The phospholipase A2
) superfamily of enzymes cleave phospholipids at the sn-2 position to release free fatty acids and lysophospholipids. These are the precursors to a multitude of lipid signaling molecules including the eicosanoids, which are metabolites of arachidonic acid (AA) and have important roles in inflammation and inflammatory diseases. Cytosolic phospholipase A2
α) is the only PLA2
enzyme with high specificity for phospholipids carrying AA at the sn-2 position, placing it as an important upstream regulator of eicosanoid production [1
]. When activated by extracellular stimuli, cPLA2
α undergoes Ca++
-dependent translocation from the cytoplasm to intracellular membranes and becomes predominantly localized to the peri-nuclear region of the cell [2
]. This is where metabolism of AA by the cyclo-oxygenase (COX) and lipo-oxygenase (LOX) pathways typically occurs, producing prostaglandins and thromboxane A2
), or leukotrienes, hydroxyeicosatetraenoic acids (HETEs), and hydroperoxyeicosatetraenoic acids (HPETEs), respectively. The importance of cPLA2α for stimulus-induced eicosanoid production and the pathogenesis of inflammation has been demonstrated by gene silencing both in vitro [5
] and in animal models [7
], and from the use of specific inhibitors of cPLA2
α in preclinical models of inflammatory diseases, as was recently reviewed by Nikolaou et al. [12
]. Examples include the use of the indole-derivative ZPL-5212372 in asthma and atopic dermatitis [13
], the pyrrolidine-based compound RSC-3388 in a Streptococcus pneumonia
infection model [14
], and the ω-3 polyunsaturated fatty acid (PUFA) derivatives AVX001 and AVX002 in collagen-induced arthritis [15
Plaque psoriasis (psoriasis vulgaris) is a disease with a chronic inflammatory phenotype that drives the hyperproliferation and aberrant differentiation of the epidermis [16
]. Chronic inflammation in psoriasis is associated with higher expression of PLA2
] and increased levels of eicosanoids [20
]. Evidence for the involvement of eicosanoids in psoriasis is supported by mouse models of the disease—the leukotriene B4
) receptor 1 and TxA2
receptor have critical roles in imiquimod-induced skin inflammation [24
], and prostaglandin E2
) acting at prostaglandin receptors EP2 and EP4 is important for Th17-dependent inflammation in interleukin 23 (IL-23)-induced psoriasis [27
]. Suppression of eicosanoid production is therefore an interesting prospect for treating psoriasis.
Non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit AA metabolism via the COX pathway are commonly used for their analgesic, anti-inflammatory, and antithrombotic actions; however, their use is associated with many adverse gastrointestinal and cardiovascular effects (as reviewed in [28
]) and they can induce or exacerbate psoriasis. The latter effect at least is postulated to be attributable to a skewed eicosanoid profile and the accumulation of leukotrienes (reviewed in [29
]). Thus, it has been hypothesized that creating a more balanced suppression of eicosanoids using either dual COX-LOX inhibitors or by suppression of AA production using PLA2
inhibitors (reviewed in [12
]) would provide a better and safer therapeutic option.
α inhibitor AVX001 is a ω-3 PUFA-derivative developed by Avexxin (now Coegin Pharma) that was demonstrated to be highly selective and to inhibit the in vitro activity of cPLA2
α with an IC50
of 120nM, being more potent than either docosahexaenoic acid (DHA) or the ω-6 PUFA derivative arachidonyl trifluoromethyl ketone (AACOCF3
, ATK) [15
]. A topical application of AVX001 was trialed in a randomized, double-blind, placebo-controlled, dose-escalation first-in-man study to assess its safety and efficacy in patients with mild to moderate plaque psoriasis [33
]. AVX001 showed significant efficacy and was well tolerated up to the maximum dose tested of 5%, supporting the targeting of cPLA2
α as a safe therapeutic strategy. The specificity and potency of cPLA2
α inhibition by AVX001 has been demonstrated [15
], however, its mode of action in psoriasis remains to be determined. Given the documented role of cPLA2
α in mediating inflammatory signals in monocytes and keratinocytes [34
] and the more recent interest in cPLA2
α as a driver of cellular proliferation [38
], we sought to study potential modes of action of AVX001 in psoriatic skin by investigating its effects on inflammation and proliferation using human peripheral blood mononuclear cells (PBMC) and keratinocytes.
