corticosteriods is associated with a large number of side effects and drug intolerance 27 . Though new topical calcineurin inhibitors have been used for the treatment of AD recently, their efficacy was not sufficient to treat AD successfully 27 . Therefore, further evaluation is warranted to heighten the potential for developing safer and more efficacious therapies against AD. The application of herbal medicines has a long history due to their use in a folk medicine, and most well-known medications are derived from plants 28 , some of which have been reported as alternative therapeutics for anti-AD treatment 29 . Recent studies have reported that PB, isolated from herbal medicine pseudolarix, has a negative effect on T lymphocytes through inhibiting inflammation and pro-inflammatory factors, leading to a suppression of differentiation, proliferation, and inflammatory responses of T cells 6,17,30,31 , which indicated PB might be regarded as an anti-inflammatory agent for the treatment of AD.
For objective assessment, we attempted to evaluate the therapeutic effects of PB using a multidisciplinary approach. Firstly, visual analysis was performed macroscopically using the clinical skin severity score for AD 32 . PB significantly resulted in the elimination of AD-like clinical symptoms in NC/Nga mice, including skin lesions and mast cell infiltration beneath hypodermis. Then, we focused on the effect of PB on IgE alteration, and found that PB treatment significantly inhibited serum IgE production. Moreover, PB markedly increased body weight compared to PD-treated mice and AD control, suggesting that oral application of PB was safe and effective.
It's well known that inflammatory cell skin infiltrate is common in all AD lesions. The most frequent cell type make up the Th cells. A number of abnormalities play critical roles in the pathogenesis of AD including Th2-skewed cytokine expression in acute AD 33 . Afterwards, environmental triggers could provoke a mixed immune response of Th1 in the progression of AD and the resulting cytokine signaling elicited subsequent inflammation unrelated to the early Th2 response, suggesting Th1 cytokines such as TNF-α and IL-1β play a more dominant role than Th2 cytokines in the chronic status of AD 21 . Therefore, we focused on the effects of PB on TNF-α and IL-1β.The results showed that PB significantly reduced the levels of IL-1β and TNF-α in lesional skin tissues and serum of NC/Nga mice.
Th17 cells are characterized by the production of IL-17, which have been implicated in the pathogenesis of AD because IL-17 is crucial for the up-regulation of numerous inflammation-related genes in keratinocytes, fibroblasts, leading to the increased production of chemokines, antimicrobial peptides, and cytokines like TNF-α and IL-1β. These IL-17 activities could promote tissue fibrosis, chronicity of the inflammatory process, and the evolution of chronic inflammatory cutaneous lesions that contribute to the development of AD 11,34 . Besides, Th22 cells represent a distinct T cell subset involved in epidermal immunity and remodeling, which also play very important roles during the pathogenesis of AD 35,36 . The discovery of Th17 and Th22 cells has opened up a new avenue for research into the etiology and treatment of AD. In this study, PB obviously suppressed serum IL-17/IL-22 levels and the expression of IL-17/IL-22 related genes in the lesional skin tissues. However, PB might counteract inflammatory response much more through suppressing IL-17 than IL-22. Based on the above analysis, we thus investigated the mechanism of PB on IL-17. Our results showed that PB treatment significantly reduced IL-17-induced inflammatory response in RAW264.7 cells, indicating the therapeutic effects of PB on AD might be mediated by IL-17 partly. The ability of PB to inhibit IL-17 production and Th17 differentiation was further assessed in NC/ Nga mice. These findings suggested that PB might act as an antagonist of IL-17 to attenuate AD-like skin lesions.
