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Background: Regulatory T cells (T regs ) are protective in atherosclerosis but reduced during disease progression due to cell death and loss of stability. However, the mechanisms of T reg dysfunction remain unknown. Oxidized phospholipids are abundant in atherosclerosis and can activate innate immune cells, but little is known regarding their impact on T cells. Given T reg loss during atherosclerosis progression and oxidized phospholipid levels in the plaque microenvironment, we investigated whether oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), an oxidized phospholipid associated with atherosclerotic plaques, alters T reg differentiation and function., Methods: CD4 + T cells were polarized to T reg , T helper (Th) 1, and Th17 cells with or without oxPAPC and assessed by flow cytometry. Gene expression in oxPAPC-treated T regs was analyzed by bulk RNA sequencing. Functional studies of oxPAPC-induced T regs were performed by coculturing T regs with CellTrace Violet-labeled cells in vitro, and by adoptively transferring T regs to hyperlipidemic Ldlr -/- mice to measure atherosclerosis progression., Results: Compared with controls, oxPAPC-treated T regs were less viable, but surviving cells expressed higher levels of the Th1-associated markers T-bet, CXCR3, and IFN (interferon)-γ. Th1 and Th17 skewing cultures were unaltered by oxPAPC. IFN-γ is linked to T reg instability, thus T reg polarization experiments were repeated using Ifngr1 -/- CD4 + T cells. IFNγR1 (INF gamma receptor 1) deficiency did not improve cell viability in oxPAPC-treated T regs ; however, T-bet and IFN-γ expression was not increased in surviving cells suggesting a role for IFN-γsignaling. OxPAPC-treated T regs were less suppressive in vitro, and adoptive transfer studies in hyperlipidemic Ldlr -/- mice showed that oxPAPC-induced T regs possessed altered tissue homing and were insufficient to inhibit atherosclerosis progression., Conclusions: OxPAPC elicits T reg -specific changes altering T reg differentiation and inducing a Th1-like phenotype in surviving cells partially through IFN-γ signaling. This is biologically relevant as oxPAPC-treated T regs do not reduce atherosclerosis progression in Ldlr -/- mice. This study supports the role of oxidized phospholipids in negatively impacting T reg differentiation and atheroprotective function., Competing Interests: Disclosures None.

T cells in systemic lupus erythematosus (SLE) exhibit multiple metabolic abnormalities. Excess iron can impair mitochondria and may contribute to SLE. To gain insights into this potential role of iron in SLE, we performed a CRISPR screen of iron handling genes on T cells. Transferrin receptor (CD71) was identified as differentially critical for T H 1 and inhibitory for induced regulatory T cells (iT regs ). Activated T cells induced CD71 and iron uptake, which was exaggerated in SLE-prone T cells. Cell surface CD71 was enhanced in SLE-prone T cells by increased endosomal recycling. Blocking CD71 reduced intracellular iron and mTORC1 signaling, which inhibited T H 1 and T H 17 cells yet enhanced iT regs . In vivo treatment reduced kidney pathology and increased CD4 T cell production of IL-10 in SLE-prone mice. Disease severity correlated with CD71 expression on T H 17 cells from patients with SLE, and blocking CD71 in vitro enhanced IL-10 secretion. T cell iron uptake via CD71 thus contributes to T cell dysfunction and can be targeted to limit SLE-associated pathology.

Autoimmune disease has presented an insurmountable barrier to restoration of durable immune tolerance. Previous studies indicate that chronic therapy with metabolic inhibitors can reduce autoimmune inflammation, but it remains unknown whether acute metabolic modulation enables permanent immune tolerance to be established. In an animal model of lupus, we determined that targeting glucose metabolism with 2-deoxyglucose (2DG) and mitochondrial metabolism with metformin enables endogenous immune tolerance mechanisms to respond to tolerance induction. A 2-week course of 2DG and metformin, when combined with tolerance-inducing therapy anti-CD45RB, prevented renal deposition of autoantibodies for 6 months after initial treatment and restored tolerance induction to allografts in lupus-prone mice. The restoration of durable immune tolerance was linked to changes in T cell surface glycosylation patterns, illustrating a role for glycoregulation in immune tolerance. These findings indicate that metabolic therapy may be applied as a powerful preconditioning to reinvigorate tolerance mechanisms in autoimmune and transplant settings that resist current immune therapies.

A Western-style, high-fat diet promotes cardiovascular disease, in part because it is rich in choline, which is converted to trimethylamine (TMA) by the gut microbiota. However, whether diet-induced changes in intestinal physiology can alter the metabolic capacity of the microbiota remains unknown. Using a mouse model of diet-induced obesity, we show that chronic exposure to a high-fat diet escalates Escherichia coli choline catabolism by altering intestinal epithelial physiology. A high-fat diet impaired the bioenergetics of mitochondria in the colonic epithelium to increase the luminal bioavailability of oxygen and nitrate, thereby intensifying respiration-dependent choline catabolism of E. coli In turn, E. coli choline catabolism increased levels of circulating trimethlamine N -oxide, which is a potentially harmful metabolite generated by gut microbiota., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Purpose of Review: Accelerated atherosclerosis is a significant comorbidity and the leading cause of death for patients with systemic lupus erythematosus (SLE). It is now apparent that SLE-accelerated atherosclerosis is not driven solely by traditional cardiovascular risk factors, adding complexity to disease characterization and mechanistic understanding. In this review, we will summarize new insights into SLE-accelerated atherosclerosis evaluation, treatment, and mechanism., Recent Findings: Recent work highlights the need to incorporate inflammatory biomarkers into cardiovascular disease (CVD) risk assessments. This is especially true for SLE patients, in which mechanisms of immune dysfunction likely drive CVD progression. There is new evidence that commonly prescribed SLE therapeutics hinder atherosclerosis development. This effect is achieved both by reducing SLE-associated inflammation and by directly improving measures of atherosclerosis, emphasizing the interconnected mechanisms of the two conditions., Summary: SLE-accelerated atherosclerosis is most likely the consequence of chronic autoimmune inflammation. Therefore, diligent management of atherosclerosis requires assessment of SLE disease activity as well as traditional cardiovascular risk factors. This supports why many of the therapeutics classically used to control SLE also modulate atherosclerosis development. Greater understanding of the mechanisms underlying this condition will allow for the development of more targeted therapeutics and improved outcomes for SLE patients., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Helper T cell activity is dysregulated in a number of diseases including those associated with rheumatic autoimmunity. Treatment options are limited and usually consist of systemic immune suppression, resulting in undesirable consequences from compromised immunity. Hedgehog (Hh) signaling has been implicated in the activation of T cells and the formation of the immune synapse, but remains understudied in the context of autoimmunity. Modulation of Hh signaling has the potential to enable controlled immunosuppression but a potential therapy has not yet been developed to leverage this opportunity., Methods: In this work, we developed biodegradable nanoparticles to enable targeted delivery of eggmanone (Egm), a specific Hh inhibitor, to CD4 + T cell subsets. We utilized two FDA-approved polymers, poly(lactic-co-glycolic acid) and polyethylene glycol, to generate hydrolytically degradable nanoparticles. Furthermore, we employed maleimide-thiol mediated conjugation chemistry to decorate nanoparticles with anti-CD4 F(ab') antibody fragments to enable targeted delivery of Egm., Results: Our novel delivery system achieved a highly specific association with the majority of CD4 + T cells present among a complex cell population. Additionally, we have demonstrated antigen-specific inhibition of CD4 + T cell responses mediated by nanoparticle-formulated Egm., Conclusion: This work is the first characterization of Egm's immunomodulatory potential. Importantly, this study also suggests the potential benefit of a biodegradable delivery vehicle that is rationally designed for preferential interaction with a specific immune cell subtype for targeted modulation of Hh signaling., Competing Interests: Professor Charles C Hong and Dr Charles H Williams report a US Patent #: US 10,329,304 B2 issued. Professor Todd D Giorgio reports patent materials and methods for the treatment of immune cells pending. The authors report no other conflicts of interest in this work., (© 2020 Haycook et al.)

