MDMs were isolated from a blood buffy coat of healthy blood donors. Briefly, buffy coats were transferred into 50 ml tubes and centrifuged at 3,000 rpm for 30 min. The WBC layer was collected and then diluted 1:1 in PBS-EDTA (1 mM). Diluted WBCs were gently overlaid on top of Ficoll-Paque solution (density 1.077 g/ml, GE Healthcare, Chicago, USA) at a 1:1 ratio and then centrifuged at 3,000 rpm for 30 min. The peripheral blood mononuclear cell (PBMC) layer at the interface was transferred to a new 50 ml tube and washed once with 40 ml PBS-EDTA by centrifugation at 1500 rpm for 5 min. The PBMCs were then diluted in 15 ml PBS-EDTA and overlaid on 46% Percoll solution (density 1.131 g/ml, GE Healthcare, Chicago, USA) at a 1:1 ratio followed by centrifugation at 3,000 rpm for 30 min. Then monocyte layer at the interface was collected into a new 50 ml tube, washed 3-5 times in 40 ml PBS-EDTA, and cultured in desired conditions. The purity of MDMs on days 0-6 was shown in Supplementary Figure 1 .

MDMs were cultured in RPMI 1640 supplemented with 10% human serum from buffy coats of healthy donors and 1% antibiotic-antimycotic (Gibco™, USA #15240062) at 37°C with 5% CO₂. The M0 macrophages condition, monocytes were grown in a complete medium without stimulation for 6 days. In the M1 macrophages, monocytes were cultured in the presence of 50 ng/ml of GM-CSF (ImmunoTools GmbH, Germany) for 6 days. In the M2 macrophages, monocytes were cultured in 50 ng/ml M-CSF (ImmunoTools GmbH, Germany) for 6 days. Cell culture mediums were replaced every 3 days in all conditions. On day 6, M0 macrophages were incubated in complete medium alone for 24 h to serve as naïve macrophage or negative control cells. M1 macrophages were polarized with 20 ng/ml IFN-γ (ImmunoTools GmbH, Germany) and 10 ng/ml LPS of Escherichia coli O55:B5 (Sigma Aldrich, MO, USA) for 24 h. M2 macrophages were polarized using 20 ng/ml IL-4 (ImmunoTools GmbH, Germany) for 24 h. For experimental groups, M0, M1, and M2 macrophages (day 6) were cultured in desired conditions plus oleamide for 24 h.

THP-1 cells obtained from ATCC were maintained in RPMI-1640 supplemented with 10% FBS and 1% Antibiotic-Antimycotic (Gibco™, USA #15240062) at 37°C with 5% CO₂. THP-1 were differentiated into macrophages by incubating with 100 nM phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) for 24 h and then replaced with PMA-free complete medium for 72 h. Following differentiation, cells were polarized into M1 macrophages by incubation with 20 ng/ml IFN-γ (ImmunoTools GmbH, Germany) and 10 ng/ml LPS (Sigma Aldrich, MO, USA) for 24 h. M2 macrophages were obtained by incubation with 20 ng/ml IL-4 (ImmunoTools GmbH, Germany) for 24 h. M0 macrophages were incubated with a complete medium only for 24. For experimental groups, M0, M1, and M2 macrophages were cultured in desired conditions plus oleamide for 24 h.

Isolated monocytes (Day 0) were seeded at a density of 4 x 10⁵ cells/well in 12-well plates and differentiated for 6 days in complete medium. Cell culture medium was replaced every 3 days. On day 6, MDMs were stimulated with 100 ng/ml LPS for 3 h. Cells were then stimulated with oleamide (10-40 µg/ml) (Sigma Aldrich, St. Louis, MO, USA) or adenosine triphosphate (ATP) (Sigma-Aldrich, St. Louis, MO, USA) as the positive control for 1 and 3 h. After stimulation, supernatants were collected, and cytokine levels were measured using an ELISA. Cells were collected, and qRT-PCR was used to examine mRNA expression.

