Published online before print October 13, 2008, doi: 10.1161/CIRCULATIONAHA.108.793869
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(Circulation. 2008;118:1837-1847.)
© 2008 American Heart Association, Inc.
Molecular Cardiology |
Increased Inflammatory Gene Expression in ABC Transporter–Deficient Macrophages
Free Cholesterol Accumulation, Increased Signaling via Toll-Like Receptors, and Neutrophil Infiltration of Atherosclerotic Lesions
From the Division of Molecular Medicine, Department of Medicine, Columbia University, New York, NY (L.Y.-C., C.W., T.A.P., M.R., S.H., M.I., R.L., N.W., A.R.T.); and Department of Physiological Chemistry, Genentech, South San Francisco, Calif (M.L.).
Correspondence to Laurent Yvan-Charvet, PhD, Division of Molecular Medicine, Department of Medicine, Columbia University, 630 W 168th St, New York, NY 10032. E-mail ly2159{at}columbia.edu
Received May 22, 2008; accepted August 27, 2008.
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Abstract |
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Background— Two macrophage ABC transporters, ABCA1 and ABCG1, have a major role in promoting cholesterol efflux from macrophages. Peritoneal macrophages deficient in ABCA1, ABCG1, or both show enhanced expression of inflammatory and chemokine genes. This study was undertaken to elucidate the mechanisms and consequences of enhanced inflammatory gene expression in ABC transporter–deficient macrophages.
Methods and Results— Basal and lipopolysaccharide-stimulated thioglycollate-elicited peritoneal macrophages showed increased inflammatory gene expression in the order Abca1–/–Abcg1–/–>Abcg1–/–>Abca1–/–>wild-type. The increased inflammatory gene expression was abolished in macrophages deficient in Toll-like receptor 4 (TLR4) or MyD88/TRIF. TLR4 cell surface concentration was increased in Abca1–/–Abcg1–/–>Abcg1–/–> Abca1–/–> wild-type macrophages. Treatment of transporter-deficient cells with cyclodextrin reduced and cholesterol-cyclodextrin loading increased inflammatory gene expression. Abca1–/–Abcg1– bone marrow–derived macrophages showed enhanced inflammatory gene responses to TLR2, TLR3, and TLR4 ligands. To assess in vivo relevance, we injected intraperitoneally thioglycollate in Abcg1–/– bone marrow–transplanted, Western diet–fed, Ldlr-deficient mice. This resulted in a profound inflammatory infiltrate in the adventitia and necrotic core region of atherosclerotic lesions, consisting primarily of neutrophils.
Conclusions— The results suggest that high-density lipoprotein and apolipoprotein A-1 exert anti-inflammatory effects by promoting cholesterol efflux via ABCG1 and ABCA1 with consequent attenuation of signaling via Toll-like receptors. In response to a peripheral inflammatory stimulus, atherosclerotic lesions containing Abcg1–/– macrophages experience an inflammatory "echo," suggesting a possible mechanism of plaque destabilization in subjects with low high-density lipoprotein levels.
