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, 7 (8), e42971

Inducible microRNA-223 Down-Regulation Promotes TLR-triggered IL-6 and IL-1β Production in Macrophages by Targeting STAT3

Affiliations

Inducible microRNA-223 Down-Regulation Promotes TLR-triggered IL-6 and IL-1β Production in Macrophages by Targeting STAT3

Qingyun Chen et al. PLoS One.

Abstract

MicroRNAs are small non-coding RNA molecules that regulate gene expression by either translational inhibition or mRNA degradation. MicroRNAs play pivotal roles in the regulation of both innate and adaptive immune responses, including TLR-triggered inflammatory response. Here we reported that the expression of microRNA-223 (miR-223) was significantly decreased in murine macrophages during activation by lipopolysaccharide (LPS) or poly (I∶C) stimulation. The inducible miR-223 down-regulation resulted in the activation of signal transducer and activator of transcription 3 (STAT3), which is directly targeted by miR-223, thus promoting the production of pro-inflammatory cytokines IL-6 and IL-1β, but not TNF-α. Interestingly, IL-6 was found to be a main factor in inducing the decrease in miR-223 expression after LPS stimulation, which formed a positive feedback loop to regulate IL-6 and IL-1β. Herein, our findings provide a new explanation characterizing the molecular mechanism responsible for the regulation of IL-6 production after TLR-triggered macrophage activation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Down-regulation of miR-223 expression in RAW264.7 cells and primary macrophages stimulated with TLR agonists.
(A–D) RAW264.7 and bone marrow-derived macrophages were stimulated with 100 ng/ml LPS or 10 µg/ml poly (I∶C) for the indicated time points. The expression of miR-223 was measured by q-PCR and normalized to the expression of U6. (E–F) RAW264.7 cells were stimulated with different concentrations of LPS (E) or poly (I∶C) (F) for 24 h. The expression of miR-223 was determined by qPCR and normalized to the expression of U6. Data are the mean ± SD (n = 3) of three independent experiments. *** p<0.001; ** p<0.01; * p<0.05.
Figure 2
Figure 2. MiR-223 inhibits IL-6 mRNA expression but not TNF-α, in TLR-triggered macrophages.
RAW264.7 cells were transfected with mimics or inhibitors of miR-223 and their controls at a final concentration of 30 nM. 24 h later, cells were stimulated with or without LPS (100 ng/ml) or poly (I∶C) (10 µg/ml). IL-6 (A, C) and TNF-α (B, D) mRNA levels were measured at 3 h and 6 h post-stimulation by qPCR. Data are the mean ± SD (n = 3) of three independent experiments. *** p<0.001; ** p<0.01; * p<0.05; NS, not significant.
Figure 3
Figure 3. MiR-223 negatively regulates LPS or poly (I∶C)-triggered IL-6 and IL-1β production in macrophages.
Raw264.7 cells were transfected with mimics or inhibitors of miR-223 and their controls at a final concentration of 30 nM. 24 h later, cells were stimulated with or without LPS (100 ng/ml) or poly (I∶C) (10 µg/ml). (A–F) Supernatants were collected after 12 h to measure IL-6, TNF-α, IL-1β by ELISA. Data are the mean ± SD (n = 3) of three independent experiments. *** p<0.001; ** p<0.01; * p<0.05; NS, not significant.
Figure 4
Figure 4. MiR-223 negatively regulates LPS-induced STAT3 activation in macrophages.
(A) RAW264.7 cells were transfected with control and mimics of miR-223 at a final concentration of 30 nM, then stimulated with LPS (100 ng/ml) for the indicated time points. P-IκB-α, p-ERK1/2 and p-P38 were analyzed by immunoblotting. (B) RAW264.7 cells were treated as described above. Phosphorylated and total STAT3 were detected by immunoblotting. (C) RAW264.7 cells were stimulated with LPS for the indicated time points and STAT3 protein expression was measured by immunoblotting. (D) RAW264.7 cells were transfected with miR-223 inhibitors at a final concentration of 30 nM, and stimulated with LPS for the indicated time points, then the phosphorylated and total STAT3 protein were determined. The data shown represent three independent experiments.
Figure 5
Figure 5. MiR-223 targets mouse STAT3 for translational inhibition but not mRNA degradation.