Peroxisome proliferators activated receptors (PPARs) belong to the nuclear receptor superfamily, which appear to be essential for maintaining skin barrier permeability, inhibit keratinocyte cell growth, promote keratinocyte terminal differentiation and regulate skin inflammation 18 . Four different subtypes have been identified: PPARα, PPARβ/δ and PPARγ. Recently, PPARγ and its agonists were found to have anti-inflammatory activity and immunoregulatory function in patients and animal models with both irritant and allergic skin diseases 37 . The activation of PPARγ could cause the repression of pro-inflammatory gene expression, and ameliorate AD-like skin lesion in NC/Nga murine model 38 . Moreover, PPARγ could selectively suppress Th17 differentiation via the inhibition of RORγt expression, confirming it as a negative regulator of RORγt 39 . Our previous studies have found that PB could promote PPARγ expression and affect the balance of Th17/Treg cells in a murine model of allergic contact dermatitis 6 . In the present study, we further demonstrated that PB could suppress RORγ-mediated Il17 promoter activation in Jurkat cells, and inhibit RORγt expression in NC/Nga mice by enhancing PPARγ activation, which results suggested the amelioration of PB on AD-like skin lesions would be involved in its mechanism to suppress IL-17 through increasing PPARγ transactivation and activation.
MicroRNAs (miRNAs) are a novel class of short (21-25 nucleotides), non-coding, evolutionary conserved RNAs that regulate post-transcriptional gene expression through incomplete base pairing with the 3′-untranslated region (3′-UTR) of target mRNAs to degrade the target mRNAs or inhibit translation 40 . Numerous evidences have indicated that miRNAs are critically involved in regulating immune system, among which miR-155 plays an important role in inflammatory and immune reactions. Moreover, miR-155 has been reported to be over-expressed in the patients with AD through modulating the differentiation and function of Th17 21 . For further insight into the role of PB on the relationship between miR-155 and IL-17 during the pathogenesis of AD, we analyzed the expression of miR-155 in AD-like skin lesions of NC/Nga mice, and proved that PB exerted a significant inhibitory effect on miR-155 expression. Then, we further verified the inhibition of PB on miR-155 in IL-17-stimulated RAW264.7 cells. The results would provide a more detailed insight into the mechanism of PB in alleviating inflammation of AD.
NF-κB is a master switch of inflammatory gene expression and regulates the levels of IL-17, IL-1β, TNF-α and so on. Uncontrolled activation of NF-κB is associated with various human diseases including inflammatory skin   diseases. In unstimulated cells, NF-κB is sequestered in the cytoplasm through interaction with inhibitory κB (IκB) proteins. In response to inflammatory stimuli, a cascade of phosphorylation events increases the activity of the IκB kinase (IKK) complex; IKK acts to phosphorylate IκBα, which results in its degradation by the proteasome and, consequently, its dissociation from the NF-κB dimers. The release of IκBα promotes the translocation of NF-κB subunits to the nucleus, where it initiates transcription of pro-inflammatory genes 41 . Recent studied reported that miR-155 and IL-17 could aggravate the pathological process of AD by activating NF-κB signaling pathway 21,42 . We and several pieces of literatures have correlated the anti-inflammatory effects of PB with the down-regulation of NF-κB pathway 6, 24, 30, 43 . In the present study, PB was confirmed to ameliorate AD-like skin lesions through a PPARγ-dependent NF-κB pathway.  (1.0 μmol/l) for 1 h before added with PB (0.5 μmol/l). After 21 h, cultured cells were stimulated with IL-17 (10 ng/ml) for 3 h. Cells were harvested and total RNA was used to quantify the level of miR-155 mRNA by qRT-PCR. Data are presented as mean ± SD. Each measurement was performed in duplicate and all experiments were repeated three times. *P < 0.05, **P < 0.01.
In conclusion, we have demonstrated the therapeutic efficacy of PB in an AD-like murine model, which mechanisms might be related to inhibit IL-17-induced inflammation, block NF-κB pathway and diminish miR-155 expression in a PPARγ-dependent manner (Fig. 11). Thus, the successful treatment of AD skin lesions is likely to require the use of drugs like PB with broad targets rather than the specific inhibition of single molecule. However, future studies are needed to define more precisely the mechanism of PB responsible for AD. Our results raise the possibility that PB could be a potential therapeutic candidate of herbal origin for AD and other inflammatory skin diseases.
