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J Clin Invest. Nov 1, 2012; 122(11): 4190–4202.
Published online Oct 8, 2012. doi:  10.1172/JCI61716
PMCID: PMC3484435

MicroRNA-155 promotes atherosclerosis by repressing Bcl6 in macrophages

Abstract

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.

Introduction

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 (610). 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 (1315). 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 (2124). 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 (3032). 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.

Results

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 (Figure1A1A 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 (Figure1B1B and Supplemental Table 3). Moreover, stimulation with LPS and IFN-γ suppressed the expression of 26 miRs in BMDMs (Figure (Figure1B1B and Supplemental Table 4).

Figure 1
Expression of miR-155 and miR-147 in atherosclerotic lesions and in inflammatory macrophages.

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 Figure1C).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 (Figure1D).1D). Expression of miR-155 was also higher in human carotid plaques than in the artery wall (Figure (Figure1E).1E). Similar to miR-155, expression of miR-147 in the murine aortic wall only increased after 10 months of HFD feeding (Figure (Figure1F).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 Figure1G),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 (Figure1H).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 (Figure2A).2A). Additionally, some SMCs also expressed miR-155, as detected by combined immunostaining for miR-155 and SMA (Figure (Figure2B).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.

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 (Figure3,3, 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 (Figure3A3A and Supplemental Figures 3 and 4). Moreover, the relative SMC content tended to be decreased in Mir155+/+Apoe–/– mice harboring Mir155–/–Apoe–/– BM (Figure (Figure3C),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 (Figure3,3, D–F). The number of apoptotic macrophages in the lesions was similar in all 3 groups (Figure (Figure3G).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.

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 (Figure4A),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 (Figure4B).4B). Native LDL and moxLDL slightly increased miR-155 expression, whereas highly oxLDL reduced the expression of miR-155 (Figure (Figure4B)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 (Figure4B4B 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 (Figure4C).4C). The effect of miR-155 on CCL2 expression and secretion was confirmed at the protein level (Figure (Figure4,4, D and E). In contrast, macrophage migration and apoptosis appeared to be unchanged in the absence of miR-155 in vitro (Figure (Figure4,4, 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 (Figure5).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 (Figure5),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.
Figure 5
Role of miR-155 in CCL2 expression by lesional macrophages.

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 (Figure6,6, 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 (Figure6C).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 (Figure6D6D 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 (Figure6,6, E and F). Mutation of one of the predicted binding sites within the Bcl6 3′UTR prevented suppression by miR-155 (Figure (Figure66F).

Figure 6
Identification of miR-155 targets in BMDMs.

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 (Figure77 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 (Figure7).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.

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 (Figure8,8, 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 (Figure8,8, 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 (Figure8E).8E). Similar results were obtained at the protein level (Figure (Figure8,8, 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 (Figure8G).8G). Blocking NF-κB activation in Mir155+/+ BMDMs reduced the expression of Bcl6 mRNA 24 hours after stimulation with moxLDL and IFN-γ (Figure (Figure8H),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.

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 (Figure9A).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 (Figure9B).9B). Furthermore, Bcl6 mRNA expression was higher in carotid plaques from mice harboring Mir155–/– BM than in those from mice with Mir155+/+ BM (Figure (Figure9C).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 (Figure10A).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 (Figure10B10B 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 (Figure1010C).

Figure 9
Expression of lesional BCL6 is miR-155 dependent.
Figure 10
Role of BCL6 in miR-155–mediated atherosclerosis.

Discussion

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 (5759). 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.

Methods

See Supplemental Methods for details regarding human carotid plaque sample collection, micro–computed tomography, quantitative RT-PCR, moxLDL preparation, cell culture, migration and apoptosis assays, miR-155 target gene prediction, Western blot analysis, ELISA, and lipoprotein profiling.

Animal models of atherosclerosis.

Female Apoe–/– mice (6–8 weeks old; Jackson Laboratory) were fed HFD (21% crude fat, 0.15% cholesterol, 19.5% casein; Altromin) for 3 or 10 months. The aorta was then harvested after in situ perfusion with RNAlater (Ambion) via the left ventricle. For the control group, arteries were collected from female Apoe–/– mice (8 weeks old) fed regular chow diet.