Background and Aims: Circulating levels of oxidized lipoprotein (oxLDL) correlate with myocardial infarction risk and atherosclerosis severity. Our previous study demonstrates that oxLDL immune complexes (oxLDL-ICs) can signal through FcγRs on bone marrow-derived dendritic cells (BMDCs) and enhance their activation and inflammatory cytokine secretion. While global FcγR -/- studies have shown that activating FcγRs are proatherogenic, the role of the inhibitory FcγRIIb is unclear. We sought to determine the role of DC-specific FcγRIIb in atherosclerosis., Methods: Bone marrow chimeras were generated by rescuing lethally irradiated Ldlr -/- mice with hematopoietic cells from littermate CD11c-Cre + or CD11c-Cre - Fcgr2b fl/fl donors. Four weeks following transplant, recipients were placed on a Western diet for eight weeks. Various tissues and organs were analyzed for differences in inflammation., Results: Quantitation of atherosclerosis in the proximal aorta demonstrated a 58% increase in female CD11c-Cre + Fcgr2b fl/fl recipients, but a surprising 44% decrease in male recipients. Hepatic cholesterol and triglycerides were increased in female CD11c-Cre + Fcgr2b fl/fl recipients. This was associated with an increase in CD36 and MHC Class II expression on hepatic CD11c + CD11b + DCs in female livers. In contrast, male CD11c-Cre + Fcgr2b fl/fl recipients had decreased hepatic lipids with a corresponding decrease in CD36 and MHC Class II expression on CD11c + cells. Interestingly, both sexes of CD11c-Cre + Fcgr2b fl/fl recipients had significant decreases in serum cholesterol and TGs with corresponding decreases in liver Fasn transcripts., Conclusions: The absence of FcγRIIb expression on CD11c + cells results in sex-dependent alteration in liver inflammation influencing atherogenesis and sex-independent modulation of serum cholesterol and TGs., (Published by Elsevier B.V.)

Cardiovascular disease (CVD) is the number one cause of death in the United States and worldwide. The most common cause of cardiovascular disease is atherosclerosis, or formation of fatty plaques in the arteries. Low-density lipoprotein (LDL), termed "bad cholesterol", is a large molecule comprised of many proteins as well as lipids including cholesterol, phospholipids, and triglycerides. Circulating levels of LDL are directly associated with atherosclerosis disease severity. Once thought to simply be caused by passive retention of LDL in the vasculature, atherosclerosis studies over the past 40-50 years have uncovered a much more complex mechanism. It has now become well established that within the vasculature, LDL can undergo many different types of oxidative modifications such as esterification and lipid peroxidation. The resulting oxidized LDL (oxLDL) has been found to have antigenic potential and contribute heavily to atherosclerosis associated inflammation, activating both innate and adaptive immunity. This review discusses the many proposed mechanisms by which oxidized LDL modulates inflammatory responses and how this might modulate atherosclerosis.

All immune cells depend on specific and efficient metabolic pathways to mount an appropriate response. Over the past decade, the field of immunometabolism has expanded our understanding of the various means by which cells modulate metabolism to achieve the effector functions necessary to fight infection or maintain homeostasis. Harnessing these metabolic pathways to manipulate inappropriate immune responses as a therapeutic strategy in cancer and autoimmunity has received increasing scrutiny by the scientific community. Fine tuning immunometabolism to provide the desired response, or prevent a deleterious response, is an attractive alternative to chemotherapy or overt immunosuppression. The various metabolic pathways used by immune cells in rheumatoid arthritis, systemic lupus erythematosus and osteoarthritis offer numerous opportunities for selective targeting of specific immune cell subsets to manipulate cellular metabolism for therapeutic benefit in these rheumatologic diseases.

