miR-155 is dispensable in monosodium urate-induced gouty inflammation in mice
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The findings of a previous study by Jin et al. have shown that microRNA (miR)-155 was upregulated in patients with acute gouty arthritis and enhanced the proinflammatory cytokines. There is no direct evidence to support that miR-155 is indeed involved in monosodium urate (MSU)-induced inflammatory responses in vivo. The aim of this study was to investigate the role of miR-155 knock-out (KO) or knock-in (KI) mice in MSU-induced animal models to mimic acute gout.
MiR-155 expression in cultured bone marrow-derived macrophages (BMDMs) from miR-155 KO, miR-155 KI, and wild-type (WT) mice treated with MSU crystals in vitro was detected by real-time quantitative polymerase chain reaction (qPCR). MiR-155 KO and WT mice were used to induce an acute gouty inflammatory response with MSU crystals including models of foot pad inflammation, ankle arthritis, air pouch inflammation, and peritonitis. Furthermore, the proinflammatory interleukin (IL)-1β levels in lavage fluids from air pouch and peritoneal cavity models were measured by enzyme-linked immunosorbent assay (ELISA), and tumor necrosis factor (TNF)-α production from BMDMs of miR-155 KI mice treated with MSU were measured by flow cytometry.
MiR-155 expression was quickly upregulated in BMDMs from WT mice following MSU treatment in vitro. In comparison with WT mice in vivo, the swelling index of miR-155 KO mice showed no significant difference in the murine foot pad and ankle arthritis models for the indicated different time points. There were similar changes in total cell numbers of lavage fluids in the air pouch and peritoneal cavity models between miR-155 KO and WT mice following MSU crystal injection. Moreover, the IL-1β levels of lavage fluids in the air pouch and peritonitis models from miR-155 KO mice were almost the same as those from WT mice. TNF-α levels were comparable from BMDMs treated with MSU crystals in vitro between miR-155 KI mice and WT mice.
MiR-155 is dispensable in MSU-induced gouty inflammation in mice. Deletion of miR-155 might not be an effective therapeutic approach to relieve the inflammation in acute gout.
KeywordsMiR-155 MSU Gout Inflammation
Bone marrow-derived macrophage
Colony-stimulating factor 1 receptor
Enzyme-linked immunosorbent assay
Macrophage colony-stimulating factor
NLR family pyrin domain containing 3
Peripheral blood mononuclear cell
Quantitative polymerase chain reaction
Synovial fluid mononuclear cell
SH2 domain-containing inositol 5′-phosphatase 1
tumor necrosis factor
Gout is one of the most common forms of inflammatory arthritis disorder and is caused by deposition of monosodium urate (MSU) crystals in and around the joints . Accumulated studies indicate that at least two pathways, the Toll-like receptor (TLR)/nuclear factor (NF)-κB signaling pathway and the NLR family pyrin domain containing 3 (NLRP3) pathway, are involved in MSU crystal-induced inflammatory cytokine release from monocytes/macrophages [2, 3, 4]. However, the molecular mechanisms of gouty inflammation are still not entirely elucidated.
MicroRNAs (miRNAs) are an abundant class of small, evolutionary conserved, non-coding RNAs acting as post-transcriptional regulators. Our previous data have reported that miRNAs regulate the immune cell development and function, and control autoimmune disease development [5, 6]. Aberrant miRNA expression has been observed in a number of diseases. Recent studies suggest that miRNAs are potentially involved in the development of human acute gouty arthritis, including miR-155  and miR-146a . Dalbeth et al.  found that miR-146a expression increased in both human monocytic THP-1 cells and human peripheral blood mononuclear cells (PBMCs) using MSU crystal stimulation in vitro, but the expression of other miRNAs (including miR-155, miR-146b, miR-9, and miR-21) did not, implicating interleukin (IL)-1β regulation. Interestingly, higher levels of miR-146a were expressed in PBMCs from intercritical gout patients when compared with both control groups (normouricemic control participants/hyperuricemic control participants) and the acute gout group. Additionally, miR-146a expression was reduced during the acute flare compared with the intercritical period in the paired samples as well as in the urate peritonitis model. Those findings reflect the more complex multicellular in vivo response to MSU crystals. Numerous studies have reported that miR-155 could negatively regulate the TLR/NF-κB signaling pathway by targeting different kinds of genes , and that miR-155 deficiency reduced inflammatory responses in the colitis mouse model . MiR-155 expression, which is closely related to disease activity, is upregulated in the PBMCs and synovial membrane of rheumatoid arthritis [11, 12]. Jin et al.  showed that miR-155 was upregulated in synovial fluid mononuclear cells (SFMCs) from patients with acute gouty arthritis and MSU crystals strongly induced miR-155 expression in PBMCs in vitro. Furthermore, the increased expression of miR-155 in SFMCs led to downregulation of the SH2 domain-containing inositol 5′-phosphatase 1 (SHIP1), which activates the Akt/NF-kB pathway and enhances the production of proinflammatory cytokines, including IL-1β. Notably, miR-155 levels in PBMCs from gout patients were comparable with healthy individuals. These paradoxical data suggest that the same miRNA may play different roles in different tissues/organs and conditions. However, there is still a lack of direct evidence to further determine whether miR-155 is indeed involved in MSU-induced inflammation in vivo.
