MicroRNA-302b negatively regulates IL-1β production in response to MSU crystals by targeting IRAK4 and EphA2

THP-1 cells from American Type Culture Collection (Manassas, VA, USA) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and penicillin–streptomycin. THP-1 cells were exposed to 100 ng/ml phorbol myristate acetate (PMA; Beyotime Biotechnology, Shanghai, China) on the day before the indicated experiment.

Eight-week-old male BALB/c mice were purchased from the Dossy Experimental Animals Company (Chengdu, China). Animals were housed in a specific pathogen-free facility of the State Key Laboratory of Biotherapy, Sichuan University, Chengdu, China. All animal studies were approved by the Ethics Committee of the State Key Laboratory of Biotherapy, Sichuan University.

A total of 38 human serum samples were obtained from 18 patients diagnosed with acute gouty arthritis during an acute gout flare, and 20 healthy subjects were randomly selected from healthy individuals who participated in a physical examination at the West China Hospital from June 2017 and July 2017 (Additional file 1: Table S1). Once serum samples were collected, they were stored immediately at −80 °C. Written informed consents were provided by all participants, and the study was approved by the Ethics Committee of the West China Hospital, Sichuan University.

MSU crystals were purchased from InvivoGen (San Diego, CA, USA) and used freshly for in-vitro treatment by dissolving in phosphate buffered saline (PBS). Antibodies for cleaved caspase-1 (p20), phospho-NF-κB p65 (ser536), and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Human and mouse mature IL-1β were measured using the IL-1β ELISA kit (Neobioscience, Shenzhen, China). PerCP-CD45, PE-F4/80, and APC-Gr-1 antibodies were obtained from BioLegend (San Diego, CA, USA).

THP-1 cells were seeded in six-well or 24-well plates with 100 ng/ml PMA overnight. After washing the cells once with PBS, they were transfected with negative control miRNA mimics (NS-m), miR-302b mimics (302b-m), negative control siRNA (si-NC), IRAK4 siRNA (si-IRAK4), or EphA2 siRNA (si-EphA2) purchased from RiboBio Co., Ltd (Guangzhou, China) using Lipofectamine® 3000 (Life Technologies, Grand Island, NY, USA).

Total RNA was isolated from samples with TRIzol™ Reagent (Life Technologies) and dissolved in RNase-free water. For human serum samples, Caenorhabditis elegans miRNA cel-miR-39 was added as a synthetic spike-in control RNA. For mature miRNA detection, reverse transcription was performed using a miScript II RT Kit (Qiagen, Valencia, CA, USA), and real-time qPCR was performed using a miScript SYBR® Green PCR Kit (Qiagen). For mRNA detection, reverse transcription was performed using a PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China), and real-time qPCR was performed using SYBR® Premix Ex Taq™ II (Takara). The miRNA and mRNA detections were performed on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), and the data were analyzed with CFX Manager™ software version 3.1 (Bio-Rad). The levels of mature miRNA were normalized against the control U6 snRNA (human source cell samples), sno202 (mouse source cell samples), or cel-miR-39 (human source serum samples). The levels of EphA2 and IRAK4 were normalized against GAPDH. The primers used in this study are presented in Additional file 1: Table S2.

THP-1 cells were cotransfected with miRNA (NS-m or 302b-m) and the luciferase reporter vector containing wild-type or point-mutated 3′ UTR (WT UTR or mutant UTR) of IRAK4 and EphA2 using Lipofectamine® 3000 (Life Technologies). Luciferase expression levels were measured at 24 h post transfection using a dual-luciferase reporter assay system according to the manufacturer’s instructions (Promega, Madison, WI, USA).

The antibodies for β-actin (1:1000), phosphor-NF-κB p65 (1:1000), and cleaved caspase-1 p20 (1:1000) were used for western blot analysis. The quantitative analysis for the results of the western blot analysis was performed using the Gel-Pro analyzer 4.0 (Media Cybernetics, Bethesda, MD, USA).