Oxidized low-density lipoprotein (oxLDL) is known to activate inflammatory responses in a variety of cells, especially macrophages and dendritic cells. Interestingly, much of the oxLDL in circulation is complexed to Abs, and these resulting immune complexes (ICs) are a prominent feature of chronic inflammatory disease, such as atherosclerosis, type-2 diabetes, systemic lupus erythematosus, and rheumatoid arthritis. Levels of oxLDL ICs often correlate with disease severity, and studies demonstrated that oxLDL ICs elicit potent inflammatory responses in macrophages. In this article, we show that bone marrow-derived dendritic cells (BMDCs) incubated with oxLDL ICs for 24 h secrete significantly more IL-1β compared with BMDCs treated with free oxLDL, whereas there was no difference in levels of TNF-α or IL-6. Treatment of BMDCs with oxLDL ICs increased expression of inflammasome-related genes Il1a , Il1b , and Nlrp3 , and pretreatment with a caspase 1 inhibitor decreased IL-1β secretion in response to oxLDL ICs. This inflammasome priming was due to oxLDL IC signaling via multiple receptors, because inhibition of CD36, TLR4, and FcγR significantly decreased IL-1β secretion in response to oxLDL ICs. Signaling through these receptors converged on the adaptor protein CARD9, a component of the CARD9-Bcl10-MALT1 signalosome complex involved in NF-κB translocation. Finally, oxLDL IC-mediated IL-1β production resulted in increased Th17 polarization and cytokine secretion. Collectively, these data demonstrate that oxLDL ICs induce inflammasome activation through a separate and more robust mechanism than oxLDL alone and that these ICs may be immunomodulatory in chronic disease and not just biomarkers of severity., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Objective: Antiatherosclerotic effects of tumor necrosis factor-α (TNF-α) blockade in patients with systemic inflammatory states are not conclusively demonstrated, which suggests that effects depend on the cause of inflammation. Macrophage LRP1 (low-density lipoprotein receptor-related protein 1) and apoE contribute to inflammation through different pathways. We studied the antiatherosclerosis effects of TNF-α blockade in hyperlipidemic mice lacking either LRP1 (MΦLRP1(-/-)) or apoE from macrophages., Approach and Results: Lethally irradiated low-density lipoprotein receptor (LDLR)(-/-) mice were reconstituted with bone marrow from either wild-type, MΦLRP1(-/-), apoE(-/-) or apoE(-/-)/MΦLRP1(-/-)(DKO) mice, and then treated with the TNF-α inhibitor adalimumab while fed a Western-type diet. Adalimumab reduced plasma TNF-α concentration, suppressed blood ly6C(hi) monocyte levels and their migration into the lesion, and reduced lesion cellularity and inflammation in both wild-type→LDLR(-/-) and apoE(-/-)→LDLR(-/-) mice. Overall, adalimumab reduced lesion burden by 52% to 57% in these mice. Adalimumab reduced TNF-α and blood ly6C(hi) monocyte levels in MΦLRP1(-/-)→LDLR(-/-) and DKO→LDLR(-/-) mice, but it did not suppress ly6C(hi) monocyte migration into the lesion or atherosclerosis progression., Conclusions: Our results show that TNF-α blockade exerts antiatherosclerotic effects that are dependent on the presence of macrophage LRP1., (© 2016 American Heart Association, Inc.)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes atherosclerosis by increasing low-density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptor (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque is unknown. To study the local effect of human PCSK9 (hPCSK9) on atherosclerotic lesion composition, independently of changes in serum cholesterol levels, we generated chimeric mice expressing hPCSK9 exclusively from macrophages, using marrow from hPCSK9 transgenic (hPCSK9tg) mice transplanted into apoE(-/-) and LDLR(-/-) mice, which were then placed on a high-fat diet (HFD) for 8 weeks. We further characterized the effect of hPCSK9 expression on the inflammatory responses in the spleen and by mouse peritoneal macrophages (MPM) in vitro. We found that MPMs from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. We detected hPCSK9 in the serum of mice transplanted with hPCSK9tg marrow, but did not influence lipid levels or atherosclerotic lesion size. However, marrow-derived PCSK9 progressively accumulated in lesions of apoE(-/-) recipient mice, while increasing the infiltration of Ly6C(hi) inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b- and Ly6C(hi) -positive cell numbers in spleens of apoE(-/-) mice. In vitro, expression of hPCSK9 in LPS-stimulated macrophages increased mRNA levels of the pro-inflammatory markers Tnf and Il1b (40% and 45%, respectively) and suppressed those of the anti-inflammatory markers Il10 and Arg1 (30% and 44%, respectively). All PCSK9 effects were LDLR-dependent, as PCSK9 protein was not detected in lesions of LDLR(-/-) recipient mice and did not affect macrophage or splenocyte inflammation. In conclusion, PCSK9 directly increases atherosclerotic lesion inflammation in an LDLR-dependent but cholesterol-independent mechanism, suggesting that therapeutic PCSK9 inhibition may have vascular benefits secondary to LDL reduction., (Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Objective: Accelerated atherosclerosis is a major source of morbidity in systemic lupus erythematosus (SLE). However, the cause of SLE-accelerated atherosclerosis remains unclear., Methods: CD4(+) T cells from C57/Bl/6 (B6) or SLE-susceptible B6.Sle1.2.3 (B6.SLE) mice were transferred into LDLr(-/-), Rag(-/-) mice. T cells were examined for cytokine production and expression of interleukin-10 receptor (IL-10R) and functional markers. T cells were isolated based on FoxP3(GFP) expression and transferred to LDLr(-/-), Rag(-/-) mice to establish a role for B6.SLE effector T cells (Teff) in atherosclerosis., Results: Mice receiving whole B6.SLE CD4(+) T cells displayed no other SLE phenotype; however, atherosclerosis was increased nearly 40%. We noted dysregulated IL-17 production and reduced frequency of IL-10R expression by B6.SLE regulatory T cells (Treg). Functional assays indicated resistance of B6.SLE Teff to suppression by both B6.SLE and B6 Treg. Transfer experiments with CD4(+)FoxP3(-) Teff and CD4(+)FoxP3(+) Treg from B6.SLE and B6 mice, respectively, resulted in increased atherosclerosis compared with B6 Teff and Treg recipients. Treg isolated from mice receiving B6.SLE Teff with B6 Treg had increased production of IL-17 and fewer expressed IL-10R compared with B6 Teff and Treg transfer., Conclusions: Transfer of B6.SLE Teff to LDLr(-/-), Rag(-/-) mice results in accelerated atherosclerosis independent of the source of Treg. In addition, the presence of B6.SLE Teff resulted in more IL-17-producing Treg and fewer expressing IL-10R, suggesting that B6.SLE Teff may mediate phenotypic changes in Treg. To our knowledge, this is the first study to provide direct evidence of the role of B6.SLE Teff in accelerating atherosclerosis through resistance to Treg suppression., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Expression of molecules involved in lipid homeostasis such as the low density lipoprotein receptor (LDLr) on antigen presenting cells (APCs) has been shown to enhance invariant natural killer T (iNKT) cell function. However, the contribution to iNKT cell activation by other lipoprotein receptors with shared structural and ligand binding properties to the LDLr has not been described. In this study, we investigated whether a structurally related receptor to the LDLr, known as LDL receptor-related protein (LRP), plays a role in iNKT cell activation. We found that, unlike the LDLr which is highly expressed on all immune cells, the LRP was preferentially expressed at high levels on F4/80+ macrophages (MΦ). We also show that CD169+ MΦs, known to present antigen to iNKT cells, exhibited increased expression of LRP compared to CD169- MΦs. To test the contribution of MΦ LRP to iNKT cell activation we used a mouse model of MΦ LRP conditional knockout (LRP-cKO). LRP-cKO MΦs pulsed with glycolipid alpha-galactosylceramide (αGC) elicited normal IL-2 secretion by iNKT hybridoma and in vivo challenge of LRP-cKO mice led to normal IFN-γ, but blunted IL-4 response in both serum and intracellular expression by iNKT cells. Flow cytometric analyses show similar levels of MHC class-I like molecule CD1d on LRP-cKO MΦs and normal glycolipid uptake. Survey of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages and no homeostatic disruption as evidenced by the absence of programmed death-1 and Ly-49 surface receptors. Mixed bone marrow chimeras showed that the inability iNKT cells to make IL-4 is cell extrinsic and can be rescued in the presence of wild type APCs. Collectively, these data demonstrate that, although MΦ LRP may not be necessary for IFN-γ responses, it can contribute to iNKT cell activation by enhancing early IL-4 secretion.

