microRNA-155 deficiency results in decreased macrophage inflammation and attenuated atherogenesis in apoE^−/− mice

Abstract

Objective

microRNA-155 (miR155) plays a critical role in immunity and macrophage inflammation. We aim to investigate the role of miR155 in atherogenesis.

Approach and Results

Quantitative real-time PCR showed that miR155 was expressed in mouse and human atherosclerotic lesions. miR155 expression in macrophages was positively correlated with proinflammatory cytokine expression. Lentivirus-mediated overexpression of miR155 in macrophages enhanced their inflammatory response to LPS through targeting SOCS-1, and impaired cholesterol efflux from acetylated LDL-loaded macrophages, whereas deficiency of miR155 blunted macrophage inflammatory responses, and enhanced cholesterol efflux possibly via enhancing lipid loading-induced macrophage autophagy. We next examined the atherogenesis in apoE^−/− and miR155^−/−/apoE^−/− (DKO) mice fed a western diet. Compared with apoE^−/− mice, the DKO mice developed less atherosclerosis lesion in aortic root, with reduced neutral lipid content and macrophages. Flow cytometric analysis showed that there were increased number of regulatory T cells, and reduced numbers of Th17 cells and CD11b+/Ly6C^high cells in the spleen of DKO mice. Peritoneal macrophages from the DKO mice had significantly reduced pro-inflammatory cytokine expression and secretion both in the absence and presence of LPS stimulation. To determine whether miR155 in leukocytes contributes to atherosclerosis, we performed bone marrow transplantation study. Deficiency of miR155 in bone marrow-derived cells suppressed atherogenesis in apoE^−/− mice, demonstrating that hematopoietic cell-derived miR155 plays a critical role.

Conclusion

miR155 deficiency attenuates atherogenesis in apoE^−/− mice by reducing inflammatory responses of macrophages, enhancing macrophage cholesterol efflux and resulting in an anti-atherogenic leukocyte profile. Targeting miR155 may be a promising strategy to halt atherogenesis.

Atherosclerosis is both a lipid storage disease and a chronic inflammatory process^1–6. While lipid accumulation in the intimal space and local inflammation are closely linked in atherogenesis, macrophages are pivotal players in the process through both maintaining vessel wall lipid homeostasis and orchestrating inflammatory responses⁷. Numerous molecules take part in the pathological transformation of macrophages in atherogenesis; and the complex signaling network underlying macrophage inflammation in the context of atherogenesis is far from clear.

MicroRNAs (miRs) are short non-coding RNA molecules that regulate gene expression post-transcriptionally through base pairing with mRNAs, resulting in either translational repression or mRNA degradation⁸. They are estimated to regulate up to a third of all human genes⁹ and play critical roles in many physiological or pathological processes, including cell differentiation, immunity development and cancer transformation. Therefore, miRs are emerging as new targets for the diagnosis and therapy of human diseases¹⁰, including dyslipidemia and atherosclerosis. MicroRNA-155 (miR155) is encoded in the B cell integration cluster (Bic) gene¹¹ and is prominently expressed in many hematopoietic cell types¹²; it has been demonstrated to be oncogenic and play a crucial role in immune response regulation^13–15. In macrophages, a microarray analysis found that miR155 was among the few miRs that were substantially up-regulated by Toll-like receptor (TLR) ligands^{16, 17}. Although the functional relevance of macrophage miR155 expression is unclear, studies indicated that miR155 may enhance inflammatory response by stimulating the release of inflammatory mediators by targeting several negative feedback molecules including SOCS-1 and SHIP-1. However, it may also attenuate inflammatory response under other circumstances^18–22. Given the critical role of macrophage inflammation in atherogenesis, it is imperative to define the impact of miR155 expression and regulation on atherogenesis. Very recently, two studies showed opposite results regarding the effects of bone marrow cell miR155 deficiency on atherosclerosis. In one study, Donners et al. reported that bone marrow miR155 deficiency increased atherosclerosis in LDLR^−/− mice fed a high-fat diet by generating a more pro-atherogenic immune cell profile and a more pro-inflammatory monocyte/macrophage phenotype²³. In the other study, Nazari-Jahantigh and colleagues showed that miR155 promoted atherosclerosis in a partial carotid ligation apoE^−/− mouse model by repressing Bcl6 in macrophages thus enhancing vascular inflammation²⁴.

