Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Feb 3.
Published in final edited form as: Circ Res. 2011 Dec 22;110(3):416–427. doi: 10.1161/CIRCRESAHA.111.253377

NR4A1 (Nur77) Deletion Polarizes Macrophages Towards an Inflammatory Phenotype and Increases Atherosclerosis

Richard N Hanna 1, Iftach Shaked 1, Harper G Hubbeling 2, Jennifer A Punt 2, Runpei Wu 1, Erica Herrley 1, Claudia Zaugg 1, Hong Pei 1, Frederic Geissmann 3, Klaus Ley 1, Catherine C Hedrick 1
PMCID: PMC3309661  NIHMSID: NIHMS350316  PMID: 22194622

Abstract

Rationale

NR4A1 (Nur77) is a nuclear receptor that is expressed in macrophages and within atherosclerotic lesions, yet its function in atherosclerosis is unknown.

Objective

Nur77 regulates the development of monocytes, particularly patrolling Ly6C− monocytes that may be involved in resolution of inflammation. We sought to determine how absence of NR4A1 in hematopoietic cells impacted atherosclerosis development.

Methods and Results

Nur77−/− chimeric mice on a Ldlr−/− background showed a 3-fold increase in atherosclerosis development when fed a Western diet for 20 weeks, despite having a drastic reduction in Ly6C− patrolling monocytes. In a second model, mice deficient in both Nur77 and ApoE (ApoE−/−Nur77−/−) also showed increased atherosclerosis after 11 weeks of Western diet. Atherosclerosis was associated with a significant change in macrophage polarization towards a pro-inflammatory phenotype, with high expression of TNFα and nitric oxide, and low expression of Arginase-I. Moreover, we found increased expression of TLR4 mRNA and protein in Nur77−/− macrophages as well as increased phosphorylation of the p65 subunit of NFκB. Inhibition of NFκB activity blocked excess activation of Nur77−/− macrophages.

Conclusions

We conclude that the absence of Nur77 in monocytes and macrophages results in enhanced TLR signaling and polarization of macrophages towards a pro-inflammatory M1 phenotype. Despite having fewer monocytes, Nur77−/− mice developed significant atherosclerosis when fed a Western diet. These studies indicate that Nur77 is a novel target for modulating the inflammatory phenotype of monocytes and macrophages and may be important for regulation of atherogenesis.

Keywords: monocyte, atherosclerosis, nuclear receptors, macrophage, toll-like receptors

Introduction

Nur77 is an orphan nuclear receptor that, along with Nurr1 and NOR-1, constitutes the NR4A subfamily of orphan nuclear receptors in the steroid/thyroid receptor family1. They are designated as ‘orphans’ because their ligands are currently unknown. There is some question as to whether these nuclear receptors actually bind ligand, as all 3 receptors appear to have a closed ligand-binding pocket 2. This has led some to postulate that these receptors are solely regulated in a ligand-independent fashion either transcriptionally, or post-translationally, by kinases and phosphatases 1, 3.

All three NR4A family members are expressed within atherosclerotic lesions4. In macrophages, all 3 family members have been identified, and appear to be induced rapidly by multiple stimuli, including LPS, TNFα, and oxidized LDL5. Induction of NR4A nuclear receptors leads to rapid induction of transcription of a host of anti-inflammatory genes1, 3, 6. These findings have led to the hypothesis that these nuclear receptors act to resolve inflammation 5. Recently, Nurr1 has been shown to serve as a repressor of NFκB activation in microglia and astrocytes 7. NOR-1 deficiency has been shown to inhibit vascular injury and atherosclerosis in mice 8,9. However, the contribution of Nur77 to atherosclerosis development has not yet been reported, although de Vries and colleagues reported that Nur77 agonism inhibited vascular injury6.

Monocytes serve as a first line of defense in innate and adaptive immune reactions. They are rapidly recruited to sites of injury or inflammation, where after differentiation, they can present antigen to T lymphocytes 10 and secrete cytokines 11–13. As critical as monocytes are to respond and resolve inflammation, they are also critically involved in mediating chronic diseases, such as atherosclerosis and rheumatoid arthritis. Counterparts to human monocyte subsets have been identified in mice 11, 14, 15. Two primary monocyte subsets in mice have now been well-characterized. One subset is termed as ‘inflammatory’ and the other subset is considered as ‘surveillance’ or ‘resident’. The inflammatory monocytes are Ly6C+CCR2+CX3CR1loCD62L+, and are selectively recruited to inflamed tissues and lymph nodes 10, 16. The Ly6C+ inflammatory monocyte subset has been shown to egress from the bone marrow within hours of a bacterial infection in mice, and is CCR2-dependent 14, 16–19. The second monocyte subset is characterized as Ly6C−CCR2−CX3CR1hiCD62L−. The Ly6C− monocytes patrol the endothelium of blood vessels, but may not readily migrate in response to inflammation, except during tissue injury 20. Several groups have demonstrated that both populations of monocytes are recruited to inflammation or injury sites20–22. Both Ly6C+ monocytes23 and Ly6C− monocytes24 can also participate in the resolution of inflammation and tissue repair.

Nurr1 and NOR-1 have been shown to participate in homeostasis of myeloid progenitors 25. We recently reported that Nur77 drives expression of the Ly6C− monocyte subset through regulation of differentiation from a myeloid dendritic precursor26. Mice deficient in Nur77 showed almost complete loss of Ly6C− monocytes in blood, bone marrow, and spleen 26.

There has been a flurry of reports showing polarization of macrophages in response to tissue injury or infection27–32. In many cases, there is probably a continuum of macrophage phenotypes; however, two primary phenotypes have emerged and are delineated as ‘M1’ or ‘M2’. M1 or classically activated macrophages typically are considered ‘pro-inflammatory’, and produce IL-12, TNFα, and nitric oxide. In contrast, M2 ‘alternatively-activated’ macrophages express distinct markers such as resistin-like molecule α (RELMα), CD301, CD206, Arginase-I, YM-1, Fizz, and may secrete IL-10 or VEGF, among others. Although Ly6C+ and Ly6C− monocytes can differentiate into either M1 or M2-like macrophages, several studies show that Ly6C+ monocytes tend to polarize towards a M1 phenotype in several models20, 22, whereas Ly6C− monocytes tend to differentiate into M2-like macrophages 33. These Ly6C− monocyte-derived macrophages may be involved in resolution of inflammation and wound repair. Interestingly, a recent study indicates that in addition to recently recruited monocytes, macrophages that proliferate locally within tissues can also exhibit a M2 phenotype and control the inflammatory response34.

In the current study, we examined the impact of loss of Ly6C− monocytes in Nur77−/− mice on atherosclerosis development. We report that loss of Nur77 in hematopoietic cells enhances atherosclerosis development, most likely due to differential and enhanced macrophage polarization towards a M1 pro-inflammatory phenotype.

Methods

Detailed, expanded methods can be found online at http://circres.ahajournals.org. C57BL/6J wild-type mice (000664), ApoE−/− (002052), Ldlr−/− (002207), and Nur77−/− mice on a congenic C57BL/6J background (006187) were from The Jackson Laboratory. Nur77−/− mice were subsequently backcrossed onto the ApoE−/− background. Nur77−/− and wild-type mice were used as donors for bone marrow transplants (BMTs) into Ldlr−/− recipients as described in the online supplement. Atherosclerosis was quantified in the aorta using en face analysis, aortic root analysis, and Oil Red O staining as described previously1,2.