Key Words: ABC transporters • atherosclerosis • cholesterol • inflammation • lipoproteins
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Introduction |
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High-density lipoprotein (HDL) levels are inversely related to the incidence of cardiovascular disease,1 and increasing HDL can reduce atherosclerosis in animal models.2,3 A principal antiatherogenic property of HDL is thought to be its ability to promote cholesterol efflux from macrophage foam cells. In addition, HDL has been described to have various anti-inflammatory properties, including its ability to bind lipopolysaccharide, to remove oxidized lipids from low-density lipoprotein (LDL), and to carry enzymes such as platelet-activating factor acetylhydrolase and paraoxonase that inactivate oxidized phospholipids.4
Clinical Perspective p 1847
The ability of HDL and its apolipoproteins to mediate macrophage cholesterol efflux depends in large part on the macrophage ABC transporters ABCA1 and ABCG1. Whereas ABCA1 promotes cholesterol efflux to lipid-poor apolipoprotein (apo) A-1, ABCG1 induces cholesterol efflux to HDL particles.5,6 Thus, these 2 transporters have complementary activity in mediating cholesterol efflux.7,8 Accordingly, in vivo measurements of macrophage reverse cholesterol transport have shown additive effects of ABCA1 and ABCG1,9 and genetic knockout of both ABCA1 and ABCG1 in hematopoietic cells leads to massive cholesteryl ester accumulation in peritoneal macrophages, prominent foam cell accumulation in organs, and accelerated atherogenesis in a susceptible background.10,11 In double knockout bone marrow recipients, atherosclerotic lesions and heart showed increased numbers of apoptotic cells, as well as an infiltration of inflammatory cells and foam cells.11 Peritoneal macrophages from Abca1–/–Abcg1–/– mice and Abcg1–/– mice and to a lesser extent Abca1–/– mice showed increased expression and secretion of a variety of inflammatory cytokines (tumor necrosis factor [TNF]-
, interleukin [IL]-6, IL-1β, and IL-12p70) and chemokines (macrophage inflammatory protein [MIP]-1, MIP-2, monocyte chemoattractant protein [MCP]-1).11–13 Abcg1–/– mice accumulate foam cells and other inflammatory cells in the lung,14–16 but, surprisingly, transplantation of Abcg1–/– bone marrow into atherosclerosis-susceptible recipients has resulted in either no change or reduced atherosclerosis in most studies.10,17,18 These observations suggested that accumulation of cholesterol, oxysterols, or other lipids in ABC transporter–deficient macrophages leads to increased inflammatory gene expression.11,16 The purpose of the present study was to determine the mechanisms responsible for the increased expression of inflammatory genes in Abcg1–/– and Abca1–/–Abcg1–/– macrophages and to further explore the consequences of ABCG1 deficiency in the inflammatory response of atherosclerotic lesions.
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Methods |
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For details, see the online-only Data Supplement.
Animals and Diets
Abca1–/–, Abcg1–/–, and Abca1–/–Abcg1–/– littermates in a mixed C57BL/6 x DBA background were as described previously.11 C3H/HeJ mice carrying a Toll-like receptor 4 mutation were obtained from The Jackson Laboratory (Bar Harbor, Me). Bone marrow transplantation was performed as described previously.19 The atherosclerosis studies were conducted in female C57BL/6 Ldlr–/– mice transplanted with C57BL/6 Abcg1–/– bone marrow fed a Western diet (TD 88137, Harlan Teklad) for 12 weeks.18 At the end of the study, Abcg1–/– recipients were challenged for 3 days with a single intraperitoneal injection of thioglycollate (38 g/L), and 1 group of mice was allowed to recover for 2 weeks after injection. All mice were housed at Columbia University Medical Center according to animal welfare guidelines. Animals had ad libitum access to both food and water.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Macrophages Deficient in ABCA1 and/or ABCG1 Show Increased Expression of Inflammatory Cytokines in Response to Lipopolysaccharide
After exposure of elicited macrophages to lipopolysaccharide, an exaggerated cytokine response was seen in Abca1–/– Abcg1–/– macrophages (granulocyte colony-stimulating factor [G-CSF], MIP-1
, MIP-2, TNF-, IL-6, IL-1β) and Abcg1–/– macrophages (all cytokines tested except IL-6), whereas Abca1–/– macrophages showed slight increases only in some cytokines (G-CSF, MIP-2, and TNF-) (Figure 1A). Although different macrophage preparations showed different degrees of increase in cytokine gene expression, the pattern was always as follows: Abca1–/–Abcg1–/–>Abcg1–/–>Abca1–/–> wild-type (WT). Secretion patterns of cytokines were parallel to mRNA responses (not shown). A time course experiment showed a greater initial increase as well as more sustained cytokine gene expression in Abcg1–/– compared with WT macrophages after lipopolysaccharide exposure (Figure 1B.) We also evaluated inflammatory cytokine secretion in bone marrow–derived macrophages after 10 days in cell culture. An enhanced response to lipopolysaccharide was seen in Abcg1–/– and double knockout macrophages but not in Abca1–/– macrophages (Figure 1C). Notably, under these conditions no detectable basal secretion of cytokines was found in either WT or Abc transporter–deficient macrophages (Figure 1C), suggesting that the exaggerated cytokine expression of thioglycollate-elicited macrophages represented a response to exogenous factors such as lipopolysaccharide. Lipopolysaccharide is a known contaminant of thioglycollate and is likely present in trace amounts in freshly elicited peritoneal macrophages.20 These observations indicated a markedly increased cytokine response to lipopolysaccharide in Abca1–/–Abcg1–/– and Abcg1–/– macrophages, with a much smaller effect in Abca1–/– cells.