In the current study, we first show that miR155 expression was increased in mouse and human aorta atherosclerotic lesions, and that miR155 expression was positively correlated with proinflammatory cytokine expression under various conditions. Furthermore, increased miR155 expression conferred macrophages a pro-atherogenic phenotype, including enhanced inflammatory responses to LPS and impaired cholesterol efflux upon cholesterol loading; while miR155 deficiency rendered macrophage less inflammatory. Moreover, we compared the atherosclerosis development in apoE^−/− and apoE^−/−/miR155^−/− mice, and showed that miR155 deficiency in apoE^−/− mice attenuated atherogenesis by reducing macrophage inflammation. This result was confirmed by bone marrow transplantation study showing that deficiency of miR155 in bone marrow-derived cells reduced atherogenesis in apoE^−/− mice.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement

Results

miR155 is up-regulated in mouse and human aortic atherosclerotic lesions

We first examined miR155 expression in mouse aorta atherosclerotic lesions by qRT-PCR. miR155 expression was determined in aorta segments from 4-month-old wild-type C57BL/6 mice and 3–7-month-old apoE^−/−/LDLR^−/− mice. The aortas of the apoE^−/−/LDLR^−/− mice were dissected into atherosclerotic and non-lesion (normal) segments under microscope. Total RNAs (including microRNAs) were extracted from the aorta segments for qRT-PCR analysis of miR155 and TNFα expression. Supplemental Figure 1A shows that the atherosclerotic segments of aortas from apoE^−/−/LDLR^−/− mice expressed higher levels of miR155 and TNFα, compared to the non-atherosclerotic aorta segments of the same apoE^−/−/LDLR^−/− mice, as well as aortas from wild-type mice.

We further examined miR155 expression in human aorta atherosclerotic lesions by qRT-PCR. Lesional tissue and the surrounding normal tissue from human aortas were dissected and total RNA was extracted for measurement of human miR155 and TNFα expression. Supplemental Figure 1B shows that compared to surrounding normal aortic tissue, human aortic atherosclerotic lesion had 2-fold increase in miR155 expression, along with a 7-fold increase in TNFα expression.

miR155 expression is up-regulated in macrophages by TLR4 activation and is correlated with inflammatory cytokines

It is known that a toll-like receptor (TLR) 4 ligand, lipopolysacchride (LPS), stimulates miR155 expression in macrophages¹⁶. We first examined whether a more physiologically relevant atherogenic TLR4 ligand, minimally oxidized LDL (mmLDL), induces miR155 expression in macrophages. LDL was minimally oxidized by incubation with copper sulfate (10 µM, 3 h) to generate mmLDL. The mmLDL was used to stimulate thioglycollate-elicited peritoneal macrophages from wild-type C57Bl/6 mice, apoE^−/−/LDLR^−/− mice and TLR4^−/− mice. Cells were also stimulated with LPS as a positive control. The macrophages were incubated for 6 h with serum-free DMEM, with the addition of either mmLDL (100 µg/ml) or LPS (100 ng/ml). qPCR was performed to quantify miR155 expression. Supplemental Figure 2A shows that both mmLDL and LPS significantly increased macrophage miR155 expression in a TLR4-dependent manner, and the responses were significantly enhanced in macrophages from apoE^−/−/LDLR^−/− mice than in those from wild-type C57Bl/6 mice. We further measured miR155 expression levels in macrophages from mice of different genotypes, including WT C57Bl/6, LDLR^−/−, apoE^−/−, apoE^−/−/LDLR^−/−, and TLR4^−/−, and subjected to various treatments, including DMEM control, mmLDL or LPS stimulation, or ace-LDL loading. Concurrently, we also measured the expression levels of inflammatory cytokines in these macrophages. In Supplemental Figure 2B, we plotted the correlation of miR155 levels and TNFα or IL-6 mRNA levels in the macrophages, showing a clear positive correlation between miR155 expression levels and TNFα or IL-6 levels.