Results

Nur77 deficiency in bone marrow-derived cells increases atherosclerosis

We examined atherosclerosis in ApoE−/−Nur77−/− mice fed a Western diet for 11 weeks. ApoE−/− mice that were deficient in Nur77 showed a significant 4-fold increase in atherosclerosis development by en face staining of the aorta (Figure 1A, B). We also observed a 2-fold induction in atherosclerotic lesion area in aortic roots of the ApoE−/−Nur77−/− mice (Figure 1C). Histological analysis of atherosclerotic lesions showed increased macrophage and lipid content in plaques from ApoE−/−Nur77−/− mice compared to controls (Figure 1D, E). Total cholesterol levels were not different between the two groups. There was a trend towards higher plasma triglyceride levels in ApoE−/−Nur77−/− mice but this did not reach statistical significance (Online Figure I).

Figure 1. Increased atherosclerotic plaque area in ApoE−/−Nur77−/− mice.

Figure 1

Open in a new tab

(A) Quantification of plaque area as % of aortic surface in ApoE−/− Nur77−/− mice and ApoE−/− control mice after 11 weeks of Western diet feeding. P < 0.01 (Mann-Whitney test) (B) Representative Oil Red O staining (red) in aortic arches of ApoE−/−Nur77−/− mice and ApoE−/− control mice after 11 weeks of Western diet feeding. (C) Quantification of aortic root lesion area in ApoE−/− Nur77−/− mice and ApoE−/− control mice after 11 weeks of Western diet feeding. P < 0.02 (Student’s t-test) (D) Oil Red O (red, top) and CD68+ macrophage (green, bottom) staining of aortic root sections from ApoE−/− (left) and ApoE−/−Nur77−/− (right) mice on 11 weeks of Western diet feeding under low magnification (10x, left panel of each group) and high magnification (40x, right panel of each group). (E) Quantification of Oil Red O (left) and macrophage (CD68, right) staining expressed as percentage of total plaque area. *P < 0.05 (unpaired Student’s t-test) Data are representative of two independent experiments (n = 12 per group A and B, n = 7 per group for C, and n=10 per group D and E; mean and s.e.m).

We previously reported that Nur77−/− mice lack Ly6C− monocytes in vivo26. We confirmed loss of the Ly6C− monocyte subset in these double knockout mice on the ApoE−/− background. As expected, we found almost complete loss of Ly6C− monocytes in blood and spleens of ApoE−/−Nur77−/− mice fed a Western diet (Figure 2). In Nur77-deficient mice fed a Western diet, we observed a slight increase in Ly6C+ monocytes compared to WT, thus due to the increase of this subset, overall total monocyte levels were not altered between the two groups after Western diet feeding (Figure 2). The T cell compartment was completely normal in Nur77−/− mice (Online Figure II) as has been described 35. In addition, no differences in numbers of B cells, granulocytes or other immune cells were detected in blood from Nur77−/− mice compared to controls after 11 weeks of Western diet feeding (Online Figure II).

Figure 2. Reduction in the Ly6C− monocyte population in ApoE−/−Nur77−/− mice fed a Western diet.

Figure 2

Open in a new tab

Representative flow scatterplots of Ly6C+ and Ly6C− monocyte populations in the spleen and blood of ApoE−/−Nur77−/− mice and ApoE−/− control mice after 11 weeks of Western diet feeding (left). Numbers next to gates show percentages of cell population in plot. Quantification of Ly6C+ and Ly6C− monocyte populations as a percentage of all live cells in the spleen and blood (right). *P < 0.01 (unpaired Student’s t-test). Data are representative of two independent experiments (n = 8 per group; mean and s.e.m.)

These findings led us to ask whether loss of Nur77 in bone marrow monocyte-derived cells contributes to the increased atherosclerosis observed in these mice. To address this question, we performed bone marrow transplant studies in which wild-type (WT) or Nur77-deficient (Nur77−/−) marrow was transplanted into atherosclerosis-susceptible Ldlr−/− mice. Mice were allowed to reconstitute the bone marrow compartment for 6 weeks and then were placed on a Western diet for 20 weeks. We observed a 3-fold increase in atherosclerosis development in mice that received Nur77−/− bone marrow (Figure 3). We also observed a 3-fold increase in the amount of macrophages per aorta in mice that received Nur77−/− bone marrow (Figure 3C). Thus, absence of Nur77 in bone marrow-derived cells contributes to atherogenesis and increased inflammatory macrophage content, despite the fact that these mice possess significantly fewer Ly6C− monocytes.

Figure 3. Increased atherosclerotic plaque area and macrophage content in Nur77−/− bone marrow transplanted mice.

Figure 3

Open in a new tab

Ldlr−/− recipient mice were reconstituted for six weeks with either Nur77−/− or Nur77+/+ (WT) bone marrow before being fed a Western diet for 20 weeks. (A) Quantification of plaque area as % of aortic surface in Ldlr−/− mice receiving Nur77−/− bone marrow (Nur77−/−) and Ldlr−/− mice receiving C57BL/6J control bone marrow (WT) after 20 weeks of Western diet feeding. P < 0.001 (Mann-Whitney test). (B) Representative Oil Red O staining of plaques (red) in aortic arches of Ldlr−/− mice receiving Nur77−/−bone marrow (Nur77−/−) and Ldlr−/− mice receiving C57BL/6J control bone marrow (WT). (C) Representative flow plot of CD11b+F4/80+ macrophages in aortas of Ldlr−/− mice receiving Nur77−/−bone marrow (Nur77−/−) and Ldlr−/− mice receiving C57BL/6J control bone marrow (WT) after Western diet feeding as measured by flow cytometry after gating on live CD45+ cells (left). Quantification of CD11b+F4/80+ macrophages in aorta after BMT and Western diet feeding as measured by flow cytometry (right). *P < 0.01 (unpaired Student’s t-test). Data are representative of four independent experiments (n = 15 per group in A and B, n = 4 per group in C).

Monocytes and macrophages from Nur77−/− mice display a pro-inflammatory phenotype and show increased lipid accumulation