2. Materials and Methods
Cell culture media and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, US) unless stated otherwise. A23178, naproxen, celecoxib calcipotriol hydrate, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. Nordihydroguaiaretic acid (NDGA) was from Cayman chemicals (Ann Arbor, MI, USA) Recombinant human epidermal growth factor (EGF) and tumour necrosis factor (TNF)-α were from R&D systems (Abdingdon, UK). The fluoroketone AVX001 was synthesized and characterized according to Holmeide and Skattebol [39
], and provided by Dr. Inger Reidun Aukrust and Dr. Marcel Sandberg (Synthetica AS, Oslo, Norway). AVX001 was stored at −80 °C as a 20 mM stock solution in dimethyl sulphoxide (DMSO) under argon gas to minimize oxidation.
2.2. PBMC Isolation and Treatment
Blood was recruited from healthy donors at St. Olavs Hospital HF, the Bloodbank (project approved by Regional Ethical Committee of Mid-Norway; #2016/553). Peripheral blood mononuclear cells (PBMC) were isolated using SepMate separation tubes with LymphoPrep density gradient medium from STEMCELL Technologies (Cambridge, UK), according to the manufacturer’s recommendations. For experiments, 1 x 106 cells per well were plated in 1 mL Roswell Park Memorial Insitute (RPMI) medium supplemented with 5% fetal bovine serum (FBS), 0.3 mg/mL glutamine, and 0.1 mg/mL gentamicin. Inhibitors were added 2 h prior to the addition of the Ca++ ionophore A23178 (30 µM, 15 min) to activate cPLA2α or lipopolysaccharide (LPS) (10 ng/mL, 72 h) as a potent inducer of inflammation. Following treatment, the cell suspensions were centrifuged to isolate the supernatant from the cell fraction. Samples were stored at −80 °C until analysis.
2.3. Enzyme-Linked Immunoassay Detection of Eicosanoids
Cell supernatant samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for PGE2 (Cayman #514435), LTB4 (Cayman #10009292), TxB2 (Cayman #501020), or 12S-HETE (Enzo Lifesciences #ADI-900-050) according to the manufacturers’ protocols. Cell supernatants were assayed at dilutions of 1:100 for PGE2, except supernatants from non-LPS-treated PBMC that were assayed undiluted in all assays. Supernatants were hybridized overnight, and the enzymatic conversion of the substrate was read at OD420 nm. Data were processed using a 4-parameter logistic fit model.
2.4. Culture of HaCaT Keratinocytes
The spontaneously immortalized skin keratinocyte cell line HaCaT [40
] was kindly provided by Prof. N. Fusenig (Heidelberg, Deutsches Krebsforschungszentrum, Germany). These cells are commonly used to study proliferative and inflammatory responses in psoriasis research [41
], as they express epidermal growth factor receptor (EGFR) and can proliferate both independently of, as well as in response to, stimulation with growth factors [47
]. HaCaT were maintained in Dulbecco’s modified Eagle Medium (DMEM) supplemented with 5% (v
) FBS, 0.3 mg/mL glutamine, and 0.1 mg/mL gentamicin (DMEM-5) at 37 °C with 5% CO2
in a humidified atmosphere at sub-confluency to prevent differentiation. Treatments were carried out in DMEM supplemented with 0.5% (v
) FBS and 0.3 mg/mL glutamine (DMEM-0.5)
2.4.2. Eicosanoid Release
For analysis of eicosanoid release, we plated HaCaT in 12-well plates at 5 × 104 cells per well in DMEM-5 and cultured them for 3 days until reaching approximately 50% confluency, when the media was replaced with DMEM-0.5. The following day, the cells were stimulated with tumour necrosis factor (TNF)-α (30 ng/mL, 72 h), EGF (30 ng/mL, 24 h), or calcipotriol (10 nM, 72 h).
2.5. [3H]-Arachidonic Acid Release Assay
At 2 days post-confluency, we labelled HaCaT for 18 h with 3H-AA (0.4 μCi/mL) in DMEM/0.5% FBS. After labelling, the cells were washed twice with phosphate-buffered saline (PBS) containing fatty acid-free bovine serum albumin (BSA) (2 mg/mL) in order to remove unincorporated radioactivity. After stimulation (EGF 100 ng/mL, 60 min), the supernatants were cleared of detached cells by centrifugation (13,000 rpm, 10 min). The release of 3H-AA from the cells was assessed by liquid scintillation counting in a LS 6500 Multi-Purpose Scintillation Counter (Beckman Coulter, Brea, CA, USA). Adherent cells were dissolved in 1M NaOH in order to determine incorporated 3H-AA in the cells by liquid scintillation counting. The results are given as released 3H-AA in the supernatants relative to total 3H-AA incorporated into the cells.