Overexpression of miR155 alters macrophage function

To further investigate the relationship between miR155 expression and macrophage function, we generated a lentiviral vector for miR155 overexpression. A 419-bp DNA fragment containing the mouse pre-miR155 stem-loop was amplified by PCR from the C57BL/6 mouse macrophage cDNA and sub-cloned into the bi-cistronic lentiviral vector PWPI to generate the PWPI-miR155-GFP construct. This bic cDNA fragment is very similar to the fragment that Costinean et al. used for the generation of the B-lymphocyte specific miR155 transgenic mouse model²⁵. The expression of miR155 and GFP is under the control of the universal promoter EF-1α (Suppl. Fig. 3A). The transduction efficiency for primary macrophages is over 90% when a MOI of 30 is used, as demonstrated by flow cytometric analysis of GFP expression (data not shown). The overexpression of mature mouse miR155 was confirmed in PWPI-miR155-GFP lentivirus transduced mouse peritoneal macrophages by qPCR (increased up to 30-fold compared to PWPI-GFP lentivirus transduced cells) (Suppl. Fig. 3B). We transduced peritoneal macrophages from WT C57Bl/6 mice with PWPI-miR155-GFP or control PWPI-GFP lentiviruses and examined inflammatory cytokine expression. Interestingly, we found that miR155 overexpression alone did not increase TNFα expression in macrophages. However, when stimulated with LPS, the PWPI-miR155-GFP lentivirus-transduced macrophages displayed a 3-fold increase in TNFα expression compared to the control group (p=0.022) (Figure 1A).

Figure 1. miR155 enhances macrophage inflammatory responses by targeting SOCS-1.

A. Mouse macrophages were transduced with PWPI-GFP or PWPI-miR155-GFP lentiviruses. The transduced cells were treated with LPS (50 ng/ml) for 6 h before mRNA was extracted for qPCR measurement of TNFα expression. N=3 for each group. B. The transduced cells were treated with LPS (50 ng/ml) for 6–24 h. Cell lysates were used for detection of SOCS-1 and SHIP-1 levels by western blotting. C. Western blots in three experiments were analyzed for the relative protein levels (*p<0.05).D–F. WT and miR155^−/− mouse peritoneal macrophages were treated with lipopolysaccharide (LPS, 100ng/ml), Pam3CSK4 (500ng/ml), FSl-1 (100ng/ml), poly (I:C) (10 ug/ml), ODN1668 (1uM) for 6h, respectively. Total RNA was extracted and reverse transcribed into cDNA as template to detect the inflammatory cytokines (D. TNFα; E. IL-6; F. IL-1β) by qPCR. n=3; *p<0.05, **p<0.01.

To investigate the molecular mechanisms through which miR155 enhances macrophage inflammatory responses to pro-inflammatory stimuli, protein levels of SOCS-1 and SHIP-1 were detected in miR155-overexpressing macrophages. It has been reported that SOCS-1 and SHIP-1, two negative regulators of the TLR4-mediated inflammatory pathway, are miR155 target proteins in monocytes and macrophages. Therefore, we examined whether miR155 overexpression reduced protein levels of SOCS-1 and SHIP-1 in mouse peritoneal macrophages. The transduced macrophages were stimulated with LPS (100 ng/ml) for 0, 6 or 24 h. Protein levels of SOCS-1 and SHIP-1 were examined by western blot using β-actin as a loading control. The results showed that mouse miR155 overexpression substantially reduced SOCS-1 levels in mouse peritoneal macrophages, while SHIP-1 levels were not changed (Figs. 1B and 1C).