Based on our previous findings that Nur77 regulates the monocyte compartment in vivo, and based on the strong association of monocytes and macrophages with atherosclerosis, we focused the remaining aspects of our study on understanding how Nur77 regulates monocyte and macrophage function. First, we examined monocyte recruitment to tissues in vivo using a thioglycolate assay in the peritoneal cavity. We did not observe a difference in total populations of resident or thioglycolate-elicited peritoneal macrophages in Nur77−/− compared to control mice, implying that there is not a major change in macrophage recruitment in the absence of Nur77 (Online Figure III). Second, we examined the inflammatory responsiveness of Ly6C+ monocytes from WT and Nur77−/− mice. Splenic CD115+CD11b+ cells were gated on Ly6C expression and sorted as described previously26. Monocytes were used untreated or were stimulated with LPS overnight. Expression of TNFα was quantified in the cells using FACS. Using TNFα protein expression as an indicator of inflammation, we observed that Ly6C+ monocytes from Nur77-deficient mice showed an exacerbated response to LPS (Figure 4A). To examine if this exaggerated LPS response in Ly6C+ monocytes could be possibly related to an absence of Nur77 in this subset, we examined the ability of wild-type Ly6C+ cells to upregulate Nr4A1 family members in response to inflammatory stimuli and upon adhesion-induced differentiation. As macrophages have previously been shown to rapidly upregulate Nr4a1 family members in response to LPS, we confirmed this phenomenon in peritoneal macrophages (Online Figure IV), and also determined that circulating Ly6C+ monocytes likewise upregulate Nur77 and NOR1, but not Nurr1, in response to LPS stimulation (Figure 4B). Thus, Nur77 is induced in Ly6C+ monocytes upon inflammatory stimulation, which suggests that Nur77 has a function in the stimulated monocyte. As the inflammatory response is exacerbated in the Ly6C+ monocyte lacking Nur77, this suggests that at least one function of Nur77 in the activated Ly6C+ monocyte may be to resolve or control the inflammatory response of this cell during inflammation. In addition, monocytes adhere and will differentiate into macrophages when incubated on plastic. Using a new reporter transgenic mouse model36 where induction of the Nr4a1 promoter drives green fluorescent protein (GFP) expression (Nur77-GFP), we previously observed that most CD11b+F4/80+Ly6C− monocytes circulating in the blood and spleen of these reporter mice expressed abundant GFP expression, whereas most of the Ly6C+ monocytes expressed low levels of GFP26. We sorted GFP−CD11b+Ly6C+ cells from this reporter mouse and incubated these monocytes on plastic to differentiate them into macrophages. As shown in Figure 4C, incubation of Ly6C+ cells on plastic caused a significant induction in GFP expression, suggesting that Nur77 expression is induced in macrophages upon their differentiation, and thus may be important for their normal function.

Figure 4. TNFα production and induction of Nur77 in Ly6C+ monocytes.

Figure 4

Open in a new tab

(A) Sorted Ly6C+ monocytes from ApoE−/− (WT) and ApoE−/−Nur77−/− (Nur77−/−) mice were cultured for 18 hours in the absence (UN) or presence of 50 ng/ml LPS (LPS). Cells were stained intracellularly for TNFα and analyzed by flow cytometry. Data are expressed as fold change in TNF mean fluorescent intensity over Ly6C+ WT monocytes set as 1. Data are representative of two independent experiments (n = 3 per group; mean and s.e.m.) *P < 0.01 (unpaired Student’s t-test). (B) Induction of Nr4A1 family members Nur77, NOR1 and Nurr1 in C57BL/6J control Ly6C+ blood monocytes in response to LPS (100 ng/ml) stimulation for 1.5 hours (n = 4 per group; mean and s.e.m.) *P < 0.001 (unpaired Student’s t-test). Quantification of data expressed as percent change in mRNA of LPS treated over untreated expression for each gene set as 100%. (C) Isolated Nur77-GFPlowCD11b+Ly6C+ inflammatory monocytes with almost no initial GFP expression (Untreated), achieved increased GFP expression after 2 hours incubation on plastic. Inflammatory Nur77-GFPlowCD11b+Ly6C+ monocytes were sorted from blood using a FACS Aria II cell sorter, and isolated cells were incubated for 2 hours at 37° C, 5% CO2 on a plastic surface. Negative control is the same monocyte population from a GFP− mouse. Data are representative of three independent experiments.

We next measured the ability of Ly6C+ monocytes to accumulate lipid. Ly6C+ monocytes isolated from Ldlr−/− recipients that had received Nur77−/− bone marrow showed increased neutral lipid accumulation as measured by Nile Red staining (Figure 5A). Ly6C+ monocytes lacking Nur77 showed a slight but significant increase in expression of SR-A, but not CD36 (Figure 5B). Culturing the Ly6C+ monocytes overnight with Dil-OxLDL resulted in increased uptake of OxLDL in the Nur77−/− monocytes compared to wild-type (Figure 5C). Thus, Ly6C+ monocytes lacking Nur77 have enhanced cytokine production and enhanced uptake of OxLDL, indicating a pro-inflammatory phenotype.

Figure 5. Increased lipid accumulation in Ly6C+ cells from Nur77−/− mice.

Figure 5

Open in a new tab

Ldlr−/− recipient mice were reconstituted for six weeks with either Nur77−/− or Nur77+/+ (WT) bone marrow before being placed on Western diet for 20 weeks. (A) Representative histogram (left) and quantification (right) of Nile Red staining (neutral lipid content) by FACS in blood Ly6C+ monocytes from Ldlr−/−Nur77−/− (Nur77−/−, blue) or Ldlr−/− control (WT, red) mice after 20 weeks of Western diet feeding. Data are expressed as fold change in Nile Red mean fluorescent intensity over Ly6C+ WT monocytes set as 1. (B) Change in scavenger receptor CD36 and SRAI/II surface expression in Ly6C+ monocytes isolated from bone marrow (BM), blood and spleen of Ldlr−/− (WT) and Ldlr−/−Nur77−/− mice measured by flow cytometry. Data are expressed as fold change in receptor mean fluorescent intensity over Ly6C+ WT bone marrow monocytes set as 1. (C) Uptake of Dil-OxLDL by CD115+CD11b+ splenocytes from Ldlr−/−Nur77−/− (Nur77−/−) or Ldlr−/− control (WT) mice detected by flow cytometry. Isolated splenocytes from Ldlr−/− bone marrow transplant mice on high fat diet for 20 weeks were incubated overnight with 10 µg/ml Dil-OxLDL. Quantification of data expressed as fold change in Nile Red mean fluorescent intensity over CD115+CD11b+ WT myeloid cells set as 1 (left). Representative staining of Dil-OxLDL (red) uptake and DAPI (blue) in CD115+CD11b+ cells (right). *P < 0.01 (unpaired Student’s t-test). Data are representative of three independent experiments (n = 4 per grouping a–c; mean and s.e.m.).

We then examined the phenotypes of macrophages in these mice. We isolated resident macrophages from peritoneal lavage of 11-week Western-diet fed ApoE−/− and ApoE−/−Nur77−/− mice (gating strategy, Online Figure V) and found enhanced expression of IL-12 and reduced expression of Arginase-I (ArgI) in ApoE−/−Nur77−/− macrophages compared to ApoE−/− (WT) resident macrophages (Figure 6A). Stimulation of these macrophages from 11-week Western diet-fed mice for 2 hours with Kdo Lipid A (KLA), the active component of LPS 37, caused further induction of IL-12, TNF and iNOS in WT cells (Figure 6A). The IL-12 and iNOS expression in the stimulated macrophages was tripled in the absence of Nur77. ArgI expression remained lower in the ApoE−/−Nur77−/− macrophages, even when treated with KLA. MHCII expression was significantly higher on peritoneal macrophages isolated from ApoE−/−Nur77−/− mice compared to ApoE−/− (Figure 6B), supporting the notion of increased macrophage activation in the absence of Nur77. We also observed increased Dil-Ox-LDL uptake in bone marrow-derived macrophages cultured in GM-CSF from Nur77−/− mice (Figure 6C). Finally, to confirm that macrophages in aorta of ApoE−/−Nur77−/− mice showed a similar pro-inflammatory phenotype, F4/80+ macrophages from aorta were analyzed for CD36, SR-A and TNFα expression. We observed a significant 2-fold induction of TNFα, a 2.5 fold induction of SR-A, and a 5-fold induction of CD36 in ApoE−/−Nur77−/− macrophages obtained from aorta (Figure 6D), supporting the presence of pro-inflammatory macrophages and increased lipid uptake in the aorta.

Figure 6. Increased inflammatory phenotype and Oxidized-LDL uptake of macrophages from Nur77−/− mice.

