Background
Gout is a prevalent disease manifesting most commonly as episodes of acute and extremely painful arthritis. Monosodium urate (MSU) crystals, a crystallized form of uric acid, deposit in joints and other tissues and induce the production of pro-inflammatory factors such as interleukin 1-beta (IL-1β), resulting in aseptic inflammation [
1,
2].
MicroRNAs (miRNAs) constitute an abundant class of small, evolutionary conserved non-coding RNAs that function as post-transcriptional regulators [
3]. Increasing evidence has demonstrated that miRNAs regulate gene expression by triggering translational inhibition and/or degradation of the targeted messenger in infectious and autoimmune diseases [
4,
5]. One miRNA in particular, miR-146a, has been shown to act as a negative-feedback effecter in the inflammatory signaling pathway initiated by NF-κB [
6], and it directly downregulates the production of pro-inflammatory cytokines by targeting TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase (IRAK1), which are components of the cascade downstream of toll-like receptors and act as critical mediators of inflammation via impairment of NF-κB activity, to regulate innate immunity [
6,
7]. Although the function and mechanism of miR-146a in many immune and rheumatic diseases has been investigated [
6‐
9], little is known about its role in gouty arthritis. One report from Dalbeth et al. shows that miR-146a functions as a transcriptional break that is lost during acute inflammatory responses triggered by the presence of MSU crystals [
10]. Unfortunately, the specific mechanisms of miR-146a regulates gouty arthritis were not explored in this study. Here, we sought to determinate the role of miR-146a and its mechanism of action in gouty arthritis using a miR-146a-deficient animal model.
Methods
Animals
MiR-146a knockout (KO) and B6 (wild-type (WT)) mice were housed at 24 ± 2 °C under a 12-h light/12-h dark cycle in a pathogen-free facility. Handing of mice and experimental procedures were in accordance with requirements of the Institutional Animal Care and Use Committee and this study was granted permission by the Ethics Committee of the Affiliated Hospital of North Sichuan Medical College.
Preparing mouse bone marrow-derived macrophages (BMDMs) and MSU
Bone marrow cells were isolated from the femurs and tibias of the mice by flushing the medullary cavity with PBS containing 2% fetal calf serum (FCS). After one wash in the same solution, cells were seeded in petri dishes in PRIM-1640 medium supplemented with 20% FCS, 100 μg/mL streptomycin, 100 IU/mL penicillin, and 30 ng/mL macrophage colony-stimulating factor (BioSource International, Camarillo, CA, USA) at 37 °C for 7–9 days. Macrophages were then assessed by flow cytometry using a FACScan (Becton Dickinson Biosciences, San Jose, CA, USA) and staining with phycoerythrin (PE)-conjugated anti-F4/80 (eBioscience, San Diego, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated anti-CD11b (eBioscience).
MSU crystals were prepared under pyrogen-free conditions. Briefly, 1 g uric acid (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 200 mL of boiling water containing 6 mL of 1 N NaOH. The pH value of the final solution was adjusted to 7.2 through the addition of HCl. The solution was cooled and stirred at room temperature and then stored overnight at 4 °C. The precipitate was filtered from the solution and dried under low heat. The crystals were weighed under sterile conditions and suspended in PBS at a concentration of 25 mg/mL [
11].
MSU-induced inflammation in vivo
Mice were injected intraperitoneally with 3 mg MSU crystals in 0.5 mL PBS. After 2.5 h and 5 h, the peritoneal cavities were washed with 2 mL PBS. The number of peritoneal exudate cells was counted using a hemocytometer. The cells were resuspended in PBS and subjected to staining and flow cytometric analysis.
RNA extraction and quantitative reverse-transcribed PCR (qRT-PCR)
A total of 1 × 10
6 BMDMs were treated with 0.25 mg/mL MSU crystals in a 24-well plate. At 0, 4, and 8 h after stimulation, total RNA was isolated from BMDMs using a combination of QIAzol lysis reagent and a miRNeasy Mini kit (Qiagen, Germantown, MD, USA) with some modifications. The RNA was reverse-transcribed (RT) using a TaqMan miRNA Reverse Transcription Kit (Applied Biosystems, Foster City, USA). Following pre-amplification, the gene expression was assessed on a 7900HT Fast Real-Time PCR System (Applied Biosystems), using the manufacturer’s recommended protocol. For analysis of gene expression, SYBR green gene expression assays were used for quantitative RT-PCR of IL-1β, TNF-α and NALP3. The primer sequences are shown in Table
1.