The field of immunometabolism is burgeoning, with hundreds of papers published on the topic each year. Our understanding of the contribution of immune cells to metabolic regulation has expanded from a simple idea of innate immune cells, such as macrophages, altering adipose tissue function in obesity, to an awareness of the complex role of adaptive immunity in many different organ systems. Recent findings have clearly demonstrated the presence of adaptive lymphocytes, such as T and B cells, in adipose tissue. Furthermore, these data demonstrated T-cell accumulation and limited T-cell receptor repertoire diversity in obese adipose tissue, indicating that an antigen-specific immune response may occur within this tissue. In a recently published paper, we reported that a mouse model of weight cycling resulted in increased T-cell accumulation in adipose tissue. In the current commentary, we discuss the possibility that this increase in adipose tissue T-cell number could represent a local secondary immune response to self-antigens exposed in adipose tissue during obesity. If further experimentation indicates that this hypothesis is true, these data will fortify the concept that obesity is a complex immune-mediated disease and would emphasize the importance of designing therapies to maintain weight loss.

Objective: MCPIP1 is a newly identified protein that profoundly impacts immunity and inflammation. We aim to test if MCPIP1 deficiency in hematopoietic cells results in systemic inflammation and accelerates atherogenesis in mice., Approach and Results: After lethally irradiated, LDLR(-/-) mice were transplanted with bone marrow cells from either wild-type or MCPIP1(-/-) mice. These chimeric mice were fed a western-type diet for 7 weeks. We found that bone marrow MCPIP1(-/-) mice displayed a phenotype similar to that of whole body MCPIP1(-/-) mice, with severe systemic and multi-organ inflammation. However, MCPIP1(-/-) bone marrow recipients developed >10-fold less atherosclerotic lesions in the proximal aorta than WT bone marrow recipients, and essentially no lesions in en face aorta. The diminishment in atherosclerosis in bone marrow MCPIP1(-/-) mice may be partially attributed to the slight decrease in their plasma lipids. Flow cytometric analysis of splenocytes showed that bone marrow MCPIP1(-/-) mice contained reduced numbers of T cells and B cells, but increased numbers of regulatory T cells, Th17 cells, CD11b+/Gr1+ cells and CD11b+/Ly6C(low) cells. This overall anti-atherogenic leukocyte profile may also contribute to the reduced atherogenesis. We also examined the cholesterol efflux capability of MCPIP1 deficient macrophages, and found that MCPIP1 deficiency increased cholesterol efflux to apoAI and HDL, due to increased protein levels of ABCA1 and ABCG1., Conclusions: Hematopoietic deficiency of MCPIP1 resulted in severe systemic and multi-organ inflammation but paradoxically diminished atherogenesis in mice. The reduced atheroegensis may be explained by the decreased plasma cholesterol levels, the anti-atherogenic leukocyte profile, as well as enhanced cholesterol efflux capability. This study suggests that, while atherosclerosis is a chronic inflammatory disease, the mechanisms underlying atherogenesis-associated inflammation in arterial wall versus the inflammation in solid organs may be substantially different.

Background: Elevated cholesterol and triglycerides in blood lead to atherosclerosis and fatty liver, contributing to rising cardiovascular and hepatobiliary morbidity and mortality worldwide., Methods and Results: A cell-penetrating nuclear transport modifier (NTM) reduced hyperlipidemia, atherosclerosis, and fatty liver in low-density lipoprotein receptor-deficient mice fed a Western diet. NTM treatment led to lower cholesterol and triglyceride levels in blood compared with control animals (36% and 53%, respectively; P<0.005) and liver (41% and 34%, respectively; P<0.05) after 8 weeks. Atherosclerosis was reduced by 63% (P<0.0005), and liver function improved compared with saline-treated controls. In addition, fasting blood glucose levels were reduced from 209 to 138 mg/dL (P<0.005), and body weight gain was ameliorated (P<0.005) in NTM-treated mice, although food intake remained the same as that in control animals. The NTM used in this study, cSN50.1 peptide, is known to modulate nuclear transport of stress-responsive transcription factors such as nuclear factor kappa B, the master regulator of inflammation. This NTM has now been demonstrated to also modulate nuclear transport of sterol regulatory element-binding protein (SREBP) transcription factors, the master regulators of cholesterol, triglyceride, and fatty acid synthesis. NTM-modulated translocation of SREBPs to the nucleus was associated with attenuated transactivation of their cognate genes that contribute to hyperlipidemia., Conclusions: Two-pronged control of inflammation and dyslipidemia by modulating nuclear transport of their critical regulators offers a new approach to comprehensive amelioration of hyperlipidemia, atherosclerosis, fatty liver, and their potential complications.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against self-antigens such as double-stranded DNA and phospholipids. Classical comorbidities of SLE include glomerulonephritis, infection, cardiovascular disease, arthritis, skin disorders, and neurological disease. In addition to these classical comorbidities, there is emerging evidence that SLE patients are at higher risk of developing insulin resistance and other components of the metabolic syndrome. Visceral adipose tissue inflammation is a central mediator of insulin resistance in the obese setting, but the mechanism behind the pathogenesis of metabolic disease in the SLE patient population is unclear. We hypothesize that lupus-associated changes in the adaptive immune system are associated with disruption in glucose homeostasis in the context of SLE. To test this hypothesis, we assessed the metabolic and immunological phenotype of SLE-prone B6.SLE mice. B6.SLE mice fed a low-fat diet had significantly worsened glucose tolerance, increased adipose tissue insulin resistance, increased β-cell insulin secretion, and increased adipocyte size compared with their respective B6 controls. Independently of diet, B cells isolated from the white adipose tissue of B6.SLE mice were skewed toward IgG production, and the level of IgG1 was elevated in the serum of SLE-prone mice. These data show that B6.SLE mice develop defects in glucose homeostasis even when fed a low-fat diet and suggest that B cells may play a role in this metabolic dysfunction.

Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease characterized by increased serum autoantibody levels and tissue damage. With improved diagnosis and more effective treatment of the resultant kidney disease, accelerated atherosclerosis has become a major cause of morbidity in patients suffering from SLE. Although the exact mechanisms for SLE-accelerated atherosclerosis are unknown, multiple factors have been established as potential players in this process. Among these potential players are dysregulation of T and B cell populations and increased circulating levels of inflammatory cytokines. In addition, SLE patients exhibit a proatherogenic lipid profile characterized by low HDL and high LDL and triglycerides. Recent therapeutic approaches have focused on targeting B cells, the producers of autoantibodies, but most studies do not consider the effects of these treatments on atherosclerosis. Evidence suggests that T cells play a major role in SLE-accelerated atherosclerosis. Therefore, therapies targeted at T cells may also prove invaluable in treating SLE and atherosclerosis.

Rationale: Recent clinical and preclinical studies have demonstrated that systemic lupus erythematosus (SLE) is associated with an increased risk for cardiovascular disease (CVD). However, unlike in the general population, little is known regarding the efficacy of atheroprotective interventions in patients with SLE. The current study aims to determine the benefit of lymphocyte inhibition on reducing the atherosclerotic burden in SLE-susceptible LDLr-deficient mice., Methods: Female LDLr(-/-) mice were lethally irradiated and reconstituted with bone marrow from C57Bl/6 mice (LDLr.B6) or the SLE-susceptible B6.Sle1.2.3 mice (LDLr.Sle). At 16 weeks post transplant, mice were treated with atorvastatin (10 mg/kg), mycophenolate mofetil (MMF; 40 mg/kg), or both (MMF-A) for 8 weeks, after which the extent of atherosclerosis and the presence of SLE were assessed., Results: Following 8 weeks of treatment, we observed that atorvastatin-mediated reduction in cholesterol levels attenuated atherogenesis in LDLr.B6 mice but failed to significantly reduce atherosclerotic lesion size in LDLr.Sle mice, in spite of a significant reduction in serum cholesterol levels. Treatment with MMF and MMF-A attenuated atherogenesis in LDLr.B6 and LDLr.Sle mice. In addition, MMF-containing regimens inhibited recruitment of CD4+ T cells to atherosclerotic lesions in LDLr.Sle mice. In these mice, MMF also reduced the proportion of activated splenic T cells, as well as interleukin 10 secretion by T cells. With regard to lupus activity, MMF had no overt effect on anti-double-stranded DNA (dsDNA) antibody titres or kidney function and pathology., Conclusions: The current study demonstrates that reduction of cholesterol levels alone is not atheroprotective in lupus-mediated atherogenesis. This is the first study to demonstrate that MMF reduces the atherosclerotic burden in a model of lupus-accelerated atherosclerosis. Our results suggest that MMF treatment may prove beneficial in preventing CVD in patients with SLE.

The mechanisms for increased risk of chronic kidney disease (CKD) in obesity remain unclear. The renin-angiotensin system is implicated in the pathogenesis of both adiposity and CKD. We investigated whether the angiotensin type 1 (AT(1)) receptor, composed of dominant AT(1a) and less expressed AT(1b) in wild-type (WT) mice, modulates development and progression of kidney injury in a high-fat diet (HFD)-induced obesity model. WT mice had increased body weight, body fat, and insulin levels and decreased adiponectin levels after 24 wk of a high-fat diet. Identically fed AT(1a) knockout (AT1aKO) mice gained weight similarly to WT mice, but had lower body fat and higher plasma cholesterol. Both obese AT1aKO and obese WT mice had increased visceral fat and kidney macrophage infiltration, with more proinflammatory M1 macrophage markers as well as increased mesangial expansion and tubular vacuolization, compared with lean mice. These abnormalities were heightened in the obese AT1aKO mice, with downregulated M2 macrophage markers and increased macrophage AT(1b) receptor. Treatment with an AT(1) receptor blocker, which affects both AT(1a) and AT(1b), abolished renal macrophage infiltration with inhibition of renal M1 and upregulation of M2 macrophage markers in obese WT mice. Our data suggest obesity accelerates kidney injury, linked to augmented inflammation in adipose and kidney tissues and a proinflammatory shift in macrophage and M1/M2 balance.

The atherosclerotic process is accelerated in patients with systemic lupus erythematosus (SLE). In addition to a robust lipid-lowering effect, various immunomodulatory functions have been ascribed to statins. By virtue of the latter they may be able to reduce atherosclerotic vascular disease in SLE by inhibiting immune activation within the arterial wall and by attenuating lupus activity. The effects of statins on SLE as well as on lupus-mediated atherogenesis in vivo are discussed in this viewpoint.