Mir155–/– mice (Jackson Laboratory) were crossed with Apoe–/– mice to obtain double-deficient Mir155–/–Apoe–/– mice. BM cells from Mir155+/+Apoe–/– and Mir155–/–Apoe–/– mice (5–10 × 106 cells) were injected into the tail vein of Mir155+/+Apoe–/– recipients treated with an ablative dose of whole-body irradiation (2 × 6.5 Gy). In HFD-fed mice, atherosclerotic plaques were induced 4 weeks after BM transplantation by partial carotid ligation (41). In brief, mice were anesthetized with ketamin and xylazine, and the left external carotid, internal carotid, and occipital arteries were ligated. This partial carotid ligation resulted in acutely reduced blood flow velocity in the common carotid artery due to restricted outflow through the superior thyroid artery. Perivascular treatment of the carotid artery with Bcl6 siRNA (Accell siRNA, 4 nmol/treatment; Dharmacon) was performed once weekly starting 2 weeks after partial ligation. In the control group, the partially ligated carotid arteries were treated with nontargeting siRNA (Dharmacon). The siRNA was mixed with transfection reagent (DharmaFECT 4; Dharmacon) and dissolved in pluronic gel (35%; Sigma-Aldrich) as described previously (19). The left and right carotid arteries were harvested 6 weeks after partial ligation following in situ perfusion with RNAlater (Ambion), paraformaldehyde, or PAXgene Tissue Fix (PAXgene Tissue Containers; Qiagen) through the left ventricle.

Laser capture microdissection.

Serial sections (20 μm thick) of the carotid and innominate arteries and the aortic root were mounted on membrane-mounted metal frame slides (Molecular Machines and Industries), deparaffinized under RNase-free conditions, and completely dried. Laser capture microdissection was performed using a laser microdissection system (MMI cellcut plus; Molecular Machines and Industries) attached to an inverted microscope (IX71; Olympus). At least 40 sections of plaque tissue or morphologically normal vessel wall tissue were collected from each mouse. RNA was isolated using the RecoverAll total nucleic acid isolation kit (Life Technologies) according to the manufacturer’s instructions.

miR real-time PCR array.

Total RNA was isolated from carotid arteries and cultured BMDMs using the mirVana kit (Invitrogen), and the quality of the RNA was determined using an Agilent 2100 Bioanalyzer. Reverse transcription and preamplification was performed using the Megaplex RT & Preamp Rodent Pool Set (Life Technologies) according to the manufacturer’s instructions. Samples were loaded onto preconfigured 384-well microfluidic cards (TaqMan Array MicroRNA Cards) for real-time analysis of 518 mouse miRs (Sanger miRBase v10) using a 7900HT RT-PCR System (Life Technologies). Data were analyzed using StatMiner software (Integromics) according to the ΔΔCt method using multiple internal control genes. The most stable combination of internal controls was determined using the Genorm algorithm. A Ct value less than 40 was defined as the limit of detection of the individual assays. The fold change compared with the control group was calculated and logarithmically transformed (log10). Array data were deposited in GEO ( http://www.ncbi.nlm.nih.gov/geo/; accession nos. GSE26555 and GSE33453).

Immunostaining and histology.

Serial sections (5 μm thick) from the common carotid artery were collected within 1 mm of the area of bifurcation and stained (3–5 sections per mouse) with elastic van Gieson (EVG) stain. Images were obtained under a brightfield microscope (Leica DMLB) connected to a CCD camera (JVC). The plaque area was quantified by planimetry (Diskus software; Hilgers, Bonn).

Quantitative immunostaining for TAGLN (rabbit polyclonal; Abcam); SMA, MAC2, and CD3E (rabbit polyclonal; Dako); MPO (rabbit polyclonal; Abcam); and collagen type I (rabbit polyclonal; Cedarlane) was performed on carotid artery sections. The primary antibody was detected with a fluorescently labeled secondary antibody. The percentage of positively stained area per total plaque area (2–3 sections/mouse, 50- to 100-μm distance between sections) was determined using image analysis software (ImageJ), with the threshold set according to the background of negative control staining. Combined immunostaining for CCL2/MAC2 or BCL6/MAC2 was performed by sequential incubation of the carotid sections with either an anti-CCL2 antibody (goat polyclonal; Santa Cruz Biotechnology) or an anti-BCL6 antibody (rabbit polyclonal; Abcam) followed by a FITC-labeled secondary antibody and an anti-MAC2 antibody, which was visualized using a Dylight549-conjugated anti-rat antibody (KPL).