Animals and experimental design. Specific pathogen-free male 6 weeks old NC/Nga mice were obtained from Riken BioResource center, Japan. Mice were maintained on a dark/light cycle (12/12 h) in air-conditioned rooms (22.5 ± 0.5 °C, 50 ± 5% humidity) and were adapted to local conditions for one week before the beginning of the study. All animal experiments were conducted according to national and international laws and policies (Guide for the Care and Use of Laboratory Animals) and were approved by the Institutional Animal Care and Use Committee of Logistics University of the Chinese People's Armed Police Force.
As shown in Fig. 1A, DNFB sensitization were evoked by the topical application of 100 μl of 0.15% DNFB dissolved in acetone/olive oil (3:1, v/v) to the shaved dorsal skin of NC/Nga mice once on days 1, 4 and 7. On days 10 and 13, sensitized mice were challenged with 100 μl of 0.2% DNFB to the dorsal skin surfaces. A total of 48 NC/Nga mice were randomly divided into six groups with 8 mice in each: vehicle group (intact mice with saline treatment containing 0.5% DMSO), AD model group (DNFB-sensitized and challenged mice), three PB group Evaluation of dermatitis severity. The severity of AD-like symptoms was evaluated by the SCORAD method 44 , and features such as development of edema, erythema, oozing, crust, excoriation, and lichenification were scored as 0 (none), 1 (mild), 2 (moderate) or 3 (severe). Overall dermatitis score (minimum 0, maximum 12) was determined from the sum of all individual scores. Assessment was performed by an investigator who was blinded to the treatment groups.
Histopathological analysis. By the end of the study period, mice were sacrificed and their lesional skin tissues were harvested and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4). Skin sections (3-5 μm) were stained with hematoxylin and eosin (H&E) for histopathological analysis and with toluidine blue for analysis of mast cells 45 . All images were captured with an Olympus BX41 microscope equipped with a video camera and analyzed using Image-Pro-Plus software (version 6.0, Media Cybernetics, MD, USA). A morphological evaluation of all the skin sections was carried out in a blinded fashion. Numbers of inflammatory cells and mast cells were expressed as average total counts in four fields of 100 μm 2 .
ELISA. The serum of mice was collected and stored at −80 °C until use. Moreover, the lesional skin tissues of mice were separated and homogenized with ice-cold lysis buffer containing protein inhibitor. The protein concentration of the supernatant was measured using the BCA protein assay kit according to the manufacturer's instructions. The levels of IgE, IL-17, IL-22, IL-1β and TNF-α were measured using ELISA kits according to the manufacturer's protocol. The absorbance was measured at 450 nm using a microplate reader. A concentration below the lowest detectable value of the ELISA standard curve was assigned the mean value of the lowest concentration of the ELISA standard curve and zero.
Quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA from lesional skin tissues was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. The purity of RNA samples was was assessed by inspecting the 28 S and 18 S bands after 1.5% agarose gel electrophoresis; a 260/280 absorbance ratio was between 1.9 and 2.0. Total RNA was transcribed into cDNA using a reverse transcription system (Promega, Madison, WI, USA), and the primers were purchased from Shanghai Sangon Biological Engineering Technology Company (Table 1). Amplification conditions were set to heat activation at 95 °C for 10 min, and followed by 40 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 1 min. All melting curve analysis was performed between 50 °C and 95 °C. The qRT-PCR reactions were performed using the PerfeCTa SYBR Green SuperMix (Quanta BioSciences) on an ABI PRISM 7300 sequence detection system (Applied Biosystems, USA). The expression of target genes was normalized to β-actin, and the fold change of each sample was calculated using the comparative CT method (2 −△Ct ). All reactions were run in triplicates, and all experiments were performed three times.
