MicroRNA-155 promotes atherosclerosis by repressing Bcl6 in macrophages

Macrophages in atherosclerotic plaques drive inflammatory responses, degrade lipoproteins, and phagocytose dead cells. MicroRNAs (miRs) control the differentiation and activity of macrophages by regulating the signaling of key transcription factors. However, the functional role of macrophage-related miRs in the immune response during atherogenesis is unknown. Here, we report that miR-155 is specifically expressed in atherosclerotic plaques and proinflammatory macrophages, where it was induced by treatment with mildly oxidized LDL (moxLDL) and IFN-γ. Leukocyte-specific Mir155 deficiency reduced plaque size and number of lesional macrophages after partial carotid ligation in atherosclerotic (Apoe^–/–) mice. In macrophages stimulated with moxLDL/IFN-γ in vitro, and in lesional macrophages, loss of Mir155 reduced the expression of the chemokine CCL2, which promotes the recruitment of monocytes to atherosclerotic plaques. Additionally, we found that miR-155 directly repressed expression of BCL6, a transcription factor that attenuates proinflammatory NF-κB signaling. Silencing of Bcl6 in mice harboring Mir155^–/– macrophages enhanced plaque formation and CCL2 expression. Taken together, these data demonstrated that miR-155 plays a key role in atherogenic programming of macrophages to sustain and enhance vascular inflammation.

Macrophages are the principal effector cells in atherosclerosis, in which they perpetuate the vascular inflammatory response to lipid overloading (1, 2). The subendothelial deposition of lipoproteins precedes infiltration by macrophages, which take up modified lipoproteins in an unrestricted manner via scavenger receptors (2). The lysosomal degradation of lipoproteins generates free cholesterol, which is then converted to, and stored as, cholesterol esters (1). Macrophages can dispose of cholesterol by reverse transport to the liver through several mechanisms of HDL-mediated cholesterol efflux (3, 4). In atherosclerosis, the influx of lipoproteins into macrophages exceeds cholesterol efflux, resulting in the intracellular accumulation of cholesterol esters within lipid droplets, a hallmark of macrophage-derived foam cells (5). Although lipid-handling macrophages show an antiinflammatory phenotype characterized by activation of PPARγ and NR1H3/NR1H2, excessive intracellular lipid storage can induce inflammatory activation by increasing free cholesterol or by inducing the formation of cholesterol crystals (6–10). Conversely, stimulation with inflammatory mediators (such as TNF-α, IL-1β, or the Th1 cytokine IFN-γ) and activation of TLR4 (e.g., by minimally modified lipoproteins) not only induces an inflammatory response, but also enhances intracellular lipid accumulation in macrophages (11, 12). NF-κB–dependent signaling pathways are of crucial importance in the proinflammatory activation of macrophages; however, in vivo studies on the role of the myeloid NF-κB module in atherosclerosis show inconsistent results, indicative of the complexity of the different NF-κB signaling pathways that control both the activation and the resolution phases of inflammation (13–15). Genetic deficiency of TLR4 or its downstream signaling molecule, MYD88, both of which are potent activators of classical NF-κB signaling, limits atherosclerosis by reducing macrophage recruitment and is also associated with reduced expression of chemokines, such as CCL2 (16, 17). In atherosclerosis, macrophage-derived CCL2 acts in concert with endothelial CXCL1 to induce additional macrophage accumulation through a positive feedback regulatory mechanism (18, 19).

BCL6 is primarily expressed in B cells during the germinal center phase of differentiation and acts as a proto-oncogene in diffuse larger B cell lymphomas by compromising the cells’ ability to sense DNA damage (20). The effect of BCL6, at least in part, on the germinal center reaction is mediated by counter-regulation of NF-κB activation (20). Several mechanisms of BCL6-mediated inhibition of NF-κB signaling have been described, including direct repression of p50, inhibitory binding of BCL6 to NF-κB proteins, and opposing regulation of NF-κB target genes (21–24). In macrophages, Bcl6 is transiently induced in a NF-κB–dependent manner and directly represses Ccl2 expression, which indicates that BCL6 inhibits the acute inflammatory response (23, 24).

MicroRNAs (miRs) play an important role in macrophage biology by regulating their differentiation from precursor cells and modifying their inflammatory capacity (25). The typical effect of an individual miR lowers the protein level by less than 2-fold, primarily by destabilizing the target mRNA (26). However, an miR can effectively control the function of its target, either by switch or by fine-tuning interactions (27). In macrophages, functional polarization is associated with the upregulation of a distinct set of miRs (28, 29). TLR4-mediated activation of NF-κB induces a negative feedback loop by upregulating miRs such as miR-21, miR-147, miR-210, miR-34a, and miR-146, which dampen TLR-induced signaling and cytokine expression (30–32). In contrast, miR-155 shows both anti- and proinflammatory effects by regulating TAB2 and SOCS1, respectively (29, 32, 33). Furthermore, upregulation of miR-29b and miR-125a-5p in inflammatory macrophages promotes the expression of proinflammatory cytokines, suggestive of feed-forward regulation (29). Interestingly, treatment of macrophages with oxidized LDL (oxLDL) appears to suppress several miRs induced after inflammatory stimulation, such as miR-146a, miR-155, and miR-21 (34, 35). oxLDL-mediated downregulation of miR-146a enhances both lipid uptake and TLR4 signaling (34). However, oxLDL can also upregulate miR-125a-5p and miR-155, which reduce the accumulation of lipids and cytokine secretion in macrophages (36, 37). miRs that are crucial for the inflammatory macrophage response, such as miR-21, miR-210, miR-146a, miR-34a, miR-147, and miR-125a-5p, are significantly upregulated in human atherosclerotic lesions, which indicates that macrophage-derived miRs contribute to atherogenesis (38, 39). In addition, miR-33 promotes the progression of atherosclerosis and impairs ABCA1-dependent cholesterol removal from lesional macrophages (40). Although these data suggest that miRs play a key role in regulating macrophage function at the intersection of lipid-handling and inflammatory activation, it is still unclear how miRs affect macrophages in atherosclerosis.

Here, we found that miR-155 was upregulated in inflammatory macrophages and in macrophages in atherosclerotic lesions. Increased expression of miR-155 induced CCL2 in macrophages stimulated with mildly oxLDL (moxLDL) and IFN-γ via direct suppression of Bcl6, a transcription factor that counter-regulates NF-κB activation. Furthermore, we demonstrated that deficiency of Mir155 in hematopoietic cells reduced advanced atherosclerotic plaque formation and decreased CCL2 expression in lesional macrophages by derepressing Bcl6. In summary, we identified what we believe to be a novel miR-155–dependent inflammatory pathway in macrophages that plays a role in atherosclerosis.

