Polyandric Acid A, a Clerodane Diterpenoid from the Australian

Jan 8, 2014 - Sansom Institute for Health Research, University of South Australia, Frome ... Chuulangun Aboriginal Corporation, PMB 30, Cairns Mail Ce...
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Polyandric Acid A, a Clerodane Diterpenoid from the Australian Medicinal Plant Dodonaea polyandra, Attenuates Pro-inflammatory Cytokine Secretion in Vitro and in Vivo Bradley S. Simpson,*,†,‡ Xianling Luo,§ Maurizio Costabile,† Gillian E. Caughey,† Jiping Wang,†,§ David J. Claudie,⊥ Ross A. McKinnon,†,‡ and Susan J. Semple† †

Sansom Institute for Health Research, University of South Australia, Frome Road, Adelaide, 5000, South Australia Flinders Centre for Innovation in Cancer, Flinders University, Bedford Park, 5042, South Australia § Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade Parkville, Victoria, 3052, Australia ⊥ Chuulangun Aboriginal Corporation, PMB 30, Cairns Mail Centre, Cairns, Queensland, 4870, Australia ‡

ABSTRACT: Dodonaea polyandra is a medicinal plant used traditionally by the Kuuku I’yu (Northern Kaanju) indigenous people of Cape York Peninsula, Australia. The most potent of the diterpenoids previously identified from this plant, polyandric acid A (1), has been examined for inhibition of pro-inflammatory cytokine production and other inflammatory mediators using well-established acute and chronic mouse ear edema models and in vitro cellular models. Topical application of 1 significantly inhibited interleukin-1β production in mouse ear tissue in an acute model. In a chronic skin inflammation model, a marked reduction in ear thickness, associated with significant reduction in myeloperoxidase accumulation, was observed. Treatment of primary neonatal human keratinocytes with 1 followed by activation with phorbol ester/ionomycin showed a significant reduction in IL-6 secretion. The present study provides evidence that the anti-inflammatory properties of 1 are due to inhibition of pro-inflammatory cytokines associated with skin inflammation and may be useful in applications for skin inflammatory conditions including psoriasis and dermatitis.

efficacy of current treatments but have improved safety profiles are needed. Pro-inflammatory cytokines are key regulators in the pathogenesis of both psoriasis and dermatitis; however, differing cytokine profiles have been observed in lesions from each of these conditions.7−10 Polyandric acid A (1) is the most active diterpenoid found in D. polyandra, and this study aimed to identify the molecular pathways, namely, the proinflammatory cytokines that may be modulated by this compound, using both acute and chronic mouse ear edema models and in vitro cellular models. Additionally, the effects of 1 on nitric oxide, a key modulator of pro-inflammatory cytokine production and neutrophil infiltration, were quantified. This report describes findings that support the anti-inflammatory properties of 1 in attenuating a number of pro-inflammatory cytokines in vivo and in vitro and discusses its potential as a therapeutic agent for the treatment of skin inflammatory disorders.

Dodonaea polyandra Merr. & L.M Perry (Sapindaceae) is a traditional medicinal plant used by the Kuuku I’yu people from Cape York Peninsula in northern Australia for pain and inflammation of the mouth. Clerodane diterpenoids found in extracts of D. polyandra have been reported previously to have anti-inflammatory properties in an acute model of skin inflammation in mice.1 The results from this experimental model suggest that products from this plant might be useful in applications for inflammatory skin conditions including psoriasis and dermatitis.2,3 These skin conditions cause significant morbidity, with psoriasis affecting between 0.3% and 3% of the population (depending on ethnicity)4 and dermatitis affecting on average approximately 10% of the global population. Common treatments for these conditions may include topical use of steroid-based therapeutics and other products (including topical calcineurin inhibitors, salicylic acid, vitamin D3 and vitamin A analogues, and phototherapy).5,6 A major drawback of current steroid-based therapies is the risk of adverse side effects that can lead to exacerbation of the underlying condition (e.g., thinning of the skin and delayed wound healing). Therefore, novel therapeutics that retain the © 2014 American Chemical Society and American Society of Pharmacognosy