miR155 deficiency attenuates macrophage inflammatory response

To confirm the function of miR155 in macrophage inflammatory responses, we compared the macrophages from WT and miR155^−/− mice. We treated WT and miR155^−/− mouse macrophages with different toll-like receptor ligand, including LPS (TLR4 ligand), Pam3CSK4 (TLR1/2 ligand), Fsl-1 (TLR2/6 ligand), Poly(I:C) (TLR3 ligand) and ODN1668 (TLR9 ligand). We found that miR155 deficiency diminished the expression of TNFα, IL-6 and IL-1β induced by all of above toll-like receptor ligands, indicating that miR155 deficiency dampened macrophage inflammatory signaling triggered by multiple toll-like receptor activation, including TLR2, TLR4, TLR3 and TLR9 (Fig. 1C).

miR155 expression affects macrophage cholesterol efflux

It has been well demonstrated that macrophage inflammation and cholesterol homeostasis are intimately connected. The contact point is the PPAR-LXR axis. To examine if miR155 affects macrophage cholesterol homeostasis, we first measured the cholesterol efflux from the macrophages transduced with PWPI-miR155-GFP lentiviruses. Figure 2A shows that, compared to control PWPI-GFP lentivirus transduced macrophages, miR155-overexpressing macrophages displayed impaired cholesterol efflux to apoAI (a 10% decrease, p=0.035), but efflux to DMEM (baseline cholesterol diffusion) was not changed. We next used peritoneal macrophages from miR155^−/− or WT mice to measure cholesterol efflux. We found that miR155 deficiency significantly increased the cholesterol efflux to apoAI (by 45%, p=0.0002), whereas cholesterol efflux to HDL was not significantly different (Fig. 2B). To examine molecular mechanisms underlying the increased cholesterol efflux in miR155^−/− macrophages, we loaded WT and miR155^−/− macrophages with ace-LDL for 48 h, qPCR was used to quantify the mRNA expression, and western blot was performed to compare the protein expression, of ABCA1 and ABCG1. Surprisingly we found that ABCA1 and ABCG1 in miR155^−/− macrophages were comparable to those in WT macrophages (Figs. 2C and 2D), indicating increased cholesterol efflux in miR155^−/− macrophages was not due to increased expression of cholesterol efflux genes, ABCA1 and ABCG1. Recent studies have shown that macrophage autophagy enables cholesterol efflux via presenting free cholesterol to cell membrane^{26, 27}. We thus examined whether miR155 deficiency increases autophagy of macrophages, especially under cholesterol loading condition. We performed western blot to measure LC3 protein in macrophages, and found both forms of LC3 (LC3-I and LC3-II) were significantly increased in miR155^−/− macrophages, both in unloaded and ace-LDL loaded cells, compared to WT macrophages (Figs. 2E and 2F), indicating miR155 deficiency enhances macrophage autophagy.

Figure 2. miR155 modulates macrophage cholesterol efflux.

A. The lentivirus transduced macrophages from WT mice were loaded with ace-LDL for 72 h and cholesterol efflux to DMEM or apoAI was measured. B. Macrophages from WT or miR155^−/− mice were loaded with ace-LDL for 72 h. Cholesterol efflux to DMEM, apoAI or HDL was measured. n=3 for each group. C and D. Macrophages from WT or miR155^−/− mice were incubated in the absence or presence of ace-LDL for 48 h. Total RNA was extracted for qPCR analysis of ABCA1 and ABCG1 (C), or the cell lysates were used for western blotting analysis of ABCA1 and ABCG1 (D). The experiments were repeated once and the results were similar. E. With or without loaded with ace-LDL for 48 h, macrophage cell lysates were used for western blotting analysis of LC3. F. Quantitative analysis of three independent experiments.

miR155 deficiency attenuates atherogenesis in mice

Since macrophage miR155 deficiency attenuated inflammatory responses and enhanced cholesterol efflux, we next examined if miR155 deficiency protects mice from developing atherosclerotic lesions. Female apoE^−/− and miR155/apoE DKO mice at 8 weeks of age were fed a western type diet to raise plasma cholesterol levels and to induce atherosclerosis. After 12 weeks on the western diet, the mice were sacrificed. Body weight, plasma cholesterol and triglyceride levels, as well as FPLC lipoprotein profiles at the end point were comparable between both groups of mice (Suppl. Fig. 4). As visualized by H&E staining, mean lesion area was 0.48 ± 0.03 mm² in aortic roots of apoE^−/− mice, and 0.30 ± 0.04 mm² in DKO mice, reflecting a 37% reduction (Fig. 3A). However, atherosclerotic lesion area in en face aortas was not statistically significantly different between apoE^−/− and DKO mice (Suppl. Fig. 5).