MiR expression profile in atherosclerosis and inflammatory macrophages. To study the miR expression signature in atherosclerosis, flow-mediated plaque formation was induced in Apoe^–/– mice by partial carotid ligation (41). When combined with high-fat diet (HFD) feeding, this process resulted in advanced stenotic plaques within 6 weeks, as detected by micro–computed tomography (42) and immunostaining (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI61716DS1). We found that 33 miRs, including miR-155 and miR-147, were increased, and 83 miRs were decreased, in carotid arteries with plaques versus those without (Figure 1A and Supplemental Tables 1 and 2). Notably, miR-155 and miR-147 were also among the 6 upregulated miRs identified in BM-derived macrophages (BMDMs) after stimulation with LPS and IFN-γ compared with unstimulated BMDMs (Figure 1B and Supplemental Table 3). Moreover, stimulation with LPS and IFN-γ suppressed the expression of 26 miRs in BMDMs (Figure 1B and Supplemental Table 4).

Figure 1

Expression of miR-155 and miR-147 in atherosclerotic lesions and in inflammatory macrophages. (A) Expression profile of miRs in carotid plaques from Apoe^–/– mice 6 weeks after partial ligation in the contralateral carotid artery (n = 6). (B) Expression profile of miRs in LPS- and IFN-γ–stimulated and unstimulated BMDMs (n = 5). (C and F) Expression of miR-155 (C) and miR-147 (F) in the aortic vessel wall of Apoe^–/– mice fed regular diet (ctrl) or HFD for 3 or 10 months (n = 3–5 per group). *P < 0.05 vs. ctrl and 3 months. (D and G) Expression of miR-155 (D) and miR-147 (G) in plaque samples laser-microdissected from the aortic root of Apoe^–/– mice fed HFD for 3 or 10 months (n = 3–4 per group). The miR expression profile of these plaques was compared with that in samples from arteries without plaques within the same mouse (control). *P < 0.05 vs. control. (E and H) Expression of miR-155 (E) and miR-147b (the human homolog of mouse miR-147; H) in human carotid plaque samples and vessel walls (control). n = 3–4 per group. *P < 0.05. Data represent mean (A and B) or mean ± SEM (C–H).

To further study the role of miR-155 and miR-147 expression during atherosclerosis, we quantified their expression in a mouse model of diet-induced atherosclerosis. Expression of miR-155 in the aortic wall of Apoe^–/– mice was significantly higher after 10 months of HFD feeding than after 3 months of HFD or regular chow diet (P = 0.0111; Figure 1C). However, miR-155 expression in plaque samples of Apoe^–/– mice was already increased after 3 months of HFD feeding compared with that in the normal arterial wall (Figure 1D). Expression of miR-155 was also higher in human carotid plaques than in the artery wall (Figure 1E). Similar to miR-155, expression of miR-147 in the murine aortic wall only increased after 10 months of HFD feeding (Figure 1F). Although miR-147 expression was not significantly increased in murine plaques (P = 0.6161 at 3 months and P = 0.3567 at 10 months; Figure 1G), the expression of miR-147b — the human homolog of murine miR-147 — was increased in human carotid plaques compared with that in the vessel wall (Figure 1H). Thus, upregulation of miR-155 was consistently observed in different mouse models of atherosclerosis and in human plaques, whereas miR-147 expression was more variable.

Macrophages play a key role in the inflammatory response during atherosclerosis, and M1 polarized macrophages have been described in atherosclerotic lesions. Therefore, we performed in situ hybridization to identify the cellular source of miR-155 in atherosclerotic lesions. First, we verified the specificity of in situ hybridization for miR-155 using Mir155^–/– mice as negative controls (Supplemental Figure 2). In atherosclerotic lesions, additional immunostaining for the macrophage marker MAC2 demonstrated that the majority of lesional macrophages expressed miR-155 (Figure 2A). Additionally, some SMCs also expressed miR-155, as detected by combined immunostaining for miR-155 and SMA (Figure 2B). Therefore, both hematopoietic and vessel wall–derived cells in atherosclerotic lesions expressed miR-155.

Figure 2

miR-155 is expressed in macrophages and SMCs in atherosclerotic lesions. (A) In situ hybridization for miR-155 (red) and immunostaining for galectin-3 (MAC2; green) was performed in murine carotid artery plaques. Arrows denote macrophages expressing miR-155. (B) In situ hybridization for miR-155 (red) and immunostaining for SMA (green) was performed in murine carotid artery plaques. Arrows denote SMCs expressing miR-155. Scale bars: 20 μm.

Effect of miR-155 on atherosclerosis. To study the function of miR-155 in atherogenesis, partial carotid ligation was performed in Mir155^+/+Apoe^–/– mice transplanted with Mir155^–/–Apoe^–/– BM cells and in Mir155^–/–Apoe^–/– mice transplanted with Mir155^+/+Apoe^–/– BM. Mir155^+/+Apoe^–/– mice repopulated with Mir155^+/+Apoe^–/– BM cells were used as controls. A significant reduction in plaque size and in the number of macrophages per plaque was found in Mir155^+/+Apoe^–/– mice harboring Mir155^–/–Apoe^–/– BM compared with the other 2 experimental groups (Figure 3, A and B). However, the medial area, external elastic lamina length, relative macrophage plaque content (determined as macrophage number or MAC2⁺ area normalized to the plaque area), and macrophage size were not different among the groups (Figure 3A and Supplemental Figures 3 and 4). Moreover, the relative SMC content tended to be decreased in Mir155^+/+Apoe^–/– mice harboring Mir155^–/–Apoe^–/– BM (Figure 3C), whereas the relative lesional CD3⁺ T cell and MPO⁺ neutrophil counts and the collagen type I content were not substantially affected by Mir155 deficiency (Figure 3, D–F). The number of apoptotic macrophages in the lesions was similar in all 3 groups (Figure 3G). No differences were observed in the serum lipoprotein profile among the groups (Supplemental Figure 5). A combination of immunostaining for MAC2 and Oil red O staining demonstrated that lipid accumulation in Mir155^–/– macrophages in atherosclerotic lesions was significantly less than that in Mir155^+/+ macrophages (P = 0.0466; Supplemental Figure 6). These results indicate that the expression of miR-155 in lesional macrophages promotes atherosclerosis by increasing the number of lesional macrophages and critically regulating their function.