For microRNA-155 (miR-155) detection, RNA was isolated using the QIAGEN miRNeasy mini kit according to the manufacturer's instructions (QIAGEN Inc., Valencia, CA, USA). Reverse transcription reaction was performed with specific miRNA primers ( Table 1). All reactions were performed using TaqMan ® MicroRNA Reverse Fluorescence-activated cell sorting (FACS) analysis. Cells from the draining lymph nodes of mice were prepared as described previously 6 . Red blood cells were lysed with erythrocyte lysis buffer (0.15 M NH 4 Cl, 10 mM NaHCO 3 and 0.1 mM EDTA; pH 7.3). The cell phenotype of Th17 cells was defined using the appropriate antibodies according to the manufacturer's protocol. Briefly, the cells (2 × 10 5 cells) were stimulated with immobilized anti-CD3ε mAb (2 μg/ml) for 6 h and incubated with 10 μg/ml Brefeldin A, 50 ng/ml PMA, and 1 mmol/l ionomycin. The stimulated cells were then stained with FITC-conjugated anti-CD4 antibody, fixed with 4% paraformaldehyde, and made permeable with permeabilization wash buffer. Finally, the cells were stained with PE-Cyanine7-conjugated anti-IL-17A mAb. Isotype controls were used to enable correct compensation and to confirm antibody specificity. The data were acquired on a FACScan flow cytometer (CytomicsTM FC 500, Beckman Coulter, USA) and analyzed using the FlowJo7 software (Treestar, Ashland, OR, USA).
Cell culture and cytotoxicity analysis. RAW 267.4 cells were obtained from the American Type Culture Collection (ATCC, TIB-71), and maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin and then plated into 12-well plates. PB was dissolved in DMSO and the final DMSO concentration was <0.1% in all experiments in vitro. To examine whether PB could affect cell viability, RAW264.7 cells were incubated with increasing concentrations of PB (0, 0.125, 0.25, 0,5, 1.0, 2.0, 4.0 μmol/l) for 24 h. Cytotoxic effect of PB was estimated by using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) following the manufacturer's instructions.

Measurement of pro-inflammatory cytokines expression.
For pro-inflammatory cytokine assay, RAW264.7 cells were incubated with PB (0.5 μmol/l) for 21 h and stimulated with 10 ng/ml of recombinant murine IL-17 (PeproTech Inc., Rocky Hill, NJ) for 3 h. Then, total RNA was extracted from RAW264.7 cells to determine the expression of IL-1β and TNF-α mRNA. For experiments involving PPARγ, RAW267.4 cells were pre-incubated with GW9662 (1.0 μmol/l) for 1 h before the addition of PB. Transient transfection and reporter gene assay. PPARγ transactivation assay was done to explore the mechanism of PB based on the previously protocol with some modifications 30 . PPARγ-induced transcriptional activity was evaluated by transient transfection of a TK-PPRE3x luciferase reporter plasmid driven by the PPRE-containing acyl-CoA oxidase promoter (kindly provided by Dr. R.M. Evans, The Salk Institute, Howard Hughes Medical Institute, CA). RAW264.7 cells were seeded in 6-well culture plates (2 × 10 6 cells/well). 24 h after seeding, cells were transiently transfected with 0.5 μg of TK-PPRE3x and 0.05 μg of pRL-TK using X-tremeGene ™ HP (Roche, Mannheim, Germany) and OPTI-MEN (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. Six h later, the transfection medium was replaced with DMEM medium containing no PB, or PB (0.5μmol/l), or GW9662 (1.0 μmol/l) for 1 h before PB incubation. Cells were then cultured for 21 h, and were stimulated with IL-17 (10 ng/ml) for 3 h. Finally, the cells were lysed and the luciferase activities were determined with a luminometer according the instruction of Dual Luciferase II reporter assay kit. The values of luciferase activity were normalised with the pRL-TK values to correct the differences caused by unequal transfection efficiency. All values were normalized to control wells to calculate relative luciferase activity.  Table 1. Primer sequences in this study.