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International Immunopharmacology 25 (2015) 235–241

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International Immunopharmacology

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Curcumin relieves TPA-induced Th1 inflammation in K14-VEGFtransgenic mice

Jun Sun a,1, Yi Zhao b,1, Hairong Jin c,1, Jinhong Hu c,⁎a Department of Pharmacy, General Hospital of Jinan Military Command, Jinan 250031, Chinab Department of Pharmacy, General Hospital of Lanzhou Military Command, Lanzhou 730050, Chinac Department of Pharmacy, Changhai Hospital, The Second Military Medical University, Shanghai 200433, China

⁎ Corresponding author. Tel./fax: +86 2131162302.E-mail address: 434772224@qq.com (J. Hu).

1 All of the three authors contributed equally to this stu

http://dx.doi.org/10.1016/j.intimp.2015.02.0071567-5769/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

Article history:Received 24 August 2014Received in revised form 25 January 2015Accepted 4 February 2015Available online 12 February 2015

Keywords:CurcuminInflammationTPATransgenicMouse

Curcumin has been confirmed to have anti-inflammatory properties in addition to the ability to decrease the ex-pression of pro-inflammatory cytokines in keratinocytes. It was suggested that the interleukin-23 (IL-23)/IL-17Acytokine axis played a critical role in the pathogenesis of 12-O-tetradecanoyl phorbol 12-myristate 13-acetate(TPA)-induced K14-VEGF transgenic psoriasis-like mice model. Here, we report that topical use of a curcumingel formulation inhibited TPA-induced Th1 inflammation in K14-VEGF transgenicmice ears but not Th17 inflam-mation as expected. Real-time PCR showed that mRNA levels of IL-23, IL-17A, IL-22, IL-6 and TNFα cytokinesfailed to increase after TPA-induction inK14-VEGF transgenicmice ear skin; but themRNA level of IFNγ increasedsignificantly at the same time. Furthermore, TPA-induction up-regulated the TCRγδ protein but failed to impactthe CCR6 protein, whichmeans that the proliferation of γδ T cells is incapable of IL-17A production. We find thatcurcumin is capable of relieving TPA-induced inflammation by directly down-regulating IFNγ production. Inconclusion, curcumin inhibits TPA-induced Th1 inflammation in K14-VEGF transgenic mice which has notbeen previously described.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Psoriasis is a sort of immune-mediated chronic inflammatory skindisease characterized by hyperproliferative keratinocytes and the infil-tration of leukocytes [1]. The most specific histopathological changesthat distinguish psoriasis from other inflammatory dermatogic diseasesare the dramatic hyperplasia of the epidermiswith a loss of the granularlayer, regular elongation of the rete ridges, thickening of the cornifiedlayer and incomplete keratinocyte (KC) differentiation, infiltration ofmany different leukocytes and increased vascularity in the dermis [2].Although the pathogenesis of psoriasis is not very clear, there is growingevidence to indicate that the dermal γδ T cells play a critical role in dis-ease development [3–6]. Many immune-derived cytokines, includinginterleukin-23 (IL-23), IL-17A, IL-20, IL-22, IL-1β, IL-6, and TNFα, areinvolved and interact as a network in the pathogenesis of psoriasis[3–6].

A number of animal models have been developed that possess someof the characteristic features of human psoriasis. Delivery of vascularendothelial growth factor (VEGF) to the mice's skin results inover-expression of VEGF in epidermis, and thus produces a phenotype

dy.

with more or less hallmarks of human psoriasis, such as hyper-plasticdermal blood vessels, epidermal thickening with aberrant keratinocytedifferentiation, and leukocyte infiltration [7]. A mouse model withover-expression of VEGF in the epidermal basal layer of skin has beendeveloped using a transgenic technique — the expression of murineVEGF-A164 has been combined and controlled by the human keratin14 (K14) promoter [7]. The transgenic K14-VEGF mice can developinflammatory skin conditions similar to human psoriasis after induc-tion with 12-O-tetradecanoyl phorbol 12-myristate 13-acetate(TPA), characterized by increased dermal micro-vascular densityand epidermal hyperplasia with aberrant keratinocyte differentia-tion [8]. This model is convenient for the study of anti-psoriasistherapies.

