The human monocyte THP-1 cell line (TIB-202) was purchased from ATCC (Manassas, VA, USA) and was maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 1% glutamine, 1% sodium pyruvate (Invitrogen), and 50 µg/ml gentamicin (Mediatech) in a tissue culture incubator at 37°C with 5% CO₂ and 95% relative humidity. WFA was purchased from ChromaDex. Adenosine triphosphate (ATP), Nigericin, monosodium urate (MSU), and phorbol myristate acetate (PMA) were from Sigma-Aldrich; LPS was purchased from eBioscience. Anti-NLRP3 and ASC antibodies were purchased from Millipore and AdipoGen, respectively. Antibodies to NF-κB, p65 and Histone H3 were obtained from Abcam; antibodies to IL-1β, IL-18, caspase-1, β-actin, and secondary antibodies were purchased from Santa Cruz Biotechnology.

Human cytokine profiling was performed using a Proteome Profiler Human XL Cytokine Array Kit (R&D Systems), which detects 102 human soluble cytokines. THP-1 cells were seeded at a density of 5 × 10⁵ cells per well in a six-well plate in the presence of PMA (500 ng/mL) to differentiate the cells into macrophages (21, 22). Following overnight incubation and addition of LPS (200 ng/mL) for 2 h, ATP (1 mM) was added to the cultured cells to induce cytokine production in the presence or absence of WFA (20 µM). Following incubation for 4 h, culture supernatant was collected, and the cells were lysed to isolate RNA. Human XL Cytokine Arrays were incubated overnight at 4°C with 1.5 mL of the cell culture supernatant, and the procedure was performed according to the manufacturer’s instructions. Following incubation with a detection antibody cocktail, antibody conjugation, and recommended washes, the immunoblots on the membrane were developed with Chemiluminescent Substrate Reagent Kit (Invitrogen) and were exposed to X-ray film. Cytokine array TIF file images were analyzed using ImaGene 8.0 software (BioDiscovery, Inc.). After uploading images into the software, a grid file was manually over imposed on them to assign spot gene identities. In the next step, spot finding and automated segmentation-proprietary algorithms were used to determine both signal and background intensities. For background correction, median background intensities were subtracted from mean signal intensities. To further calculate the differential expression, the mean signal intensity of the cytokine/chemokine spots was divided by the mean signal intensity of the reference spots (n = 6) for each membrane.

Based on Figure 1E, the gene coding promoter regions for some of the cytokines/chemokines, IL-1β, GM-CSF, B-cell activating factor (BAFF), sex hormone binding globulin (SHBG), chemokine (C-C motif) ligand (CCL2/MCP-1, and CCL17/TARC), cystatin-3 (CST3), thrombospondin-1 (TSP-1), relaxin-2 (RLN2), tumor necrosis factor receptor superfamily 8 (TNFRSF8/CD30), and adiponectin (ACRP30), were screened for NF-κB transcription factor binding sites using MatInspector software 8.4 with Matrix Library 10.0 from the Genomatix software suite (http://www.genomatix.de/cgi-bin/eldorado/main.pl). The parameters for searching the database for NF-κB binding sites were set at core similarity of 1.00 (maximum) with optimized matrix similarity.

Total RNA was isolated from the THP-1 cells using an RNA isolation kit (RNeasy Mini Kit, Qiagen), and cDNA was synthesized by reverse transcriptase (Superscript II; Invitrogen). The level of mRNA expression of the differentially expressed cytokines was performed using FAST SYBR Master Mix on StepOnePlus Real-Time PCR (Applied Biosystems). The primer sequences to amplify the cytokine gene products are given in the Table S1 in Supplementary Material.

Cell lysates were prepared with RIPA buffer (Sigma-Aldrich) using the standard protocol. Nuclear and cytoplasmic fraction analysis from the THP-1 cells was performed with nuclear and cytoplasmic extraction reagents (Thermo Scientific) according to the manufacturer’s protocol. Briefly, the harvested cells were suspended in lysis buffer containing a proteinase inhibitor cocktail (Sigma-Aldrich) and centrifuged at 16,000 × g for 5 m. The supernatant was saved for cytoplasmic extracts, and the pellet was re-suspended in a special buffer (NER) for the nuclear fraction. After being centrifuged at 16,000 × g for 10 m, the supernatant was collected for nuclear extract. The collected supernatant from the total cell lysates or the cytosol and nucleus were subjected to SDS-PAGE western blot analysis and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membranes were incubated with the indicated primary antibodies at a dilution of 1:500 and HRP-conjugated secondary antibodies (dilution 1:2,000). The immune-reactive bands were visualized using a chemiluminescent substrate (Invitrogen) and were exposed to X-ray film.