Received: August 30, 2013 Published: January 8, 2014 85

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compared to the group receiving only TPA (Figure 2B). Treatment with betamethasone, a potent steroidal antiinflammatory agent, resulted in a 54.2% decrease in edema relative to the TPA group. No change in edema was observed in the solvent control group. The ears of mice treated with 1 (Figure 3C) showed a marked reduction in redness, scaling,

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RESULTS AND DISCUSSION Effect of Polyandric Acid A (1) on in Vivo TNF-α and IL-1β Levels in Mouse Ear Tissue. Treatment with 1 at a dose of 0.9 μmol/ear, the same dose previously shown to significantly reduce ear edema,1 resulted in a significant reduction in interleukin (IL)-1β levels. A 78% reduction in IL-1β was observed in the group treated with 1 relative to the 12-O-tetradeconylphorbol 13-acetate (TPA) group (p < 0.05) after subtracting basal IL-1β levels (in untreated mouse ear) from both groups (Figure 1). In the acute mouse ear inflammation model, no tumor necrosis factor (TNF)-α was detected in the solvent control, control-treated, or compound 1-treated groups.

Figure 3. Visual representation of the effect of topical treatment of 1 on TPA-induced chronic mouse ear inflammation. (A) Normal ear. (B) TPA-only treated ear. (C) 1 + TPA treated ear. (D) Betamethasone + TPA treated ear.

and psoriatic-like (flaky) appearance when compared to TPAtreated mice (Figure 3B), with the group treated by 1 tending toward resolution of the inflammatory response with an appearance more like a normal ear (Figure 3A). Effect of Polyandric Acid (1) on Neutrophil Accumulation. Topical application of TPA results in a marked increase in neutrophil infiltration into the inflamed tissue.11 Following treatment of 1 for four days the accumulation of myeloperoxidase (MPO) (a marker of neutrophil infiltration) in mouse ear

Figure 1. IL-1β levels in ear tissue homogenate from mice treated with 1 (0.9 μmol/ear) compared to TPA-treated mice. Means ± SEM, n = 5, *p < 0.05, vs TPA group, Student’s t test.

Effect of Polyandric Acid A (1) in a Chronic Model of Mouse Ear Edema. Treatment with 1 in the chronic model of mouse ear edema over a four-day period was associated with a statistically significant (p < 0.001) decrease (41.1%) in edema

Figure 2. Effect of 1 on chronic mouse ear edema induced by repeated applications of TPA. (A) Full view; (B) exploded view of days 7−10. (●) TPA control (2.5 μg/ear); (■) 1 (0.9 μmol/ear); (▲) betamethasone-17,21-dipropionate (0.9 μmol/ear); and (▼) solvent vehicle control. Each data point represents the means ± SEM (n ≥ 6). **p < 0.01, ***p < 0.001 compared to TPA control group (one-way ANOVA followed by Dunnett’s post hoc test). (Note: error bars for the solvent group are not visible due to the small variability.) 86

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Cell Viability. Polyandric acid A (1) did not significantly affect cell viability at concentrations below 60 μM. On the basis of this observation, the maximum concentration used for assessing the effect of 1 on cytokine production in THP-1 and nHEKs and nitrite production in RAW264.7 cells was 30 μM. Effect of Polyandric Acid A (1) on IL-1β, pro-IL-1β, and TNF-α Production by THP-1 Cells. In order to establish the efficacy of treatment by 1 on pro-inflammatory cytokine production (IL-1β, proIL-1β, and TNF-α, IL-6) in immune cells, THP-1 monocytes were used as a model. Interleukin-6 was not detected under the experimental conditions used. After 24 h of TPA/ionomycin stimulation, extracellular IL-1β levels were reduced significantly (p < 0.05) by 26.3% and 18% at concentrations of 30 and 3 μM 1, respectively. A slightly lower effect was observed for TNF-α at 30 μM, which was inhibited by 19.7% (p < 0.05). Intracellular levels of IL-1β and its precursor, pro-IL-1β, were also inhibited by 15% and 17%, respectively, at a concentration of 30 μM 1 (p < 0.05). Production of these cytokines is known to involve the NFκB signaling cascade. To confirm the stimulus was acting through NF-κB activation, the known NF-κB inhibitor BAY-11-7082 was also tested and found to inhibit significantly (p < 0.05) production of each cytokine when tested at 5 μM (Figure 5). Effect of Polyandric Acid A (1) on TNF-α, IL-1α, IL-1β, IL-6, and IL-8 Production by nHEKs. Subsequent experiments were conducted using primary neonatal human epidermal keratinocytes (nHEKs). As with THP-1 cells, 1 was not cytotoxic to nHEKs at the highest dose used (30 μM)