Figure 3

Role of miR-155 in atherosclerosis. (A) Lesion area and medial area 6 weeks after partial carotid ligation in Mir155^+/+Apoe^–/– mice harboring Mir155^+/+Apoe^–/– or Mir155^–/–Apoe^–/– BM (Mir155^+/+ BM/Mir155^+/+ [n = 7] and Mir155^–/– BM/Mir155^+/+ [n = 8] respectively) and in Mir155^–/–Apoe^–/– mice harboring Mir155^+/+Apoe^–/– BM (Mir155^+/+ BM/Mir155^–/– [n = 6]). Representative images are shown. (B) The number of MAC2⁺ macrophages in carotid lesions was determined by immunostaining. Representative images are shown. (C–F) Lesional content of SMCs, T cells, neutrophils, and collagen, assessed by immunostaining for SMA (C), CD3 (D), MPO (E), and collagen type I (F). (G) Apoptotic macrophages were quantified in carotid lesions by combined TUNEL staining and MAC2 immunostaining. n = 6–8 mice per group. *P < 0.05. Scale bars: 100 μM. Data are mean ± SEM.

Role of miR-155 in proinflammatory macrophage activation. miR-155 is implicated in the regulation of proinflammatory M1 macrophage polarization (43); however, its functional role in the development of atherosclerotic plaque containing activated macrophages is unclear. Our analysis of macrophage-specific miR-155 expression, which was downregulated during macrophage differentiation (Figure 4A), showed that polarization of BMDMs into proinflammatory M1-type macrophages upon stimulation with LPS and IFN-γ (in contrast to antiinflammatory M2-type polarization induced by IL-4) induced miR-155 expression (Figure 4B). Native LDL and moxLDL slightly increased miR-155 expression, whereas highly oxLDL reduced the expression of miR-155 (Figure 4B) and induced expression of the M2 marker Mrc1 (Supplemental Figure 7). This indicates that the degree of LDL oxidation plays a role in the effect of modified LDL on miR-155 expression. Similar to LPS and IFN-γ, stimulation with moxLDL and IFN-γ induced miR-155 together with M1-type markers, such as Nos2 and Tnf (Figure 4B and Supplemental Figure 7). Thus, moxLDL-stimulated macrophages also required IFN-γ, which is highly expressed in atherosclerotic plaques (44), to develop a M1 phenotype and to substantially upregulate miR-155 expression. In contrast to moxLDL- and IFN-γ–stimulated Mir155^+/+ BMDMs, Mir155^–/– BMDMs mainly showed reduced expression of Ccl2 mRNA (and also some reduction in Tnf mRNA), whereas no substantial difference in Il1b and Nos2 expression was detectable between Mir155^+/+ and Mir155^–/– BMDMs (Figure 4C). The effect of miR-155 on CCL2 expression and secretion was confirmed at the protein level (Figure 4, D and E). In contrast, macrophage migration and apoptosis appeared to be unchanged in the absence of miR-155 in vitro (Figure 4, F and G). To study the effect of miR-155 on CCL2 expression in lesional macrophages, combined immunostaining for CCL2 and MAC2 was performed (Figure 5). In line with the results obtained for BMDMs, the number of CCL2-expressing macrophages was substantially reduced in carotid lesions in Mir155^+/+Apoe^–/– mice harboring Mir155^–/–Apoe^–/– BM compared with that in Mir155^+/+Apoe^–/– and Mir155^–/–Apoe^–/– mice harboring Mir155^+/+Apoe^–/– BM (Figure 5), which may lead to impaired macrophage infiltration into the plaques in Apoe^–/– mice with Mir155-deficient leukocytes.

Figure 4

Role of miR-155 in atherogenic macrophage activation. (A) miR-155 expression by quantitative RT-PCR in murine BM cells cultured in L929-conditioned medium to induce macrophage differentiation. (B) Regulation of miR-155 expression in BMDMs by modified LDL was quantified and compared with classical and alternative macrophage activation by quantitative RT-PCR. (C) Expression of inflammatory macrophage markers was analyzed by quantitative RT-PCR in BMDMs from Mir155^+/+ and Mir155^–/– mice after stimulation with moxLDL and IFN-γ. (D and E) CCL2 protein in cell lysates (D) and in the cell culture medium (E) of BMDMs from Mir155^+/+ and Mir155^–/– mice after stimulation with moxLDL and IFN-γ, quantified by ELISA. (F) Migration of Mir155^+/+ and Mir155^–/– BMDMs across a Transwell filter toward CCL2. (G) Apoptosis of BMDMs from Mir155^+/+ and Mir155^–/– mice, determined by TUNEL assay. *P < 0.05, ***P < 0.001 vs. all other groups; ^#P < 0.05 vs. control, oxLDL, IL-4, LPS plus IFN-γ, and moxLDL plus IFN-γ; ^‡P <0.05 vs. control, native LDL, moxLDL, oxLDL, and IL-4; ^§P < 0.05 vs. Mir155^+/+. 3 independent experiments were conducted per group. Data are mean ± SEM.

Figure 5

Role of miR-155 in CCL2 expression by lesional macrophages. Expression of CCL2 (green) was assessed in MAC2⁺ macrophages (red) from plaques 6 weeks after partial carotid ligation in Apoe^–/– mice harboring Mir155^+/+Apoe^–/– or Mir155^–/–Apoe^–/– BM and in Mir155^–/–Apoe^–/– mice harboring Mir155^+/+Apoe^–/– BM by double immunostaining. Representative overlays of CCL2 and MAC2 immunostaining are shown (top). CCL2 expressed in macrophages is shown in yellow. The percentage of CCL2⁺ macrophages was determined. Adjacent sections were stained with EVG to demonstrate lesion morphology (bottom). n = 6–8 per group. *P < 0.05. Scale bars: 20 μm. Data are mean ± SEM.

Mechanism of miR-155–mediated cytokine expression. Several targets for miR-155 have been described in macrophages, including Socs1 and Sfpi1, which regulate inflammatory and developmental processes, respectively (33, 45). To identify the mechanism of miR-155–induced CCL2 expression, we studied 7 potential miR-155 targets that were increased in either unstimulated Mir155^–/– BMDMs or in Mir155^–/– BMDMs stimulated with moxLDL and IFN-γ (Figure 6, A and B) using EIF2C2-specific IP. miR-155 was highly enriched in EIF2C2-containing miR-induced silencing complexes (mRISCs) in Mir155^+/+, but not Mir155^–/–, BMDMs (Figure 6C). Of the 7 potential miR-155 targets in BMDMs, Rela, Tcf7l2, Socs1, Bcl6, and Sfpi1 were associated with EIF2C2 in Mir155^+/+, but not Mir155^–/–, BMDMs (Figure 6D and Supplemental Figure 8), indicative of direct repression by miR-155, in moxLDL- and IFN-γ–stimulated macrophages. Although 2 binding sites for miR-155 are predicted to reside within the 3′UTR of Bcl6, to our knowledge, targeting Bcl6 has not been described previously. Therefore, using luciferase reporter assays, we verified the direct targeting of the 3′UTR of Bcl6 by miR-155 (Figure 6, E and F). Mutation of one of the predicted binding sites within the Bcl6 3′UTR prevented suppression by miR-155 (Figure 6F).