Figure 3. Lesion areas in aortic root of apoE^−/− mice and DKO mice.

The mice were fed a western diet for 12 weeks. The aortic roots were embedded in OCT medium and frozen in −20 °C immediately after being cut away from aorta. For analysis of atherosclerosis, 10 micron thick sections were collected in the region of the proximal aorta starting from the end of the aortic sinus. A. The lesion area of the frozen sections were detected by H&E staining and quantified by Image-Pro Plus 6.0. The quantitative analysis and representative images are shown. B. The lipid content in lesions was determined by ORO staining. Quantitative analysis and representative images are shown. C. The areas of macrophages in lesions were determined by immunostaining for MOMA-2 (a macrophage marker). Quantitative analysis and representative images are shown. Scale bar, 250 µm.

We further stained the lesions for neutral lipid with Oil Red-O (ORO). The results showed that lipid staining area in the lesions of DKO mice (0.31 ± 0.02 mm²) was significantly smaller than that in apoE^−/− mice (0.20 ± 0.02 mm²), reduced by 36% (Fig. 3B). We stained lesional macrophages using a mouse macrophage specific antibody MOMA-2, and found the DKO mice displayed a ~48% less macrophage area (0.26±0.03 mm²) in aortic root atherosclerotic lesions compared to apoE^−/− mice (0.13 ± 0.02 mm²) (Fig. 3C). However, collagen content in lesions, as determined by Movat’s Pentachrome staining was not significant different between the two groups (Suppl. Fig. 6).

miR155 is profoundly involved in immunity, modulating immune cell differentiation and maturation. We thus examined whether miR155 deficiency altered leukocyte populations in our atherosclerotic mouse model using flow cytometry. Our data showed that in DKO mouse spleen, the percentages of T cells (CD3+/CD4+, and CD3+/CD8+) and Th17 cells (CD4+/IL17+) were decreased, and those of B cells (CD19+) and regulatory T cells (CD4+/FoxP3+) were increased, compared to those in apoE^−/− mouse spleens. Even though the percentage of CD11+/Ly6C+ cells were not different in apoE^−/− or DKO mouse spleens, the percentage of CD11+/Ly6C^high subpopulation was significantly reduced in DKO mouse spleens (Fig. 4).

Figure 4. Flow cytometric analysis of mouse splenocytes.

A. The percentages of different splenocyte population. Data were presented as the mean ± SEM of 8–9 mice. *p<0.05; **p<0.01. B and C. Representative examples of flow cytometric analysis of splenocytes using CD4 and FoxP3 antibodies for detection of regulatory T cells (B) or using CD11b and Ly6C antibodies to detect CD11b+/Ly6C+ monocytes (C).

At the end-point, we obtained peritoneal macrophages from apoE^−/− and DKO mice, and treated the macrophage with vehicle or LPS (100 ng/ml) for 6 or 24 h. We found that, both in the absence and presence of LPS stimulation, DKO macrophages expressed TNFα, IL-6 and IL-1β mRNAs at significantly lower levels than apoE^−/− macrophages after 6 h of stimulation (Fig. 5A). ELISA analysis showed that after 24 h of incubation, DKO macrophage conditioned medium contained significantly lower levels of TNFα and IL-6, compared to the conditioned medium of apoE^−/− macrophages (Fig. 5B). These data suggest that miR155 deficiency in lipoprotein-loaded apoE^−/− macrophages diminished their inflammatory responses. We also measured TNFα and IL-6 concentrations in mouse plasma, and found that the concentration of IL-6 was significantly reduced in DKO mice than in apoE^−/− mice; however, the plasma levels of TNFα showed no difference in two groups of mice.