Table 1
The primers used for quantitative PCR
IL-1β | GGGCCTCAAAGGAAAGAATC | CTCTGCTTGTGAGGTGCTGA |
TNF-α | ACAAAGGTGCCGCTAACCACATGT | ATGCTGCTGTTTCAGTCGAAGGCA |
NALP3 | CGTGGTTTCCTCCTTTTGTATT | CGACCTCCTCTCCTCTCTTCTT |
ASC | TCACAGAAGTGGACGGAGTG | TGTCTTGGCTGGTGGTCTCT |
Caspase-1 | CGTGGAGAGAAACAAGGAGTG | AATGAAAAGTGAGCCCCTGAC |
TRAF6 | ATTTCATTGTCAACTGGGCA | TGAGTGTCCCATCTGCTTGA |
IRAK1 | GAGACCCTTGCTGGTCAGAG | GCTACACCCACCCACAGAGT |
GAPDH | GGTGAAGGTCGGTGTGAACG | TGTAGACCATGTAGTTGAGGTCA |
ELISA
IL-1β levels in the BMDM culture supernatants were detected using a mouse IL-1β ELISA Ready-SET-Go! Kit (eBioscience), following the recommended protocol.
Analyses of MSU-induced arthritis
A total of 1 mg MSU in 40 μL PBS or 0.5 mg MSU in 20 μL PBS was injected into the foot pads and synovial space of the right knee, respectively, of WT and miR-146a KO mice, while the same volume of PBS injected into the contralateral limb served as a control. The swelling index is expressed as the MSU-injected joint/PBS-injected joint ratio, and a ratio >1.15 indicated inflammation. Paw swelling and the size of the joint were measured with an electronic caliper by a researcher blinded to the intervention, at the indicated time points [
12].
Flow cytometric analysis
Single BMDM suspensions and peritoneal cells were incubated with mAb 2.4G2 for 30 min at 4 °C to block non-specific binding sites and then stained with mAbs anti-F4/80(eBioscience) and anti-CD11b(eBioscience) for 30 min at 4 °C. For intracellular staining, peritoneal cells were incubated with 100 μL IC fixation buffer (eBioscience) at 4 °C for 30 min in the dark. After incubation, cells were washed twice with 2 mL 1 × permeabilization buffer (eBioscience) and centrifuged and decanted. Next, intracellular staining was performed in 100 uL of 1 × permeabilization buffer using anti-TNF-a (eBioscience). After incubation for 30 min in the dark at room temperature, cells were washed twice with 1 × permeabilization buffer and resuspended in 150 μL 1 × PBS and filtered for flow cytometric analysis. Data were analyzed using a FACSAriaTMII (BD Biosciences, Franklin Lakes, NJ, USA) and Flowjo 7.6 DH software.
Western blot analysis
The cells were disrupted in lysis buffer, and the concentrations of the extracted proteins were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). The concentrations of the extracted proteins were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific). The samples were separated on 10% SDS-PAGE and then electro-transferred at 90 V to an Immun-Blot polyvinylidene fluoride (PVDF) membrane for 2 h. Membranes were then blocked in I-BlockTM Protein-Based Blocking Reagent for 30 min at room temperature and then incubated with primary antibodies overnight at 4 °C. Blots were washed extensively in TBST and incubated with secondary antibodies for 2 h at room temperature. The signal was detected using an enhanced chemiluminescence method (ECL kit; Amersham Pharmacia Biotech, Piscataway, NJ, USA). All antibodies used were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Data analysis
Data were analyzed using Prism 5 software (GraphPad Software, La Jolla, CA, USA). The results of gene expression were analyzed using the 2-△△Ct method. Data from three to five individual experiments were pooled for analysis using the t test.
Discussion
This study has shown that miR-146a KO mice suffer more severe gouty arthritis than WT mice and has indicated that miR-146a-deficient mice lose the repression of TRAF6 and IRAK1, leading to enhanced production and secretion of pro-IL-1β. To our knowledge, the current study is the first to identify the importance of miR-146a in gouty arthritis using a KO mouse model and to explore the potential mechanism underlying this relation.
