MicroRNA-155 is induced during the macrophage inflammatory response

Abstract

The mammalian inflammatory response to infection involves the induction of several hundred genes, a process that must be carefully regulated to achieve pathogen clearance and prevent the consequences of unregulated expression, such as cancer. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators that has also been linked to cancer. However, the relationship between inflammation, innate immunity, and miRNA expression is just beginning to be explored. In the present study, we use microarray technology to identify miRNAs induced in primary murine macrophages after exposure to polyriboinosinic:polyribocytidylic acid or the cytokine IFN-beta. miR-155 was the only miRNA of those tested that was substantially up-regulated by both stimuli. It also was induced by several Toll-like receptor ligands through myeloid differentiation factor 88- or TRIF-dependent pathways, whereas up-regulation by IFNs was shown to involve TNF-alpha autocrine signaling. Pharmacological inhibition of the kinase JNK blocked induction of miR-155 in response to either polyriboinosinic:polyribocytidylic acid or TNF-alpha, suggesting that miR-155-inducing signals use the JNK pathway. Together, these findings characterize miR-155 as a common target of a broad range of inflammatory mediators. Importantly, because miR-155 is known to function as an oncogene, these observations identify a potential link between inflammation and cancer.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Microarray analysis of miRNAs induced during the macrophage antiviral response. (A) WT murine macrophages were stimulated with medium (m), 2 μg/ml poly(I:C) [p(I:C)], or 1,000 units/ml IFN-β for 6 h. RNA was extracted and used to conduct a microarray analysis to determine the expression levels of 200 mammalian miRNAs. Data are presented on a scatter plot showing log10-transformed signal intensities for each probe on both channels for the Cy3-labeled media controls and samples stimulated with Cy5-labeled IFN-β (Left) or poly(I:C) (Right). (B) RNA used in A was analyzed by qPCR to assay expression of miR-155 and in a separate experiment by Northern blot analysis under the same conditions. (C) RNA used in A was assayed by qPCR to detect expression of the small nuclear RNA U6 as a loading control or IP10 mRNA to ensure equivalent stimulation by poly(I:C) and IFN-β. (D) A portion of the macrophages generated in A were stimulated with medium, poly(I:C), or IFN-β and assayed for CD11b and CD86 expression by using FACS to ensure proper macrophage development and activation, respectively.

Fig. 2.

Kinetics of poly(I:C) and IFN-β induction of BIC mRNA and mature miR-155. (A) Unscaled depiction of the genomic structure of the human BIC noncoding RNA gene and location of miR-155 (155) in exon 3. E, exon; I, intron. (B) After stimulation with 2 μg/ml poly(I:C) or 1,000 units/ml IFN-β, macrophage expression of BIC mRNA was analyzed over a 48-h time course by reverse transcription with an oligonucleotide dT primer followed by detection using PCR and agarose gel electrophoresis. Primers were designed to target BIC sequences extending outside of miR-155. L32 mRNA detection is included as a control. (C) RNA from B was also used to assay mature miR-155 by qPCR over a 48-h time course.

Fig. 3.

TLRs induce miR-155 expression through MyD88- or TRIF-dependent signaling pathways. (A) WT (Wt) murine macrophages were stimulated with medium (m), 2 μg/ml poly(I:C) [p(I:C)], 5 ng/ml LPS, 2 μg/ml Pam3CSK4 (P3C), or 100 nM CpG for 6 h and assayed by Northern blot analysis with a miR-155-specific probe. (B) WT, MyD88−/−, or TRIF−/− macrophages were stimulated with medium, poly(I:C), LPS, CpG, or Pam3CSK4 for 6 h, and miR-155 expression was assayed by qPCR. (C) WT or IFNAR−/− macrophages were stimulated with medium, poly(I:C), LPS, CpG, or Pam3CSK4 for 6 h, and miR-155 expression was assayed by qPCR.

Fig. 4.

IFNs induce miR-155 expression through TNF-α autocrine/paracrine signaling. (A) WT (Wt) and TNFR1−/− murine macrophages were stimulated with medium (m), 1,000 units/ml IFN-β, 50 ng/ml IFN-γ, or 10 ng/ml TNF-α for 6 h, and miR-155 was assayed by qPCR. (B) (Left) WT macrophages were stimulated with medium, IFN-β, or IFN-γ for 6 h, and TNF-α mRNA was analyzed by qPCR. (Right) WT and TNFR1−/− macrophages were stimulated with IFN-β for 6 h and assayed for IP10 mRNA expression by qPCR. (C) (Left) WT macrophages were stimulated with medium or 2 μg/ml poly(I:C) for 6 h and assayed for TNF-α expression by qPCR. (Right) WT and TNFR1−/− macrophages were stimulated with medium or poly(I:C) for 6 h, and miR-155 was assayed by qPCR.

Fig. 5.

Pharmacological inhibition of JNK blocks poly(I:C) and TNF-α induction of miR-155. WT murine macrophages were pretreated for 30 min with DMSO or sp600125 at 5 or 25 μg/ml and subsequently stimulated with medium, 2 μg/ml poly(I:C), or 10 ng/ml TNF-α in the presence of the vehicle or inhibitor. After 4 h, RNA was collected, and miR-155 expression was assayed by qPCR.