Figure 6

Open in a new tab

(A) IL-10, IL-12, Arginase (Arg1), TNFα (TNF), iNOS, and TGFβ1 mRNA production in peritoneal macrophages from ApoE−/−Nur77−/− (Nur77−/−) mice and ApoE−/− control (WT) mice fed a Western diet for 11 weeks (left) and after stimulation for 2 hours with 30 µg/ml KLA (right). Quantification of data expressed as percent change in Nur77−/− mRNA over WT expression for each gene. (B) Representative flow scatter plot (left) and quantification (right) of MHCII expression in peritoneal macrophages from ApoE−/−Nur77−/− (Nur77−/−) mice and ApoE−/− control (WT) mice after 11 weeks of Western diet feeding. Quantification of data expressed as fold change in MHCII mean fluorescent intensity over WT macrophages set as 1. (C) Bone marrow-derived macrophages from Nur77−/− and wild-type control (WT) mice were incubated with Dil-OxLDL for 6h , and then analyzed for uptake of Oxidized-LDL (OxLDL) using FACS. (D) Measurement of TNFα (TNF), CD36 and SRA-1 mRNA expression in F4/80+ macrophages sorted from aortae of ApoE−/−Nur77−/− (Nur77−/−) mice and ApoE−/− control (WT) mice after 11 weeks of Western diet feeding. Quantification of data expressed as percent change in Nur77−/− mRNA over WT expression for each gene. *P < 0.01 (unpaired Student’s t-test) Data are representative of three independent experiments (n =6 a,b and n=3 c,d per grouping; mean and s.e.m.).

A recent study by Herzenberg and colleagues reported the presence of small versus large macrophages in the peritoneal cavity 38. Although Jenkins et al. have recently reported that macrophages can proliferate in tissue rather than necessarily being recruited from blood or spleen 34, many monocytes are indeed recruited to tissues in response to inflammatory stimuli. Small macrophages are thought to be derived from recruited Ly6C+ monocytes, whereas large macrophages tend to be the resident macrophages. In the peritoneal cavities of ApoE−/−Nur77−/− mice, we found fewer large macrophages and more small macrophages (Online Figure VI).

Abnormal TLR expression and NFκB mediated signaling in Nur77−/− macrophages

Interestingly, we found that expression of several toll-like receptors was increased in ApoE−/−Nur77−/− macrophages fed chow and Western diets, particularly TLR4 and TLR9 (Figure 7A). Concomitant with increased TLR4 mRNA expression, we found increased TLR4 surface protein expression in macrophages from ApoE−/− mice lacking Nur77 by FACS (Figure 7B). Nur77-deficient macrophages also showed increased expression of IL-12, and iNOS mRNA after 2h of KLA incubation in vitro (Figure 7C). There was a small increase in TNFα expression although it was not significant. These data point to polarization of macrophages toward a M1 phenotype in the absence of Nur77. Moreover, Nur77-deficient macrophages exhibited a blunted ‘M2-like’ response, with little induction of ArgI expression in response to KLA treatment (Figure 7C). Similarly, Nur77-deficient macrophages showed enhanced responsiveness to TLR agonists, including the TLR4 agonist KLA, the TLR2 agonist Pam3CSK4, and the TLR7 agonist R-848 (Figure 7D). Treatment with these TLR agonists uniformly increased TNFα, IL-12, and nitric oxide production (Figure 7D).

Figure 7. Increased TLR expression and NFκB mediated inflammatory cytokine production by Nur77−/− macrophages.

Figure 7

Open in a new tab

(A) Expression of TLR2, 4, 7 and 9 mRNA levels in peritoneal macrophages from ApoE−/−Nur77−/− (Nur77−/−) mice and ApoE−/− control (WT) mice on chow (left), after 11 weeks of Western diet feeding (center) or after Western diet feeding with incubation for an additional 2 hours with 30 µg/ml KLA (right). Quantification of data expressed as percent change in Nur77−/− mRNA over WT expression for each gene. (B) Representative flow plot (left) and quantification (right) of TLR4-MD2 expression on peritoneal macrophages from ApoE−/−Nur77−/− (Nur77−/−) mice and ApoE−/− control (WT) mice measured by flow cytometry. Quantification of data expressed as fold change in TLR4-MD2 mean fluorescent intensity over WT macrophages set as 1; *p<0.01. (C) IL-12, TNFα (TNF), iNOS, and Arginase (Arg1) mRNA expression in peritoneal macrophages from Nur77−/− mice and C57BL/6J wildtype control (WT) mice on chow diet, unstimulated (UN) or stimulated for 2 or 24 hours with 30 µg/ml KLA (right) measured by quantitative real-time PCR. Quantification of data expressed as percent change in mRNA over untreated WT expression for each gene. (D) TNFα, IL-12, and nitric oxide production in production by peritoneal macrophages from Nur77−/− mice and C57BL/6J wild-type control (WT) mice on chow diet, unstimulated (UN) or stimulated for 18 hours with either 30 µg/ml KLA, 5 µg/ml R848, or 30 ng/ml PAM2. TNFα and IL-12 proteins were measured by ELISA, and nitric oxide was measured by the Griess assay. (E) Intracellular staining of p65 activation in peritoneal macrophages from Nur77−/− mice and C57BL/6J wild-type control (WT) mice on chow diet unstimulated (UN) or stimulated for 2 hours with either 30 µg/ml KLA by flow cytometry using a p65 phosphoserine 529 (pS529) antibody. Data are representative of two independent experiments (F) IL-12, iNOS and TNFα mRNA expression in peritoneal macrophages from Nur77−/− mice and wild-type control (WT) mice unstimulated (UN), pretreated for 1 hour with the NFκB inhibitor Bay11-7082 (Bay), stimulated with 100 ng/ml LPS (LPS) for 2 hours, or stimulated with LPS after Bay11-7082 pretreatment (LPS+Bay), measured by quantitative real-time PCR. Quantification of RNA data expressed as percent change in Nur77−/− mRNA over WT expression for each gene. (G) Quantification of TNFα protein levels secreted in media by peritoneal macrophages treated under same conditions as in A. *P < 0.01 (unpaired Student’s t-test) (n =6 per group; mean and s.e.m.). (n =6 a–c,f,g; d and e n=4 per grouping; mean and s.e.m.).

We also discovered that, similar to what we previously reported for monocytes, Nur77-deficient macrophages had increased phosphorylation of the p65 subunit of NFκB, suggesting enhanced activation of NFκB (Figure 7E). Phosphorylation of p65 helps stabilize NFκB in the nucleus for gene transcription39, 40. To determine if the enhanced inflammatory signaling in Nur77-deficient macrophages is NFκB-dependent, we conducted similar peritoneal macrophage activation experiments in the presence of the NFκB inhibitor, BAY11-7082 (BAY), which selectively and irreversibly inhibits NF-κB activation by blocking phosphorylation and degradation of IκB-α. The increased inflammatory activity observed in the Nur77-deficient macrophages was ablated by BAY-mediated inhibition of NF-κB (Figure 7F,G). Thus, Nur77-deficient macrophages show enhanced NFκB-dependent inflammatory activation.