tissue was reduced significantly by 81.5% relative to TPA control group (p < 0.0001) (Figure 4). While betamethasone at

Figure 4. Effect of 1 on MPO accumulation in mouse ear tissue in a chronic edema model induced by repeated applications of TPA (BM = betamethasone). Each bar represents the means ± SEM (n = 3 for TPA, n = 6 for other groups). ****p < 0.0001 compared to TPA control group (one-way ANOVA followed by Dunnett’s post hoc test).

the same concentration also showed a significant reduction (90.0%) in the amount of MPO present, there was no statistical difference between the treatments with 1 and betamethasone. The reduction in MPO supports histological analysis performed on ear tissue that showed significant reduction in the influx of neutrophils to the mouse ear skin following treatment with 1 (data not shown).

Figure 5. Effect of 1 on extracellular (ex) and intracellular (in) cytokine production by THP-1 cells after 24 h. (A) exIL-1β; (B) inIL-1β; (C) pro-IL1β; and (D) exTNF-α. Data expressed as means ± SEM, n = 6, *p < 0.05, vs TPA/ionomycin control. BAY = Bay-11-7082. 87

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Figure 6. Effect of 1 on secretion of extracellular cytokines in nHEKs in vitro. (A) exTNF-α secretion after 6 h (solid bars) and 24 h (pattern bars). (B) exIL-6 secretion after 6 and 24 h. Data expressed as means ± SEM, n = 6, *p < 0.05, vs TPA/ionomycin control. BAY = Bay-11-7082.

under the experimental conditions employed. Treatment with 1 inhibited both TNF-α and IL-6 secretion. TNF-α secretion was inhibited by 26.2% (p > 0.05) with treatment of 3 μM 1 after 6 h (Figure 6A, solid bars) with no effect observed after 24 h (Figure 6A pattern bars). Higher concentrations failed to inhibit TNF-α secretion. After 6 and 24 h stimulation, IL-6 was inhibited significantly by 33.2% and 41.5% (p < 0.05), respectively, with 30 μM 1 (Figure 6B). There was no significant effect by 1 on IL-1α or IL-8 secretion (data not shown). Interestingly, IL-1β was not secreted at detectable levels even after TPA/ionomycin treatment. Keratinocytes are the most abundant cell type in the skin (∼95%), and it is now recognized widely that HEKs play a key role in the initiation and maintenance of inflammatory skin conditions through secretion of proinflammatory cytokines.12,13 Activation of HEKs with TPA/ionomycin has been shown to induce proinflammatory cytokines including TNF-α, IL-1β, IL-6, and IL8.14−17 While HEKs are known to secrete IL-1β following activation by certain stimuli (e.g., UV-B light),18 treatment with TPA/ionomycin did not stimulate the production of IL-1β in this study. The multiplex analysis of 13 cytokines by flow cytometry revealed that only IL-6 and TNF-α were produced at 6 and 24 h by nHEKs under the experimental conditions used. Polyandric acid A (1) did not significantly inhibit TNF-α, but inhibition of IL-6 was observed at a similar magnitude, as determined by ELISA (at 30 μM). Effect of Polyandric Acid A (1) on NO Production in RAW264.7 Cells. In addition to establishing the effects of treatment by 1 on cytokine production, it was also sought to evaluate whether nitric oxide (NO) production could be inhibited due to the role that NO plays as an inflammatory mediator. Lipopolysaccharide (LPS) is a known activator of iNOS expression in the RAW264.7 mouse macrophage cell line, leading to an increase in NO production.19 The amount of nitric oxide was measured indirectly colorimetrically using the Greiss assay. Treatment of the LPS-stimulated mouse macrophage cell line RAW264.7 with 1 did not result in any decrease in NO production. Addition of the NF-κB inhibitor BAY-117082 decreased significantly (p < 0.001) the amount of NO produced due to the direct role of the transcription factor NFκB complex in LPS-mediated iNOS expression.20 The results of this study demonstrate that the diterpenoid polyandric acid A (1) mediates its anti-inflammatory effects via inhibition of the pro-inflammatory cytokine IL-1β, together with reduced neutrophil infiltration, resulting in reduced ear edema in the mouse models studied. The anti-inflammatory

