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Cited by 45

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MicroRNA-223 Suppresses IL-1β and TNF-α Production in Gouty Inflammation by Targeting the NLRP3 Inflammasome

Zhang, Quan-Bo, Zhu, Dan, Dai, Fei, Huang, Yu-Qin, Zheng, Jian-Xiong, Tang, Yi-Ping, Dong, Zeng-Rong, Liao, Xia, Qing, Yu-Feng·2021·Frontiers in Pharmacology
DOI 10.3389/fphar.2021.637415PMC PMC8085756
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Abstract

Introduction: MicroRNA-223 (MiR-223) serves as an important regulator of inflammatory and immune responses and is implicated in several auto-inflammatory disorders. Here, we measured miR-223 expression in acute and intercritical gout patients, after which we used RAW264.7 macrophages transfected with a miR-223 mimic/inhibitor to determine the function of miR-223 in monosodium urate (MSU)-induced gouty inflammation. Methods and Results: MiR-223 was detected among 122 acute gout patients (AG), 118 intercritical gout patients (IG), and 125 healthy subjects (HC). RAW264.7 macrophages were cultured and treated with MSU. Over-expression or under-expression of miR-223 was inducted in RAW264.7 macrophages to investigate the function of miR-223. Real-time quantitative PCR, ELISA and western blotting were used to determine the expression levels of miR-223, cytokines and the NLRP3 inflammasome (NLRP3, ASC, and caspase-1). MiR-223 expression was significantly decreased in the AG group in comparison with the IG and HC groups (p < 0.001, respectively). Up-regulated expression of miR-223 was observed after acute gout remission in comparison with that observed during gout flares in 30 paired cases (p < 0.001). The abundance of the NLRP3 inflammasome and cytokines was significantly increased after RAW264.7 macrophages were treated with MSU (p < 0.01, respectively), while that of miR-223 was significantly reduced (p < 0.01). Up-regulation of miR-223 decreased the concentrations of IL-1β and TNF-α, as well as the NLRP3 inflammasome expression (p < 0.01, respectively), while IL-37 and TGF-β1 levels were unchanged (p > 0.05, respectively). Under-expression of miR-223 increased the concentrations of IL-1β and TNF-α, as well as NLRP3 inflammasome expression (p < 0.01, respectively), while IL-37 and TGF-β1 levels were not influenced (p > 0.05, respectively). Conclusion: These findings suggest that miR-223 provides negative feedback regulation of the development of gouty inflammation by suppressing production of IL-1β and TNF-α, but not by regulating IL-37 and TGF-β1. Moreover, miR-223 regulates cytokine production by targeting the NLRP3 inflammasome.