As a multi-target molecule, curcumin exhibits various pharmacologi-cal effects, such as anti-oxidant, anti-inflammatory, anti-microbial andanti-carcinogenic activities [9], that are mediated by its inhibitory effectson NF-κB, MAPK, and cytokines [10–12]. For example, curcumin down-regulates the production of many proinflammatory cytokines (such asTNFα, IL-1, IL-2, IL-6, IL-8, and IL-12) by inactivating NF-κB [13,14].Therefore, we believe that curcumin is potentially valuable for the treat-ment of psoriasis [15].

This studywasdesigned to investigate the effect of a curcumin trans-dermal gel on a TPA-induced psoriasis-like model in transgenicK14-VEGFmice. In doing so, we expected to obtain supporting evidencefor a role of curcumin in treating psoriasis.

Table 1The treatments performed in experiment.

TimeGroup

0 days 1 day 2 days 12 days

Control Acetone + HPC gel HPC gel Acetone + HPC gel HPC gelTPA TPA + HPC gel HPC gel TPA + HPC gel HPC gelTPA + CUR TPA + CUR CUR TPA + CUR CURTPA + CLO TPA + CLO CLO TPA + CLO CLOCUR Acetone + CUR CUR Acetone + CUR CUR

TPA or acetone treatment was performed every two days and other treatments wereperformed twice daily. CUR, curcumin; CLO, clobetasol.

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2. Materials and methods

2.1. Chemicals

The chemicals used in this study include curcumin (Sigma, St. Louis,MO, USA), azone (Shanghai Health-well Chemical, China), TPA (Sigma,St. Louis, MO, USA), hydroxypropylcellulose (HPC)-MF (HerculesIncorporated-Aqualon Division, USA), 0.02% clobetasol propionatecream (Shanghai General Pharmaceutical Co., LTD., China), anti-RORγ an-tibody (Abcam, Cambridge, UK), anti-CCR6 antibody (Abcam, Cambridge,UK), anti-T cell receptor (TCR) γδ antibody (Abcam, Cambridge, UK),anti-TCR γδ antibody conjugated to biotin (Abcam, Cambridge, UK),streptavidin-peroxidase polymer (Sigma, St. Louis, MO, USA), goat

Fig. 1. Curcumin inhibited TPA-induced psoriasis-like inflammation. A, The thickness (Mean ±ANOVA followed by Student–Newman–Keuls test, N = 6). B, H&E staining of the mouse ear skskin. CUR, curcumin; CLO, clobetasol.

anti-rabbit IgG conjugated to biotin (Santa Cruz Biotechnology, USA),and the REAL™ EnVision™ detection system using peroxidase/DAB+and rabbit/mouse antibodies (Dako, Denmark). Mouse IL-17A/F, IL-22,IL-27 p28/IL-30 and IFNγ ELISA kits (R&D Systems, Minneapolis, USA),TRIzol reagent (Invitrogen, Carlsbad, USA), and PrimeScript RT reagentkit and SYBR® premix Ex Taq™ II (Takara, Dalian, China) were alsoused. All other chemicals used were of analytical grade.

2.2. Preparation of curcumin transdermal gel

The 1% curcumin gel formulation used in this study was preparedfrom a formulation used in a previous study [15].

2.3. Transgenic animals

The K14-VEGF transgenic mice used in this studywere replicated byCyagen Biosciences Inc. according to methods described by MichaelDetmar [16]. Homozygous K14-VEGF mice were used in this study.