Figure 7. Effect of 1 on nitrite production. LPS-stimulated (1 μg/mL) RAW264.7 cells were treated with 1 at either 3 or 30 μM or the NF-κB inhibitor BAY-11-7082. Data are expressed as means ± SEM, n = 8, ***p < 0.001, vs LPS control.

effects observed with 1 were comparable to or greater than that observed by topical treatment of the currently used steroid therapy betamethasone. We attempted to elucidate the mechanism of these effects by conducting a series of studies using in vitro cellular models, whereby inhibition of extracellular and intracellular IL-1β, in addition to its precursor pro-IL-1β, resulted. While production of TNF-α was not evident in the mouse models studied, its synthesis was inhibited by 1 in both the THP-1 and nHEK in vitro cellular models. Furthermore, this diterpenoid also inhibited IL-6 synthesis using the nHEK cellular model. No inhibition of NO was observed on treatment with 1. Interleukin-1β is a potent inflammatory mediator in the skin and performs roles in both the initiation and amplification stages, where it activates dermal endothelial cells and production of the leucocyte adhesion molecules ICAM-1 and VCAM-1.12,21 The major cell type of the skin is the keratinocyte, constituting around 95% of the cell mass of human epidermis.12 Keratinocytes are known to be a source of IL-1β as well as expressing both type I and II IL-1 receptors and IL-1 receptor antagonist13 in whole animal systems. It was hypothesized that 1 acts on keratinocytes within the mouse ear tissue, leading to the observed reduction of IL-1β. However, on examination of primary nHEKs treated with 1 and stimulated with TPA, it was found that synthesis of IL-1β protein was absent. There are conflicting studies in the literature reporting the production of IL-1β by TPA-induced (cultured) keratinocytes.14,22−24 The present findings support the 88