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FIGURE 1 Expression of miR-223 in gout patients. (A) Expression of miR-223 in peripheral blood mononuclear cells from patients with acute gout (n = 122), patients with intercritical gout (n = 118) and healthy control subjects (n = 125), one-way ANOVA F = 83.811, p < 0.001. ***p < 0.001 for the Bonferroni test for the comparison between groups. (B) Relative expression of miR-223 in patients with gout flare and after resolution. Paired t-test (n = 30). t = −15.649, p < 0.001.
FIGURE 1 Expression of miR-223 in gout patients. (A) Expression of miR-223 in peripheral blood mononuclear cells from patients with acute gout (n = 122), patients with intercritical gout (n = 118) and healthy control subjects (n = 125), one-way ANOVA F = 83.811, p < 0.001. ***p < 0.001 for the Bonferroni test for the comparison between groups. (B) Relative expression of miR-223 in patients with gout flare and after resolution. Paired t-test (n = 30). t = −15.649, p < 0.001.CC BY · PubMed Central
FIGURE 2 Altered expression of miR-223, NLRP3 inflammasome and cytokines in MSU crystal-induced RAW264.7 murine macrophage inflammation. (A, B) IL-1β and TNF-α levels were dramatically increased in cultural supernatants after RAW264.7 macrophages were treated with MSU for 6 h (n = 5). (C) Altered expression of NLRP3 mRNA and miR-223 upon stimulation of RAW264.7 cells in comparison with that of unstimulated control cultures (n = 5). NLRP3 mRNA up-regulation of approximately 4.88-fold after MSU stimulation for 3 h, 2.39-fold after 6 h, and 1.92-fold after 12 h. MiR-223 down-regulation of approximately 3.71-fold after MSU stimulation for 3 h, 3.39-fold after 6 h, and 2.50-fold after 12 h. (Di, Dii) Altered expression of NLRP3 protein upon stimulation of RAW264.7 cells in comparison with that of unstimulated control cultures (n = 5). NLRP3 protein up-regulation of approximately 2.85-fold after MSU stimulation for 3 h, 3.62-fold after 6 h, and 2.99-fold after 12 h. (E–H) Altered expression of ASC, caspase-1 and IL-1β mRNA and protein in RAW264.7 macrophages stimulated with MSU for 6 h *p < 0.05, **p < 0.01.
FIGURE 2 Altered expression of miR-223, NLRP3 inflammasome and cytokines in MSU crystal-induced RAW264.7 murine macrophage inflammation. (A, B) IL-1β and TNF-α levels were dramatically increased in cultural supernatants after RAW264.7 macrophages were treated with MSU for 6 h (n = 5). (C) Altered expression of NLRP3 mRNA and miR-223 upon stimulation of RAW264.7 cells in comparison with that of unstimulated control cultures (n = 5). NLRP3 mRNA up-regulation of approximately 4.88-fold after MSU stimulation for 3 h, 2.39-fold after 6 h, and 1.92-fold after 12 h. MiR-223 down-regulation of approximately 3.71-fold after MSU stimulation for 3 h, 3.39-fold after 6 h, and 2.50-fold after 12 h. (Di, Dii) Altered expression of NLRP3 protein upon stimulation of RAW264.7 cells in comparison with that of unstimulated control cultures (n = 5). NLRP3 protein up-regulation of approximately 2.85-fold after MSU stimulation for 3 h, 3.62-fold after 6 h, and 2.99-fold after 12 h. (E–H) Altered expression of ASC, caspase-1 and IL-1β mRNA and protein in RAW264.7 macrophages stimulated with MSU for 6 h *p < 0.05, **p < 0.01.CC BY · PubMed Central
FIGURE 3 Effect of miR-223 on the secretion of cytokines from RAW264.7 murine macrophages treated with MSU. (A) Transfection of miR-223 mimics (100 nM) for 24 h increased miR-223 expression approximately 1000-fold in RAW264.7 macrophages. (B) Transfection of miR-223 inhibitors (100 nM) for 24 h significantly decreased miR-223 expression by at least 80% in RAW264.7 macrophages. (C, D) Increased miR-223 abundance suppressed production of IL-1β and TNF-α (n = 5, t = −18.67, −11.214), while decreased miR-223 abundance enhanced IL-1β and TNF-α production compared to that of the NC cells (n = 5, t = 24.45,19.07), ***p < 0.001. (E, F) Secretion of TGF-β1 and IL-37 was not affected by increased (n = 5, t = 1.041, 0.001; p > 0.05, respectively) or decreased (n = 5, t = 0.146, 2.126; p > 0.05, respectively) expression of miR-223 in RAW264.7 cells in comparison with NC cells. The cells were transfected with 100 nM of a miR-223 mimic or miR-223 inhibitor for 24 h, then stimulated with 200 μg/ml MSU for 12 h, and finally evaluated of TNFa, IL-1, TGF-β1 and IL-37 in supernatant culture.
FIGURE 3 Effect of miR-223 on the secretion of cytokines from RAW264.7 murine macrophages treated with MSU. (A) Transfection of miR-223 mimics (100 nM) for 24 h increased miR-223 expression approximately 1000-fold in RAW264.7 macrophages. (B) Transfection of miR-223 inhibitors (100 nM) for 24 h significantly decreased miR-223 expression by at least 80% in RAW264.7 macrophages. (C, D) Increased miR-223 abundance suppressed production of IL-1β and TNF-α (n = 5, t = −18.67, −11.214), while decreased miR-223 abundance enhanced IL-1β and TNF-α production compared to that of the NC cells (n = 5, t = 24.45,19.07), ***p < 0.001. (E, F) Secretion of TGF-β1 and IL-37 was not affected by increased (n = 5, t = 1.041, 0.001; p > 0.05, respectively) or decreased (n = 5, t = 0.146, 2.126; p > 0.05, respectively) expression of miR-223 in RAW264.7 cells in comparison with NC cells. The cells were transfected with 100 nM of a miR-223 mimic or miR-223 inhibitor for 24 h, then stimulated with 200 μg/ml MSU for 12 h, and finally evaluated of TNFa, IL-1, TGF-β1 and IL-37 in supernatant culture.CC BY · PubMed Central
