Re-polarization of tumor-associated macrophages to pro-inflammatory M1 macrophages by microRNA-155

Abstract

Dear Editor, A significant macrophage population has been detected within almost all human and mouse tumors. These tumor-associated macrophages (TAMs) are critical regulators of the tumor microenvironment and directly affect multiple steps in tumor development, including the growth, survival, invasion, and metastasis of tumor cells. Macrophages are highly versatile, multifunctional cells, and their phenotype closely depends on the physiological or pathological context. In response to microenvironmental signals, macrophages undergo different activation, including the ‘classic’ pro-inflammatory phenotype (also called M1) and the ‘alternative’ activated M2 polarization associated with an anti-inflammatory profile (Mosser, 2003). Classically activated M1 macrophages and alternatively activated M2 macrophages represent two extreme macrophage phenotypes. TAMs closely resemble ‘alternative’ (M2) macrophages, which produce high amounts of interleukin (IL)-10 but not IL-12, express scavenger receptors, and exhibit anti-inflammatory and tissue repair functions (Sica et al., 2008). In contrast, M1 macrophages, activated by interferon (IFN)-γ or other microbial products, produce large amounts of pro-inflammatory cytokines, express high levels of major histocompatibility complex molecules, and are potent killers of pathogens and tumor cells (Mills et al., 2000). The molecular mechanisms underlying TAM polarization to different phenotypes are the focus of intense investigation. Here, we present the first evidence that microRNA-155 (miR-155) is akey molecule controlling macrophage polarization. We found that the overexpression of miR-155 could re-program anti-inflammatory, pro-tumorial M2 TAMs to pro-inflammatory, antitumor M1 macrophages. Given the fact that the discovery of miRNAs, a class of ∼22-nucleotide-long non-coding RNAs, has revealed a new layer of gene regulation in almost every aspect of the biological process, including immune systems (Lodish et al., 2008), it is logic to hypothesize that miRNAs are involved in modulating macrophage polarization. To identify alterations in the miRNA expression profile during macrophage polarization, we prepared standard M1 and M2 phenotype macrophages. In this experiment, bone marrow-derived macrophages (BMDMs) were isolated from BALB/c mice and treated with lipopolysaccharide (LPS) and IFN-γ (for M1 polarization) or IL-4 (for M2 polarization) (Pelegrin and Surprenant, 2009). We used tumour necrosis factor-alpha (TNF-α), Nos2, and IL-12 as markers for the M1 phenotype and four other proteins, Arg1, Ym1, Msr2, Fizz1, and IL-10, as markers for the M2 phenotype. As shown in Supplementary Figure S1A, the marker gene assay clearly indicated that LPS and IFN-γ induced BMDMs to the M1 phenotype, whereas IL-4 induced BMDMs to the M2 phenotype. BMDMs treated with LPS/IFN-γ had significantly higher TNF-α and Nos2 levels but lower levels of Arg1, Ym1, Msr2, and Fizz1 than IL-4-treated BMDMs. As shown in Supplementary Figure S2B and C, the enzyme-linked immunosorbent assay (ELISA) indicated that BMDMs treated with LPS/IFN-γ had a significantly higher IL-12 level but lower IL-10 level than IL-4-treated BMDMs. Employing a low-density miRNA microarray assay, we compared the miRNA expression profiles in BMDMs, LPS/IFN-γ-induced M1, and IL-4-induced M2 macrophages (Supplementary Table S1). The microarray data showed a dramatic differential expression of miRNAs in M1 and M2 macrophages (Figure 1A). In particular, when BMDMs were induced to M1 macrophages, a panel of miRNAs, including miR-155, miR-147, miR-214, and miR-455, were significantly up-regulated, whereas the up-regulation of these miRNAs was not observed when BMDMs were induced to the M2 phenotype. TaqMan probe-based quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays further showed that, among the miRNAs we tested, miR-155 was the most up-regulated (>120-fold) when BMDMs polarized to the M1 phenotype (Figure 1B). Differential expression of miR-155 during macrophage polarization and its role in re-polarizing TAMs to M1 macrophages. (A) The miRNA expression profile of macrophages after M1 or M2 polarization. Microarray assays were performed three times. (B) qRT-PCR assays confirmed that miR-155 was the most up-regulated miRNA during the M1 polarization of BMDMs. (C) miR-155 levels in macrophages following M1-to-M2 re-polarization by IL-4 for 10 h. (D) miR-155 levels in macrophages following M2-to-M1 re-polarization by LPS and IFN-γ for 10 h. (E) Re-polarization of M1 macrophages to M2 macrophages by depleting miR-155. (F) Re-polarization of M2 macrophages to M1 macrophages through the overexpression of miR-155. (G) Macrophages were transfected with an anti-miR-155 ASO or a control oligonucleotide and then stimulated with LPS and IFN-γ for M1 polarization. (H) Macrophages were transfected with pre-miR-155 prior to IL-4-mediated M2 polarization. (I) The M2-like phenotype of TAMs isolated from mice implanted with