Human CD14dimCD16+ monocytes express Nur77

Three distinct subsets of human monocytes have been identified based on their surface markers: CD14+CD16−, CD14+CD16+ and CD14dimCD16+ (Figure 8A). Murine Ly6C+ monocytes are analogous to CD14+ monocytes in humans, which exhibit a strong inflammatory response to LPS, and Ly6C− mouse monocytes are most analogous to CD14dimCD16+ human monocytes, which patrol blood vessels41. The function of the newly identified CD14+CD16+ subset is unclear 11, 42. We quantified Nur77 mRNA expression in these 3 human monocytes subsets using real-time PCR, and found a pattern of expression similar to what we observe in the mouse 26. CCL3 is highly expressed in CD14hi monocytes and is shown here as a control for separation of the subsets. Expression of NR4A1 is quite high in CD14dimCD16+ human monocytes, the counterpart to patrolling Ly6C− monocytes in mice (Figure 8B). Nur77 was expressed in CD14+CD16+ human monocytes, but at much lower levels. In contrast, Nur77 was barely detectable in the CD14+ subset (analogous to Ly6C+ monocytes in mice). Thus, Nur77 most likely plays a functional role in the CD14dimCD16+ monocyte subset in humans under basal conditions, similar to mice.

Figure 8. CD14dimCD16+ human monocytes express high levels of Nur77.

Figure 8

Open in a new tab

Monocytes were isolated from human blood of normal donors, and monocytes isolated by cell sorting using CD14 and CD16 antibodies. (A) representative scatter plot of human blood monocyte populations based on CD14 and CD16 expression (B) Quantification of mRNA expression for CCL3 and Nur77 in the three human monocyte subsets, ***p<0.0001 by ANOVA.

Discussion

The NR4A family of nuclear receptors plays an important role in inflammation and vascular injury3, 4. Although studies have shown that NOR-1 is important in atherogenesis8, 9, the role of Nur77 in atherogenesis has remained unclear. We recently reported a role for Nur77 in regulating monocyte development26, and others have reported a role for NR4A family members in regulating inflammatory responses in macrophages and microglia5–7. In the current study, we show that mice deficient in Nur77 display greater atherosclerosis development than do control mice when fed a Western diet. Our data indicate that this is associated with: 1) a lack of Ly6C− patrolling monocytes and 2) enhanced polarization of Ly6C+ monocytes and derived macrophages towards a pro-inflammatory M1-like phenotype, which may be responsible for exacerbated lesion development. How enhanced M1 polarization is related to loss of Ly6C− monocytes remains to be elucidated.

Based on our prior work that showed the almost complete loss of Ly6C− monocytes in Nur77−/− bone marrow, blood, and spleen26, one hypothesis is that the increased macrophage activation and phenotypic pro-inflammatory M1 polarization of macrophages in Nur77−/−mice is due to loss of this protective Ly6C− monocyte subset. Lack of this subset may impair the resolution of inflammation and would lead to enhanced pro-inflammatory signaling in macrophages, thus, diminishing an M2-like inflammatory response by macrophages. A second possibility for the observed inflammatory macrophage phenotype is that Ly6C+ monocytes from Nur77−/− mice may give rise to a more pro-inflammatory activated M1-like macrophage phenotype, suggesting that Nur77 plays a cell-autonomous role in regulating the inflammatory responsiveness of the Ly6C+ monocyte subset. This scenario would be independent of any regulation or suppression by Ly6C− monocytes. In support of this concept is the fact that Nur77, although highly expressed in Ly6C− monocytes, is also present in Ly6C+ monocytes26, particularly upon their activation (Figure 4B). Therefore, Nur77 likely becomes important in activated monocytes and macrophages in a cell-autonomous manner to keep the inflammatory process in check. In the absence of a Nur77-mediated negative feedback mechanism, these macrophages appear to be somewhat constitutively activated and hypersensitive to inflammatory stimuli. However, we cannot rule out an important role solely for the Ly6C− monocyte subset in resolving the inflammatory response in vivo, particularly in that the known function of this subset is to patrol the vasculature in search of pathogens. This subset could act to directly dampen an inflammatory response that is mediated by the Ly6C+ subset in vivo. Future studies will hopefully allow us to discern the roles of each of these monocyte subsets on atherogenesis as well as to define how Nur77 influences the function of each subset.

In the current study, we observed increased inflammatory activities mediated via activation of the NFκB and TLR signaling pathways in Nur77−/− macrophages, which are blocked by NFκB inhibitors (Figure 7). We do not find any evidence of Nur77 activation of NFκB signaling by induction of IKK expression as has been reported by Pei et al. 43. The studies from Pei et al. were Nur77 overexpression studies performed in macrophage cell lines and not in primary cells or in vivo, which may partially explain the differing results. As of yet, we do not know the exact mechanisms for how Nr4a1 functions in these cells to regulate NFκB. We believe the increased inflammatory activity is at least partially due to the absence of Nur77's ability to produce the NFκB inhibitory protein, IκB-α, as we report in monocytes and others have reported in cell lines26, 44. Nur77 has been demonstrated to positively regulate expression of IκB-α via a defined and functional Nur77 response element in the IκB-α promoter 44. Glass and colleagues have also previously shown that another NR4A family member, Nurr1, inhibits NFκB through the recruitment of corepressor CoREST complexes, in microglia7. This Nurr1-CoREST negative feedback loop results in NFκB turnover and loss of activated gene expression. Whether Nur77 interacts with CoREST in macrophages is currently unknown. Additionally, the ability of Nur77 to heterodimerize with other nuclear receptors such as RXR allows for varying cellular responses and likely will make the underlying mechanism of Nur77 regulation of NFκB and other pro-inflammatory transcription factors more difficult to discern.

As a transcription factor, Nur77 functions to regulate many cellular processes, including cell growth, activation, proliferation, and apoptosis1, 4, 45. TLR-mediated NFκB signaling is one of several pathways that may be modulated by this nuclear receptor. Increased NFκB activity in the absence of the Nur77 most likely induces expression of a number of pro-inflammatory cytokines due to the loss of Nur77 transrepression. Indeed, studies have suggested that Nur77 negatively regulates several cytokines that may be important in inflammation46–48. Previous studies have demonstrated that Nur77 expression inhibits scavenger receptor-A and CD36 expression to reduce lipid loading in cultured macrophages, implying a role in regulating lipid uptake 48. We confirm this important role of Nur77 in regulating lipid uptake by demonstrating increased scavenger receptor expression and lipid uptake in macrophages in the absence of Nur77 in vivo. We also find a role for Nur77 in regulating scavenger receptor-A expression and lipid uptake in monocytes. Interestingly, we did not observe variation of CD36 expression in monocytes as we did in macrophages. One possible explanation of this result is that there may be differential regulation of these scavenger receptors in monocytes versus macrophages. In fact, it has been demonstrated that CD36 is strongly upregulated with monocyte to macrophage differentiation and M-CSF stimulation 49. In addition, Nur77 can function to directly induce expression of anti-inflammatory genes. One example of this is that Nur77 has been shown to induce ABCA1, a protein involved both in cholesterol efflux and macrophage inflammation 43, 50, 51. ArgI is a well-known marker of macrophage polarization whose expression is increased in anti-inflammatory M2 macrophages. As such, we find reduced expression of ArgI in Nur77−/− macrophages (Figure 7). Analysis of the murine ArgI promoter using Jaspar and MacVector software packages revealed the presence of 8 putative Nur77 NBRE elements and one NBRE:RXR element located within a 3 kb fragment upstream of the transcriptional start site of the murine ArgI gene (data not shown). Thus, Nur77 may directly regulate ArgI transcription, either alone or through RXR dimerization, which would directly impact the polarization of macrophages.