In the present study, we took advantage of miR-155 knock-out (KO) mice to investigate the role of miR-155 in MSU-induced gout in vivo. Four MSU-induced gout mouse models, including foot pad inflammation, ankle arthritis, peritonitis, and air pouch inflammation, were used. The proinflammatory cytokine IL-1β levels in lavage fluids from the peritoneal cavity and air pouch models and tumor necrosis factor (TNF)-α levels from bone marrow-derived macrophages (BMDMs) of miR-155 knock-in (KI) mice treated with MSU crystals were measured.
MiR-155−/−C57BL/6 knock-out (miR-155 KO) mice, Csf1r+155Tg/Tg knock-in (miR-155 KI) mice, and Csf1r−155Tg/Tg wild-type (WT) mice were kindly provided by the CBR Institute for Biomedical Research, Harvard Medical School, Boston, USA. C57BL/6 as WT mice were purchased from the Jackson Laboratory. Colony-stimulating factor 1 receptor (Csf1r), also known as macrophage colony-stimulating factor (M-CSF) receptor, controls the production, differentiation and function of macrophages. As we know, macrophages play crucial roles (such as phagocytosis and trigging the inflammatory response) in acute gout including in the initial phase, in development, and in remission. The mice were housed at 24 ± 2 °C under 12-h light/12-h dark cycles in a pathogen-free facility; 8- to 10-week-old males were used to perform the experiments. The handling of mice and experimental procedures in this study were performed in accordance with the requirements of the Institutional Animal Care and Use Committee of Henry Ford Health System.
Preparation of MSU crystals
Briefly, 1.0 g uric acid (Sigma-Aldrich) was dissolved in 200 ml boiling distilled water containing 6.0 ml 1 M NaOH. After adjusting the pH of the solution to 7.2 with HCl, crystals that formed were sterilized by heating at 180 °C for 2 h . The solution was gradually cooled by stirring at room temperature and stored overnight at 4 °C. The precipitate was filtered from the solution, dried under low heat, and suspended in phosphate-buffered saline (PBS) at a concentration of 50 mg/ml. All reagents were prepared under pyrogen-free conditions.
BMDM culture and MSU stimulation
Bone marrow cells obtained from the femoral bones of C57BL/6(WT), miR-155 KO, Csf1r+155Tg/Tg (miR-155 KI), and Csf1r−155Tg/Tg (WT) mice, respectively, were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100 μg/ml). To induce the proliferation and differentiation of myeloid progenitors to macrophages, the medium was supplemented with 30 ng/ml M-CSF (#0914245, Peprotech). The cells were washed and received fresh medium with M-CSF every 2–3 days. After 7 days the cells were harvested with 0.25% trypsin. Dead cells were first gated out by propidium iodide (PI) staining. The phenotypic validation of BMDMs was performed by flow cytometry with staining using fluorescein isothiocyanate (FITC)-conjugated anti-CD11b and phycoerythrin (PE)-conjugated anti-F4/80 antibodies (both diluted 1:200). The BMDMs are double-positive for CD11b and F4/80. According to the experimental protocol, the MSU crystal suspension (MSU 2.5 mg/ml concentration) was added to the incubated BMDMs (MSU 100 μg/ml final concentration) for 4 or 8 h and the ratio of TNF-α production from BMDMs treated with MSU for 2 or 4 h were measured.