Figure 5. Expression of inflammatory cytokines in mouse macrophages and their concentrations in mouse plasma.

A. Residential peritoneal macrophages were obtained from apoE^−/− and DKO mice at the time of sacrifice. The cells were treated with vehicle or LPS (100 ng/ml) for 6 h before total RNAs were extracted for qPCR quantification of inflammatory cytokine expression. *p<0.01, n=8. B. Macrophages from the mice were cultured with or without addition of LPS (100 ng/ml) in the medium for 24 h; the cytokine concentrations in the medium were measured by ELISA. n=5. C. Concentrations of cytokines in mouse plasma at the end point were determined by ELISA. n=8.

Bone marrow miR155 deficiency reduces atherosclerosis in mice

To further dissect the role of myeloid cell miR155 expression in atherogenesis, we performed a bone marrow transplantation study. We transplanted bone marrow cells from apoE^−/− or DKO mice to lethally irradiated apoE^−/− mice of 12 weeks of age. After 4 weeks, mice were fed a western diet for 16 weeks. As in the study using whole body knock-outs, apoE^−/− recipient mice with miR155 deficiency in bone marrow-derived cells did not have plasma lipid alteration compared to their miR155-WT chimeric controls (data not shown), but had significantly reduced atherosclerosis in aortic root (Figs. 6A–6F). Using laser capture microdissection, we collected atherosclerotic lesions from the aortic root. Supplemental Figure 7 shows images of aortic root section before and after the atherosclerotic lesion was removed by LCM. Quantitative real-time PCR confirmed that in the lesions of the recipients of DKO bone marrow, the mRNA expression of miR155 targets Bcl-6 and SOCS-1was significantly increased compared to that in recipients of apoE^−/− BM (Fig. 6G). Pro-inflammatory chemokine CCL2 expression was decreased in DKO bone marrow recipients; however, TNFα expression showed no difference between apoE^−/− bone marrow and DKO bone marrow recipients (Fig. 6C).

Figure 6. Bone marrow deficiency of miR155 attenuates atherogenesis in mice.

A, C and E. Representative proximal aorta lesions visualized by H&E (A), ORO (C) or MOMA-2 (E) staining in apoE^−/− mice received apoE^−/− (EKO) or apoE/miR155 DKO (DKO) bone marrow. Scale bar, 250 µm. B, D and F. Quantitative results of atherosclerotic lesion size measured by H&E (B), ORO (D) or MOMA-2 (F) staining. X-axis shows the genotype (EKO or DKO) of the bone marrow received by apoE^−/− mice and the gender (Female or Male) of the recipients. G. Atherosclerotic lesion tissues obtained by LCM from the mouse aortic root were analyzed using qPCR for the expression of indicated genes. Open column: mice received EKO bone marrow; Black column: mice received DKO bone marrow.

Discussion

miR155 is a unique microRNA in that it is coded only by one gene, called bic, and is the only product of this gene^28–30. miR155 deficient mice are the first microRNA knockout mouse model^{13, 14}, providing a unique opportunity to investigate the role of a microRNA in pathophysiology. miR155 has been demonstrated to play important roles in immunity and inflammation, especially macrophage inflammatory responses, implying that it may also be involved in atherogenesis. Indeed, two recent studies have shown that miR155 deficiency in bone marrow cells affected atherosclerosis. However, the results are contradictory. While one report showed that western diet-fed LDLR^−/− mice transplanted with miR155 deficient bone marrow developed more atherosclerotic lesions than mice received WT bone marrow, indicating in this model miR155 is atheroprotective²³; the other showed that bone marrow miR155 deficiency reduced plaque size after partial carotid ligation in apoE^−/− mice, suggesting miR155 is pro-atherogenic²⁴. The primary reason for this contradiction was that miR155 deficiency enhanced macrophage inflammation in hypercholesterolemic LDLR^−/− mice as shown in the first study²³ while attenuated macrophage inflammation in another hypercholesterolemic mouse model (apoE^−/− mice) as shown in the second study²⁴. Our current study provided comprehensive data and demonstrated that whole body or bone marrow miR155 deficiency profoundly reduced macrophage inflammation and atherosclerosis in the aortic root region in western diet-fed apoE^−/− mice. However, atherosclerosis in aortic arch and descending aortic regions were not different between the two genotypes. Region specific effects have been reported in many atherosclerosis studies^{31, 32}. In apoE^−/− mice fed a western diet for 3 months, atherosclerotic lesions are most profound in the aortic root, whereas lesions are relatively scant in the descending region. It is possible that lack of significant difference in the descending region was due to relatively small lesions in this region. However, the definitive mechanism of this region specific effect remains to be defined.