We focused on macrophages in this study because of our recent finding that Nur77 regulates monocyte development. In this current study, we find increased macrophage inflammation in atherosclerotic plaques of Nur77−/− mice, confirming the importance of Nur77 in regulating inflammatory events in myeloid cells. However, Nur77 also influences T cell receptor (TCR) affinity36, thereby possibly regulating T cell activation or even thymocyte selection. Several groups have implicated Nur77 in thymocyte selection52, 53, although the Nur77−/− mouse shows normal T cell development35. We observed no obvious differences in numbers of circulating T cells or other immune cells in Nur77−/− mice fed a Western diet (Online Figure II), implying that the increase in atherosclerosis is most likely myeloid cell-specific. Furthermore, under normal conditions Nur77 is most highly expressed in Ly6C− monocytes, with little expression in other immune cell types 26, 36. Nur77 may be up-regulated in lymphocytes including T cells, NKT and T regs with antigen stimulation, but not with inflammatory stimuli, implying that there might not be a major role of Nur77 in T cell-mediated inflammation 36. Although our results clearly point to a critical role for Nur77 in monocytes and macrophages, we cannot definitively rule out a role for Nur77 in T cells or other immune cells on atherosclerosis development. The use of conditional knockout mice will be important to delineate the roles of lymphocyte versus myeloid-selective Nur77 functions in atherogenesis.

The fact that we observed high expression of Nur77 in CD14dim human monocytes, the equivalent to the Ly6C− patrolling monocyte subset in mice, suggests perhaps a unique function for Nur77 in this human monocyte subset. Polymorphisms in NR4A1 that change Nur77 expression could be important for both acute and chronic inflammatory responses in human disease. Several SNPs have been reported for the human NR4A1 gene and several of these are located within the known promoter region of the gene54. Future studies of these polymorphisms in population studies will be important for determining whether therapies targeting Nur77 expression could be effective for inflammatory diseases.

In summary, we have identified that absence of Nur77 in hematopoietic cells amplifies atherosclerosis development in mice. Our data suggest that Nur77 functions to regulate macrophage phenotypes in vivo, and that absence of Nur77 in macrophages skews the profile of macrophages towards a pro-inflammatory, pro-atherogenic phenotype.

Novelty and Significance.

What is Known?

  • -

    Monocyte-derived macrophages and foam cells are the primary cell types that constitute atherosclerotic plaques

  • -

    Multiple studies have shown that the inflammatory phenotype of macrophages or foam cell formation increases atherosclerotic plaque size.

  • -

    The orphan nuclear receptor Nur77 has the ability to suppress inflammatory activity and is involved in regulation of monocyte and macrophage production

What New Information Does This Article Contribute?

  • -

    Mice transplanted with Nur77-deficient bone marrow-derived cells show increased aortic lipid accumulation associated with increased macrophage content in plaques.

  • -

    Nur77-deficient macrophages have increased inflammatory activity and exhibit increased lipid uptake.

  • -

    Nur77-deficient macrophage inflammatory activity is NFκB-dependent

Monocytes are the primary inflammatory cell type that infiltrates early atherosclerotic plaques, and their recruitment into plaques drives disease progression. We have found that the orphan nuclear receptor Nur77 functions as a master regulator of the differentiation and survival of a unique monocyte subset (Ly6C− in mice), which patrols the vasculature and participates in the resolution of inflammation. Here, we report that mice deficient in Nur77 show increased inflammatory activity and atherosclerotic development. Nur77-deficient macrophages exhibited a pro-inflammatory M1-like phenotype, in which inhibiting NFκB could block their activation. These findings suggest that hematopoietic expression of Nur77 can suppress polarization of macrophages to a pro-inflammatory phenotype and that absence of the patrolling monocytes exacerbates inflammation, most likely due to an inability to resolve the inflammatory response. Thus, our study identifies Nur77 as a novel target for modulating the inflammatory phenotype of monocytes and macrophages in cardiovascular diseases.

Supplementary Material

1

Acknowledgements

The authors would like to acknowledge Amy Blatchley and Deborah Yoakum for mouse colony management and Jennifer Pattison and Dr Joseph L. Witztum for assistance with plasma lipoprotein analysis.

Sources of Funding

This work was funded in part by NIH R01 HL071141 (to C.C.H.)

Non-standard Abbreviations and Acronyms

IL-12

interleukin-12

KLA

Kdo Lipid A

LPS

lipopolysaccharide

MFI

mean fluorescence intensity

NR4A1

Nuclear receptor family 4 member A1

OxLDL

oxidized low density lipoprotein

TLR

Toll-like receptor

TNF

tumor necrosis factor

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Disclosures

No financial relationships to disclose.