FIGURE 4 Effect of miR-223 on RAW264.7 macrophage apoptosis. Effect of miR-223 mimics (A), miR-223 inhibitors (B) and the scrambled control (C) on RAW264.7 macrophage apoptosis. Representative results are shown. (D) Comparison of the percentage of apoptotic cells among the three groups. The percentage of apoptotic cells (annexin V-positive/PI-negative) was 0.061 om0.028%, 0.056 .00.027% and 0.058 020.026%, respectively, in the miR-223 mimic group, miR-223 inhibitor group and scrambled control group. The differences among the groups were not statistically significant (n = 5, p > 0.05).
FIGURE 4 Effect of miR-223 on RAW264.7 macrophage apoptosis. Effect of miR-223 mimics (A), miR-223 inhibitors (B) and the scrambled control (C) on RAW264.7 macrophage apoptosis. Representative results are shown. (D) Comparison of the percentage of apoptotic cells among the three groups. The percentage of apoptotic cells (annexin V-positive/PI-negative) was 0.061 om0.028%, 0.056 .00.027% and 0.058 020.026%, respectively, in the miR-223 mimic group, miR-223 inhibitor group and scrambled control group. The differences among the groups were not statistically significant (n = 5, p > 0.05).CC BY · PubMed Central
FIGURE 5 Effect of miR-223 on expression of the NLRP3 inflammasome and IL-1β in RAW264.7 macrophages treated with MSU. Over-expression of miR-223 significantly decreased the mRNA and protein expression levels of the NLRP3 inflammasome (NLRP3, ASC and caspase-1) and IL-1β in comparison with the miRNA negative controls in RAW264.7 macrophages. In contrast, miR-223 inhibitors significantly increased the mRNA and protein expression levels of the NLRP3 inflammasome and IL-1β in comparison with the miRNA negative controls in RAW264.7 macrophages (*p < 0.05, **p < 0.01).
FIGURE 5 Effect of miR-223 on expression of the NLRP3 inflammasome and IL-1β in RAW264.7 macrophages treated with MSU. Over-expression of miR-223 significantly decreased the mRNA and protein expression levels of the NLRP3 inflammasome (NLRP3, ASC and caspase-1) and IL-1β in comparison with the miRNA negative controls in RAW264.7 macrophages. In contrast, miR-223 inhibitors significantly increased the mRNA and protein expression levels of the NLRP3 inflammasome and IL-1β in comparison with the miRNA negative controls in RAW264.7 macrophages (*p < 0.05, **p < 0.01).CC BY · PubMed Central
FIGURE 6 The potential mechanisms by which miR-146a and miR-223 influence gouty arthritis. On the left, the schematic shows how miR-146a might decrease IL-1β production by inhibition of TRAF6 and IRAK1 function. On the right, the schematic shows how miR-223 reduces IL-1β production by inhibiting NLRP3 function. ASC, apoptosis-associated speck-like protein; IL-1β, interleukin-1β; IL-1R, IL-1 Receptor; IRAK1, IL-1 receptor-associated kinase 1; MSU, monosodium urate; NLRP3, NACHT-LRR-PYD-containing protein; Pro-IL-1β, precursor of IL-1β; TLR, toll-like receptor; TRAF6, TNF receptor-associated factor 6.
FIGURE 6 The potential mechanisms by which miR-146a and miR-223 influence gouty arthritis. On the left, the schematic shows how miR-146a might decrease IL-1β production by inhibition of TRAF6 and IRAK1 function. On the right, the schematic shows how miR-223 reduces IL-1β production by inhibiting NLRP3 function. ASC, apoptosis-associated speck-like protein; IL-1β, interleukin-1β; IL-1R, IL-1 Receptor; IRAK1, IL-1 receptor-associated kinase 1; MSU, monosodium urate; NLRP3, NACHT-LRR-PYD-containing protein; Pro-IL-1β, precursor of IL-1β; TLR, toll-like receptor; TRAF6, TNF receptor-associated factor 6.CC BY · PubMed Central

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Authors
Zhang, Q.-B., Zhu, D., Dai, F., Huang, Y.-Q., Zheng, J.-X., Tang, Y.-P., Dong, Z.-R., Liao, X., & Qing, Y.-F.
Venue
Frontiers in Pharmacology
Publisher
Frontiers Media SA
Published
2021-04-16
Volume
12
ISSN
1663-9812
DOI
10.3389/fphar.2021.637415
PMC
PMC8085756
Cited by
45
References
30
Licence
creativecommons.org/licenses/by/4.0/ (vor)

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CoChat. (2026). Citation check: MicroRNA-223 Suppresses IL-1β and TNF-α Production in Gouty Inflammation by Targeting the NLRP3 Inflammasome. https://app.cochat.ai/papers/10.3389/fphar.2021.637415
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Zhang, Q.-B., Zhu, D., Dai, F., Huang, Y.-Q., Zheng, J.-X., Tang, Y.-P., Dong, Z.-R., Liao, X., & Qing, Y.-F. (2021). MicroRNA-223 Suppresses IL-1β and TNF-α Production in Gouty Inflammation by Targeting the NLRP3 Inflammasome. Frontiers in Pharmacology. https://doi.org/10.3389/fphar.2021.637415

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