Cell viability was determined using the Methyl Thiazol Tetrazolium (MTT) assay. MDMs (Day 6) were seeded at a density of 2.0 x 10⁴ cells/well in a 96-well plate and incubated with the serial concentration of oleamide for 48 h. The MTT salt solution (Thermo Fisher Scientific, Waltham, MA, USA) was then added and incubated for 3 h at 37°C. To dissolve the formazan crystal, 100 µl of dimethyl sulfoxide (DMSO) (VWR International, West Chester, PA, USA) was added to each well. Plates were gently shaken for 5-10 min. The absorbance was measured at 570 nm using a microplate reader (PerkinElmer, Inc., USA).

After stimulation, MDMs were harvested using Trypsin-EDTA for 15 min followed by a cell scraper. Cells were washed once with ice-cold PBS and resuspended in ice-cold FACS buffer (0.5% BSA and 0.05% sodium azide in PBS). Cells (2 x 10⁵ cells) were stained with the fluorochrome-conjugated anti-human antibodies, including CD80-FITC, CD163-PE, and CD206-FITC (BioLegend, San Diego, CA, USA), CD14-PE, CD16-FITC, and isotype control (ImmunoTools GmbH, Germany) for 30 min at 4°C in FACS buffer. The cells were then washed three times in ice-cold PBS and fixed with 2% formaldehyde (Sigma Aldrich, MO, USA) at 4°C for 10 min. After fixation, cells were washed once in PBS, resuspended in FACS buffer, and stored at 4°C in the dark until used. Cell surface expressions were detected using CytoFlex S (Beckman Coulter Life Sciences, Indiana, USA) and analyzed with CytoExpert software.

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized using Tetro cDNA Synthesis Kit (Bioline, Tennessee, USA). The resulting cDNAs were amplified with different primers ( Supplementary Table 1 ) using the SensiFAST™ SYBR ^® No-ROX Kit (Bioline USA Inc., Taunton, MA, USA). Results were analyzed using the relative gene expression method. Briefly, the relative expression of the target genes was calculated by relating the Ct-value of the target gene to unstimulated M0 macrophage. The quantitative cycle (Ct) values were used to calculate relative variations in gene expression between groups. These values were normalized to a housekeeping gene (β-actin) in the same sample (ΔCt) and expressed as the fold-change over control (2^−ΔΔCt). Real-time fluorescence detection was performed using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad).

MDM cells were plated into 12-cell well plate containing sterile coverslips. After differentiation, samples were fixed with 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (1.14 g NaH₂PO₄, 1.69 g Na₂HPO₄ in a 100-ml final volume of ddH₂O, pH 7.4) for 30 min at 4°C. Samples were washed three times for 5 min each in 0.1 M phosphate buffer and then dehydrated with graded ethanol (EtOH) series beginning with 25%, 50%, 75%, 95%, and 100% EtOH (5 min for each). Then, the cells were further dehydrated with 1:1 [Hexamethyldisilazane (HMDS): ETOH] for 5 min and 100% HMDS two times for 5 min at room temperature. Samples were then coated with gold particles and imaged with an Apreo 2 SEM (Thermo Fisher Scientific) using a 5 kV incident beam.

Levels of TNF-α, IL-6, IL-1ß and IL-18 cytokines were measured by a sandwich ELISA Kit according to the manufacturer’s instructions (Sino Biological Inc., China). Absorbance of the reaction was measured at 450 nm using an EnSpire^® Multimode microplate reader (PerkinElmer, Inc., MA, USA).

The release of lactate dehydrogenase (LDH) was measured using the CyQUANT LDH Cytotoxicity Assay (Thermo Fisher, Waltham, USA). MDMs were plated into a 96-well plate at a density of 2.0 x 10⁴ cells/well overnight, and then incubated with LPS for 3 h followed by oleamide (10 - 40 µg/ml) or ATP (30 mM) for 3 h. After incubation, 50 µl of culture medium was transferred to a new 96-well plate and 50 µl of LDH reaction mixture was added, mixed, and incubated for 30 min at room temperature. Then, 50 µl of stop solution was added to terminate the reactions. Absorbances of the reaction mixtures were measured at 490 nm and 680 nm.