2.4. Mice and treatment

Mice were housed under specific pathogen-free conditions and pro-vided with food and water ad libitum. Animals were between 8 and15weeks oldwhen experimentswere started. Based on a previous report

SD) of the right ear skin was measured on the days indicated (*P b 0.05, vs. TPA group,in of different treatment groups (200×, N = 6). Arrowheads denote the inside of the ear

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[8], inductionwith TPA started at day 0with the application of 20 μL 0.01%TPA suspended in acetone to the inside of the right ear. TPA treatmentwas repeated on days 2, 4, 6 and 8. For therapy, the TPA-inductionmouse model was treated twice daily with topical 50 mg/cm2 curcuminHPC gel or 40 mg/cm2 clobetasol propionate cream. Clobetasol was usedas a positive control drug in this study. Mice smearedwith acetone insidethe right ear and treated similarly with HPC gel (without curcumin andazone) were used as the control group; and treated with curcumin gelas a baseline (Table 1). All protocols were approved by the experimentalanimal ethics committee of the Second Military Medical University andmet the national guidelines for the care and use of experimental animals.The experimental animal center of the SecondMilitaryMedical Universitywas certified by the “International Association for Assessment and Ac-creditation of Laboratory Animal Care International”.

2.5. Measurement of ear thickness

The ear thickness was measured using a thickness gauge (GuangluDigital Caliper Manufacturer Co., Ltd., China) at 0, 2, 4, 6, 8, 10, 12 days.

Fig. 2. The levels ofmRNAmeasured using real-time PCR (Part 1). The figures show themean va4 d, 8 d, and 12 days (*P b 0.05,Wilcoxon scores test, N= 6). A, IL-23. The changes in IL-23mRNbetween groupswerenot statistically significant. C, IL-22. The changes in IL-22mRNAbetween ggroup was increased significantly and was inhibited in the curcumin-/clobetasol-treated grouTNFα. The changes in TNFα mRNA between groups were not statistically significant. CUR, curc

The increase in ear thicknesswas used to indicate the extent of inflamma-tion. Experiments were conducted in triplicate, and the data were aver-aged and used to evaluate epidermal proliferation and inflammation.

2.6. Histology, immunohistochemistry and real-time quantitative PCR

At experimental day 12, ear samples from eachmouse in each groupwere fixed in 4% paraformaldehyde and embedded in paraffin. Sections(thickness = 4 μm) were stained using hematoxylin and eosin (H&E).Immunohistochemistry and real-time quantitative PCRwere performedas described in our previous study [15]. The primer sequences used inreal-time PCR are listed in Supplemental Table 1.

2.7. Measurements of RORγ, TCR γδ, CCR and cytokines in ear skin

Samples from all groups of K14-VEGF animals terminated at day 12were investigated. To determine the levels of RORγ, TCR γδ and CCR6expression in the mouse ear samples, the tissues were cleaned usingTris-buffered saline (TBS), cut into pieces and homogenized. Total

lue± SD (fold) of themeasuredmRNA inmouse ear tissue of different treatment groups atA between groupswere not statistically significant. B, IL-17A. The changes in IL-17AmRNAroupswere not statistically significant. D, IL-1β. The level of IL-1βmRNA in theTPA-treatedps. E, IL-6. The changes in IL-6 mRNA between groups were not statistically significant. F,umin; CLO, clobetasol.

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proteins were extracted and detected by Western blotting as describedelsewhere [17]. Nine days following the start of experiment, the mouseauricular specimens (averageweight 30mg)werewashedwith PBS to re-move blood, and then homogenized by a homogenizer in ice-cold PBS(30 mg tissue: 0.5 mL PBS) containing a protease-inhibitor cocktail. Thetissue homogenates were stored −20 °C, and underwent two freezingand thawing cycles to disrupt the cell membrane. All tissue extractswere centrifuged (10,000 g, 4 °C) for 20 min and the supernatants werecollected and assayed by ELISA. The standard protocol from themanufac-turer was followed. Two experiments were pooled and data were aver-aged for analysis. ELISA results were normalized as pg/g tissue ultimately.

2.8. Statistics

Themeans and standard deviations were calculated. The significanceof the differences between the treatments was determined using eitherANOVA analysis or Wilcoxon scores (rank sums)/Kruskal–Wallis test.