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conclusions of previous work,22−24 while other reports challenge this result.14 Therefore, this indicates that following processing of IL-1β there are other mechanisms required for the release of IL-1β from TPA-stimulated HEKs in vivo,25 if these cells are the source of IL-1β detected in the mouse model. On the contrary, other cell types such as infiltrating monocytes can also contribute as a source of this cytokine. This highlights the possibility that the particular in vitro cellular model used may not be suitable for the investigation of IL-1β-mediated inflammatory processes. In the same series of experiments with nHEKs, there was a modest but statistically insignificant reduction in TNF-α. Synthesis of IL-6 was also observed in the HEKs, which was inhibited significantly by 1. Quantitative analysis of IL-6 was not performed on mouse ear tissue. However, the significance of this in vitro result may aid in interpreting the effects observed during this study using the chronic model of mouse ear inflammation as well as the involvement of IL-6 in cutaneous diseases such as psoriasis. Interleukin-6 is a pleiotropic cytokine and like other cytokines (IL-1 and TNFα) is attracting interest in its role in driving chronic inflammation. An important effect of IL-6 on keratinocytes is its ability to potently induce keratinocyte proliferation.26,27 Psoriasis is characterized by hyperplasia of the dermis and inflammatory infiltrate, resulting in a scaly (flaky) appearance as a result of hyperproliferating, highly differentiated keratinocytes. As illustrated by the photographs from the chronic mouse ear inflammation study shown in Figure 3, the morphology of the inflamed skin induced with TPA resembles closely that of psoriatic lesions. Indeed, this model is recognized as one of many that reflect some of the pathological characteristics of psoriasis,28 which may be related to the involvement of protein kinase C (PKC), of which TPA is a potent activator and known to be stimulatory for keratinocyte differentiation and proliferation markers.29 In the present study, topical application of 1 showed evidence for reduced hyperplasia, redness, and edema in mouse ear skin. These results are also significant from the viewpoint of the interplay that exists between IL-1 and IL-6 in the inflammatory response. IL-1 is a known inducer of IL-6 production,15,30 and they can also act synergistically.31 In TPAinduced THP-1 monocytes, treatment with 1 resulted in inhibition of pro-IL-1β, IL-1β, and TNF-α. Collectively, these results suggest that the pathways leading to IL-1 and IL-6 production, in particular, appear to be inhibited by 1. These findings do not rule out the possibility that 1 could be acting on other skin cell types including dermal dendritic cells, fibroblasts, epidermal Langerhans cells, melanocytes, and leukocytes, which are known to be sources and targets of cytokines.13 The TPA-induced mouse ear edema model is also characterized by an increase in neutrophil infiltration, which was significantly inhibited by 1 in the chronic ear edema model. Recruitment of neutrophils to the site of inflammation is also influenced by the potent chemotactic factor interleukin-8.32 Interestingly, although IL-8 was screened for in the keratinocyte assays, there was no inhibition of IL-8 observed from nHEKs treated with 1. However, the reduction in MPO supports histological analysis performed on ear tissue (data not shown), which showed a significant reduction in the influx of neutrophils to the mouse ear skin following treatment with 1. Polyandric acid A (1) did not inhibit nitric oxide production from RAW264.7 cells stimulated with LPS. Nitric oxide is a pleiotropic chemical messenger showing contradictory physiological functions that are dependent on the generation and

regulation within different cell types.33 Excess NO appears to mediate acute and chronic inflammation. For example, it is cytotoxic to both the cells that produce it and neighboring cells, where it can trigger apoptotic events of cell death. In addition, it can result in the generation of free-radical adducts with DNA, fatty acids, and smaller molecular species, further cascading events of cellular damage19 In contrast, the known NF-κB inhibitor BAY11-7082 significantly suppressed NO production, suggesting that the anti-inflammatory effects of 1 are acting independently of the NF-κB pathway. There are a small number of studies that report clerodane diterpenoids as having anti-inflammatory properties.34−38 Of these, secretory phospholipase A2 and 5-lipoxygenase have been identified as molecular targets of these natural products. Interestingly, inhibitory activity of cyclooxygenase (COX) 1 and 2 is not apparent,34 which is in agreement with our own testing of 1 on COX-1 and -2 (unpublished data). A structurally similar clerodane diterpenoid from the species Dodonaea viscosa, hautriwaic acid, has recently been reported for its anti-inflammatory activity in the TPA-induced acute mouse ear edema model,39 the same as used in this study. Although its structure had been elucidated some 40 years earlier,40 it remains today as the only other diterpenoid isolated from a species of Dodonaea to display anti-inflammatory effects. In summary, the present study has demonstrated the clerodane diterpenoid polyandric acid A (1), from D. polyandra, inhibits the production of inflammatory cytokines that are known to be fundamental in mediating processes associated with chronic skin inflammation. These data provide a basis for more in-depth studies exploring the signaling pathways leading to expression of the cytokines that are modulated by 1. The effects demonstrated by 1 in reducing psoriasis-like skin inflammation warrant further investigation of the potential development of such compounds as therapies for treating these conditions.