References

  • 1.Martinez-Gonzalez J, Badimon L. The nr4a subfamily of nuclear receptors: New early genes regulated by growth factors in vascular cells. Cardiovasc Res. 2005;65:609–618. doi: 10.1016/j.cardiores.2004.10.002. [DOI] [PubMed] [Google Scholar]
  • 2.Wang Z, Benoit G, Liu J, Prasad S, Aarnisalo P, Liu X, Xu H, Walker NP, Perlmann T. Structure and function of nurr1 identifies a class of ligand-independent nuclear receptors. Nature. 2003;423:555–560. doi: 10.1038/nature01645. [DOI] [PubMed] [Google Scholar]
  • 3.Pols TW, Bonta PI, de Vries CJ. Nr4a nuclear orphan receptors: Protective in vascular disease? Curr Opin Lipidol. 2007;18:515–520. doi: 10.1097/MOL.0b013e3282ef77d1. [DOI] [PubMed] [Google Scholar]
  • 4.Bonta PI, Pols TW, de Vries CJ. Nr4a nuclear receptors in atherosclerosis and vein-graft disease. Trends Cardiovasc Med. 2007;17:105–111. doi: 10.1016/j.tcm.2007.02.001. [DOI] [PubMed] [Google Scholar]
  • 5.Pei L, Castrillo A, Chen M, Hoffmann A, Tontonoz P. Induction of nr4a orphan nuclear receptor expression in macrophages in response to inflammatory stimuli. J Biol Chem. 2005;280:29256–29262. doi: 10.1074/jbc.M502606200. [DOI] [PubMed] [Google Scholar]
  • 6.Pires NM, Pols TW, de Vries MR, van Tiel CM, Bonta PI, Vos M, Arkenbout EK, Pannekoek H, Jukema JW, Quax PH, de Vries CJ. Activation of nuclear receptor nur77 by 6-mercaptopurine protects against neointima formation. Circulation. 2007;115:493–500. doi: 10.1161/CIRCULATIONAHA.106.626838. [DOI] [PubMed] [Google Scholar]
  • 7.Saijo K, Winner B, Carson CT, Collier JG, Boyer L, Rosenfeld MG, Gage FH, Glass CK. A nurr1/corest pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell. 2009;137:47–59. doi: 10.1016/j.cell.2009.01.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Nomiyama T, Zhao Y, Gizard F, Findeisen HM, Heywood EB, Jones KL, Conneely OM, Bruemmer D. Deficiency of the nr4a neuron-derived orphan receptor-1 attenuates neointima formation after vascular injury. Circulation. 2009;119:577–586. doi: 10.1161/CIRCULATIONAHA.108.822056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Zhao Y, Howatt DA, Gizard F, Nomiyama T, Findeisen HM, Heywood EB, Jones KL, Conneely OM, Daugherty A, Bruemmer D. Deficiency of the nr4a orphan nuclear receptor nor1 decreases monocyte adhesion and atherosclerosis. Circ Res. 2010;107:501–511. doi: 10.1161/CIRCRESAHA.110.222083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Palframan RT, Jung S, Cheng G, Weninger W, Luo Y, Dorf M, Littman DR, Rollins BJ, Zweerink H, Rot A, von Andrian UH. Inflammatory chemokine transport and presentation in hev: A remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues. J Exp Med. 2001;194:1361–1373. doi: 10.1084/jem.194.9.1361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Cros J, Cagnard N, Woollard K, Patey N, Zhang SY, Senechal B, Puel A, Biswas SK, Moshous D, Picard C, Jais JP, D'Cruz D, Casanova JL, Trouillet C, Geissmann F. Human cd14dim monocytes patrol and sense nucleic acids and viruses via tlr7 and tlr8 receptors. Immunity. 2010;33:375–386. doi: 10.1016/j.immuni.2010.08.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Geissmann F, Auffray C, Palframan R, Wirrig C, Ciocca A, Campisi L, Narni-Mancinelli E, Lauvau G. Blood monocytes: Distinct subsets, how they relate to dendritic cells, and their possible roles in the regulation of t-cell responses. Immunol Cell Biol. 2008;86:398–408. doi: 10.1038/icb.2008.19. [DOI] [PubMed] [Google Scholar]
  • 13.Woollard KJ, Geissmann F. Monocytes in atherosclerosis: Subsets and functions. Nat Rev Cardiol. 2010;7:77–86. doi: 10.1038/nrcardio.2009.228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Randolph GJ. Emigration of monocyte-derived cells to lymph nodes during resolution of inflammation and its failure in atherosclerosis. Curr Opin Lipidol. 2008;19:462–468. doi: 10.1097/MOL.0b013e32830d5f09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Tacke F, Randolph GJ. Migratory fate and differentiation of blood monocyte subsets. Immunobiology. 2006;211:609–618. doi: 10.1016/j.imbio.2006.05.025. [DOI] [PubMed] [Google Scholar]
  • 16.Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 2003;19:71–82. doi: 10.1016/s1074-7613(03)00174-2. [DOI] [PubMed] [Google Scholar]
  • 17.Boring L, Gosling J, Chensue SW, Kunkel SL, Farese RV, Jr, Broxmeyer HE, Charo IF. Impaired monocyte migration and reduced type 1 (th1) cytokine responses in c-c chemokine receptor 2 knockout mice. J.Clin.Invest. 1997;100:2552–2561. doi: 10.1172/JCI119798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Drevets DA, Dillon MJ, Schawang JS, Van Rooijen N, Ehrchen J, Sunderkotter C, Leenen PJ. The ly-6chigh monocyte subpopulation transports listeria monocytogenes into the brain during systemic infection of mice. J Immunol. 2004;172:4418–4424. doi: 10.4049/jimmunol.172.7.4418. [DOI] [PubMed] [Google Scholar]
  • 19.Sunderkotter C, Nikolic T, Dillon MJ, Van Rooijen N, Stehling M, Drevets DA, Leenen PJ. Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. J Immunol. 2004;172:4410–4417. doi: 10.4049/jimmunol.172.7.4410. [DOI] [PubMed] [Google Scholar]
  • 20.Auffray C, Fogg D, Garfa M, Elain G, Join-Lambert O, Kayal S, Sarnacki S, Cumano A, Lauvau G, Geissmann F. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science. 2007;317:666–670. doi: 10.1126/science.1142883. [DOI] [PubMed] [Google Scholar]
  • 21.Landsman L, Varol C, Jung S. Distinct differentiation potential of blood monocyte subsets in the lung. Journal of immunology. 2007;178:2000–2007. doi: 10.4049/jimmunol.178.4.2000. [DOI] [PubMed] [Google Scholar]
  • 22.Nahrendorf M, Swirski FK, Aikawa E, Stangenberg L, Wurdinger T, Figueiredo JL, Libby P, Weissleder R, Pittet MJ. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions. J Exp Med. 2007;204:3037–3047. doi: 10.1084/jem.20070885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Shechter R, London A, Varol C, Raposo C, Cusimano M, Yovel G, Rolls A, Mack M, Pluchino S, Martino G, Jung S, Schwartz M. Infiltrating blood-derived macrophages are vital cells playing an anti-inflammatory role in recovery from spinal cord injury in mice. PLoS Med. 2009;6:e1000113. doi: 10.1371/journal.pmed.1000113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Nahrendorf M, Swirski FK, Aikawa E, Stangenberg L, Wurdinger T, Figueiredo JL, Libby P, Weissleder R, Pittet MJ. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions. J Exp Med. 2007;204:3037–3047. doi: 10.1084/jem.20070885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Mullican SE, Zhang S, Konopleva M, Ruvolo V, Andreeff M, Milbrandt J, Conneely OM. Abrogation of nuclear receptors nr4a3 and nr4a1 leads to development of acute myeloid leukemia. Nat Med. 2007;13:730–735. doi: 10.1038/nm1579. [DOI] [PubMed] [Google Scholar]
  • 26.Hanna RN, Carlin LM, Hubbeling HG, Nackiewicz D, Green AM, Punt JA, Geissmann F, Hedrick CC. The transcription factor nr4a1 (nur77) controls bone marrow differentiation and the survival of ly6c(-) monocytes. Nat Immunol. 2011 doi: 10.1038/ni.2063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 2004;25:677–686. doi: 10.1016/j.it.2004.09.015. [DOI] [PubMed] [Google Scholar]
  • 28.Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nature reviews. Immunology. 2008;8:958–969. doi: 10.1038/nri2448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Zhang X, Mosser DM. Macrophage activation by endogenous danger signals. J Pathol. 2008;214:161–178. doi: 10.1002/path.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Satoh T, Takeuchi O, Vandenbon A, Yasuda K, Tanaka Y, Kumagai Y, Miyake T, Matsushita K, Okazaki T, Saitoh T, Honma K, Matsuyama T, Yui K, Tsujimura T, Standley DM, Nakanishi K, Nakai K, Akira S. The jmjd3-irf4 axis regulates m2 macrophage polarization and host responses against helminth infection. Nat Immunol. 2010;11:936–944. doi: 10.1038/ni.1920. [DOI] [PubMed] [Google Scholar]