A Boyden chamber with an 8-μm porous membrane (Corning) in the 24-well plate was used for the migration assay. Briefly, THP-1 cells were transfected with NS-m, 302b-m, si-NC, or si-EphA2 for 48 h. The cell numbers were counted with a hemocytometer and resuspended with RPMI 1640 medium without serum. Then 500 μl cell suspension containing the indicated cell number was loaded into the Boyden chamber, whereas 1 ml RPMI 1640 medium with 5% serum was placed in the bottom compartment. After incubating at 37 °C for 24 h, cells on the upper side of membranes were removed. The migratory cells on the lower side of the membrane were stained with crystal violet and then counted under light microscope.

THP-1 cells were transfected with NS-m or 302b-m and si-EphA2 or NC respectively for 48 h, and then the cells were treated with MSU for another 1 h. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton-X 100. Rhodamine phalloidin plus DAPI were diluted into PBS, and the cells were incubated at room temperature in the dark for 30 min. Rhodamine phalloidin-labeled F-actin (red) and DAPI-labeled nuclei (blue) were detected using confocal microscopy (Nikon TI-DH, Japan).

All statistical analyses were conducted with SPSS 21.0 software. Data are presented as the mean ± SDEVs or SEMs. Statistical analysis was performed using Student’s t test for comparing two groups. Differences in the mean values were considered to be significant at p < 0.05.

PMA-induced differentiation of THP-1 monocytes into macrophages was used as an in-vitro experimental model. Since IL-1β is an indicator of gout inflammation [12], we first explored a rational MSU treatment for subsequent assays by detecting the expression of IL-1β. The PMA-induced THP-1 cells were treated with 150 μg/ml MSU at different time points from 0 to 48 h. The results showed that the IL-1β protein level was increased in a time-dependent manner upon MSU stimulation (Fig. 1a). Then we analyzed miR-302b expression in THP-1 cells with/without MSU treatment, showing that miR-302b expression was significantly upregulated at 3 h post MSU treatment (Fig. 1b; Additional file 1: Table S2). Based on the established mice air pouch model, the air membranes were dissected to detect the possible alteration for the miR-302b expression. As shown in Fig. 1c, the expression of miR-302b in mouse air membranes was also significantly increased in the MSU-treated group, suggesting that miR-302b might be involved in MSU-induced gouty arthritis.

To determine the potential role of miR-302b in MSU treatment, we examined the effect of miR-302b on MSU-induced inflammatory cytokine gene expression in THP-1 cells. We first showed by qRT-PCR that the chemically synthesized miRNA mimics (302b-m) could strikingly elevate the expression level of miR-302b (Fig. 1d) and inhibit MSU-induced IL-1β mRNA expression compared to a miRNA mimic negative control (NS-m) (Fig. 1e). Consistent with this result, ELISA was able to show that the protein level of IL-1β in cells overexpressing miR-302b was also lower compared to the cells transfected with the NS-m (Fig. 1f).

Given the correlation between miR-302b and IL-1β expression, we next sought to investigate the role of miR-30b in a widely used MSU-induced mouse air pouch inflammatory model [13‐15]. miR-302b agomir (302b-a) or a negative control miRNA agomir (NS-a) was reconstituted with PBS and air pouch administration (1 nmol/mouse/per dose) 2 and 4 days prior to MSU treatment (Fig. 2a). miR-302b expression was first examined in mouse air pouch membrane tissues. Two doses after 302b-a injection, miR-302b was found to be increased more than 700 times in the presence of NS-a (Fig. 2b). As expected, the IL-1β expression level in air pouch fluid was reduced in the 302b-a group compared with that of NS-a after MSU stimulation (Fig. 2c). Furthermore, it has been reported that MSU could induce macrophage and neutrophil infiltration into the air pouch [16]. To further verify the relationship between the reported phenomenon and IL-1β expression, we next assessed the number of macrophages and neutrophils infiltrating the air pouch by staining the cells with macrophage marker F4/80 and neutrophil marker Gr-1. As shown in Fig. 2d, MSU enhanced the infiltration of macrophage and neutrophils into the air pouch, but this was dramatically inhibited by 302b-a. Notably, there was no difference between the ratio of infiltrating macrophages and neutrophils in the 302b-a and NS-a groups (Fig. 2d). In addition, 302b-a also attenuated leukocyte infiltration in the air pouch tissues induced by MSU, as shown by the histological examination (Fig. 2e). Thus, these findings suggest that miR-302b played a negative regulatory role in regulating the expression of MSU-induced inflammatory cytokines in gout.