In agreement with the recently published study²⁴, we found miR155 expression was increased in both mouse and human atherosclerotic lesions. We confirmed that minimally oxidized LDL and LPS stimulated macrophage miR155 expression via TLR4-dependent pathway. Moreover, we showed a positive correlation between miR155 expression level and proinflammatory cytokine expression level in macrophages of various genotypes and under varied treatment including cholesterol loading. Furthermore, using both gain-of-function and loss-of-function approaches, we showed that baseline expression of miR155 substantially affected inflammatory responses of macrophages to TLR ligands. Additionally, consistent with previous reports, we found that miR155 suppressed the expression of SOCS-1, a negative feedback protein of macrophage inflammation ^{21, 33}. This was also confirmed in vivo by laser capture microdissection and qPCR. In atherosclerotic lesions of apoE/miR155 DKO bone marrow recipient mice, the expression of SOCS-1 and another miR155 target, Bcl6, was significantly increased, compared to that in the lesions of apoE^−/− bone marrow recipient mice. Our data strongly suggest that in macrophages pro-inflammatory cytokines and miR155 may reciprocally regulate each other and thus miR155-targeting strategies may lead to macrophage inflammation suppression. Donners et al. showed that in normal conditions, miR155 ablation attenuated inflammatory responses in macrophages, whereas in oxLDL-loaded macrophage foam cells, miR155 deficiency paradoxically promoted a more pro-inflammatory phenotype²³. We however found that macrophages from western diet-fed apoE/miR155 DKO mice produced less pro-inflammatory cytokines either at baseline or under LPS stimulation, compared to the macrophages from western diet-fed apoE^−/− mice. The reason for this discrepancy is unknown, possibly due to an unknown alteration caused in their study by in vitro oxLDL loading or in vivo excessive cholesterol over-loading in western diet fed LDLR^−/− mice whose plasma total cholesterol level may reach ~1500 mg/dl²³, which is exceptionally high (in our study, the plasma cholesterol level of the mice at the end-point was 700–750 mg/dl). In addition, the status of apoE may significantly alter the function of miR155 in macrophages. The exact reasons for the difference in the two different models warrant further investigation.

Macrophage inflammation and cholesterol accumulation are closely linked together in atherogenesis³⁴. Imbalance between cholesterol uptake and efflux in the context of hyperlipidemia leads to macrophage foam cell formation, which is an obligatory step of atherogenesis. In this study, we first time demonstrated that overexpression of miR155 compromised while miR155 deficiency improved cholesterol efflux from acetylated LDL-loaded macrophages. Mechanistically, we demonstrated that the enhanced cholesterol efflux by miR155 deficiency in macrophages was not due to increased expression of cholesterol transporters ABCA1 and ABCG1, rather at least partially due to increased autophagy. Autophagy is a cellular process that assists the cell to achieve a homeostasis under stress. Recently, elegant work by Ira Tabas and colleagues has shown that macrophage autophagy plays an anti-atherogenic role³⁵; and Yves Marcel and colleagues further demonstrated that autophagy was activated in macrophage foam cells and facilitated ABCA1-mediated cholesterol efflux^{26, 27}. Previous studies have shown that miR155 knockout impaired the activation of Akt³⁶. Inhibition of Akt pathway has been shown to induce autophagy³⁷. Moreover, Akt1 inhibits miR155 expression in macrophages, which would constitute a negative feedback loop^{21, 38}. However, the detailed mechanism by which miR155 regulates macrophage autophagy and cholesterol efflux warrants further investigation. Consistent with the cholesterol efflux result, the atherosclerotic lesions in DKO mice contained less oil red-O stained region and fewer macrophages than those in apoE^−/− mice. While we were revising this manuscript, two groups reported that miR155 may modulate macrophage autophagy upon Mycobacteria infection. One report showed forced miR155 expression accelerated the autophagic response in macrophages³⁹, whereas the other study found miR155 inhibited IFNγ-induced autophagy⁴⁰. These results indicated that the role of miR155 in macrophage autophagy is complex and context-dependent.