  • 31.Krausgruber T, Blazek K, Smallie T, Alzabin S, Lockstone H, Sahgal N, Hussell T, Feldmann M, Udalova IA. Irf5 promotes inflammatory macrophage polarization and th1-th17 responses. Nature immunology. 2011;12:231–238. doi: 10.1038/ni.1990. [DOI] [PubMed] [Google Scholar]
  • 32.Ley K, Miller YI, Hedrick CC. Monocyte and macrophage dynamics during atherogenesis. Arteriosclerosis, thrombosis, and vascular biology. 2011;31:1506–1516. doi: 10.1161/ATVBAHA.110.221127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Tacke F, Alvarez D, Kaplan TJ, Jakubzick C, Spanbroek R, Llodra J, Garin A, Liu J, Mack M, van Rooijen N, Lira SA, Habenicht AJ, Randolph GJ. Monocyte subsets differentially employ ccr2, ccr5, and cx3cr1 to accumulate within atherosclerotic plaques. J Clin Invest. 2007;117:185–194. doi: 10.1172/JCI28549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Jenkins SJ, Ruckerl D, Cook PC, Jones LH, Finkelman FD, van Rooijen N, MacDonald AS, Allen JE. Local macrophage proliferation, rather than recruitment from the blood, is a signature of th2 inflammation. Science. 2011;332:1284–1288. doi: 10.1126/science.1204351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Lee SL, Wesselschmidt RL, Linette GP, Kanagawa O, Russell JH, Milbrandt J. Unimpaired thymic and peripheral t cell death in mice lacking the nuclear receptor ngfi-b (nur77) Science. 1995;269:532–535. doi: 10.1126/science.7624775. [DOI] [PubMed] [Google Scholar]
  • 36.Moran AE, Holzapfel KL, Xing Y, Cunningham NR, Maltzman JS, Punt J, Hogquist KA. T cell receptor signal strength in treg and inkt cell development demonstrated by a novel fluorescent reporter mouse. J Exp Med. 2011;208:1279–1289. doi: 10.1084/jem.20110308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Raetz CR, Garrett TA, Reynolds CM, Shaw WA, Moore JD, Smith DC, Jr, Ribeiro AA, Murphy RC, Ulevitch RJ, Fearns C, Reichart D, Glass CK, Benner C, Subramaniam S, Harkewicz R, Bowers-Gentry RC, Buczynski MW, Cooper JA, Deems RA, Dennis EA. Kdo2-lipid a of escherichia coli, a defined endotoxin that activates macrophages via tlr-4. J Lipid Res. 2006;47:1097–1111. doi: 10.1194/jlr.M600027-JLR200. [DOI] [PubMed] [Google Scholar]
  • 38.Ghosn EE, Cassado AA, Govoni GR, Fukuhara T, Yang Y, Monack DM, Bortoluci KR, Almeida SR, Herzenberg LA. Two physically, functionally, and developmentally distinct peritoneal macrophage subsets. Proceedings of the National Academy of Sciences of the United States of America. 2010;107:2568–2573. doi: 10.1073/pnas.0915000107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Hu J, Haseebuddin M, Young M, Colburn NH. Suppression of p65 phosphorylation coincides with inhibition of ikappabalpha polyubiquitination and degradation. Mol.Carcinog. 2005;44:274–284. doi: 10.1002/mc.20142. [DOI] [PubMed] [Google Scholar]
  • 40.Hu J, Nakano H, Sakurai H, Colburn NH. Insufficient p65 phosphorylation at s536 specifically contributes to the lack of nf-kappab activation and transformation in resistant jb6 cells. Carcinogenesis. 2004;25:1991–2003. doi: 10.1093/carcin/bgh198. [DOI] [PubMed] [Google Scholar]
  • 41.Cros J, Cagnard N, Woollard K, Patey N, Zhang SY, Senechal B, Puel A, Biswas SK, Moshous D, Picard C, Jais JP, D'Cruz D, Casanova JL, Trouillet C, Geissmann F. Human cd14dim monocytes patrol and sense nucleic acids and viruses via tlr7 and tlr8 receptors. Immunity. 2010;33:375–386. doi: 10.1016/j.immuni.2010.08.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Wildgruber M, Lee H, Chudnovskiy A, Yoon TJ, Etzrodt M, Pittet MJ, Nahrendorf M, Croce K, Libby P, Weissleder R, Swirski FK. Monocyte subset dynamics in human atherosclerosis can be profiled with magnetic nano-sensors. PloS one. 2009;4:e5663. doi: 10.1371/journal.pone.0005663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Pei L, Castrillo A, Tontonoz P. Regulation of macrophage inflammatory gene expression by the orphan nuclear receptor nur77. Mol Endocrinol. 2006;20:786–794. doi: 10.1210/me.2005-0331. [DOI] [PubMed] [Google Scholar]
  • 44.You B, Jiang YY, Chen S, Yan G, Sun J. The orphan nuclear receptor nur77 suppresses endothelial cell activation through induction of ikappabalpha expression. Circulation research. 2009;104:742–749. doi: 10.1161/CIRCRESAHA.108.192286. [DOI] [PubMed] [Google Scholar]
  • 45.Maxwell MA, Muscat GE. The nr4a subgroup: Immediate early response genes with pleiotropic physiological roles. Nucl Recept Signal. 2006;4:e002. doi: 10.1621/nrs.04002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Kasler HG, Verdin E. Histone deacetylase 7 functions as a key regulator of genes involved in both positive and negative selection of thymocytes. Molecular and cellular biology. 2007;27:5184–5200. doi: 10.1128/MCB.02091-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Bonta PI, Matlung HL, Vos M, Peters SL, Pannekoek H, Bakker EN, de Vries CJ. Nuclear receptor nur77 inhibits vascular outward remodelling and reduces macrophage accumulation and matrix metalloproteinase levels. Cardiovasc Res. 2010;87:561–568. doi: 10.1093/cvr/cvq064. [DOI] [PubMed] [Google Scholar]
  • 48.Bonta PI, van Tiel CM, Vos M, Pols TW, van Thienen JV, Ferreira V, Arkenbout EK, Seppen J, Spek CA, van der Poll T, Pannekoek H, de Vries CJ. Nuclear receptors nur77, nurr1, and nor-1 expressed in atherosclerotic lesion macrophages reduce lipid loading and inflammatory responses. Arteriosclerosis, thrombosis, and vascular biology. 2006;26:2288–2294. doi: 10.1161/01.ATV.0000238346.84458.5d. [DOI] [PubMed] [Google Scholar]
  • 49.Huh HY, Pearce SF, Yesner LM, Schindler JL, Silverstein RL. Regulated expression of cd36 during monocyte-to-macrophage differentiation: Potential role of cd36 in foam cell formation. Blood. 1996;87:2020–2028. [PubMed] [Google Scholar]
  • 50.Tang C, Liu Y, Kessler PS, Vaughan AM, Oram JF. The macrophage cholesterol exporter abca1 functions as an anti-inflammatory receptor. J Biol Chem. 2009;284:32336–32343. doi: 10.1074/jbc.M109.047472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Zhu X, Lee JY, Timmins JM, Brown JM, Boudyguina E, Mulya A, Gebre AK, Willingham MC, Hiltbold EM, Mishra N, Maeda N, Parks JS. Increased cellular free cholesterol in macrophage-specific abca1 knock-out mice enhances pro-inflammatory response of macrophages. J Biol Chem. 2008;283:22930–22941. doi: 10.1074/jbc.M801408200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Calnan BJ, Szychowski S, Chan FK, Cado D, Winoto A. A role for the orphan steroid receptor nur77 in apoptosis accompanying antigen-induced negative selection. Immunity. 1995;3:273–282. doi: 10.1016/1074-7613(95)90113-2. [DOI] [PubMed] [Google Scholar]
  • 53.Zhou T, Cheng J, Yang P, Wang Z, Liu C, Su X, Bluethmann H, Mountz JD. Inhibition of nur77/nurr1 leads to inefficient clonal deletion of self-reactive t cells. J Exp Med. 1996;183:1879–1892. doi: 10.1084/jem.183.4.1879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Mussig K, Machicao F, Machann J, Schick F, Claussen CD, Stefan N, Fritsche A, Haring HU, Staiger H. No association between variation in the nr4a1 gene locus and metabolic traits in white subjects at increased risk for type 2 diabetes. BMC Med Genet. 2010;11:84. doi: 10.1186/1471-2350-11-84. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS350316-supplement-1.pdf (1MB, pdf)

RESOURCES