Culture supernatant was harvested from the THP-1 cells following treatments, and ELISA assays for human caspase-1, IL-1β (R&D Systems), and IL-18 (MBL) were performed using ELISA kits following manufacturer’s instructions.

THP-1 cells were seeded with 50% confluence on a coverslip in six-well plates. While in the wells, the cells were treated with nigericin with or without WFA. After removing the medium, cells were washed with phosphate-buffered saline (PBS) and then fixed with 2% paraformaldehyde for 10 min. Following subsequent washing and blocking with 5% bovine serum albumin (BSA) in PBS, the cells were incubated with ASC or NLRP3 antibody in 1% BSA for 60 min. After rinsing with 1% BSA in PBS, the cells were incubated with secondary antibody (1:500) Alexa Fluor 488 (green-fluorescent dye from Life Technologies) and Cyanine Cy^TM3 (red-fluorescent dye from Immune Research) for 60 min. Cells were rinsed with 1% BSA in PBS, and then incubated with DAPI for nuclear staining for 5 min at room temperature. The cells were rinsed, and mounted using fluorescence mounting medium (Dako). The slides were analyzed under the confocal scanning laser microscope (Zeiss LSM 510 Meta).

We used the “t-test” of unequal variance, and one-way analysis of variance (ANOVA) to calculate the differences among treatments. For the ANOVA, the comparisons between each treatment was analyzed using LPS treatment as control, and corrected for multiple comparisons by applying Dunnett’s test. All the statistical analyses were performed using GraphPad Prism 6 software.

We used a membrane-based sandwich immunoassay (Proteome Profiler Human XL Cytokine Array) to examine the expression profile of inflammatory cytokines. Culture supernatant from the human THP-1 cell derived macrophages treated with or without WFA was hybridized on cytokine array membranes. The cytokines/chemokines spot intensity signals are shown in Figure 1 (Figure 1A: LPS control, Figure 1B: ATP primed cells, and Figure 1C: WFA treatment). Adenosine triphosphate (ATP), an activator of the NLRP3 inflammasome, was used for promoting cytokine secretion by the THP-1 cells. Following image analysis and the quantification of pixel intensity for each spot, 41 cytokine genes showed differential expression of >1.5-fold between THP-1 cells treated with or without WFA (Figures 1B,C). The names of these differentially expressed 41 cytokines/chemokines are presented on a schematic grid that corresponds to the spot positions on the membrane (Figure 1D). Of these 41 cytokines, 38 (IL-1α, IL-1β, IL-2, IL-3, IL-5, IL-10, IL-12p70, IL-18BPα, IL-13, IL-23, IL-33, IL-34, IP-10, GM-CSF, PDFG-AA, CCL2/MCP-1, CCL17/TARC, SDF1α/CXCL12, CCL20/MIP-3α, KLK3, angiopoientin-1, IGFBP-2, TFF3, BAFF, BDNF, FLT3LG, IGFBP-3, ACRP30/adiponectin, GH, Leptin, LIF, SHBG, aggrecan, angiogenin, HGF, NGAL, TSP-1, and CST3) were downregulated, and 3 (MPO, RLN2, and TNFRSF8/CD30) were upregulated in ATP-stimulated WFA treated THP-1 cells (Figure 1E).

To understand the differential expression of these cytokines and to identify a common target, we screened the gene coding promoters of the randomly selected 11 cytokines/chemokines. Transcriptional analysis showed that 6 (IL-1β, GM-CSF, CCL2/MCP-1, BAFF, SHBG, and ACRP30/adiponectin) of the 9 downregulated cytokines contain NF-κB binding sites (Table 1). Three cytokines (CST3, CCL17/TARC, and TSP-1) lack NF-κB binding sites on their promoter region; however, analysis of CCL17/TARC and TSP-1 showed the c-Rel transcriptional factor, which co-cites with NF-κB. In contrast, two upregulated cytokine genes (RLN2 and TNFRSF8/CD30) neither co-cite with nor contain NF-κB sites. This analysis leads to the hypothesis that a collective decrease in production of a group of cytokines/chemokines may be regulated via a common factor, NF-κB.