These results have shown that miR-302b can reduce MSU-induced IL-1β expression at both mRNA and protein levels, indicating that miR-302b may be involved in the transcriptional and maturation process of IL-1β. It is well known that TLR/NF-κB signaling regulates the transcriptional activity of IL-1β, and MSU-induced NLRP3/Caspase-1 activation is involved in the maturation of IL-1β [5, 11]. We next elucidated the direct effects of miR-302b on the activation of NF-κB and caspase-1 during MSU stimulation. Subsequently, the effect of miR-302b on phosphorylation of the NF-κB p65 subunit in THP-1 cells was measured, indicating that the phosphorylation of NF-κB p65 subunit was majorly eliminated in 302b-m-transfected THP-1 cells when compared with the cells transfected with NS-m (Fig. 3a). Active caspase-1 heterodimer is formed by autoproteolysis-mediated pro-caspase-1 activation following inflammasome assembly, and this cleavage is the hallmark of inflammasome activation, which is required for pyroptosis and IL-1β secretion [17]. In our setting, we found that the level of cleaved caspase-1 p20 subunit was suppressed by miR-302b, thereby indicating the identical tendency between the cleaved caspase-1 p20 and the cleavage of precursor IL-1β-produced mature IL-1β (Fig. 3a).

To determine whether miR-302b has any effect on NF-κB signaling and inflammasome activation in vivo, we then dissected the air pouch membranes used for Fig. 2 to detect the phosphorylation of the NF-κB p65 subunit and the cleaved caspase-1 p20 subunit. As shown in Fig. 3b, enforced expression of miR-302b via agomir in air pouch membranes reduced the endogenous level of the phosphorylation of NF-κB p65 and cleaved caspase-1 p20 (Fig. 3b). These data suggest that the inhibitory role of miR-302b in MSU-induced proinflammatory gene expression is due to its effects on the TLR signaling pathway from downregulating the activation of NF-κB and the NLRP3 inflammasome signaling pathway by impairing the activation of capase-1.

We further used bioinformatics tools, such as miRDB, TargetScan, and miRanda, to predict the molecular targets of miR-302b. Then we paid attention to the recurrent genes associated with inflammation in all three applications, and interleukin-1 receptor-associated kinase 4 (IRAK4) and Eph receptor A2 (EphA2) were the hot targets of miR-302b. The three-prime untranslated region (3′-UTR) of IRAK4 and EphA2 was predicted to be bound by the “seed sequence” of miR-302b (Fig. 4a). IRAK4 is an important upstream molecule of the NF-κB signaling pathway and has been shown to regulate proinflammatory gene expression in a bacterial infection model [11]. Additionally, EphA2 is a member of the Eph receptor tyrosine kinase (RTK) family and regulates inflammation and immune cell trafficking [18, 19]. RT-qPCR results were firstly used to confirm that IRAK4 and EphA2 were the targets of miR-302b target (Fig. 4b, c). We further determined that IRAK4 and EphA2 expression was enhanced upon MSU treatment, found in MSU-stimulated THP-1 cells (Fig. 4b, c), and these results indicated that IRAK4 and EphA2 may function in MSU-induced inflammation. To further confirm the target genes of miR-302b, luciferase constructs containing mutated 3′ UTR of IRAK4 and EphA2 were used in the luciferase reporter gene experiments. As shown in Fig. 4d, overexpression of miR-302b inhibited the activity of a luciferase reporter construct containing IRAK4 3′ UTR and EphA2 3′ UTR, but not the mutated 3′ UTR of IRAK4 and EphA2 (Fig. 4d).