Fig. 3. The levels ofmRNAmeasured using real-time PCR (Part 2). The figures show themean va9 d (*P b 0.05, Wilcoxon scores test, N = 6). A, IL-27. The level of IL-27 mRNA in the TPA-treaterespectively. B, INFγ. The level of INFγmRNA in the TPA-treated groupwas sharply increased anchanges in IL-12 mRNA between groups were not statistically significant. D, IL-2. The changes inmRNA in the TPA-treated groupwas sharply increased andwas inhibited in the curcumin- and clogroup was sharply increased and was inhibited in the curcumin- and clobetasol-treated groups,

All experiments were performed in triplicate using a minimum of 3replicates. A P-value of 0.05 was considered statistically significant.

3. Results

3.1. Effects of curcumin on TPA-induced mouse ear incrassation andinflammation

The maximum increase in ear thickness in the TPA-treated groupwas 0.553 ± 0.065 mm on day 10; the TPA and curcumin-treatedgroup was 0.487 ± 0.049 mm on day 4. On day 12, the ear thicknessincrease in TPA and curcumin-treated group was 0.360 ± 0.045 mm,compared with the TPA-treated group (0.493 ± 0.031 mm, P b 0.05).The increase in ear thickness of the clobetasol-treated group was lessthan that of the TPA-treated and TPA and curcumin-treated group(between 0.315±0.014mmand 0.378±0.035mm). The ear thicknessof the control group and curcumin treated control group did not show

lue± SD (fold) of themeasuredmRNA inmouse ear tissue of different treatment groups atd group was increased and was inhibited in the curcumin- and clobetasol-treated groups,dwas inhibited in the curcumin- and clobetasol -treated groups, respectively. C, IL-12. TheIL-2 mRNA between groups were not statistically significant. E, CXCL9. The level of CXCL9

betasol-treated groups, respectively. F, CXCL10. The level of CXCL10mRNA in the TPA-treatedrespectively. CUR, curcumin; CLO, clobetasol.

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any changes during the experiment (Fig. 1A). On days 2–4 after theinitiation of TPA treatment, the ear skin of the TPA-treated mice beganto display signs of thickening, erythema, andmild scalingwith continu-ously increased severity that peaked on days 6–10. Similar results wereobserved in the TPA and curcumin-treated groups, but the symptomswere milder than those observed in the TPA-treated group.

Results from H&E-staining of the TPA-treated ear skin showedincreased subcutaneous tissue and mild epidermal thickness comparedwith the control group. This increased thickness was due to theincreased inflammatory cells in the subcutaneous tissue. Curcumininhibited the TPA-induced increased thickness of subcutaneous tissue,whereas clobetasol significantly inhibited the increase in thickness. Noabnormal phenotype was observed in the control group and curcumintreated control group, as shown in Fig. 1B.

3.2. The mRNA levels of IL-23, IL-17A, IL-22 and other pro-inflammatorycytokines in ear samples of the TPA-induced psoriasis-like mouse model

Results from real-time PCR measurements of cytokines in ear tissueare shown in Fig. 2. In this experiment, three time points (4, 8, 12 days)were used to test for cytokine mRNA levels. Compared with the controlgroup, themRNA levels of IL-23, IL-17A and IL-22 changed insignificantlyin all samples from TPA-treated mouse ear tissue. And the mRNA levelsof IL-6 and TNFα changed insignificantly in all groups as well. However,mRNA levels of IL-1β increasedmildly in TPA-treated group and could bedecreased dramatically by curcumin. Similar results were observed inthe clobetasol-treated group.

3.3. Curcumin decreased the high mRNA levels of IL-27, IFNγ, IL-12, IL-2,CXCL9 and CXCL10 in ear samples of the TPA-induced psoriasis-like mousemodel

On day 9 after the initiation of TPA treatment, the ear tissue mRNAlevels of cytokines and chemokines were measured by real-time PCR(Fig. 3). Compared with the control group, the mRNA levels of IL-27,IFNγ, CXCL9 and CXCL10 increased significantly (P b 0.05) in samplesof TPA-treated mouse ear tissue. In contrast, the mRNA levels of IL-27,

Fig. 4. ELISA detection of cytokines expression in skin. IL-17A, IL-22, IL-27 and IFNγ productionswsuccessfully inhibited by both curcumin and clobetasol. IL-17A and IL-22 productions did not shtest). CUR, curcumin; CLO, clobetasol.