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Knockdown of Abc transporters increases signaling via Toll-like receptor 4 (TLR4) and MyD88/TRIF in macrophages. Lipopolysaccharide is known to signal via TLR4. To test the hypothesis that the exaggerated cytokine response of elicited Abcg1–/– peritoneal macrophages was due to enhanced TLR4 signaling, we performed a small interfering RNA (siRNA) knockdown of Abcg16 in macrophages obtained from mice with genetic deficiency of TLR4. This genetic deficiency resulted in almost undetectable basal cytokine expression and prevented the inflammatory response induced by Abcg1 knockdown (Figure 1D). Deficiency of MyD88, a downstream signaling molecule of TLR4, also abolished the lipopolysaccharide response in WT and Abcg1-deficient macrophages (Figure IA and IB in the online-only Data Supplement). Similar results were obtained after knockdown of both Abcg1 and Abca1 by siRNA18 in Myd88–/–Trif–/– macrophages (Figure IC in the online-only Data Supplement). Moreover, an inhibitor of lipopolysaccharide activation of TLR4 markedly reduced the increase in cytokine expression and secretion in freshly isolated thioglycollate macrophages from Abcg1–/–,Abca1–/–, and Abca1–/–Abcg1–/– macrophages (not shown). Consistent with a role of TLR4 signaling, Abcg1–/– macrophages exhibited an increased content of nuclear p65 nuclear factor (NF)-
B after lipopolysaccharide exposure (Figure IIA in the online-only Data Supplement), and inhibitors of NF-B or p38 mitogen-activated protein kinase markedly reduced the increased cytokine gene expression in these cells (Figure IIB and IIC in the online-only Data Supplement). These studies show that increased lipopolysaccharide signaling via TLR4 is responsible for increased expression of inflammatory cytokines in elicited Abcg1–/– macrophages. However, total levels of TLR4 in membranes and cytosol were not altered in Abcg1–/– macrophages (Figure IIA in the online-only Data Supplement).
Increased inflammatory gene expression does not involve liver X receptor (LXR) transrepression, endoplasmic reticulum (ER) stress, or activation of inflammasome. We performed further studies to determine the mechanism of the increase in NF-B–mediated gene expression in Abcg1–/– macrophages. An important form of signaling pathway cross talk involves transrepression of NF-B responses as a result of activation of nuclear receptors such as LXRs or peroxisome proliferator-activated receptors (PPARs).21 Abcg1–/– macrophages accumulate sterols, notably 7-ketocholesterol and desmosterol after feeding of a high-cholesterol diet (Table) as well as 27-OH cholesterol (0.26±0.04 versus 0.44±0.04 ng/mg protein in macrophages from WT and Abcg1–/– mice fed a chow diet, respectively; P<0.05). Thus, we considered the possibility that Abcg1–/– macrophages might accumulate endogenous sterols that bind LXRs but fail to mediate transrepression of NF-B responses. The synthetic LXR activator T-0901317 was able to reduce expression of inflammatory cytokines in WT and Abcg1–/– macrophages, indicating that the transrepression mechanism by LXR was intact in these cells (Figure IIIA in the online-only Data Supplement). Similar findings were observed after treatment of macrophages with natural LXR ligands such as 27-OH cholesterol or desmosterol (Figure IVA in the online-only Data Supplement). By contrast, addition of 7-ketocholesterol to WT and Abcg1–/– cells reduced the mRNA levels of some cytokines but did not abolish the differential response of Abcg1–/– and WT macrophages, inconsistent with a transrepression mechanism involving this particular oxysterol (Figure IVB in the online-only Data Supplement). We also assessed the effects of exposure of macrophages to oxidized phospholipids or oxidized LDL before lipopolysaccharide treatment. However, oxidized phospholipids and oxidized LDL both caused a reduced inflammatory response in WT and Abcg1–/– macrophages (Figure IVB in the online-only Data Supplement) along with an induction of the PPAR-
target gene, CD36 (data not shown). These responses most likely reflect PPAR- activation and transrepression of NF-B responses.21 Finally, knockdown of both LXR and LXRβ by siRNA (
70% efficiency) did not alter the profile of inflammatory cytokine expression in WT or Abcg1–/– macrophages (Figure IIIB in the online-only Data Supplement), although it was sufficient to reduce expression of LXR target genes such as SREBP-1c (data not shown). These data are inconsistent with a mechanism involving failure of transrepression by LXRs.