2.6. Resazurin Assay
HaCaT were seeded in 96-well plates in DMEM-5 at a density of 3000 cells per well. Following 72 h of cultivation, when cells reached a density of approximately 50%, we replaced the medium with DMEM-0.5. The following day, the cells were treated with AVX001, in a series of eight wells per treatment, for 24h. Resazurin (RnD systems, Abingdon, United Kingdom) was added according to the manufacturer’s instructions and left to incubate for 2h at 37 °C with 5% CO2 in a humidified atmosphere. Fluorescence was read at 544 nm excitation and 590 nm emission wavelengths using the Cytation 5 cell imaging multimode reader (Biotek Instruments, Winooski, VT, USA).
2.7. High Throughput Microscopy Assay for Population Analysis of Cell Cycle and Apoptosis
Cells were seeded in Greiner Bio-one CELLSTAR 96-well flat clear flat-bottomed plates (BioNordika, Oslo, Norway) in DMEM-5 at a density of 3000 cells per well. After 72 h, when cells reached a density of approximately 50%, we replaced the medium with DMEM-0.5. The following day, cells were treated with vehicle, AVX001, or etoposide (10 µM) in DMEM-0.5 for 24 h. We then followed the manufacturer’s guidelines for the Click-iT 5-ethyl-2′-deoxyuridine (EdU) Alexa Fluor 594 imaging kit (ThermoFisher Scientific, Waltham, MA, USA) using a final concentration of 10 µM EdU per well incubated for a further 2 h at 37 °C, 5% CO2. Following incubation with EdU, we removed the media and replaced it with the CellEvent Caspase 3/7 Green detection reagent (ThermoFisher Scientific) prepared at 2 µM in Dulbecco’s (D)-PBS +5% FBS. The cells were incubated for a further 45 min, then the reagent was removed, and the cells were immediately fixed using 4% formaldehyde in D-PBS for 20 min on ice. Permeabilization was carried out using 0.1% Triton-X 100 in D-PBS and the Click-iT reaction was performed according to the manufacturer’s guidelines using the Alexa-594 picoyl azide to label incorporated EdU. Finally, the cells were counterstained by incubation with 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) (ThermoFisher Scientific) in D-PBS for 5 min. DAPI solution was removed and replaced with D-PBS for imaging. Plates were stored in the dark at 4 °C. All steps were performed at room temperature unless otherwise stated. Automated imaging was carried out on the Cytation5 cell imaging multimode reader (Biotek Instruments) at 4× magnification using DAPI, TexasRed, and GFP filter sets to image the DAPI, Alexa-594, and CellEvent Green signals, respectively. Four images were taken per well and 3 wells per treatment were used for the analysis.
Image analysis was performed in the freeware CellProfiler version 3.1.9 [48
]. Firstly, nuclei were segmented from DAPI images using an Ostu 2-class thresholding approach, and were then counted. In further analyses, filters were employed to remove images with fewer than 50 cells. For cell cycle analysis, the total DAPI intensity and total EdU staining were then measured per nuclei from 12 images per treatment group and the freeware Flowing version 2.5.1 (Perttu Terho, Turku Centre for Biotechnology) was used to identify cells in G1, G2, and S-phases of the cell cycle, with gating based on log10
total EdU intensity vs. total DNA intensity. Apoptotic cells were identified on the basis of robust-background thresholding of the CellEvent Green signal and reported as a percentage of the total number of cells per image. Four images were taken per well and data were based on 3 wells per treatment group. To calculate the proliferative index, we segmented EdU-positive cells using an Ostu 2-class thresholding approach and reported them as a proportion of the total number of cells per image. Four images were taken per well and data were based on 3 wells per treatment group.