In addition to macrophage functional alteration due to miR155 deficiency, we also found that miR155 deficiency resulted in an overall anti-atherogenic spleen leukocyte profile, evidenced by increased regulatory T cell and reduced CD11b+/Ly6C^high cell percentages. miR155 has been suggested to confer competitive fitness to regulatory T cells³³; the increased regulatory T cells in miR155^−/− mice in our study reflect the possibility that the effects of miR155 on regulatory T cell may be altered by hyperlipidemia. Many studies have shown that regulatory T cells are anti-atherogenic^{41, 42}, whereas CD11b+/Ly6C^high cells are pro-atherogenic^{32, 43}. However, our study does not provide direct evidence whether the increased regulatory T cells by miR155 deficiency contribute to reduced atherosclerosis, and it is currently unknown why our observations were different from those in a previous study²³.

Recent studies, including ours, suggested that miR155 modulates macrophage polarization, and miR155 deficiency rendered macrophage prone to M2 polarization through activating STAT6 and CEBP/β pathways^44–46, providing additional evidence that miR155 is pro-inflammatory in macrophages. miR155 is also expressed in vascular endothelial cells and smooth muscle cells besides myeloid cells, and the role in atherogenesis may be conflicting^{24, 47–49}. Actually, recent studies have shown that miR155 affected endothelium-dependent vasorelaxation by targeting eNOS³⁹, and attenuated angiotensin II-induced endothelial cell damage and apoptosis⁴⁸. The role of miR155 expression in endothelial cells and smooth muscle cells in atherogenesis warrant further investigation, although Nazari-Jahantigh and colleagues’ study suggested that vascular miR155 seemed not to contribute to atherogenesis²⁴. In our current study, we conclude that the myeloid cell miR155 expression is pro-atherogenic. Inhibition of myeloid cell miR155 expression would yield anti-atherogenic benefit through suppressing macrophage inflammatory responses, enhancing macrophage cholesterol efflux, and achieving an anti-atherogenic circulating leukocyte profile.

Significance.

microRNA-155 (miR155) plays a critical role in immunity and macrophage function; however, its role in atherogenesis is controversial. In this current study, miR155 was found expressed in mouse and human atherosclerotic lesions and its expression in macrophages was positively correlated with proinflammatory cytokine expression. Overexpression of miR155 in macrophages enhanced inflammatory response to LPS through targeting SOCS-1, and impaired cholesterol efflux from macrophages, whereas deficiency of miR155 blunted macrophage inflammatory responses, and enhanced cholesterol efflux possibly via enhancing lipid loading-induced macrophage autophagy. Using whole body or bone marrow miR155 knockouts, we demonstrated that both whole body and hematopoietic cell miR155 deficiency attenuated atherosclerosis in apoE^−/− mice fed a Western type diet. Flow cytometric analysis showed an overall anti-atherogenic leukocyte profile in the spleen of miR155 deficient mice. These results support an overall pro-atherogenic role of miR155 and suggest that inhibition of macrophage miR155 could be an effective anti-atherogenic strategy.

Abbreviations

FBS

fetal bovine serum

DMEM

Dulbecco’s Modified Eagle Medium

Ace-LDL

acetylated low-density lipoprotein

qPCR

quantitative real-time PCR

BMT

bone marrow transplantation

LCM

laser capture microdissection