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EXPERIMENTAL SECTION

General Experimental Procedures. Polyandric acid A (1) was isolated from the leaf resin of D. polyandra as previously described,1 with the structure and purity (>97%) confirmed by NMR spectroscopy and HPLC. Absolute ethanol and acetone were purchased from Merck (Darmstadt, Germany). Tris-hydrochloride (Trizma-HCl), EDTA disodium salt dihydrate, Triton X-100, 12-O-tetradecanoylphorbol 13-acetate, ionomycin calcium salt, and lipopolysaccharide were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mouse IL1β and TNF-α were quantified using eBioscience platinum ELISA kits (cat. nos. BMS6002 and BMS607/3, respectively). Human IL-1α (cat. no. BMS 243/2), IL-1β (cat. no. BMS 224/2), TNF-α (cat. no. BMS223/4), IL-6 (cat. no. BMS 213/2), and IL-8 (cat. no. BMS 204/ 3) were quantified using eBioscience platinum ELISA kits. An R&D Systems Quantikine ELISA kit was used for measuring human pro-IL1β (cat. no. DLBP00). A human Th1/Th2/Th9/Th17/Th22 13plex FlowCytomix multiplex kit (eBioScience cat. no. BMS817FF) was used for cytokine analysis by flow cytometry. Cell viability was determined using the CellTiter 96 Aqueous nonradioactive cell proliferation (MTS) assay (Promega, cat. no. G5421). Animals. Male Balb/C mice, seven to nine weeks of age, were housed in the Reid Animal Facility (University of South Australia), allowing acclimation for at least four days prior to initiating experiments. Experiments were conducted in accordance with ethical standards as outlined in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, ninth ed., and approved by the South Australian (SA) Pathology Animal Ethics Committee (project no. 15/11). Acute Mouse Ear Edema Model. The acute model was conducted as previously described,1 with the exception that animals 89