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We considered 2 additional mechanisms for increased NF-B responses in Abcg1–/– macrophages. First, Abcg1-/- macrophages have increased acyl-coenzyme A:cholesterol acyltransferase activity, suggesting an increased content of free cholesterol in the ER,11,22 and free cholesterol accumulation in the ER can lead to an ER stress response with subsequent NF-B activation.23 However, no increase in free cholesterol was found in the ER of Abcg1–/– macrophages (Figure VB in the online-only Data Supplement) and no induction of CHOP or other ER stress markers (Figure VA in the online-only Data Supplement). Finally, some cytokines such as IL-1β are secreted in increased amounts as a result of activity of the inflammasome.24 However, the inflammasome activator nigericin did not modify IL-1β or other cytokine secretion (Figure VC in the online-only Data Supplement), and caspase-1 cleavage, a hallmark of inflammasome activation, was unchanged in Abcg1–/– macrophages (Figure VD in the online-only Data Supplement).
Modulation of Macrophage Membrane Cholesterol Modulates Inflammatory Response: Possible Role of Lipid Rafts
ABCG1 promotes efflux of cholesterol, 7-oxysterols, and possibly phospholipids from cells.6,25 To test the hypothesis that increased inflammatory gene expression was secondary to cholesterol accumulation, we treated macrophages with cyclodextrin to deplete membrane cholesterol26 and then exposed them to lipopolysaccharide. This treatment either abolished or reduced the increase in cytokine gene expression seen in ABC transporter–deficient macrophages compared with WT (Figure 2A). In contrast, cyclodextrin-cholesterol loading led to a marked increase in cytokine gene expression in macrophages in the order Abca1–/–Abcg1–/–>Abcg1–/–> Abca1–/–>WT (Figure 2B) (ie, the same pattern as observed in basal or lipopolysaccharide-treated elicited macrophages; Figure 1A). Furthermore, treatment of macrophages with filipin, an agent that binds cholesterol in the plasma membrane,27 abolished the increased inflammatory cytokine expression in Abcg1–/– cells (Figure 2C).
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To assess the possibility that cholesterol accumulation in plasma membrane might lead to formation of increased liquid ordered domains in their plasma membranes, we incubated cells with fluorescent cholera toxin, which binds GM1 gangliosides in membrane liquid ordered domains. Confocal microscopy demonstrated significantly increased fluorescence in the plasma membrane of Abca1–/–Abcg1–/– macrophages in the basal state (Figure 3A shows representative images, and Figure 3B shows quantification of fluorescence intensity from 3-dimensional reconstruction of confocal images). After cholesterol loading, increased fluorescence was also seen in Abca1–/– and Abcg1–/– knockout macrophages (Figure 3A and 3B). Together, these data suggest that increased inflammatory responses are due to an increased content of unesterified cholesterol in the plasma membrane and may reflect increased lipid raft formation.