(A) The alignment of miR223 and its target sites in 3′-UTR of STAT3 is shown. (B) RAW264.7 cells were cotransfected with pMIR-REPORT™-STAT3-3′UTR luciferase reporter plasmid, STAT3-3′-UTR-mutant or empty pMIR-REPORT™ and pTK-RL plasmid, together with miR-223 mimics or inhibitors (30 nM). After 24 h, firefly luciferase activity was measured and normalized by Renilla luciferase activity. (C) RAW264.7 cells were transfected with the pMIR-REPORT™ STAT 3′UTR, STAT 3′UTR mutant and control plasmids. Luceferase activity in cells with or without LPS stimulation was measured. (D) RAW264.7 cells were transfected mimics or inhibitors of miR-223 and their controls (30 nM). After 24 h, STAT3 protein expression was detected by immunoblotting. β-actin served as a loading control. The data shown represent three independent experiments. (E) RAW264.7 cells were transfected with mimics or inhibitors of miR-223 and their controls, 24 h later, STAT3 mRNA levels were determined with or without LPS at 12 h post stimulation. *** p<0.001; * p<0.05; NS, not significant. Data are the mean ± SD (n = 3) of one representative experiment. Similar results were obtained in three independent experiments.
Figure 6
Figure 6. Knockdown of STAT3 inhibits LPS- and poly (I∶C)-induced IL-6 and IL-1β production.
(A) RAW264.7 cells were transfected with STAT3 siRNA or control RNA at a final concentration of 30 nM. After 24 h, STAT3 protein expression was detected by immunoblotting and densitometry analysis was shown. The data shown represent three independent experiments. (B–C) STAT3 siRNA transfected RAW 264.7 cells were stimulated with poly (I∶C) (10 µg/ml) or LPS (100 ng/ml) for 6 h, mRNA levels of IL-6 and TNF-α were determined by qPCR. (D–F) The protein levels of the IL-6, TNF-α and IL-1β at the indicated time points were analyzed by ELISA. Data are the mean ± SD (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. *** p<0.001; ** p<0.01; * p<0.05; NS, not significant.
Figure 7
Figure 7. Transfection of miR-223 mimics to STAT3-siRNA pretreated cells abolishes its ability to inhibit IL-6 production.
(A, B) RAW264.7 cells were first transfected with STAT3 siRNA or ctrl RNAs, 18 h later, cells were secondly transfected with ctrl or miR-223 mimics. After 24 h, cells were stimulated with LPS or poly (I∶C) for 3 h. IL-6 and TNF-α mRNA expression were analyzed by q-PCR. (C–E) Cells were treated as described in (A, B), IL-6, TNF-α and IL-1β production were measured by ELISA after 12 h stimulation with LPS or poly (I∶C). *** p<0.001; ** p<0.01; * p<0.05. Data are the mean ± SD (n = 3) of one representative experiment. Similar results were obtained in three independent experiments.
Figure 8
Figure 8. IL-6 acts as a main factor responsible for the down-regulation of miR-223 expression.
(A–B) RAW264.7 cells were stimulated with 10 or 20 ng/ml of recombinant IL-6 for the indicated time points. The expression of miR-223 was determined by qPCR and normalized to the expression of U6. (C) Cells were treated with a mouse IL-6-specific blocking antibody or isotype control antibody (IgG1) upon LPS challenge. MiR-223 expression was determined by qPCR at the indicated time points. * p<0.05, ** p<0.01. (D) RAW264.7 cells were transfected with STAT3 siRNA at the final concentration of 30 nM. The expression of miR-223 was analyzed by q-PCR after LPS treatment for the indicated time. * p<0.05. (E) Cells were treated with mouse IL-6 (5 ng/ml) for the indicated time points and levels of total and phosphorylated STAT3 were analyzed by immunoblotting. (F) Cells were pretreated with IL-6 neutralization or IgG1 control antibody, STAT3 protein and phosphorylation levels were determined by immunoblotting at the indicated time points after LPS (100 ng/ml) stimulation. (G, H) RAW264.7 cells were firstly transfected with miR-223 mimics or inhibitors and their controls. After 24 h, cells were treated with IL-6 and levels of total and phosphorylated STAT3 were analyzed by immunoblotting. Data are representative of three independent experiments. (I) The proposed positive regulatory loop of IL-6/miR-223/STAT3 pathway in regulating the TLR-triggered inflammatory response.

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Grant support

This work was supported by grants from the National Natural Science Foundation of China (81072405), Program for New Century Excellent Talents in University from Ministry of Education of People's Republic of China (NCET-08-0486), grant from Zhejiang Provincial Natural Science Foundation of China (R2100528) and also sponsored by Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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