Figure 6

Identification of miR-155 targets in BMDMs. (A and B) Effect of Mir155 deficiency on expression of potential miR-155 targets Hif1a (n = 4) and Rela, Socs1, Tcf7l2, Bcl6, Pparg, and Sfpi1 (n = 3 for each) in unstimulated (A) and moxLDL- and IFN-γ–stimulated (B) BMDMs. *P < 0.05 vs. Mir155^+/+. (C) Quantitative RT-PCR of miR-155 in immunoprecipitates from moxLDL- and IFN-γ–stimulated Mir155^+/+ and Mir155^–/– BMDMs. Precipitates were obtained after incubation of cell lysates with an anti-EIF2C2 or nonspecific IgG control antibody (n = 4 independent experiments per group). *P < 0.05 vs. IgG. (D) Enrichment of potential miR-155 targets in EIF2C2-IP from moxLDL/IFN-γ–stimulated Mir155^+/+ and Mir155^–/– BMDMs by quantitative RT-PCR (n = 3–4 independent experiments per group). Results are expressed as target enrichment in Mir155^–/– BMDMs normalized to that in Mir155^+/+ BMDMs. *P < 0.05 vs. Mir155^+/+. (E) Potential target sites for miR-155 in the 3′UTR of murine Bcl6 mRNA, as predicted by the miRanda prediction algorithm (sites A and B; blue). Target site A (sequence highlighted in green) was mutated in the binding region. The same sites in the human Bcl6 3′UTR are also predicted binding sites for human miR-155, differing from mouse miR-155 by 1 nucleotide. (F) Luciferase reporter assays in HEK293 cells treated with miR-155 mimics or nontargeting control mimics using the pEZX-MT01 vector containing the Bcl6 3′UTR or the Bcl6 3′UTR with mutations in predicted miR-155 binding site A (n = 3 independent experiments per group). *P < 0.05 vs. control. Data are mean ± SEM.

To study which miR-155 target regulates expression of Ccl2 and Tnf, silencing of Socs1, Bcl6, and Sfpi1 was performed using RNA interference (Supplemental Figure 9). Silencing Bcl6, but not Socs1 or Sfpi1, increased Ccl2 and Tnf mRNA expression in Mir155^–/– BMDMs (Figure 7 and Supplemental Figure 10). Upregulation of CCL2 in Mir155^–/– BMDMs by Bcl6-specific siRNA was confirmed at the protein level, whereas TNF-α protein levels only showed a tendency toward an increase after Bcl6 siRNA treatment (Figure 7). Thus, derepression of the miR-155 target Bcl6 in Mir155^–/– macrophages reduced atherogenic cytokine expression.

Figure 7

Targeting Bcl6 mediates the proinflammatory effects of miR-155. (A) Effect of siRNA-mediated silencing of Socs1 (siSocs1) in Mir155^–/– BMDMs on Ccl2 (n = 3) and Tnf (n = 3) mRNA expression was compared with that mediated by nontargeting control siRNA (siNTC) by quantitative RT-PCR. (B and C) CCL2 secretion (B; n = 3) and TNF-α protein expression (C; n = 3) in Mir155^–/– BMDMs, compared with that of control siRNA, determined by ELISA. (D) Expression of Ccl2 (n = 3) and Tnf (n = 4) mRNA after siRNA-mediated silencing of Bcl6 (siBcl6) in Mir155^–/– BMDMs, compared with that after control siRNA treatment, determined by quantitative RT-PCR. (E and F) Effect of Bcl6 siRNA treatment on CCL2 (E; n = 3) and TNF-α (F; n = 3) protein expression in Mir155^–/– BMDMs, compared with that by control siRNA. *P < 0.05 vs. control. Data are mean ± SEM.

Regulation of the antiinflammatory role of BCL6 in atherogenic macrophages. BCL6 acts as an NF-κB antagonist either by opposing the upregulation of many NF-κB–responsive target genes in stimulated macrophages or by directly inhibiting NF-κB activity (22, 23, 46). We found that blocking NF-κB activation using the NF-κB inhibitor BAY11-7985 reduced Ccl2 and Tnf mRNA expression in Bcl6 siRNA–treated Mir155^–/– macrophages (Figure 8, A and B). To determine whether autocrine stimulation by TNF-α contributes to the effect of miR-155 on CCL2 expression, TNF-α in the cell culture medium was blocked using an anti–TNF-α antibody. Although the antibody inhibited CCL2 secretion by Mir155^+/+ macrophages, blocking TNF-α did not abrogate the reduced CCL2 secretion in Mir155^–/– BMDMs compared with Mir155^+/+ macrophages (Figure 8, C and D), which indicates that miR-155 increases CCL2 secretion independently of elevated Tnf expression. In contrast to Mir155^–/– macrophages, Bcl6 mRNA in Mir155^+/+ macrophages was upregulated 24 hours after stimulation with moxLDL and IFN-γ, reducing the difference in Bcl6 expression between unstimulated Mir155^+/+ and Mir155^–/– macrophages (Figure 8E). Similar results were obtained at the protein level (Figure 8, F and G); however, upregulation of the BCL6 protein in Mir155^+/+ BMDMs was only observed 24 hours after stimulation, whereas BCL6 protein expression was still higher in Mir155^–/– than in Mir155^+/+ macrophages 6 and 10 hours after stimulation (Figure 8G). Blocking NF-κB activation in Mir155^+/+ BMDMs reduced the expression of Bcl6 mRNA 24 hours after stimulation with moxLDL and IFN-γ (Figure 8H), which indicates that upregulation via NF-κB activation offsets the suppressive effect of miR-155 on Bcl6 expression at later stages of the inflammatory macrophage response.

Figure 8

Role of NF-κB and TNF-α in regulating CCL2 and BCL6 expression during macrophage stimulation. (A and B) Role of NF-κB signaling in increased Ccl2 (A) and Tnf (B) mRNA expression in Bcl6 siRNA–treated Mir155^–/– BMDMs, as determined using the NF-κB inhibitor BAY11-7085. (C) CCL2 protein levels in the medium of Mir155^+/+ BMDMs after stimulation with moxLDL and IFN-γ, quantified by ELISA. A blocking antibody against TNF-α or an isotype control antibody was added to the medium. (D) CCL2 protein levels in the medium of Mir155^+/+ or Mir155^–/– BMDMs after stimulation with moxLDL and IFN-γ, quantified by ELISA. A blocking antibody against TNF-α (5 μg/ml) was added to the medium in both groups. (E) Effect of moxLDL and IFN-γ stimulation on Bcl6 mRNA expression in Mir155^+/+ and Mir155^–/– BMDMs. (F) BCL6 protein expression in unstimulated Mir155^+/+ and Mir155^–/– BMDMs, determined by Western blot. (G) Time course of BCL6 protein expression after moxLDL and IFN-γ stimulation of Mir155^+/+ and Mir155^–/– BMDMs, determined by Western blot. The intensity of the BCL6 bands relative to that of ACTB bands is expressed as a percentage of that in unstimulated BMBMs (F) or in Mir155^+/+ BMDMs 6 hours after stimulation (G). Lanes in G were run on the same gel but were noncontiguous (white lines). (H) Bcl6 mRNA expression in BMDMs stimulated with moxLDL and IFN-γ after treatment with BAY11-7085 or vehicle. *P < 0.05. n = 3 independent experiments per group. Data are mean ± SEM.