2.8. RNA Extraction and Real-Time Quantitative PCR
Cells were seeded in 6-well plates in DMEM-5. Following 72 h of cultivation, when cells reached a density of approximately 50%, we replaced the medium with DMEM-0.5. The following day, the cells were preincubated with AVX001 for 2 h prior to stimulation with EGF (30 ng/mL, 4 h). Total RNA was extracted with Total RNA kit I from Omega BIO-TEK (Norcross, GA, USA) according to the manufacturer′s protocol. The amount and purity of the RNA samples were quantified using a Nanodrop One/OneC Microvolume UV–VIS Spectrophotometer (ND-ONE-W) from ThermoFisher Scientific. RNA samples with absorbance (A) A260/A230 between 1.8 and 2.1 and A260/280 between 2.0 and 2.2 were accepted. Reverse transcription was carried out using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) with 1 µg of RNA per sample, according to the manufacturer’s protocol. Real-time PCR analysis was performed using the LightCycler 480 SYBR Green I Master MIX and LightCycler 96 instrument from Roche (Basel, Switzerland), according to the manufacturer’s protocol.
2.9. 3D Culture of HaCaT Keratinocytes
3D stratified HaCaT cultures were grown in Nunc cell culture inserts (0.4 µm pore size) using the 24-well carrier plates system (Thermo Fisher Scientific #141002) The culture inserts were coated using the Coating Matrix kit (Thermofisher Scientific #R-011-K) according to the manufacturer’s protocol. HaCaT were plated at a density of 0.3 × 105 cells per insert in 0.5 mL DMEM-5 and incubated for 24 h before being lifted to the air–liquid interface. The media in the lower chamber was replaced with DMEM-5 (without antibiotics) + 1 ng/mL EGF, and 5 µg/mL L-ascorbic acid in the absence or presence of AVX001 (5 µM). The media in the lower chambers and treatments were changed every 3rd day for 12 days.
The cultures were fixed in 4% paraformaldehyde (PFA) overnight, before processing for paraffin embedding. Briefly, the membranes were removed from the inserts and prepared in Tissue Clear (Sakura, Osaka, Japan) for paraffin wax embedding using the Excelsior AS Tissue processor (ThermoFisher Scientific). Paraffin embedded sections (4 μm) were cut onto SuperFrost Plus slides (ThermoFisher scientific), dried over night at 37 °C, and then baked for 60 min at 60 °C. The sections were dewaxed in Tissue Clear and rehydrated through graded alcohols to water in an automatic slide stainer (Tissue-Tek Prisma, Sakura). Next, the sections were pretreated in Target Retrieval Solution, High pH (Dako, Glostrup, Denmark, K8004) in PT Link (Dako) for 20 min at 97 °C to facilitate antigen retrieval. The staining was performed according to the manufacturer’s procedure with EnVision G|2 Doublestain System Rabbit/Mouse (DAB+/Permanent Red) kit (Dako/Agilent K5361) on the Dako Autostainer. Following their soaking in wash buffer, we quenched endogenous peroxidase and alkaline phosphatase activity with Dual Endogenous Enzyme Block (Dako). Sections were then rinsed in wash buffer and incubated with primary antibody against Ki67 (MIB1 (Dako M7240)) diluted 1:300 for 40 min. The slides were rinsed before incubating in horseradish peroxidase (HRP) - polymer and 3,3′-Diaminobenzidine (DAB) to develop the stain. After a double stain block, the sections were incubated in antibody against cytokeratin 10 (Invitrogen #MA5-13705 diluted 1:100) for 60 min. After incubation in the mouse/rabbit linker, the sections were incubated in AP- polymer and the corresponding red substrate buffer with washing between each step. Tris-buffered saline (TBS; Dako K8007) was used throughout for the washing steps. The slides were lightly counterstained with hematoxylin, completely dried, and coverslipped. Appropriate negative controls were performed; both mouse monoclonal isotype control (Biolegend, San Diego, CA, USA) and omitting the primary antibody (negative method control).
2.10. Statistical Analysis
Statistical analysis was carried out in GraphPad Prism Software, version 7, using one-way ANOVA with Dunnet’s post-analysis. For normalized data, we used the Kruskal–Wallis test with Dunn’s post-analysis.
In this study, we investigated the effects of the cPLA2α inhibitor AVX001 on inflammatory eicosanoid release and epidermal proliferation to understand its mode of action for treating psoriatic skin disease.
We demonstrate for the first time that AVX001 can significantly and dose-dependently suppress the production of both COX and LOX AA metabolites in stimulated human PBMC. The findings are consistent with the use of the cPLA2
α inhibitors pyrrophenone and WAY-196025, which similarly inhibited both PGE2
release from PBMC stimulated with A23178 [68
]. Our data thus support the fact that targeting the cPLA2
α enzyme results in a balanced suppression of inflammatory eicosanoid release.