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Increased Cell Surface Expression of TLR4
Although overall expression levels of TLR4 mRNA and protein were unaltered (Figure IIA in the online-only Data Supplement and data not shown), fluorescence-activated cell sorter analysis showed an increased cell surface expression of TLR4 in Abcg1–/– and Abca1–/–Abcg1–/– macrophages, both under basal conditions and after treatment with lipopolysaccharide (Figure 4A and 4C). Treatment with lipopolysaccharide resulted in a small but significant reduction in cell surface TLR4 concentration in WT and Abca1–/– macrophages but not in Abcg1–/– or Abca1–/–Abcg1–/– macrophages (Figure 4C). Optimal activation of the TLR4 signaling pathway by lipopolysaccharide involves the formation of a lipopolysaccharide signaling complex consisting of surface molecules such as CD14 and MD2.28 Using an antibody that selectively recognizes the TLR4/MD2 complex,29 we showed increased cell surface TLR4/MD2 complex formation in basal Abca1–/–Abcg1–/– macrophages and, after lipopolysaccharide treatment, complex formation in the order Abca1–/–Abcg1–/–>Abcg1–/–>Abca1–/–>WT (Figure 4B and 4D).
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P<0.05 vs untreated condition.
Increased Response to TLR2, TLR3, and TLR4 Ligands in Abca1–/–Abcg1–/– Cells
To assess responses to ligands of various TLRs, we treated basal or cyclodextrin-cholesterol–loaded WT or Abca1–/–Abcg1–/– bone marrow–derived macrophages with ligands to different TLRs (Figure 5). Treatment with ligands to TLR2, TLR3, and TLR4 but not TLR7 or TLR9 resulted in increased TNF- mRNA expression in Abca1–/–Abcg1–/– cells (Figure 5A). The pattern was similar for other inflammatory genes, with more restricted responses for some genes (MIP-1, G-CSF) and broader responses including to TLR7 ligand for MIP-2 (Figure 5B through 5D). Interestingly, in the absence of TLR ligands, no increase in inflammatory gene expression was found in ABC transporter–deficient cells, even though a response to cholesterol loading was present. For TLR2 and TLR3 ligands, effects of cholesterol loading and transporter deficiency were more than additive, whereas they were not for the TLR4 ligand. Although this appears different from earlier results in which clear synergy was seen (Figure 2D), this likely reflects a much smaller TLR4 stimulus because of only trace lipopolysaccharide in this earlier experiment.
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Marked Inflammatory Cell Infiltration in Lesions of Ldlr–/– Mice Transplanted With Abcg1–/– Bone Marrow
Although double knockout bone marrow recipients showed increased atherosclerosis, Abcg1–/– bone marrow–transplanted mice showed either no change or decreased atherosclerosis.10,11,17,18 This seemed to be discordant with our observations showing increased inflammatory response in thioglycollate-elicited Abcg1–/– macrophages. To further assess relevance of the inflammatory response to atherosclerosis, we examined atherosclerotic lesions of Abcg1–/– bone marrow recipients 3 days after intraperitoneal thioglycollate injection. Interestingly, a marked inflammatory cell infiltrate was found in mice transplanted with Abcg1–/– bone marrow compared with WT bone marrow–transplanted mice (Figure 6A). This was most prominent in the adventitia and in the necrotic core region of plaques. Microscopic examination of hematoxylin-eosin–stained sections indicated that many of the infiltrating cells appeared to be neutrophils with multilobed nuclei (not shown). The infiltrating cells showed abundant immunostaining for neutrophils (MCA771G) and to a much lesser extent for macrophages (Mac-3) (Figure 6A) and monocytes (CD68+) and lymphocytes (CD3+) (not shown). The neutrophil stain also reacted strongly with necrotic debris in the necrotic core region of Abcg1–/– bone marrow recipients but not in Abcg1+/+ recipients. A systematic quantification suggested an 4-fold increase in neutrophil content in the adventitia of lesions of Abcg1–/– bone marrow recipients compared with controls (Figure 6B). To determine whether longer-range consequences were present for atherosclerosis, lesions were analyzed 2 weeks after an intraperitoneal thioglycollate injection (Figure 6C). At this time, no difference was found in the neutrophil content of lesions (Figure 6B), necrotic core staining, or macrophage content (not shown), and lesions, if anything, were somewhat smaller in Abcg1–/– bone marrow recipients (Figure 6C). These observations suggest a transient intense predominantly neutrophilic inflammatory cell infiltrate associated with accumulation of necrotic debris probably derived from dead neutrophils in Abcg1–/– bone marrow recipients without an effect on lesion area. An examination of peripheral blood count in mice injected intraperitoneally with thioglycollate showed an increased neutrophil count in Abcg1–/– bone marrow recipients; 2 weeks after intraperitoneal administration of thioglycollate, neutrophil levels had returned toward baseline but were still higher in Abcg1–/– bone marrow recipients (Figure 6D).