Role of BCL6 in miR-155–mediated protection against atherogenesis. To investigate the role of BCL6 in protection against atherosclerosis in mice harboring Mir155^–/– BM cells, we analyzed BCL6 expression in lesional macrophages using a combined immunostaining approach (Figure 9A). The number of BCL6-expressing macrophages was substantially increased in Mir155^+/+Apoe^–/– mice harboring Mir155^–/–Apoe^–/– BM compared with Mir155^+/+Apoe^–/– and Mir155^–/–Apoe^–/– mice harboring Mir155^+/+Apoe^–/– BM (Figure 9B). Furthermore, Bcl6 mRNA expression was higher in carotid plaques from mice harboring Mir155^–/– BM than in those from mice with Mir155^+/+ BM (Figure 9C). Next, we studied the effect of increased Bcl6 expression in Apoe^–/– mice harboring Mir155^–/– BM on atherosclerosis by silencing Bcl6 in the carotid artery after partial ligation (Supplemental Figure 11). Treatment with Bcl6 siRNA led to a marked increase in plaque formation compared with nonspecific control siRNA, but did not affect the medial area in Apoe^–/– mice harboring Mir155^–/– BM (Figure 10A). Whereas the number of macrophages per lesion was substantially higher after treatment with Bcl6 siRNA, relative macrophage content and macrophage size were unchanged (Figure 10B and Supplemental Figure 12). Lesional SMC content tended to increase after Bcl6 siRNA treatment (Supplemental Figure 13). Of note, immunostaining of lesions showed more CCL2-expressing Mir155^–/– macrophages in arteries treated with Bcl6 siRNA than with control siRNA (Figure 10C).

Figure 9

Expression of lesional BCL6 is miR-155 dependent. (A) Combined immunostaining for Bcl6 (red) and MAC2 (green) in atherosclerotic lesions of carotid arteries after partial ligation in Mir155^+/+Apoe^–/– mice harboring Mir155^+/+Apoe^–/– or Mir155^–/–Apoe^–/– BM and in Mir155^–/–Apoe^–/– mice harboring Mir155^+/+Apoe^–/– BM. Representative images are shown. Nuclei were counterstained with DAPI (blue). Arrows denote BCL6-expressing macrophages. Scale bars: 50 μm. (B) BCL6⁺ macrophages in sections of carotid lesions immunostained for BCL6 and MAC2 (n = 6–8 per group). (C) Bcl6 mRNA expression in lesions of carotid arteries 6 weeks after partial ligation in Mir155^+/+Apoe^–/– mice harboring Mir155^+/+Apoe^–/– or Mir155^–/–Apoe^–/– BM, determined by quantitative RT-PCR (n = 4 per group). *P < 0.05. Data are mean ± SEM.

Figure 10

Role of BCL6 in miR-155–mediated atherosclerosis. Partially ligated carotid arteries from Apoe^–/– mice harboring Mir155^–/– BM were perivascularly treated with Bcl6 siRNA or nontargeting control siRNA. (A) Lesion area and medial area were determined in carotid artery sections stained with EVG by planimetry. Representative images are shown. (B) Number of macrophages in lesions from the carotid artery, analyzed by immunostaining for MAC2 (green). Representative images are shown, and the number of MAC2⁺ cells per lesion was determined. (C) Combined immunostaining for CCL2 (green) and MAC2 (red) was performed in carotid artery sections, and the percentage of CCL2⁺ macrophages (yellow) was quantified. n = 4–5 per group. *P < 0.05. Scale bars: 100 μm. Data are mean ± SEM.

Chronic, nonresolving inflammation driven by lipid-laden macrophages is crucial for atherosclerotic lesion formation. Here, we found that miR-155 promoted the inflammatory response of macrophages during atherosclerosis. Expression of miR-155 in unstimulated macrophages was essential for the upregulation of CCL2 after inflammatory stimulation by direct suppression of Bcl6 expression. Thus, miR-155 sensitized macrophages to inflammatory activation by abrogating BCL6-mediated inhibition of NF-κB signaling. The absence of miR-155 in macrophages limited atherosclerotic plaque formation by upregulating Bcl6, which decreased the expression of CCL2 in lesional macrophages. Therefore, priming of macrophages for inflammatory activation by miR-155 supported the development of advanced atherosclerotic lesions.

miRs play a crucial role in cell fate transitions and regulate the development and function of immune cells (47). Accordingly, macrophage polarization to inflammatory M1 — and alternatively activated M2 — subtypes is associated with the differential regulation of a distinct set of miRs (29). Upregulation of miR-155 and miR-147 was observed in macrophages upon stimulation of TLRs and after polarization into an M1-type (29, 30, 48); however, the functional roles of these 2 miRs in macrophages differ substantially. Whereas miR-155 primarily enhances proinflammatory cytokine expression, miR-147 is part of a negative feedback loop that constrains the inflammatory response (29, 30, 33). Our present results demonstrated that increased miR-155 and miR-147 expression was characteristic not only of M1-type macrophages, but also of atherosclerotic plaques. In addition to miR-155 and miR-147, 7 miRs — including miR-21, miR-21*, miR-34a, and miR-146a/b, which were upregulated in flow-induced carotid lesions in Apoe^–/– mice — are also increased in human atherosclerotic plaques (38, 39). Whereas miR-147 expression was also upregulated by hyperlipidemia in the walls of nonatherosclerotic arteries, increased expression of miR-155 was confined to atherosclerotic plaques, which indicates that miR-155 may affect atherosclerosis through its effects on atherogenic macrophages. Although a subset of SMCs in atherosclerotic lesions expressed miR-155, we confirmed that lesional macrophages were an abundant source of miR-155.

