Interleukin-10 Inhibits Lipopolysaccharide Induced miR-155 Precursor Stability and Maturation

Abstract

The anti-inflammatory cytokine interleukin-10 (IL-10) is essential for attenuating the inflammatory response, which includes reducing the expression of pro-inflammatory microRNA-155 (miR-155) in lipopolysaccharide (LPS) activated macrophages. miR-155 enhances the expression of pro-inflammatory cytokines such as TNFα and suppresses expression of anti-inflammatory molecules such as SOCS1. Therefore, we examined the mechanism by which IL-10 inhibits miR-155. We found that IL-10 treatment did not affect the transcription of the miR-155 host gene nor the nuclear export of pre-miR-155, but rather destabilized both pri-miR-155 and pre-miR-155 transcripts, as well as interfered with the final maturation of miR-155. This inhibitory effect of IL-10 on miR-155 expression involved the contribution of both the STAT3 transcription factor and the phosphoinositol phosphatase SHIP1. This is the first report showing evidence that IL-10 regulates miRNA expression post-transcriptionally.

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

“…Addition of LPS to these transfected cells increased firefly luciferase signal, a direct indication of increased transcription activity of the IκBζ promoter reporter. In other words, the results suggested that LPS upregulated the expression of the IκBζ gene, consistent with previous reports 7,8 . Figure 2 shows the typical results obtained in our experiments, using lipid-based transfection as an example.…”

“…The behaviours of the transfected cells were evaluated by a firefly-luciferase reporter gene harbouring the promoter region of IκBζ (pGL3-IκBζ). IκBζ expression is upregulated by the bacterial cell wall component lipopolyssacharide (LPS) 7,8 , and downregulated by the anti-inflammatory cytokine, Interleukin-10 (IL-10) 8 . To account for transfection variation among wells, we co-transfect a control plasmid containing the Renilla luciferase gene (e.g., phRL-TK) for normalization purposes.…”

The Role of Ets2 Transcription Factor in the Induction of MicroRNA-155 (miR-155) by Lipopolysaccharide and Its Targeting by Interleukin-10

“…Conflicting studies have recently reported that regulation of miR-155 does not occur at the transcriptional level (29); however, many of these studies were based heavily on the inability of LPS to drive a pri-155 luciferase reporter construct. Conversely, we and others (30,31) have repeatedly shown LPS can drive induction of the pri-155 promoter in a dose-dependent manner.…”

IL-10 Inhibits LPS-Induced Expression of miR-147 in Murine Macrophages

“…In this respect, it joins miR-155, a well studied miRNA with established roles in lymphoid and myeloid oncogenesis, as well as in the inflammatory response [41] [58] [67]. TLR signaling induces miR-155 expression in macrophages and IL-10 is able to counter-regulate that induction [55] [68]. Thus, IL-10 constrains the pro-inflammatory actions of miR-155, which include: increasing TNF production, enhancing PI3K-dependent activation of NF-κB and MAPKs, enhancing cytokine responses through JAK-STAT signaling, and potentiating Th1/Th17 cell polarization [41] [53] [55] [58] [69].…”

“…This result was followed by work showing that IL-10 counter-regulates miR-155 by first down-regulating the expression of the Ets2 transcription factor, which is required for the LPS-induced transcription of miR-155 [76]. Reminiscent of the complexities regarding how IL-10 counter-regulates protein-encoding genes, another group reported that IL-10's inhibitory effect on TLR-induced miR-155 was mediated at a post-transcriptional level via destabilizing the pri-miR-155 and pre-miR-155 transcripts [68]. It is certainly possible that IL-10 is acting on miR-155 at multiple levels, as has been shown for the mechanisms by which IL-10 can inhibit cytokine and chemokine gene expression [30]- [32] [60] [77].…”