IFNγ, CXCL9 and CXCL10 significantly decreased in the curcumin-treated group when compared with the TPA-only treated group(P b 0.05). Similar results were observed in the clobetasol-treatedgroup. Moreover, mRNA levels of IL-2 and IL-12 did not change signifi-cantly in all groups.

Our mRNA results showed that IFNγ was the most remarkablyincreased cytokine compared with IL-23, IL-17, IL-22, TNFα and IL-6 inthe TPA-treated group. Meanwhile, the mRNA levels of IL-23, IL-17,IL-22, TNFα and IL-6 failed to show any meaningful changes in allgroups.

3.4. ELISA detection the cytokines in skin tissue

The results fromELISA test of TPA-treated ear skin showed increasingproduction of IL-27 and IFNγ protein compared with other groups; bothcurcumin and clobetasol inhibited the TPA-induced high expression ofIL-27 and IFNγ (shown in Fig. 4). In all groups, IL-17A and IL-22 expres-sion failed to show any significant difference (shown in Fig. 4).

3.5. The impact of curcumin on RORγ, TCR γδ, CCR6 expression in skin

Results from western blotting of TPA-treated ear skin showedincreasing RORγ and TCR γδ expression when compared with controlgroup; both curcumin and clobetasol failed to inhibit the TPA-inducedhigh expression of RORγ and TCR γδ (as shown in Fig. 5A). In all groups,CCR6 expression failed to show any significant increase and difference(shown in Fig. 5A). Immunohistochemical results from TPA-treatedear skin showed increased TCR γδ-positive cells in skin and failed toshow CCR6-positive cells in all groups (shown in Fig. 5B). The immuno-histochemical results agreed with results from western blottingstrongly.

4. Discussion

TPA is a diester of phorbol and a potent tumor promoter oftenemployed in biomedical research to activate the signal transductionenzyme, protein kinase C (PKC). A previous study [8] has suggested

ere detected by ELISA. IL-27 and IFNγ productions increased in TPA-treatedmice andwereow any differences in all groups (*P b 0.05, ANOVA followed by Student–Newman–Keuls

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that TPA induction leads to a Th17-like response in transgenicK14-VEGF mice. Unfortunately, it is clear that our results have reacheddifferent conclusions. Concerning differences in the methods betweenthe studies,we support our conclusionswith prudent confidence. Our re-sults showed that the mRNA level of IL-27 was significantly increased inTPA-treatedmouse ear tissue. It is well known that IL-27, a IL-12-relatedcytokine, has two apparently conflicting roles in the immune response:one as an initiator of Th1 responses and the other as an attenuator ofinflammatory cytokine production— including IL-17A [18]. IL-27 inhibitsthe production of IL-17A and IL-17F in naive T cells by suppressing, in aSTAT1-dependentmanner, the expression of the Th17-specific transcrip-tion factor RORγ [19]. In this study, ear tissues from all TPA treated groupwere detected with high expression of RORγ but not CCR6. The RORγ isrequired for the development of several innate lymphoid populations,such as lymphoid tissue-inducer cells (LTi cells) and cells that secreteinterleukin 17 (IL-17) or IL-22 [20]. It has been confirmed that CCR6 isthe optimal cell surface marker for IL-17A-producing cells [3,21]. Inspite of the high RORγ expression, IL-17A mRNA failed to increaseaccordingly which may be due to the high RORγ expression nothing onIL-17A-producing cells and influence of IL-27.