Mice were placed under anesthesia (150:10 mg/kg ketamine:xylazine injected intraperitoneally) and were injected with MSU crystals into the right foot pad (1 mg in 40 μl PBS) or ankle joint (0.5 mg in 20 μl PBS). The same volume of sterile saline was injected into the left foot pad or ankle joint at the same time to serve as the control. Inflammation parameters were evaluated following MSU crystal injection at different time points (6, 24, and 48 h). Paw swelling and the size of ankle joints were measured with an electronic caliper at the indicated time points [14, 15, 16]. For an air pouch model, 5 ml sterile air was first injected subcutaneously into the back of mice to form an air pouch, and then 3 ml sterile air was injected to the air pouch at day 3 and day 5. At day 7, MSU suspension (3 mg in 1 ml PBS) was injected into the air pouch . For MSU-induced peritonitis, MSU crystals (3 mg in 0.5 ml PBS) were injected into the peritoneal cavity as part of the intraperitoneal gouty model . The total number of air pouch and peritoneal cavity exudate cells were harvested after 4 or 8 h and counted by a hemocytometer.
Real-time quantitative polymerase chain reaction
The cultured BMDMs were harvested after 7 days. Total RNA of BMDMs was extracted using Trizol reagent (Invitrogen, USA) and reverse-transcribed into cDNA using reverse transcription reagents (Invitrogen, USA) according to the manufacturer’s protocols. Real-time quantitative polymerase chain reaction (qPCR) was performed using the ABI Prism 7900HT Detection System (Applied Biosystems, USA) with Power SYBR Green PCR Master Mix (Applied Biosystems, USA). The gene primers sequences were synthesized by eurofins genomics (Louisville, USA) as follows. TNF-α: forward 5’-ACAAAGGTGCCGCTAACCACATGT-3′, reverse 5’-ATGCTGCTGTTTCAGTCGAAGGCA-3′; IL-1β: forward 5’-GGGCCTCAAAGGAAAGAATC-3′, reverse 5’-CTCTGCTTGTGAGGTGCTGA-3′; β-actin: forward 5’-CAACGAGCGGTTCCGATG-3′, reverse 5’-GCCACAGGATTCCATACCCA-3′. The β-actin as an internal control was used to normalize the gene expression. Gene expression was analyzed using the 2–ΔΔCT method.
The expression of miR-155 in BMDMs was measured using Taqman MicroRNA Assays (Applied biosystems, Foster City, CA, USA) according to the manufacturer’s protocols. The Taqman MicroRNA Assay for U6 snRNA was used to normalize the relative abundance of miRNAs.
Enzyme-linked immunosorbent assay (ELISA)
IL-1β protein levels in lavage fluids of the air pouch and peritoneal cavity models were measured by ELISA using kits from eBioscience (cat. no. 88–7013-88; San Diego, CA, USA) following the manufacturer’s instructions. The 96-well microplates were read using a VICTOR X3 plate reader.
Data were analyzed with GraphPad Prism 5. Differences between experimental groups were tested using the unpaired t test. Data are expressed as mean ± SEM. P < 0.05 was considered to denote statistical significance.
Results and discussion
Overall, our data suggest that miR-155 is dispensable in MSU-induced gouty inflammation in mice. Given that many miRNAs are regulated in monocytes/macrophages upon MSU stimulation (our unpublished data), miR-155 is more likely redundant with other regulated miRNAs during gout development.
The miR-155 expression was quickly upregulated in BMDMs from WT mice with MSU-induced acute gouty inflammation in vitro. MiR-155 deficiency did not significantly affect the phenotype in diverse murine MSU-induced gouty inflammation in vivo. Therefore, deletion of miR-155 might not be an effective therapeutic approach to relieve the inflammation in acute gout.
We thank all laboratory members for their help and encouragement.
This study was supported in part by the Henry Ford Health System Research Grants for the Immunology Program (T71016 to QM and T71016 to LZ), the National Natural Science Foundation of China (81670801 to JZ), the Education Agency of Sichuan Province Grants (15ZB0202 to QY), the Sichuan Medical Association Grants (S16027 to QY), and the National Key Research Program of China (2016YFC0903500 to YQ). The funders had no role in the study design, data collection, analysis, or interpretation of the data, or in writing the manuscript or the decision to submit the manuscript.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
QY and QZ performed the experiment. QY drafted the manuscript. YQ, LZ, QM, and JZ designed experiments and critically revised the manuscript. All authors read and approved the final manuscript.
All animal experimental procedures in the current study were approved by the Institutional Animal Care and Use Committee of Henry Ford Health System (Detroit, USA).
Consent for publication
The authors declare that they have no competing interests.
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