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with 50 μL of 1 (in RPMI-1640) for 1 h in round-bottomed 96-well microtiter plates (Corning Costar, cat. no. 3799). Cells were then stimulated with 100 μL of TPA (40 ng/mL)/ionomycin (1 μM) (final assay concentration). The ethanol concentration did not exceed 0.05% in the assay. The NF-κB inhibitor BAY-11-7082 (Cayman Chemicals) and betamethasone-17,21-dipropionate (Sigma) were included at 5 and 3 μM, respectively. Plates were incubated at 37 °C, 5% CO2 for 24 h. After 24 h, the supernatant was removed and centrifuged for 5 min at 13800g. For extracellular cytokine determination, the supernatant was stored at −80 °C until analysis. For intracellular cytokine determination, cells were washed with 200 μL/well of ice-cold PBS (endotoxin free) twice. Cells were lysed by the addition of 100 μL of ice-cold RIPA buffer (Sigma) containing protease inhibitor cocktail (Sigma) and incubated on ice for 5 min. Lysate was stored at −80 °C until ELISA analysis. Prior to quantifying samples, the lysate was centrifuged for 10 min/13800g at 4 °C. The supernatant was removed and diluted (if necessary) according to ELISA kit instructions and measured for IL-1β, TNF-α, pro-IL-1β, and IL-1β. TPA/Ionomycin-Induced Cytokine Production in Neonatal Human Epidermal Keratinocytes. Neonatal human epidermal keratinocytes were cultured (10 000 cells/200 μL/well) in a 96-well flat-bottomed tissue culture-treated microtiter plate (Corning Costar, cat. no. 3595) for 18 h at 37 °C, in 5% CO2. After removing the medium and replacing with fresh medium (50 μL), cells were preincubated for 1 h with 50 μL of 1. The known NF-kB inhibitor BAY-11-7082 (Cayman, cat. no. 10010266) was included at 5 μM. Cells were stimulated subsequently with 100 μL of TPA (40 ng/mL)/ ionomycin (1 μM) and incubated for an additional 6 and 24 h. The supernatant was removed at the appropriate time point and centrifuged at 13800g, 4 °C for 5 min and stored at −80 °C until analysis. The levels of extracellular IL-1α, IL-1β, IL-6, IL-8, and TNF-α in the supernatants were measured by ELISA. LPS-Induced Nitric Oxide Production in RAW264.7 Cells. RAW264.7 cells (100 000 cells/50 μL/well) were preincubated in the presence of 50 μL of 1 or BAY-11-7082 (in DMEM) for 1 h in roundbottomed 96-well microtiter plates (Corning Costar, cat. no. 3799). Cells were stimulated with 100 μL of LPS (final concentration 1 μg/ mL) and incubated for 24 h at 37 °C, in 5% CO2. Nitric oxide production was determined indirectly from the supernatant by measuring nitrite concentration using the Greiss assay by comparison to a sodium nitrite standard curve.42 This method was adapted to a 96well plate format. Briefly, to 100 μL of supernatant, 50 μL of 1% (w/v) sulfanilic acid (in 5% HCl) followed by addition of 50 μL of 0.1% (w/ v) N-naphthylethylenediamine dihydrochloride (in 5% HCl) were added to initiate the reaction. Plates were incubated at 37 °C for 10 min. Absorbance was measured at 540 nm using a plate reader. Flow Cytometry Assay. The supernatants collected from the cultures of keratinocytes after 6 or 24 h stimulation with TPA were assayed to determine the concentrations of secreted cytokines using a Flowcytomix multiplex kit (Bender Medsystems GmbH, Vienna, Austria), according to the manufacturer’s instructions. This kit allows for the simultaneous quantification of 13 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p70, IL-13, IL-17A, IL-22, IFN-γ, and TNF-α). Briefly, the Flowcytomix technology is based on spectrally discrete beads that are coated with an antibody to the specific analyte. The beads are differentiated by their size and different spectral addresses due to the presence of different levels of Starfire Red, a farred (685−690 nm) emitting fluorochrome. The beads are mixed with standards or test samples, and then a biotin-conjugated secondary antibody specific to each analyte is added. Streptavidin-phycoerythrin was then added, and the test samples were analyzed by flow cytometry using an Accuri C6 flow cytometer (Becton Dickinson, NJ, USA). For each analysis, altogether 10 000 events were acquired. The data were analyzed using the supplied software (Flow Cytomix Pro, eBioscience). The mean concentration of each cytokine was expressed as pg/mL. The minimum detectable concentrations of cytokines were as follows: IL-1β (4.2 pg/mL), IL-2 (16.4 pg/mL), IL-4 (20.8 pg/mL), IL-5 (1.6 pg/mL), IL-6 (1.2 pg/mL), IL-9 (1.5 pg/mL), IL-10 (1.9 pg/mL), IL-12p 70 (1.5 pg/mL), IL-13 (4.5 pg/mL), IL-17A (2.5 pg/