We further demonstrate the inhibitory effect of AVX001 on eicosanoid released in response to pro-inflammatory stimuli. The Toll-like receptor (TLR) 4 agonist LPS induced PGE2
release from PBMC, which was inhibited by AVX001. Our data using human PBMC supports the previously described involvement of cPLA2
α in LPS-stimulated PGE2
production in THP-1 monocytes [35
] reported to result from the induction of both the levels and activity of cPLA2
]. TNF-α is a pro-inflammatory cytokine and a key contributor to the pathogenesis of psoriasis [71
]. TNF-α induced a robust production of PGE2
in HaCaT, which was inhibited by AVX001. These findings support cPLA2α as a mediator of the pro-inflammatory effects of TNF-α, as proposed by Sjursen et al. [36
], however, while we demonstrated TNF-α-induced PGE2
production, Sjursen et al. reported that 6 h treatment with TNF-α preferentially induced HETE and not PGE2
production in HaCaT. It is therefore likely that stimulation of PGE2
release involves additional transcriptional upregulation of COX pathway enzymes in addition to cPLA2
α activation in these cells, as demonstrated by Seo et al. [72
Calcipotriol is a topical therapeutic for psoriasis and is known to cause skin irritation [54
]. We demonstrate that calcipotriol stimulates the release of PGE2
both in PBMC and keratinocytes, which is in agreement with the following studies [55
] and further supports the involvement of VDR/PGE2
signaling in drug-induced skin toxicity, as proposed by Shah et al. [56
]. The mechanism by which calcipotriol stimulates PGE2
production is unclear. In AVX001-treated cells, calcipotriol was unable to stimulate PGE2
release, implicating that cPLA2α activation is required. This is in contrast to studies in keratinocytes by Ravid et al. [55
], who suggest that the upregulation of COX-2 as opposed to increased AA production is responsible for the stimulation of PGE2
production. Doroudi et al. [74
] present a VDR-independent mechanism for activation of cPLA2
α by calcitriol via Ca2+
/calmodulin-dependent protein kinase II (CAMKII)-dependent phosphorylation. It will be interesting to determine how PGE2
is regulated by calcipotriol in PBMC and keratinocytes and whether direct activation of cPLA2
α by CAMKII is involved. For the treatment of psoriasis, calcipotriol is commonly combined with the potent corticosteroid betamethasone dipropionate (Daivobet), resulting in improved efficacy and tolerance [75
]. This poses the possibility that the use of AVX001 could be an interesting non-steroidal alternative combination partner for reducing inflammation and improving tolerance to calcipotriol.
Our finding that EGF-stimulated PGE2
release in keratinocytes is reduced by inhibition of cPLA2
α is in line with several reports linking EGF stimulation with AA release [76
]. Furthermore, Naini et al. describe a requirement for intact cPLA2
α/PGE2 signaling in growth factor-dependent cell cycle progression in both mouse embryonic fibroblasts (MEFs) and mesangial cells [38
]. Collectively, this puts regulation of the cPLA2
α enzyme, by means of its level and activity, in a central position to modulate growth factor-dependent responses. Thus, cPLA2
α may control both inflammatory and mitogenic processes, which are hallmarks of the pathogenesis of psoriasis.
We further show that treatment with AVX001 inhibits EGF-stimulated S-phase entry and reduces the proliferation of HaCaT keratinocytes grown both in monolayers and stratified cultures. Our findings are in agreement with the established role of cPLA2
α and eicosanoid signaling molecules as drivers of proliferation in several cancerous and non-cancerous cell types (reviewed in [80
] and [81
]). The described role of PGE2
as an autacoid growth factor [82
] and effector of EGF responses in keratinocytes [84
] make it a good candidate for mediating the effects of cPLA2
α inhibition on keratinocyte proliferation. Knockout of the PGE2
receptor, EP2, also supports a role for PGE2
in regulating keratinocyte proliferation [85
]. However, PGE2
is certainly not the only candidate, and a weakness of this study was our focus on the effects of AVX001 on AA metabolites. It is likely that cPLA2
α inhibition with AVX001 would also suppress the production of LPC and its metabolites, e.g. platelet-activating factor (PAF). Like the eicosanoids, PAF has pro-inflammatory and proliferative effects in the epidermis [46
], and PAF inhibition was found to suppress psoriasis-like skin disease progression in mice [89
]. In future studies, it will therefore be important to determine whether AVX001 can also suppress the formation of LPC metabolites, as well as to determine which lipid mediators are the most critical effectors of keratinocyte proliferation under conditions of chronic inflammation.