Background: Atherosclerosis is widely accepted as an inflammatory disease involving both innate and adaptive immunity. B cells and/or antibodies have previously been shown to play a protective role against atherosclerosis. Aside from their ability to bind to antigens, antibodies can influence inflammatory responses by interacting with various Fcγ receptors on the surface of antigen presenting cells. Although studies in mice have determined that stimulatory Fcγ receptors contribute to atherosclerosis, the role of the inhibitory Fcγ receptor IIb (FcγRIIb) has only recently been investigated., Methods and Results: To determine the importance of FcγRIIb in modulating the adaptive immune response to hyperlipidemia, we generated FcγRIIb-deficient mice on the apoE-deficient background (apoE/FcγRIIb(-/-)). We report that male apoE/FcγRIIb(-/-) mice develop exacerbated atherosclerosis that is independent of lipid levels, and is characterized by increased antibody titers to modified LDL and pro-inflammatory cytokines in the aorta., Conclusions: These findings suggest that antibodies against atherosclerosis-associated antigens partially protect against atherosclerosis in male apoE(-/-) mice by conveying inhibitory signals through the FcγRIIb that downregulate pro-inflammatory signaling via other immune receptors. These data are the first to describe a significant in vivo effect for FcγRIIb in modulating the cytokine response in the aorta in male apoE(-/-) mice., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The immune system is complex, with multiple layers of regulation that serve to prevent the production of self-antigens. One layer of regulation involves regulatory T cells (Tregs) that play an essential role in maintaining peripheral self-tolerance. Patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis have decreased levels of HDL, suggesting that apoA-I concentrations may be important in preventing autoimmunity and the loss of self-tolerance. In published studies, hypercholesterolemic mice lacking HDL apoA-I or LDLr(-/-), apoA-I(-/-) (DKO), exhibit characteristics of autoimmunity in response to an atherogenic diet. This phenotype is characterized by enlarged cholesterol-enriched lymph nodes (LNs), as well as increased T cell activation, proliferation, and the production of autoantibodies in plasma. In this study, we investigated whether treatment of mice with lipid-free apoA-I could attenuate the autoimmune phenotype. To do this, DKO mice were first fed an atherogenic diet containing 0.1% cholesterol, 10% fat for 6 weeks, after which treatment with apoA-I was begun. Subcutaneous injections of 500 μg of lipid-free apoA-I was administered every 48 h during the treatment phase. These and control mice were maintained for an additional 6 weeks on the diet. At the end of the 12-week study, DKO mice showed decreased numbers of LN immune cells, whereas Tregs were proportionately increased. Accompanying this increase in Tregs was a decrease in the percentage of effector/effector memory T cells. Furthermore, lipid accumulation in LN and skin was reduced. These results suggest that treatment with apoA-I reduces inflammation in DKO mice by augmenting the effectiveness of the LN Treg response.

Objective: In this study, we investigated whether dyslipidemia-associated perturbed invariant natural killer T (iNKT) cell function is due to intrinsic changes in iNKT cells or defects in the ability of antigen-presenting cells to activate iNKT cells., Methods and Results: We compared iNKT cell expansion and cytokine production in C57BL/6J (B6) and apolipoprotein E-deficient (apoE(-/-)) mice. In response to in vivo stimulation with alpha-galactosylceramide, a prototypic iNKT cell glycolipid antigen, apoE(-/-) mice showed significantly decreased splenic iNKT cell expansion at 3 days after injection, a profile associated with iNKT cell anergy due to chronic stimulation. This decrease in expansion and cytokine production was accompanied by a 2-fold increase in percentage of iNKT cells expressing the inhibitory marker programmed death-1 in apoE(-/-) mice compared with controls. However, in vivo and in vitro blockade of programmed death-1 using monoclonal antibody was not able to restore functions of iNKT cells from apoE(-/-) mice to B6 levels. iNKT cells from apoE(-/-) mice also had increased intracellular T cell receptor and Ly49 expression, a phenotype associated with previous activation. Changes in iNKT cell functions were cell autonomous, because dendritic cells from apoE(-/-) mice were able to activate B6 iNKT cells, but iNKT cells from apoE(-/-) mice were not able to respond to B6 dendritic cells., Conclusions: These data suggest that chronic dyslipidemia induces an iNKT cell phenotype that is unresponsive to further simulation by exogenous glycolipid and that sustained unresponsiveness is iNKT cell intrinsic.

Atherosclerosis is a chronic inflammatory disease characterized by dyslipidemia and accumulation of lipids in the arterial intima, with activation of both innate and adaptive immunity. Reciprocally, dyslipidemia associated with atherosclerosis can perturb normal immune function. Natural killer T (NKT) cells are a specialized group of immune cells that share characteristics with both conventional T cells and natural killer cells. However, unlike these cells, NKT cells recognize glycolipid antigens and produce both pro- and anti-inflammatory cytokines upon activation. Because of these unique characteristics, NKT cells have recently been ascribed a role in the regulation of immunity and inflammation, including cardiovascular disease. In addition, NKT cells represent a bridge between dyslipidemia and immune regulation. This review summarizes the current knowledge of NKT cells and discusses the interplay between dyslipidemia and the normal functions of NKT cells and how this might modulate inflammation and atherosclerosis., ((c) 2010 S. Karger AG, Basel.)

Objective: The purpose of this study was to determine the effects of an atherogenic diet on immune function in LDLr(-/-), ApoA-I(-/-) mice., Methods and Results: When LDLr(-/-), ApoA-I(-/-) (DKO), and LDLr(-/-) (SKO) mice were fed an atherogenic diet, DKO had larger peripheral lymph nodes (LNs) and spleens compared to SKO mice. LNs were enriched in cholesterol and contain expanded populations of T, B, dendritic cells, and macrophages. Expansion of all classes of LN cells was accompanied by a approximately 1.5-fold increase in T cell proliferation and activation. Plasma antibodies to dsDNA, beta2-glycoprotein I, and oxidized LDL were increased in DKO, similar to levels in diet-fed Fas(lpr/lpr) mice, suggesting the development of an autoimmune phenotype. Both LN enlargement and cellular cholesterol expansion were "prevented" when diet-fed DKO mice were treated with helper dependent adenovirus expressing apoA-I. Independent of the amount of dietary cholesterol, DKO mice consistently showed lower plasma cholesterol than SKO mice, yet greater aortic cholesterol deposition and inflammation., Conclusions: ApoA-I prevented cholesterol-associated lymphocyte activation and proliferation in peripheral LN of diet-fed DKO mice. A approximately 1.5-fold increase in T cell activation and proliferation was associated with a approximately 3-fold increase in concentrations of circulating autoantibodies and approximately 2-fold increase in the severity of atherosclerosis suggesting a common link between plasma apoA-I, inflammation, and atherosclerosis.

Macrophage low-density lipoprotein receptor-related protein (LRP) mediates internalization of remnant lipoproteins, and it is generally thought that blocking lipoprotein internalization will reduce foam cell formation and atherogenesis. Therefore, our study examined the function of macrophage LRP in atherogenesis. We generated transgenic mice that specifically lack macrophage LRP through Cre/lox recombination. Transplantation of macrophage LRP(-/-) bone marrow into lethally irradiated female LDLR(-/-) recipient mice resulted in a 40% increase in atherosclerosis. The difference in atherosclerosis was not caused by altered serum lipoprotein levels. Furthermore, deletion of macrophage LRP decreased uptake of (125)I-very-low-density lipoprotein compared with wild-type cells in vitro. The increase in atherosclerosis was accompanied by increases in monocyte chemoattractant protein type-1, tumor necrosis factor-alpha, and proximal aorta macrophage cellularity. We also found that deletion of macrophage LRP increases matrix metalloproteinase-9. This increase in matrix metalloproteinase-9 was associated with a higher frequency of breaks in the elastic lamina. Contrary to what was found with other lipoprotein receptors, deletion of LRP increases atherogenesis in hypercholesterolemic mice. Our data support the hypothesis that macrophage LRP modulates atherogenesis through regulation of inflammatory responses.