Cells were washed with ice-cold PBS and lysed with RIPA buffer containing 1X Protease/Phosphatase Inhibitor Cocktail (Sigma Aldrich, St. Louis, MO, USA) for 30 min on ice. The lysate was then transferred into microcentrifuge tubes and centrifuged at 12,000 rpm for 10 min under 4°C. The supernatant containing whole protein was collected and measured for protein concentration by the Bradford assay. Equal amounts of each sample were loaded and separated in 8-12% of SDS-PAGE at 100 V for 1.30 h. Protein was then transferred onto PVDF membrane at 100 V for 1.30 h in an icebox. The membranes were blocked with TBST containing 5% BSA for 1 h and subsequently incubated with the appropriate primary antibodies; Rabbit mAb against IL-1β, cleaved- IL-1β, caspase-1, cleaved caspase-1, NLRP3, ASC/TMS1, P2X7 receptor (Cell Signaling Technology, MA, USA) at 4°C overnight. Membranes were washed 3-5 times in TBST for 5 min each and then incubated with Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, MA, USA) for 1 h at RT. After incubation, membranes were washed in TBST 3-5 times for 5 min each and the blots were developed by chemiluminescence solution for 1 min. Then, the protein was detected using ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA, USA).

All data were expressed as mean ± standard deviation (n = 3). For comparisons of more than two groups, one-way ANOVA was performed with multiple comparison correction (Dunnett test) using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). p-values < 0.05 were considered statistically significant.

To imitate naturally occurring macrophages, monocytes were freshly isolated from the blood buffy coat of healthy donors. MDMs were cultured in complete medium containing human serum alone (M0 cells), or complete medium containing GM-CSF (M1-like cells) or M-CSF (M2-like cells) for 6 days. MDMs were further cultivated for 24 h in the same medium (M0 macrophage) or in the presence of LPS plus IFN-γ (M1 macrophage) or IL-4 (M2 macrophage) ( Figure 1A ). The presence of GM-CSF led to a majority of round adhered with short-elongated shape, whereas the presence of M-CSF enhanced more elongation. MDMs cultured in complete medium containing human serum alone exhibited dual morphology (round adherend and elongated shape), indicating the mixed phenotypes in the M0 macrophages ( Figure 1B and Supplementary Figure 3 ). Differentiation of MDMs at days 0-6 were analyzed by flow cytometry using CD80 (M1 marker) and CD163 (M2 marker). Results showed that M1-like cells or the presence of GM-CSF upregulated CD80 expression, correlating with downregulation of CD163 at days 0-6, respectively. By contrast, M2-like cells displayed upregulation of CD163 expression with downregulation of CD80. MDMs cultured in a medium containing human serum alone displayed increased expression of CD163 rather than CD80 on the cell surface ( Figure 1C ). For fully polarized macrophages, LPS plus IFN-γ treated cells displayed strong induction of CD80 and downregulation of CD163 compared to unpolarized M0 cells. Conversely, IL-4-treated cells displayed high expression of CD163 and low levels of CD80 expression ( Figure 1E ). By using an inverted microscope and a SEM, the exhibit morphology of fully M0, M1, and M2 ( Figure 1D ). Collectively, these data confirmed the M1 and M2 polarization state of LPS plus IFN-γ and IL-4-treated MDMs.

To investigate the effect of oleamide on macrophage polarization, MDMs were cultured in conditioned media for M1 (LPS + IFN-γ), M2 (IL-4), and M0 (untreated) in the presence or absence of oleamide. These cells were characterized in different conditions based on surface markers, gene expression, and cytokine production. The presence of oleamide enhanced greater elongation as spindle-like shapes of the M0 and M1 macrophages ( Supplementary Figure 4 ). Surface expressions were analyzed by flow cytometry using polarization markers including CD80 for the M1 phenotype and CD163 and CD206 for the M2 phenotype. Flow cytometry assay revealed that CD80 expression was upregulated in M0 phenotypes treated with oleamide (from 53.09% to 62.17%), whereas CD163 and CD206 were downregulated in M0 phenotypes treated with oleamide compared to the untreated control (from 58.50% to 42.10% and 63.85% to 56.24%, respectively). For the M1 phenotype, CD163 and CD206 expression levels were downregulated (from 10.72% to 5.36% and 74.14% to 54.20%, respectively), whereas CD80 was not differenced in M1 treated with oleamide compared to M1 untreated control. For the M2 phenotype, CD163 and CD206 expression levels were downregulated (from 85.32% to 59.67% and 86.04% to 71.03%, respectively), whereas CD80 was upregulated in M2 treated with oleamide compared to the control (58.21% to 64.60%) ( Figure 2 ).