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Increased Plasma G-CSF and Neutrophilia
The increased neutrophil count led us to systematically assess cell blood counts in mice of the 4 different genotypes. On a chow diet, Abca1–/–Abcg1–/– mice had an 2-fold increase in the neutrophil count (Figure 7B). In response to a high-fat diet, double knockout mice and, to a lesser extent, single knockout mice developed an increase in total white blood cell count that was not observed in control mice (Figure 7A), suggesting an interaction between hypercholesterolemia and Abc transporter deficiency. The increase in total white blood cell count was due to an increase in both neutrophils and lymphocytes (Figure 7B and 7C). Our studies in macrophages had shown a prominent increased in G-CSF mRNA and secretion in Abcg1–/– and Abca1–/–Abcg1–/– macrophages (Figure 1A and 1C). Plasma G-CSF levels were prominently increased in double knockout mice and, to a lesser extent, in single knockout mice, especially on the Western diet (Figure 7D).
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Discussion |
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Lipid accumulation and increased expression of inflammatory genes are central events in atherogenesis, but specific molecular mechanisms linking lipid accumulation to inflammation in macrophages are poorly understood. Seminal studies have indicated roles of TLR2, TLR4, and MyD88 in atherogenesis in hypercholesterolemic animal models, suggesting a link between hyperlipidemia and TLR signaling.28–32 The present study suggests that cholesterol accumulation in the plasma membrane of Abcg1–/– and Abca1–/–Abcg1–/– macrophages leads to increased levels and signaling of TLR4 and an increased inflammatory response after exposure to lipopolysaccharide. Although these findings are consistent with a recent study in Abca1–/– macrophages,33 our studies suggest that ABCG1 has a larger role in modulating macrophage inflammatory responses than ABCA1 and that ABCA1 has a compensatory role when ABCG1 is deficient.
Abca1–/–Abcg1–/– macrophages also showed increased expression of several inflammatory cytokines when treated with ligands to TLR2 and TLR3 but not TLR7 or TLR9, indicating a widespread but not general enhancement of innate immune responses. This suggests that loading of plasma and endosomal membranes with free cholesterol results in enhanced inflammatory response to several TLR ligands, a situation that may occur when macrophages ingest apoptotic cells containing bacterial or viral products. The ability of HDL and apoA-1 to promote cholesterol efflux via ABCA1 and ABCG1 may have a downmodulating effect on these innate immune responses, also likely relevant to inflammation in atherogenesis.
We initially reported increased expression and secretion of inflammatory cytokines and chemokines in peritoneal macrophages from mice deficient in Abca1 and/or Abcg1.11 Baldan et al16 found increased inflammatory gene expression in lungs and in peritoneal macrophages of Abcg1–/– mice after treatment with lipopolysaccharide, and Wojcik et al15 showed increased inflammatory gene expression in alveolar macrophages of Abcg1–/– mice. The present study has provided new insights into the mechanism of increased inflammatory gene expression in Abcg1–/– and double knockout macrophages. Several potential mechanisms such as inflammasome activation, reduced LXR transrepression of NF-B responses, ER stress, and accumulation of specific sterols such as 7-oxysterols or 27-OH cholesterol and inflammatory responses to oxidized phospholipids were largely excluded. Our data suggest that free cholesterol accumulation in the plasma membrane leads to increased levels and signaling of TLR4 via MyD88/TRIF. This may be related to increased formation of liquid ordered domains in the plasma membrane, resulting in increased cell surface expression and/or dimerization and activation of TLR4 in the presence of lipopolysaccharide. The increased TLR4/MD2 complex observed in all genotypes in the order Abca1–/–Abcg1–/–>Abcg1–/–> Abca1–/–>WT closely paralleled the inflammatory response to lipopolysaccharide and most likely reflected clustering of the TLR4/MD2 complex in rafts and increased TLR4 signaling.29,34 The lipopolysaccharide-mediated downregulation of TLR4 seen in WT and Abca1–/– cells did not occur in Abcg1–/– and Abca1–/–Abcg1–/– macrophages (Figure 4C), suggesting that reduced TLR4 internalization could also contribute to the exaggerated inflammatory response in these cells.