CD1 molecules are a family of major histocompatibility complex (MHC)-related glycoproteins that present lipid and glycolipid antigens to T cells. Interestingly, it has been demonstrated that CD1d-restricted T cells have a pathogenic role in atherosclerosis. Recent studies suggest an association between the cellular machinery that loads CD1 molecules with glycolipids and several key proteins in lipid metabolism. These proteins include the sphingolipid activator proteins (SAPs), microsomal triglyceride transfer protein (MTP) and apolipoprotein E (apoE). MTP and SAPs seem to be crucial for loading CD1d with lipids in the endoplasmic reticulum and endosomal compartments, respectively, whereas apoE facilitates efficient uptake and delivery of exogenous lipid antigens to CD1d in endosomal compartments. These studies reveal new and unexpected relationships between lipid metabolism and antigen presentation by CD1 molecules. Targeting this pathway of immune activation might have therapeutic potential for the treatment of chronic inflammatory diseases.

Patients with systemic lupus erythematosus (SLE) have accelerated atherosclerosis. The underlying mechanisms are poorly understood, and investigations have been hampered by the absence of animal models that reflect the human condition of generalized atherosclerosis and lupus. We addressed this problem by transferring lupus susceptibility to low-density lipoprotein (LDL) receptor-deficient (LDLr-/-) mice, an established model of atherosclerosis, creating radiation chimeras with NZM2410-derived, lupus-susceptible, B6.Sle1.2.3 congenic or C57BL/6 control donors (LDLr.Sle and LDLr.B6, respectively). LDLr.Sle mice developed a lupus-like disease characterized by production of double-stranded DNA autoantibodies and renal disease. When fed a Western-type diet, LDLr.Sle chimeras had increased mortality and atherosclerotic lesions. The plaques of LDLr.Sle mice were highly inflammatory and contained more CD3+ T cells than controls. LDLr.Sle mice also had increased activation of CD4+ T and B cells and significantly higher antibody to oxidized LDL and cardiolipin. Collectively, these studies demonstrate that the lupus-susceptible immune system enhances atherogenesis and modulates plaque composition.

Systemic lupus erythematosus (SLE) is increasingly recognized as a risk factor for the development of premature atherosclerosis. The inflammatory process in both of these diseases is controlled by a variety of cell types of the innate and adaptive immune systems. Recent studies from several groups, including ours, have revealed a critical role of a unique subset of lymphocytes, termed invariant natural killer T (iNKT) cells, in the development of lupus-like autoimmunity and atherosclerosis in animal models. iNKT cells appear to play complex and divergent roles in the development of SLE and atherosclerosis. Our findings suggest that alterations in iNKT cell functions during the development of SLE may be related to the increased risk of SLE patients to develop atherosclerosis and coronary heart disease. We found that iNKT cell activation with the sponge-derived glycolipid alpha- galactosylceramide generally protects against the development of lupus-like autoimmunity in mice, whereas it exacerbates atherosclerosis. Therefore, while our studies have identified iNKT cells as potential therapeutic targets for SLE, further studies are necessary to design drugs that will avoid the underlying harmful effects of iNKT cell activation on the development of atherosclerosis.

Cyclooxygenase (COX) 2 is expressed in atherosclerotic lesions. We have previously reported that selective inhibition of COX-2 reduces early atherosclerosis in LDLR deficient mice. To examine the role of COX-2 in atherosclerosis in other mouse models, we studied the effects of selective COX-2 inhibition (by rofecoxib and NS-398) and nonselective COX inhibition (by indomethacin) on early atherosclerotic lesion formation in apolipoprotein E-deficient (apoE(-/-)) mice. Selective COX-2 and nonselective COX inhibition reduced atherosclerosis in female apoE(-/-) mice by 35-38% and 38-51% in the proximal and en face aortas, respectively. Next we investigated the role of macrophage COX-2 by transplanting COX-2(-/-) fetal liver cells into C57BL/6 mice and challenging the mice with an atherogenic diet. Genetic deletion of COX-2 from hematopoietic cells reduced atherosclerosis by 51%. In addition, LPS activated COX-2(-/-) macrophages had decreased expression of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNFalpha). The results demonstrate that selective inhibition of COX-2 and elimination of COX-2 from macrophages significantly reduces early atherosclerotic lesion formation in apoE-deficient and C57BL/6 mice. These results are compatible with COX-2 expression by macrophages having a proatherogenic role, and support the potential of anti-inflammatory therapeutic approaches for atherosclerosis.

Background: Macrophage acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) and apolipoprotein E (apoE) have been implicated in regulating cellular cholesterol homeostasis and therefore play critical roles in foam cell formation. Deletion of either ACAT1 or apoE results in increased atherosclerosis in hyperlipidemic mice, possibly as a consequence of altered cholesterol processing. We have studied the effect of macrophage ACAT1 deletion on atherogenesis in apoE-deficient (apoE-/-) mice with or without the restoration of macrophage apoE., Methods and Results: We used bone marrow transplantation to generate apoE-/- mice with macrophages of 4 genotypes: apoE+/+/ACAT1+/+ (wild type), apoE+/+/ACAT1-/- (ACAT-/-), apoE-/-/ACAT1+/+ (apoE-/-), and apoE-/-/ACAT1-/- (2KO). When macrophage apoE was present, plasma cholesterol levels normalized, and ACAT1 deficiency did not have significant effects on atherogenesis. However, when macrophage apoE was absent, ACAT1 deficiency increased atherosclerosis and apoptosis in the proximal aorta. Cholesterol efflux to apoA-I was significantly reduced (30% to 40%; P<0.001) in ACAT1-/- peritoneal macrophages compared with ACAT1+/+ controls regardless of apoE expression. 2KO macrophages had a 3- to 4-fold increase in ABCA1 message levels but decreased ABCA1 protein levels relative to ACAT1+/+ macrophages. Microarray analyses of ACAT1-/- macrophages showed increases in proinflammatory and procollagen genes and decreases in genes regulating membrane integrity, protein biosynthesis, and apoptosis., Conclusions: Deficiency of macrophage ACAT1 accelerates atherosclerosis in hypercholesterolemic apoE-/- mice but has no effect when the hypercholesterolemia is corrected by macrophage apoE expression. However, ACAT1 deletion impairs ABCA1-mediated cholesterol efflux in macrophages regardless of apoE expression. Changes in membrane stability, susceptibility to apoptosis, and inflammatory response may also be important in this process.