In LPS-stimulated macrophages, AKT1-mediated suppression of miR-155 is crucial for the development of endotoxin tolerance and dampening of M1-type macrophage polarization (33, 43). The proinflammatory role of miR-155 is associated with targeting inhibitors of cytokine signalling, such as Socs1 or Inpp5d (33, 49). In addition, the antiinflammatory effect of IL-10 on macrophages has previously been linked to the inhibition of miR-155 transcription, resulting in increased expression of Inpp5d (50). However, suppression of inflammatory cytokines (such as IL-1β and TNF-α) and of NF-κB activity by miR-155 was described in monocyte-derived dendritic cells and oxLDL-treated THP-1 macrophages (51). The results of the present study clearly showed that miR-155 promoted the proinflammatory activation of macrophages by enhancing the expression of Ccl2 and Tnf. Although we found that confirmed miR-155 targets in inflammatory macrophages, such as Socs1, were indeed targeted by miR-155, the direct effects of miR-155 on Ccl2 and Tnf expression were mediated by suppression of Bcl6. Therefore, our results demonstrated that targeting of the transcriptional repressor Bcl6 by miR-155 is a key mechanism underlying the inflammatory activation of macrophages.

Accordingly, we found that targeting of Bcl6 by miR-155 in unstimulated macrophages promoted subsequent proinflammatory activation. However, once fully activated, the suppressive effect of miR-155 was offset by the NF-κB–mediated upregulation of Bcl6, which may be important for the resolution of the inflammatory response. Thus, our data suggest that miR-155–mediated suppression of Bcl6 is crucial for the acute inflammatory activation of macrophages, whereas feedback inhibition through NF-κB–induced Bcl6 expression prevails during the postactivation state.

We also found that deficiency of miR-155 in hematopoietic cells, but not in vascular cells, attenuated advanced atherosclerosis induced by acute flow disturbance and hyperlipidemia via increased BCL6 expression by macrophages. The reduction in lesion size observed in mice harboring Mir155^–/– BM was due to fewer macrophages in the plaques. Upregulation of BCL6 in lesional Mir155^–/– macrophages repressed CCL2 expression, which is known to promote the recruitment of monocytes to atherosclerotic plaques (18). In addition to its immunoregulatory functions, miR-155 can enhance or prevent the apoptosis of infected macrophages (52, 53). However, we did not observe any effect of miR-155 on the apoptosis of lesional macrophages. In contrast to our findings, increased HFD-induced early atherosclerosis and neutrophil infiltration into lesions was described in Ldlr^–/– mice harboring Mir155^–/– BM (54). Neutrophils primarily play a role during early atherogenesis by enhancing the recruitment of monocytes, whereas advanced atherosclerosis appears to be less influenced by neutrophils (55). Accordingly, we did not observe any effect of miR-155 on the neutrophil content of advanced carotid lesion that were induced by disturbed flow, which suggests that miR-155 affects atherosclerosis in a stage-dependent manner. In contrast to early atherosclerosis, in which Mir155 deficiency disturbs neutrophil homeostasis, the proinflammatory role of miR-155 in macrophage activation promoted advanced atherosclerosis. In line with our results, Bcl6 deficiency in BM cells from Ldlr^–/– mice exacerbates atherosclerosis and increases the expression of genes involved in atherogenic inflammation, such as CCL2 (56). Taken together, our findings suggest that miR-155–mediated suppression of Bcl6 sensitizes macrophages to atherogenic activation and that this is a key inflammatory mechanism in advanced atherosclerosis.

In addition to upregulating miR-155 in inflammatory macrophages, we found that both native LDL and moxLDL increased the expression of miR-155, although to a lesser extent than did LPS and IFN-γ. In contrast, profoundly oxLDL suppressed the expression of miR-155 in macrophages. Previous results from studies of the effects of oxLDL on miR-155 expression are conflicting. For example, both up- and downregulation of miR-155 in response to oxLDL has been reported in THP-1 macrophages (34, 37). Moreover, treatment with oxLDL can induce the expression of miR-155 in human primary monocytes (36). According to our present results, regulation of miR-155 expression depended on the level of oxLDL modification, which may explain the controversial findings previously reported in the literature. Interestingly, higher levels of LDL oxidation inhibited both the expression of inflammatory cytokines in macrophages and the activation of NF-κB, which is essential for the transcriptional upregulation of miR-155 (57–59). In addition to the regulation of miR-155 in macrophages by modified LDL, we found that lesional Mir155^–/– macrophages accumulated fewer lipids. This is in contrast to previous in vitro results, which report increased lipid accumulation in THP-1 macrophages following treatment with a miR-155 inhibitor (37). Interestingly, the expression of PPARγ, which plays a key role in the alternative activation and effective lipid handling of macrophages (including LDL uptake and cholesterol efflux), was increased in Mir155^–/– macrophages (10). Although our results do not indicate that miR-155 targets PPARγ in inflammatory macrophages, miR-155 suppressed PPARγ in unstimulated macrophages, which express much higher levels of PPARγ than do inflammatory macrophages (60). Alternatively, PPARγ levels might be reduced indirectly in miR-155–expressing macrophages via enhanced NF-κB activation (60, 61). Despite the reduced lipid accumulation in Mir155^–/– lesional macrophages, the size of the macrophages in atherosclerotic lesions was not altered by Mir155 deficiency. Therefore, we conclude that differences in lipid accumulation are not caused by reduced lesion formation in mice harboring Mir155^–/– macrophages.

In summary, we showed that increased miR-155 expression in proinflammatory macrophages promoted atherosclerosis by derepressing BCL6-mediated inhibition of CCL2 transcription. We therefore conclude that miR-155 plays a key role in the atherogenic programming of macrophages, which sustains and amplifies vascular inflammation.

View Supplemental data

This work was supported by the DFG (Scho1056/3-1 and FOR809), by the Interdisciplinary Centre for Clinical Research within the Faculty of Medicine at the RWTH Aachen University (to M. Nazari-Jahantigh), and by the German Centre for Cardiovascular Research (MHA VD 1.2). We thank Anna Thiemann, Stephanie Wilbertz, Melanie Garbe, and Roya Soltan for technical assistance and Pallavi Subramanian for assistance in analyzing immunostainings.

Address correspondence to: Andreas Schober, Institute for Cardiovascular Prevention, Ludwig-Maximilians University Munich, Pettenkoferstr. 9, 80336 Munich, Germany. Phone: 49.89.51605151; Fax: 49.89.51604740; E-mail: aschober@med.lmu.de.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2012;122(11):4190–4202. doi:10.1172/JCI61716.

1. Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell. 2011;145(3):341–355.
   View this article via: PubMed CrossRef Google Scholar
2. Weber C, Noels H. Atherosclerosis: current pathogenesis and therapeutic options. Nat Med. 2011;17(11):1410–1422.
   View this article via: PubMed CrossRef Google Scholar
3. Zhao Y, Van Berkel TJ, Van Eck M. Relative roles of various efflux pathways in net cholesterol efflux from macrophage foam cells in atherosclerotic lesions. Curr Opin Lipidol. 2010;21(5):441–453.
   View this article via: PubMed CrossRef Google Scholar
4. Marcel YL, Ouimet M, Wang M-D. Regulation of cholesterol efflux from macrophages. Curr Opin Lipidol. 2008;19(5):455–461.
   View this article via: PubMed CrossRef Google Scholar
5. Ouimet M, Marcel YL. Regulation of lipid droplet cholesterol efflux from macrophage foam cells. Arterioscler Thromb Vasc Biol. 2012;32(3):575–581.
   View this article via: PubMed CrossRef Google Scholar
6. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature. 1998;391(6662):79–82.
   View this article via: PubMed CrossRef Google Scholar
7. Li Y, Tabas I. The inflammatory cytokine response of cholesterol-enriched macrophages is dampened by stimulated pinocytosis. J Leukoc Biol. 2007;81(2):483–491.
   View this article via: PubMed CrossRef Google Scholar
8. Duewell P, et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature. 2010;464(7293):1357–1361.
   View this article via: PubMed CrossRef Google Scholar
9. Li Y, et al. Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-alpha and interleukin-6: model of NF-kappaB- and map kinase-dependent inflammation in advanced atherosclerosis. J Biol Chem. 2005;280(23):21763–21772.
   View this article via: PubMed CrossRef Google Scholar
10. Ricote M, Valledor AF, Glass CK. Decoding transcriptional programs regulated by PPARs and LXRs in the macrophage: effects on lipid homeostasis, inflammation, and atherosclerosis. Arterioscler Thromb Vasc Biol. 2004;24(2):230–239.
    View this article via: PubMed CrossRef Google Scholar
11. McGillicuddy FC, et al. Inflammation impairs reverse cholesterol transport in vivo. Circulation. 2009;119(8):1135–1145.
    View this article via: PubMed CrossRef Google Scholar
12. Miller YI, Choi SH, Fang L, Harkewicz R. Toll-like receptor-4 and lipoprotein accumulation in macrophages. Trends Cardiovasc Med. 2009;19(7):227–232.
    View this article via: PubMed CrossRef Google Scholar
13. Kanters E, et al. Hematopoietic NF-kappaB1 deficiency results in small atherosclerotic lesions with an inflammatory phenotype. Blood. 2004;103(3):934–940.
    View this article via: PubMed CrossRef Google Scholar
14. Vallabhapurapu S, Karin M. Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol. 2009;27:693–733.
    View this article via: PubMed CrossRef Google Scholar
15. Kanters E, et al. Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice. J Clin Invest. 2003;112(8):1176–1185.
    View this article via: JCI PubMed Google Scholar
16. Björkbacka H, et al. Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways. Nat Med. 2004;10(4):416–421.
    View this article via: PubMed CrossRef Google Scholar
17. Michelsen KS, et al. Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E. Proc Natl Acad Sci U S A. 2004;101(29):10679–10684.
    View this article via: PubMed CrossRef Google Scholar
18. Aiello RJ, et al. Monocyte chemoattractant protein-1 accelerates atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 1999;19(6):1518–1525.
    View this article via: PubMed CrossRef Google Scholar
19. Zhou Z, et al. Lipoprotein-derived lysophosphatidic acid promotes atherosclerosis by releasing CXCL1 from the endothelium. Cell Metab. 2011;13(5):592–600.
    View this article via: PubMed CrossRef Google Scholar
20. Ci W, Polo JM, Melnick A. B-cell lymphoma 6 and the molecular pathogenesis of diffuse large B-cell lymphoma. Curr Opin Hematol. 2008;15(4):381–390.
    View this article via: PubMed CrossRef Google Scholar
21. Li Z, et al. BCL-6 negatively regulates expression of the NF-kappaB1 p105/p50 subunit. J Immunol. 2005;174(1):205–214.
    View this article via: PubMed Google Scholar
22. Perez-Rosado A, Artiga M, Vargiu P, Sanchez-Aguilera A, Alvarez-Barrientos A, Piris M. BCL6 represses NFkappaB activity in diffuse large B-cell lymphomas. J Pathol. 2008;214(4):498–507.
    View this article via: PubMed CrossRef Google Scholar
23. Barish GD, et al. Bcl-6 and NF-kappaB cistromes mediate opposing regulation of the innate immune response. Genes Dev. 2010;24(24):2760–2765.
    View this article via: PubMed CrossRef Google Scholar
24. Toney LM, et al. BCL-6 regulates chemokine gene transcription in macrophages. Nat Immunol. 2000;1(3):214–220.
    View this article via: PubMed CrossRef Google Scholar
25. O’Connell RM, Zhao JL, Rao DS. MicroRNA function in myeloid biology. Blood. 2011;118(11):2960–2969.
    View this article via: PubMed CrossRef Google Scholar
26. Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs on protein output. Nature. 2008;455(7209):64–71.
    View this article via: PubMed CrossRef Google Scholar
27. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136(2):215–233.
    View this article via: PubMed CrossRef Google Scholar
28. Qi J, Qiao Y, Wang P, Li S, Zhao W, Gao C. microRNA-210 negatively regulates LPS-induced production of proinflammatory cytokines by targeting NF-kappaB1 in murine macrophages. FEBS Lett. 2012;586(8):1201–1207.
    View this article via: PubMed CrossRef Google Scholar
29. Graff JW, Dickson AM, Clay G, McCaffrey AP, Wilson ME. Identifying functional microRNAs in macrophages with polarized phenotypes. J Biol Chem. 2012;287(26):21816–21825.
    View this article via: PubMed CrossRef Google Scholar
30. Liu G, Friggeri A, Yang Y, Park YJ, Tsuruta Y, Abraham E. miR-147, a microRNA that is induced upon Toll-like receptor stimulation, regulates murine macrophage inflammatory responses. Proc Natl Acad Sci U S A. 2009;106(37):15819–15824.
    View this article via: PubMed CrossRef Google Scholar
31. Taganov KD, Boldin MP, Chang KJ, Baltimore D. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci U S A. 2006;103(33):12481–12486.
    View this article via: PubMed CrossRef Google Scholar
32. O’Neill LA, Sheedy FJ, McCoy CE. MicroRNAs: the fine-tuners of Toll-like receptor signalling. Nat Rev Immunol. 2011;11(3):163–175.
    View this article via: PubMed Google Scholar
33. Androulidaki A, et al. The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs. Immunity. 2009;31(2):220–231.