Previous studies have shown that miR-146a is upregulated in lipopolysaccharide (LPS) or MSU stimulated THP-1, a common human monocyte cell line used to study monocyte/macrophage differentiation and function [
7,
10]. In our study, miR-146a expression was dramatically upregulated in BMDMs after exposure to MSU crystals for 4 h, which is in agreement with the study by Dalbeth et al. [
10]; interestingly, we were surprised to observe that miR-146a expression was sharply downregulated at 8 h post-stimulation. Furthermore, miR-146a expression paralleled the gene expression of IL-1β, suggesting that miR-146a plays a pivotal role in MSU-induced inflammation and that its role might be closely connected with that of IL-1β. A previous study has shown that peritoneal monocyte miR-146a expression is significantly reduced at 2 h and 8 h following injection of MSU [
10]. We stained MSU-induced peritoneal cells with anti-F4/80 and anti-CD11b antibodies and obtained two macrophage subsets, CD11b
highF4/80
high and CD11b
intermediumF4/80
intermedium. Additionally, miR-146a expression was significantly upregulated at 2.5 h following injection, with normalization of expression at 5 h. These findings were in contrast with the study of Dalbeth et al. [
10], perhaps reflecting that sorted macrophages can more accurately represent the inflammatory response in MSU-induced inflammation.
To further confirm the participation of miR-146a in MSU-induced inflammation. MSU crystals were injected into the ankle joints and footpads of WT and miR-146a KO mice to mimic acute gouty arthritis in humans; the data indicated that miR-146a KO mice suffered more severe arthritis than WT mice in two types of gouty models, signifying miR-146a acts as a negative regulator during the process of gouty arthritis.
MiR-146a can directly downregulate the production of pr-inflammatory cytokines, which play critical roles in the inflammatory responses triggered by MSU crystals, by acting as a negative-feedback effecter of the inflammatory signaling pathway initiated by NF-κB [
4]. Our results showed that miR-146a deficiency led to enhanced IL-β and TNF-α expression in MSU-primed BMDMs. Meanwhile, we also found that IL-1β protein levels were much higher in miR-146a-deficient BMDMs than in WT cells, even without stimulation by MSU, implying that loss of miR-146a may lead to an autoimmune phenotype and exaggerate the inflammatory response in mice [
9,
11]. Previous studies have suggested that IL-1β acts as the central regulatory cytokine in acute gouty arthritis to recruit neutrophils into the synovium and joint [
12]. As we know now, the production and activity of IL-1β are tightly regulated via a multi-step process. The precursor of IL-1β (pro-IL-1β) is synthesized based on the activation of the toll-like receptor (TLR) pathway, and pro-IL-1β is then cleaved to a mature form by the NALP3 inflammasome [
1,
13]. It is has been proposed that miR-146a targets IRAK1 and TRAF6, two central adaptor kinases in the downstream signaling cascade of TLR, mediating the NF-κB pathway through a negative feedback regulation loop, leading to an obvious reduction in the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in mycobacteria-infected macrophages [
14‐
16]. Consistent with previous reports [
7,
14,
15], deficiency of miR-146a in BMDMs exposed to MSU led to a significant increase in gene and protein expression of both TRAF6 and IRAK1 compared with the WT control. Synthesized the results of IL-1β and TNF-α changing tendency, it is indicated that knock out miR-146a discharged the repression of TRAK6 and IRAK1 function to accelerate the production pro-inflammatory cytokines and to exacerbate the MSU-induced gouty arthritis (Fig.
3c).
MSU crystals can trigger the activation of the NALP3 inflammasome, culminating in the production of IL-1β. We were surprised to find increased mRNA levels of NALP3 inflammasome components in macrophages of miR-146a KO mice, as there are no reports indicating that miR-146a directly targets components of NALP3 inflammasome, and bioinformatics analysis has indicated that NAPL3 does not contain a binding site for miR-146a. Our results suggest the possibility that miR-146a indirectly targets the NALP3 inflammasome to improve the activation of IL-1β; alternatively, some unknown regulatory mechanisms may exist to promote this effect.