Background: Atherosclerosis is a disease marked by lipid accumulation and inflammation. Recently, atherosclerosis has gained recognition as an autoimmune-type syndrome characterized by increased activation of the innate and acquired immune systems. Natural killer T (NKT) cells have characteristics of both conventional T cells and NK cells and recognize glycolipid antigens presented in association with CD1d molecules on antigen-presenting cells. The capacity of NKT cells to respond to lipid antigens and modulate innate and acquired immunity suggests that they may play a role in atherogenesis., Methods and Results: We examined the role of NKT cells in atherogenesis and how the atherosclerotic environment affects the NKT cell population itself. The data show that CD1d-deficiency in male apolipoprotein E-deficient (apoE(0)) mice results in reduction in atherosclerosis, and treatment of apoE(0) mice with alpha-galactosylceramide, a potent and specific NKT cell activator, results in a 2-fold increase in atherosclerosis. Interestingly, we demonstrate that alpha-galactosylceramide-induced interferon-gamma responses and numbers of NKT cells in apoE(0) mice show age-dependent qualitative and quantitative differences as compared with age-matched wild-type mice., Conclusions: Collectively, these findings reveal that hyperlipidemia and atherosclerosis have significant effects on NKT cell responses and that these cells are proatherogenic.

The antiatherogenic effect of high-density lipoprotein (HDL) and its major protein component apolipoprotein A-I (apoA-I) has been largely attributed to their key roles in reverse cholesterol transport (RCT) and cellular cholesterol efflux. Substantial evidence shows that overexpression of human apoA-I reduces atherosclerosis in animal models. However, it is uncertain whether this protection is due to an increase in plasma HDL level or to a local effect in the artery wall. To test the hypothesis that expression of human apoA-I in macrophages can promote RCT in the artery wall, we used a retroviral construct expressing human apoA-I cDNA (MFG-HAI) to transduce ApoE(-/-) bone marrow cells and then transplanted these cells into ApoE(-/-) mice with preexisting atherosclerosis. ApoE(-/-) mice reconstituted with MFG-HAI marrow had a significant reduction (30%) in atherosclerotic lesions in the proximal aorta compared to control mice that received marrow expressing MFG parental virus. Peritoneal macrophages isolated from MFG-HAI mice showed a four- to fivefold increase in mRNA expression levels of both ATP-binding cassette (ABC) A1 and ABCG1 compared to controls. Our data demonstrate that gene transfer-mediated expression of human apoA-I in macrophages can compensate in part for apoE deficiency and delay the progression of atherosclerotic lesions by stimulating ABC-dependent cholesterol efflux and RCT.

Objective: Atherosclerosis is an inflammatory disease characterized by innate and adaptive immune responses. We investigated the role of B cells and antibodies in the development of atherosclerosis in low density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice., Methods and Results: Using wild-type and B cell-deficient mice as bone marrow donors, we were able to generate LDLR(-/-) mice that possessed <1.0% of their normal B cell population. B cell-deficient LDLR(-/-) mice on a Western diet showed marked decreases in total serum antibody and anti-oxidized LDL antibody. B cell deficiency was associated with a 30% to 40% increase in the lesion area in the proximal and distal aortas. Real-time reverse transcription-polymerase chain reaction and enzyme-linked immunospot analyses showed a decrease in proatherogenic (interferon-gamma) and antiatherogenic (interleukin-10 and transforming growth factor-beta) cytokine mRNA and a decrease in interleukin-4- and interferon-gamma-producing cells. Additionally, we observed a decrease in splenocyte proliferation to oxidized LDL in the B cell-deficient LDLR(-/-) mice, suggesting that B lymphocytes may play a role in the presentation of lipid antigen., Conclusions: Collectively, these data demonstrate that B cells and/or antibodies are protective against atherosclerosis and that this protection may be conferred by B cell-mediated immune regulation.

We have previously reported that the introduction of macrophage apoE into mice lacking both apoE and the LDL receptor (apoE(-)(/-)/LDLR(-)(/-)) through bone marrow transplantation (apoE(+)(/+)/LDLR(-)(/-)-->apoE(-)(/-)/LDLR(-)(/-)) produces progressive accumulation of apoE in plasma without affecting lipid levels. This model provides a tool to study the effects of physiologically regulated amounts of macrophage apoE on atherogenesis in hyperlipidemic animals. Ten-week-old male apoE(-)(/-)/LDLR(-)(/-) mice were transplanted with either apoE(+)(/+)/LDLR(-)(/-) (n = 11) or apoE(-)(/-)/LDLR(-)(/-) (n = 14) marrow. Although there were no differences between the two groups in lipid levels at baseline or at 5 and 9 weeks after transplantation, apoE levels in the apoE(+)(/+)LDLR(-)(/-)-->apoE(-)(/-)/LDLR(-)(/-) mice increased to 4 times the apoE levels of normal mice. This resulted in a 60% decrease in aortic atherosclerosis in the apoE(+)(/+)/LDLR(-)(/-)-->apoE(-)(/-)/LDLR(-)(/-) compared with the apoE(-)(/-)/LDLR(-)(/-)-->apoE(-)(/-)/LDLR(-)(/-) controls, (15957 +/- 1907 vs. 40115 +/- 8302 micro m(2) +/- SEM, respectively). In a separate experiment, apoE(+)(/+)/LDLR(-)(/-) mice were transplanted with either apoE(+)(/+)/LDLR(-)(/-) or apoE(-)(/-)/LDLR(-)(/-) marrow and placed on a high-fat diet for 8 weeks. In the absence of macrophage apoE, lesion area was increased by 75% in the aortic sinus and by 56% in the distal aorta. These data show that physiologic levels of macrophage apoE in the vessel wall are anti-atherogenic in conditions of severe hyperlipidemia and can affect later stages of plaque development.