We subsequently evaluated whether IRAK4 and EphA2 were the functional targets of miR-302b by siRNA-mediated IRAK4 and EphA2 knockdown in the THP-1 cells (Fig. 4e, h), and ELISA results showed that the IL-1β protein expression level was repressed in the supernatant of THP-1 cells transfected with IRAK4 or EphA2 siRNA compared to the controls (Fig. 4f, i). IRAK4 has been shown to play a critical role in TLR/NF-κB-dependent immune responses by inducing NF-κB phosphorylation [20]. Our western blot analysis showed that the phosphorylation level of the NF-κB p65 subunit in THP-1 cells transfected with IRAK4 siRNA (si-IRAK4) was significantly decreased compared to the cells transfected with control siRNA (si-NC) (Fig. 4g). However, there was no significant change in the cleavage of the caspase-1 p20 subunit level after IRAK4 silencing (Additional file 1: Figure S1a). Silencing EphA2 in THP-1 cells resulted in decreased cleavage of the caspase-1 p20 subunit, but did not affect the phosphorylation level of NF-κB p65 subunit, compared to si-NC transfected cells (Fig. 4j, Additional file 1: Figure S1b). Collectively, these data strongly indicated that miR-302b inhibits the NF-κB signaling-mediated IL-1β mRNA transcription by directly targeting IRAK4, and inhibits caspase-1-mediated IL-1β protein maturation by directly targeting EphA2. Thus, miR-302b regulated the production of mature IL-1β in the two aspects.

Additionally, MSU phagocytosed by resident macrophages resulted in the production of inflammatory cytokines and chemokines, including TNF-α [21]. Indeed, our data also showed the upregulated expression of TNF-α in macrophages after exposure to MSU. The expression level of TNF-α could be downregulated by miR-302b (Additional file 1: Figure S2a). However, these two targets, IRAK4 and EphA2, had no effect on the regulation of TNF-α expression (Additional file 1: Figure S2b, c).

Our results suggested that miR-302b attenuated macrophage and neutrophil infiltration (Fig. 2), and previous studies reported that EphA2 could regulate the expression of cytokines and leukocyte infiltration in a mouse model [22‐24]. To determine whether miR-302b and EphA2 influence cell migration, we next investigated the effects of miR-302b and EphA2 on the migration ability of THP-1 cells. Filament actin (F-actin) plays a very important role in cell movement and migration, and the formation of F-actin is a marker of cell transformation [25]. Phalloidin staining results showed that the formation of F-actin was largely suppressed in 302b-m or si-EphA2-transfected THP-1 cells with MSU treatment (Fig. 5a). Meanwhile, we determined THP-1 cell migration by staining the nuclei of the migratory cells on the underside of the inserted Transwell membrane. As expected, THP-1 cells transfected with NS-m or si-NC had markedly high migration ability, and the migrated cell number of miR-302b or si-EphA2 transfection decreased approximately 30% and 50%, respectively (Fig. 5b). These results suggested that miR-302b and EphA2 may be involved in the recruitment of inflammatory cells into the inflammatory site by regulating the migration of cells during the MSU-induced inflammatory response in gouty arthritis.

To examine the prognostic value of circulating miR-302b in gouty arthritis (GA), we finally determined the levels of miR-302b in the serum of GA patients during an acute gout flare. The characteristics of healthy controls and GA patients are summarized in Additional file 1: Table S1. As shown in Additional file 1: Figure S3, the levels of circulating miR-302b were changed significantly in GA patients compared to controls. These results indicated that the elevated expression of miR-302b was obviously associated with GA patients. Collectively, our findings implicate that miR-302b plays a crucial role in the development of gout flares and represents a novel strategy to prevent excessive inflammation in gouty arthritis (Fig. 6).