Combined in situ hybridization and immunostaining.

Sections (7 μm thick) from carotid arteries fixed with Paxgene (Qiagen) were hybridized with double digoxigenin-labeled probes (miR-155 and scrambled probes, 50 nM; U6 probe, 5 nM) in hybridization buffer (Exiqon) at 51°C for 1 hour in a Thermoblock (Eppendorf). After stringent washing with SSC buffer and blockade of nonspecific binding sites using TNB (Perkin Elmer) and biotin/avidin binding sites using a blocking kit (Vector Lab), sections were incubated with a peroxidase-conjugated anti-digoxigenin antibody (Fab fragments from sheep, 1:100 dilution; Roche) for 1 hour at 37°C. A tyramide-based amplification system (TSA Plus Biotin; Perkin Elmer) and Dylight 549–conjugated streptavidin (KPL) were used to visualize the probe. Sections were subsequently incubated with anti-MAC2 (rat, clone M3/38; Cederlane) or anti-SMA (mouse, clone1A4; Dako) antibodies followed by a FITC-conjugated secondary antibody.

EIF2C2 IP.

BMDMs from Mir155+/+Apoe–/– and Mir155–/–Apoe–/– mice were washed in ice-cold PBS and incubated with lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES [pH 7.0], 0.5% NP40, 5 mM DTT, 250 U/ml RNase OUT [Invitrogen], 400 μM vanadyl ribonucleoside complexes [New England Biolabs], and protease inhibitors [Complete Protease Inhibitor Cocktail Tablets; Roche]) for 15 minutes on ice. After homogenization in a Dounce homogenizer, cell extracts were centrifuged. Input RNA was extracted from the supernatant using TRIzol (Invitrogen). Before IP, protein A/G conjugated to magnetic beads (Millipore) was incubated with a mouse monoclonal anti-EIF2C2 antibody (clone 2E12-1C9; Abnova) or mouse IgG (Millipore). The antibody-conjugated beads were then incubated with the cell extracts for 5 hours at 4°C before immobilization of the precipitate with a magnetic separator (Millipore). RNA was isolated from the precipitate with TRIzol, reverse transcribed with random primers, and amplified using the SYBR Green PCR Master Mix (Fermentas). The fold enrichment (FE) of target genes in the EIF2C2-IP RNA compared with that in IgG-IP RNA was determined as follows: (a) ΔCtEIF2C2-IP was calculated as Ctinput – CtEIF2C2-IP; (b) ΔCtIgG-IP was calculated as Ctinput – CtIgG-IP; (c) ΔΔCt was calculated as ΔCtEIF2C2-IP – ΔCtIgG-IP; and (d) FE was calculated as 2ΔΔCt.

Luciferase assay.

Constructs of the pEZX-MT01 vector with or without the 3′UTR of the murine Bcl6 gene were purchased from GeneCopoeia. Mutagenesis PCR was performed at the miR-155 target site of Bcl6. HEK293 cells were transfected with 100 ng luciferase reporter or empty vector and treated with miR-155 mimic (30 nM, Ambion Pre-miR miRNA Precusor; Life Technologies) or nontargeting control oligonucleotides (30 nM, Pre-miR miRNA Precusor Negative Control) using Lipofectamine 2000 (Invitrogen). Firefly and Renilla luciferase activity were assessed using the Dual-Glo Luciferase Assay System (Promega) and a microplate reader (Tecan) 72 hours after transfection.

Statistics.

miR array data are presented as means and were compared using unpaired, moderated, 2-tailed t test (Statminer 4.2; Integromics). A false-positive discovery test (according to the Benjamini-Hochberg procedure) was applied to the array data. All other data represent mean ± SEM. 2 groups were compared using paired, 2-tailed t test; more than 2 groups were compared using 1-way ANOVA followed by Newman-Keuls post-test (Prism 5.0; GraphPad). A P value less than 0.05 was considered significant.

Study approval.

Animal experiments were reviewed and approved by the local authorities (LANUV NRW) in accordance with German animal protection laws. The Ethics Committee of the Medical Faculty (RWTH Aachen University) approved the study protocol for the collection of human atherosclerotic plaque samples, and written informed consent was obtained from all participating subjects.

Supplementary Material

Supplemental data:

Acknowledgments

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.

Footnotes

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

Citation for this article: J Clin Invest. 2012;122(11):4190–4202. doi:10.1172/JCI61716.

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