M1/M2 phenotypes were further confirmed by studying the expression of M1-associated genes (TNFα, IL-1β, IL-6, iNOS, CXCL10), along with the expression of M2-associated genes (Arg-1, CD206, and CCL22) using RT-qPCR. Results showed that M0 macrophages treated with oleamide displayed increased expression of TNF-α, IL-1β, IL-6, and iNOS genes, while M2 macrophages treated with oleamide displayed defective expression of M2-associated genes (Arg-1, CD206, and CCL22) along with increased expression of the IL-1β gene. For M1 macrophages, treatment with oleamide displayed a minor change in both M1and M2-associated genes ( Figure 3A ). To support these results, pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were determined by ELISA.

Interestingly, augment releasing of IL-1β was observed in all macrophage phenotypes after treatment with oleamide, whereas TNF-α and IL-6 increased in M0 macrophages but not significantly ( Figure 3B ). To confirm these results, cytokine production was further investigated using the THP-1 derived macrophage model. Surprisingly, increasing IL-1β production was also observed in M0 macrophages treated with oleamide, similar to the MDM model ( Figure 3C ). Therefore, this suggested that oleamide induced IL-1 β production in both MDMs and THP-1 cells. Taken together, these results indicated that oleamide mediated naïve macrophages (M0) toward the M1 phenotype and promoted IL-1β production while hindering the polarization of M2 phenotypes.

An increase of IL-1β production was observed in both MDMs and THP-1 treated with oleamide, suggesting that oleamide functioned as a secondary signal to trigger NLRP3 inflammasome activation and lead to the production of IL-1β. To explore this hypothesis, naïve macrophages (M0) were primed with LPS (signal 1) followed by oleamide treatment for 1-3 h ( Figure 4A ). Then, cell morphology was observed using a bright-field microscope( Figure 4B ). Cell-free supernatant was detected for IL-1β and IL-18 secretion and inflammasome-related genes (IL-1β, IL-18, ASC, NLRP3) were analyzed by RT-qPCR. As predicted, IL-1β and IL-18 were readily produced in LPS-primed MDMs exposed to oleamide in a dose- and time-dependent manner ( Figure 4C ). Furthermore, a high concentration of oleamide (30-40 µg/ml) induced IL-1β and IL-18 production equivalent to ATP, which is known as the NLRP3 inflammasome activation molecule. Consistent with results obtained using cell-free supernatant, oleamide induced upregulation of NLRP3, IL-1β, and IL-18 mRNA expression compared to the control cells ( Figure 4D ). Therefore, results suggested that oleamide mediated IL-1β and IL-18 production by triggering the NLRP3 inflammasome pathway.

To further investigate how oleamide impacted NLRP3 inflammasome activation and IL-1β production, LPS-primed MDMs were treated with oleamide for 3 h. Results showed that oleamide induced activation of NLRP3 and ASC speck formation similar to induction by ATP, which activated the NLRP3 inflammasome via the purinergic P2X7 receptor ( Figure 5A ). Both IL-1β and IL-18 are initially produced as biologically inactive pro-forms that require cleavage into mature cytokines. Typically, this processing is mediated by caspase-1, which is activated following the formation of an inflammasome (20). Our results showed that oleamide induced upregulation of cleaved caspase 1 and cleaved IL-1β in the supernatant, correlating to downregulation of these proteins in cell lysate ( Figure 5A ). The release of lactate dehydrogenase (LDH) induced by oleamide in LPS-primed MDMs significantly increased in a dose-dependent manner ( Figure 5B ), suggesting that oleamide increased caspase-1-mediated pyroptosis. Similar to LDH, an increase in cell death was observed in a dose-dependent manner ( Figure 5C ). These findings suggested that oleamide induced activation of the NLRP3 inflammasome pathway and mediated IL-1β production in LPS-primed MDMs.