were sacrificed after 6 h as opposed to 48 h. A 5 mm diameter biopsy of ear tissue was collected immediately from each ear of all mice using a conventional hole punch and kept on ice. Ear tissue was homogenized (MICCRA D-1, ART-moderne Labortechnik, Germany) in 500 μL of Tris-HCl (50 mM, pH 7.4)/EDTA (1 mM) buffer containing protease inhibitor cocktail (Sigma, cat. no. P8340). The homogenate was incubated on ice in the presence of 1% (v/v) Triton X-100 for 15 min and subsequently centrifuged at 4 °C for 15 min at 9500g. The supernatant was removed and stored at −80 °C pending analysis by ELISA. Chronic Mouse Ear Edema Model. Chronic skin inflammation was evaluated by a previously described protocol using ear thickness and myeloperoxidase production as the experimental end points.28 Briefly, 20 μL of TPA (125 μg/mL) was applied to the inner and outer surface of both ears on alternate days, starting on day 0. Polyandric acid A (1) (0.9 μmol/ear/20 μL ethanol vehicle) or betamethasone17,21-dipropionate (0.9 μmol/ear/20 μL ethanol vehicle) was applied topically to both ears in the morning and afternoon on days 7, 8, and 9. On day 10, mice received one further application of test compound in the morning and were euthanized subsequently by inhalation of isoflurane followed by cervical dislocation. Ear thicknesses were measured in the morning on days 0, 1, 2, 3, 4, 7, 8, 9, and 10 using a digital micrometer (±0.001 mm, Mitutoyo, Japan). Myeloperoxidase Assay. The extent of neutrophil infiltration may be indirectly determined by measuring the presence of the biochemical marker myeloperoxidase. The assay was conducted as previously described by de Young et al.41 Briefly, mouse ear tissue (hole punch biopsies, 5 mm) was placed in 750 μL of 80 mM PBS (pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide (HTAB) and homogenized for 45 s (0 °C). Homogenate was transferred to a 1.5 mL microfuge tube, and the vessel was rinsed with a second 750 μL aliquot of HTAB in PBS, which was also added to the tube and centrifuged at 11200g (4 °C for 20 min). Thirty-microliter aliquots (in duplicate) of the resulting supernatant were transferred to a 96-well flat-bottomed plate, and 200 μL of a solution mixture (containing 100 μL of PBS, 85 μL of sodium phosphate buffer, pH 5.4, and 15 μL of 0.017% H2O2) was added to the wells. The reaction was initiated by addition of 20 μL of 18.4 mM tetramethylbenzidine hydrochloride in dimethylformamide for 3 min at 37 °C. The reaction was terminated by addition of 30 μL of 1.46 M sodium acetate (pH 3.0), and absorbance measured at 600 nm. Cell Culture. THP-1 cells (provided by Dr. Maurizio Costabile, University of South Australia) were grown in RPMI-1640 medium (Sigma) containing 10% (v/v) fetal calf serum (Sigma), penicillin (200 000 units/L)/streptomycin solution (200 mg/L) (Sigma), 4 mM L-glutamine (Sigma), and 15 μL of β-mercaptoethanol (Sigma). RAW264.7 cells were grown in DMEM (endotoxin free) containing (per 500 mL) 10% (v/v) fetal calf serum, 10 mL of penicillin (12 mg/ 10 mL)/gentamicin (16 mg/10 mL), 4 mM L-glutamine, and 15 μL of β-mercaptoethanol. Primary neonatal human epidermal keratinocytes were purchased from Life Technologies (Invitrogen, cat. no. C-0015C). The culture system used included EpilLife medium (Gibco, cat. no. M-EPICF-500) containing growth supplement (Gibco, cat. no. HKGS kit S-001-K) and calcium chloride (0.06 mM final concentration), undefined for extended lifespan. Cells were cultured according to the manufacturer’s instructions and subcultured once cells reached 80% confluence. Cells were detached from culture flasks using a trypsin-like enzyme, TrypLE Express, according to the manufacturer’s instructions. Cells were not used under experimental conditions beyond passage number 3. Cell Viability (MTS) Assay. Cell viability was assessed using the MTS method according to the manufacturer’s instructions. Briefly, cells (50 000/50 μL/well) were preincubated for 1 h, to which serial half-dilutions of 1 were added (50 μL), and subsequently incubated for 24 h. After this time, 20 μL of MTS reagent was added to the wells and incubated for 1 h. Plates were measured at 492 nm on a Labsystem Multi Scan Ascent plate reader (model 354; Helsinki, Finland) using the supplied Ascent software (version 2.4.). TPA/Ionomycin-Induced Cytokine Production in THP-1 Cells. THP-1 cells (100 000 cells/50 μL/well) were preincubated 90

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mL), IL-22 (43.3 pg/mL), TNF-α (3.2 pg/mL), and IFN-γ (1.6 pg/ mL). Statistics. Data are expressed as means ± SEM. Graphpad v5.0 (Graphpad Software Inc., San Diego, CA, USA) was used for data analysis and statistical comparison purposes. Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s post hoc test or Student’s t test (where appropriate) with p values of