Furthermore, previous studies have shown that γδ T cells are capableof producing both IL-17A and IL-17F [22]. In murine skin, γδ T cells areabundant and act as dendritic epidermal T cells for local immunesurveillance.We foundmouse skinγδ TCRproteinwas increased follow-ing TPA treatment. However, our results show that CCR6 expression

Fig. 5. Result of RORγ, TCR γδ, CCR6Western blotting and immunohistochemical staining. A, ROexpression increased in TPA-treated mice and failed to be inhibited by both curcumin and clobshown are representative of 3 independent experiments and the bands' gray densitometry isexamined in samples of differently treated mouse ear skin (200×). CUR, curcumin; CLO, clobe

failed to be increased in all samples including TPA-treated mouse earskin. Taken together, these results indicate that the increased γδ Tcells after TPA induction in mouse skin might not be a function ofIL-17A-production. Therefore, we found the conclusion that TPA induc-tion leads to a Th17-like inflammation in K14-VEGF transgenic mice tobe unsupported.

As we know, IL-27 exerts a proinflammatory Th1-enhancing activityaside from its significant anti-inflammatory functions.Murugaiyan et al.[23] have found that IL-27-induced CD4+ T cells produce large amountsof IL-10 and IFNγ. In this study, ourmRNA results showed that IFNγwasthemost remarkably increased cytokine after TPA-treatment comparedwith TNFα and IL-6, which was consistent with the previous conclu-sions. As CXCL9 and CXCL10 are products of IFNγ induction, their highexpression strengthened the credibility of this observation as well.Although IL-2 is produced by all helper T cells early in their activation,our results showed that the mRNA levels of IL-2 and IL-12 have nosignificant changes after TPA induction. Additionally, evidence indicatesthat IL-27 limits IL-2 production during Th1 differentiation [24]. Thus,we have every reason to believe that TPA induction in K14-VEGF trans-genic mice leads to a Th1 inflammation rather than a Th17-likeinflammation.

Topical application of TPA on mouse skin leads to the rapid inflam-matory cell infiltration and increased cytokine production. These effectsmake the mouse skin format symptoms closely resemble human psori-asis vulgaris with respect to erythema, skin thickening, scaling,

Rγ, TCRγδ and CCR6 expressions were detected usingWestern blotting. RORγ and TCR γδetasol. CCR6 expression did not show any changes or differences in all groups. The resultslisted in Supplemental Table 2. B, TCR γδ and CCR6 immunohistochemical staining was

tasol.

241J. Sun et al. / International Immunopharmacology 25 (2015) 235–241

epidermal alteration (acanthosis, parakeratosis), and neoangiogenesisas well as the inflammatory infiltrate, including T cells and neutrophils.Although those symptomswere similarwith psoriasis, their pathogenesiswas different from psoriasis. Therefore, we should deliberate over thedecision for using TPA-induction K14-VEGF transgenic mice psoriasis-like model to evaluate treatments for psoriasis.

Extensive scientific research over the past decade has shown thatcurcuminmodulatesmultiple cellular targets and possesses therapeuticactivities against a wide variety of diseases, including psoriasis [25]. Inthis study, we find that curcumin inhibits increased thickness and in-flammation in TPA-treated mouse ear skin. The increased thickness isdue to Th1 inflammation in subcutaneous tissue of the skin. Althoughcurcumin can significantly attenuate the increase in mRNA levels ofIL-27 and IFNγ after TPA-induction of this model, it remains unclearhow curcumin exerts this effect. We infer that the above effects ofcurcumin are due to its inhibitory phosphorylation of STAT1 as anon-selective phosphorylase inhibitor (data not shown). In addition,similar effects to curcumin were observed of positive control drug —

clobetasol, which just were more stronger. In conclusion, TPA-inducedK14-VEGF transgenic psoriasis-like mice model presented Th1 typeinflammation and this Th1 inflammation was inhibited by curcumin.

For a long time, research and evaluation of psoriasis suffered fromthe lack of a suitable animal model and developed slowly as a result.There is no doubt that we hope the new data of this study will play apromoting role in future research of both curcumin and psoriasis.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.intimp.2015.02.007.

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