To validate the effect of WFA on the modulation of targeted cytokines/chemokines, we performed quantitative real-time PCR on samples harvested from THP-1 cells following treatment with or without WFA in presence of ATP. These data showed that the mRNA levels of RLN2 (P = 0.027) and TNFRSF8/CD30 (P = 0.023) in LPS-primed ATP-stimulated THP-1 cells were significantly induced by the WFA treatment, while IL-1β (P = 0.005), GM-CSF (P = 0.043), PTX3 (P = 0.008), and CST3 (P = 0.003) were significantly reduced compared to ATP-treated controls (Figure 2A). ATP-induced IL-1β downregulation due to WFA was further validated at the protein levels by ELISA assay (P = 0.001) and western blot (Figure 2B). Although non-significant, downregulation of CCL2/MCP-1 (P = 0.18) and PDGF-AA (P = 0.171) was also observed following WFA treatment. Thus, the mRNA expression levels corroborate the observations of differentially expressed cytokines/chemokines on the array as in Figure 1. The mRNA transcript level for ACRP30/adiponectin (P = 0.399) remained unchanged following WFA treatment, and it did not match the observed expression on the cytokine array. It is possible that WFA could regulate ACRP30/adiponectin expression at the protein level and may not affect the mRNA transcript; however, more studies are required to establish this observation.

In silico promoter analysis revealed that the downregulated cytokines/chemokines possess either NF-κB transcriptional binding sites or co-cites with NF-κB. Therefore, to gain an insight into collective downregulation of multiple cytokines, we examined the activity of NF-κB on nuclear and cytoplasmic fractions in LPS-primed THP-1 cells following WFA treatment. These data showed the inhibition of NF-κB translocation following WFA treatment, supporting that the collective downregulation of the identified cytokines/chemokines may be due to inhibition of NF-κB activation (Figure 3).

Using the NLRP3 inflammasome-specific stimulator nigericin, we sought to examine the effect of WFA on the NLRP3 inflammasome, which consists of three proteins (NLRP3, ASC, and pro-caspase-1). Interestingly, we did not observe any significant change in the protein expression levels of NLRP3 (P = 0.712), ASC (P = 0.319), or pro-caspase-1 (P = 0.999) (Figure 4). However, the downstream target protein pro-IL-1β was significantly reduced (P = 0.002) in a dose-dependent manner, while pro-IL-18 remained unchanged (P = 0.999). The analysis of variance (ANOVA) further revealed that the dispersion of the means across various treatments was minimal for NLRP3 (F = 0.585), ASC (F = 1.695), pro-caspase-1 (F = 0.012), and IL-18 (F = 0.033). Analysis of active and mature forms of caspase-1, IL-1β, and IL-18 revealed that WFA significantly inhibits the secretion of caspase-1 (P < 0.0001), IL-1β (P < 0.0001), and IL-18 (P < 0.0001) in THP-1 cells induced by nigericin (Figure 4). Based on the ANOVA, F-value indicates that the dispersion of the mean differences between the treatments was maximum for IL-18 (F = 1516.0) as compared to caspase-1 (F = 237.4) and IL-1β (F = 190.4). Similar results were obtained by using MSU, which is also an NLRP3 inflammasome-specific stimulator (Figure S1 in Supplementary Material).

Further examination of the localization of inflammasome complex proteins showed that NLRP3 and ASC are located both in the cytoplasm and the nucleus; however, the co-localization of ASC and NLRP3 seems outside the nucleus (Figure 5; PMA control). Following NLRP3 inflammasome activator nigericin, the co-localization of NLRP3 and ASC appeared more dominant outside around the nuclear membrane (Figure 5; Nig). This strong co-localization due to nigercin activation may be necessary for the inflammasome complex activity leading to increased secretion of caspase-1, IL-1β, and IL-18 (Figure 4). WFA treatment of the nigercin-primed THP-1 cells showed an altered distribution and disintegrated pattern of both co-localized and individual NLRP3 and ASC proteins inside and outside of the nucleus (Figure 5). Because of such unevenly distributed and fragmented localization of NLRP3 and ASC, it is likely that WFA blocks the activity of NLRP3 inflammasome complex, and hence diminishing the secretion of downstream targets (caspase-1, IL-1β, and IL-18). To further support our observations, we also analyzed the localization of NLRP3 and ASC following ATP (an activator of the NLRP3 inflammasome) stimulation of THP-1 cells with or without WFA. Similar results of altered and fragmented localization of NLRP3 and ASC proteins were observed following WFA treatment (Figure S2 in Supplementary Material).