    View this article via: PubMed CrossRef Google Scholar
34. Yang K, et al. MiR-146a inhibits oxidized low-density lipoprotein-induced lipid accumulation and inflammatory response via targeting toll-like receptor 4. FEBS Lett. 2011;585(6):854–860.
    View this article via: PubMed CrossRef Google Scholar
35. Nazari-Jahantigh M, Wei Y, Schober A. The role of microRNAs in arterial remodelling. Thromb Haemost. 2012;107(4):611–618.
    View this article via: PubMed CrossRef Google Scholar
36. Chen T, et al. MicroRNA-125a-5p partly regulates the inflammatory response, lipid uptake, and ORP9 expression in oxLDL-stimulated monocyte/macrophages. Cardiovasc Res. 2009;83(1):131–139.
    View this article via: PubMed CrossRef Google Scholar
37. Huang RS, Hu GQ, Lin B, Lin ZY, Sun CC. MicroRNA-155 silencing enhances inflammatory response and lipid uptake in oxidized low-density lipoprotein-stimulated human THP-1 macrophages. J Investig Med. 2010;58(8):961–967.
    View this article via: PubMed Google Scholar
38. Raitoharju E, et al. miR-21, miR-210, miR-34a, and miR-146a/b are up-regulated in human atherosclerotic plaques in the Tampere Vascular Study. Atherosclerosis. 2011;219(1):211–217.
    View this article via: PubMed CrossRef Google Scholar
39. Bidzhekov K, et al. microRNA expression signatures and parallels between monocyte subsets and atherosclerotic plaque in humans. Thromb Haemost. 2012;107(4):619–625.
    View this article via: PubMed CrossRef Google Scholar
40. Rayner KJ, et al. Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis. J Clin Invest. 2011;121(7):2921–2931.
    View this article via: JCI PubMed CrossRef Google Scholar
41. Nam D, et al. Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis. Am J Physiol Heart Circ Physiol. 2009;297(4):H1535–H1543.
    View this article via: PubMed CrossRef Google Scholar
42. Gremse F, et al. Virtual elastic sphere processing enables reproducible quantification of vessel stenosis at CT and MR angiography. Radiology. 2011;260(3):709–717.
    View this article via: PubMed CrossRef Google Scholar
43. Arranz A, et al. Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization. Proc Natl Acad Sci U S A. 2012;109(24):9517–9522.
    View this article via: PubMed CrossRef Google Scholar
44. McLaren JE, Ramji DP. Interferon gamma: a master regulator of atherosclerosis. Cytokine Growth Factor Rev. 2009;20(2):125–135.
    View this article via: PubMed CrossRef Google Scholar
45. Faraoni I, Antonetti FR, Cardone J, Bonmassar E. miR-155 gene: a typical multifunctional microRNA. Biochim Biophys Acta. 2009;1792(6):497–505.
    View this article via: PubMed Google Scholar
46. Natoli G, Ghisletti S, Barozzi I. The genomic landscapes of inflammation. Genes Dev. 2011;25(2):101–106.
    View this article via: PubMed CrossRef Google Scholar
47. O’Connell RM, Rao DS, Chaudhuri AA, Baltimore D. Physiological and pathological roles for microRNAs in the immune system. Nat Rev Immunol. 2010;10(2):111–122.
    View this article via: PubMed CrossRef Google Scholar
48. O’Connell RM, Taganov KD, Boldin MP, Cheng G, Baltimore D. MicroRNA-155 is induced during the macrophage inflammatory response. Proc Natl Acad Sci U S A. 2007;104(5):1604–1609.
    View this article via: PubMed CrossRef Google Scholar
49. Kurowska-Stolarska M, et al. MicroRNA-155 as a proinflammatory regulator in clinical and experimental arthritis. Proc Natl Acad Sci U S A. 2011;108(27):11193–11198.
    View this article via: PubMed CrossRef Google Scholar
50. McCoy CE, et al. IL-10 inhibits miR-155 induction by toll-like receptors. J Biol Chem. 2010;285(27):20492–20498.
    View this article via: PubMed CrossRef Google Scholar
51. Ceppi M, et al. MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells. Proc Natl Acad Sci U S A. 2009;106(8):2735–2740.
    View this article via: PubMed CrossRef Google Scholar
52. Ghorpade DS, Leyland R, Kurowska-Stolarska M, Patil SA, Balaji KN. MicroRNA-155 is required for mycobacterium bovis BCG-mediated apoptosis of macrophages. Mol Cell Biol. 2012;32(12):2239–2253.
    View this article via: PubMed CrossRef Google Scholar
53. Koch M, Mollenkopf HJ, Klemm U, Meyer TF. Induction of microRNA-155 is TLR- and type IV secretion system-dependent in macrophages and inhibits DNA-damage induced apoptosis. Proc Natl Acad Sci U S A. 2012;109(19):E1153–E1162.
    View this article via: PubMed CrossRef Google Scholar
54. Donners MM, et al. Hematopoietic miR155 deficiency enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice. PLoS One. 2012;7(4):e35877.
    View this article via: PubMed CrossRef Google Scholar
55. Drechsler M, Megens RT, van Zandvoort M, Weber C, Soehnlein O. Hyperlipidemia-triggered neutrophilia promotes early atherosclerosis. Circulation. 2010;122(18):1837–1845.
    View this article via: PubMed CrossRef Google Scholar
56. Barish GD, et al. The Bcl6-SMRT/NCoR cistrome represses inflammation to attenuate atherosclerosis. Cell Metab. 2012;15(4):554–562.
    View this article via: PubMed CrossRef Google Scholar
57. Ohlsson BG, et al. Oxidized low density lipoprotein inhibits lipopolysaccharide-induced binding of nuclear factor-kappaB to DNA and the subsequent expression of tumor necrosis factor-alpha and interleukin-1beta in macrophages. J Clin Invest. 1996;98(1):78–89.
    View this article via: JCI PubMed CrossRef Google Scholar
58. Hamilton TA, Major JA, Chisolm GM. The effects of oxidized low density lipoproteins on inducible mouse macrophage gene expression are gene and stimulus dependent. J Clin Invest. 1995;95(5):2020–2027.
    View this article via: JCI PubMed CrossRef Google Scholar
59. Gatto G, Rossi A, Rossi D, Kroening S, Bonatti S, Mallardo M. Epstein-Barr virus latent membrane protein 1 trans-activates miR-155 transcription through the NF-kappaB pathway. Nucleic Acids Res. 2008;36(20):6608–6619.
    View this article via: PubMed CrossRef Google Scholar
60. Welch JS, Ricote M, Akiyama TE, Gonzalez FJ, Glass CK. PPARgamma and PPARdelta negatively regulate specific subsets of lipopolysaccharide and IFN-gamma target genes in macrophages. Proc Natl Acad Sci U S A. 2003;100(11):6712–6717.
    View this article via: PubMed CrossRef Google Scholar
61. Necela BM, Su W, Thompson EA. Toll-like receptor 4 mediates cross-talk between peroxisome proliferator-activated receptor gamma and nuclear factor-kappaB in macrophages. Immunology. 2008;125(3):344–358.
    View this article via: PubMed CrossRef Google Scholar