Deficiency of TLR2, TLR4, and MyD88 reduces atherosclerosis in susceptible backgrounds,30–32 suggesting that TLR ligands are activating macrophages in atherosclerotic lesions. Although the nature of the relevant ligands in atheroma remains unclear,35 this indicates that our observation of enhanced signaling via TLR2, TLR3, and TLR4 in Abca1–/–Abcg1–/– macrophages is likely relevant to accelerated atherosclerosis in mice with deficiency of these 2 transporters in hematopoietic cells. One consequence of increased inflammatory gene expression was an increase in neutrophils in blood. Abca1–/–Abcg1–/– mice and, to a lesser extent, single knockout mice developed a remarkable neutrophilia after feeding of a high-cholesterol diet. Neutrophilia likely reflected increased production of G-CSF by hematopoietic cells, as levels of G-CSF and degree of neutrophilia appeared well correlated. The mechanism of neutrophilia likely involved at least in part increased G-CSF secretion by macrophages secondary to increased TLR4/MyD88 signaling. Interestingly, lymphocytosis was also found in high-cholesterol diet–fed transporter-deficient mice (Figure 5C), consistent with a recent report showing enhanced lymphocyte proliferative responses in mice deficient in LXRβ or ABCG1.36 Together, these findings emphasize the role of HDL and LXRs in dampening innate and acquired immune responses as a result of promotion of sterol efflux via ABCA1 and ABCG1.
We made the surprising observation that neutrophils became prominent in lesions of Abcg1–/– bone marrow–transplanted mice after a peripheral inflammatory stimulus (intraparenchymal thioglycollate injection), most likely reflecting exposure to trace lipopolysaccharide in the thioglycollate.20 Marked neutrophil accumulation in plaques appeared to reflect the underlying neutrophilia in these mice. In addition, macrophages of Abcg1–/– mice show increased secretion of MIP-1 and MIP-2, both potent neutrophil chemokines.37 Neutrophils are present in small numbers in atherosclerotic lesions of Ldlr–/– and apoE–/– mice, especially in the surface and adventitia of the lesion.38,39 Remarkably, a recent study showed that neutrophils accounted for approximately one fifth of the phagocytic capacity of leukocytes in lesions of apoE–/– mice.40 Neutrophils are prominent in human lesions in the context of acute coronary syndromes, but it is not clear whether they are present secondary to atherothrombosis as part of the cleaning-up process or whether they have a causative role, involving secretion of elastase, myeloperoxidase, and H2O2.41 In prospective studies, the neutrophil count is predictive of coronary heart disease and also of the outcome after an acute coronary syndrome event.42 Although the neutrophil infiltration did not result in a plaque disruption in our study, such effects may have been limited because mice are resistant to plaque breakdown and thrombus formation. We speculate that similar changes in human atherosclerotic plaques could be involved in plaque destabilization (ie, after a peripheral inflammatory stimulus, a subject with low HDL levels and low ABCG1 activity might be more susceptible to an enhanced inflammatory "echo" response in lesions, leading to plaque destabilization). Our study suggests that one of the basic antiatherogenic properties of HDL, suppression of inflammatory responses, may be in part secondary to its ability to promote cholesterol efflux via ABCG1 and, to a lesser extent, ABCA1. A variety of treatments that raise HDL levels, such as niacin and cholesteryl ester transfer protein inhibitors, may promote cholesterol efflux via ABCG143,44 and thus suppress inflammatory and chemokine gene responses in macrophage foam cells.
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Acknowledgments |
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Sources of Funding
This work was supported by grants from the National Institutes of Health (HL54591).
Disclosures
A.R. Tall reports being a consultant to Pfizer, Merck, Boehringer-Ingelheim, and Takeda Pharmaceuticals. The other authors report no conflicts.
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.793869/DC1.
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