Harnessing New Players in Atherosclerosis to Treat Heart Disease

Abstracts

A Transcriptional Network Underlies the Identity and Diversity of Tissue Macrophages
Emmanuel L. Gautier, Washington University School of Medicine, St. Louis

Macrophages play a critical role in the development and the progression of cardiometabolic diseases. We recently assessed gene expression in tissue macrophages, which do not derive from monocytes, extracted from various mouse organs and found that the diversity in gene expression among different populations of macrophages was considerable. Only a few hundred mRNA transcripts were selectively expressed by macrophages rather than dendritic cells, and many of these were not present in all macrophages. Nonetheless, well-characterized surface markers, including MerTK and FcγR1 (CD64), along with a cluster of previously unidentified transcripts, were distinctly and universally associated with mature tissue macrophages. We further demonstrated how these transcripts and the proteins they encode facilitated distinguishing macrophages from dendritic cells, and showed that they were turned on during monocyte to inflammatory macrophage differentiation. Furthermore, in support of the high diversity observed among tissue resident macrophages, the mRNAs encoding several transcription factors were associated with single macrophage populations and we provided evidence that Pparg and Gata6 specifically controls the homeostasis of resident lung and peritoneal macrophages, respectively.
 

Monocyte Behavior, Imaging and Targeting in Atherosclerosis
Matthias Nahrendorf, MD, PhD, Harvard Medical School

Monocytes and macrophages are innate immune cells that reside and accumulate in atherosclerotic lesions but also in the healthy and injured heart. The cells and their subsets pursue distinct functions in steady state and disease, and their tenure may range between hours to months. Some subsets are highly inflammatory, while others support tissue repair. The talk discusses current concepts of cell supply by the hematopoietic system, lineage relationships and systems' cross talk, highlights open questions, and describes imaging tools for studying monocyte and macrophage subsets.
 

Trapped in the Plaque: Mechanisms of Macrophage Retention in Atherosclerosis
Kathryn J. Moore, PhD, New York University Medical Center

Atherosclerotic inflammation is fueled by the accumulation of cholesterol-laden myeloid-derived cells in the artery wall, however the mechanisms by which these cells become trapped are poorly understood. We postulated that negative guidance cues quench the emigration of macrophages and other immune cells from the artery wall. This led to our discovery that netrin-1 and semaphorin 3E, molecules originally characterized as neuronal guidance cues, were expressed by macrophages in human and mouse atherosclerotic plaques, and blocked the emigration of macrophages from plaques. Using a variety of bone marrow and aortic transplant models, we have shown that these retention factors are dynamically regulated in progressing and regressing plaques, and that their targeting decreases macrophage accumulation and atherosclerosis. Collectively, these studies established causative roles for netrin-1 and semaphorin 3E in promoting chronic inflammation in the plaque, and identified these guidance cues as novel therapeutic targets in atherosclerosis.
 

Diabetes-accelerated Atherosclerosis: Mechanistic Insight Based on Mouse Models
Karin Bornfeldt, PhD, University of Washington School of Medicine

Diabetes accelerates the formation and progression of atherosclerotic lesions, which likely explains the increased risk of cardiovascular disease in diabetic humans. The accelerated atherosclerosis is driven, at least in part, by the altered function and properties of myeloid cells; cells that display and contribute to an increased inflammatory vascular state in atherogenesis. Changes in endothelial cells also likely contribute to diabetes-accelerated atherosclerosis. In mouse models of diabetes, atherosclerosis is accelerated due to increased accumulation of macrophages in the arterial wall. Based on our recent studies, the enzyme acyl-CoA synthetase 1 (ACSL1), which converts long-chain fatty acids into their acyl-CoA derivatives, has emerged as causal to the enhanced atherosclerosis associated with diabetes. ACSL1 is expressed at higher levels in myeloid cells from diabetic mice and in a small group of human subjects with type 1 diabetes, as compared to controls, and is induced by inflammatory mediators in these cells. Furthermore, deletion of ACSL1 in myeloid cells results in complete protection of these cells from the inflammatory activation associated with diabetes and from early diabetes-accelerated atherosclerosis. Interestingly, the protective effect of myeloid ACSL1-deficiency is obvious in diabetic mice, but not in non-diabetic mice, indicating that ACSL1-deficiency targets a pathway that is selectively activated by diabetes. In endothelial cells, ACSL1 is induced by TNF-α, and contributes to TNF-α-induced secretion of the important chemokine CCL2, suggesting that endothelial ACSL1 might also promote atherosclerosis. Without doubt, the most important question is whether ACSL1 and other factors identified in mouse models play equally important roles in diabetes-accelerated atherosclerosis in humans.
 

Antigen Presentation in the Atherosclerotic Mouse Aorta
Klaus Ley, MD, La Jolla Institute for Allergy and Immunology

We recently reported that dendritic cells in the atherosclerotic mouse aorta present antigen to antigen-experienced CD4 T cells isolated from atherosclerotic Apoe^−/− mice, but not from wild-type mice (Koltsova et al, JCI 2012, 122: 3114). These interactions were productive as evidenced by MHC-II-restricted live cell interactions in the aorta and production of IFN-γ, TNF and IL-17. These findings show that relevant antigen-experienced T cells exist in atherosclerotic, but not in control mice. No exogenous antigen was added, showing that the relevant (unknown) antigen was present in atherosclerotic mouse aortas. Based on these findings, we screened peptides from a candidate autoantigen, ApoB100, the core protein of low density lipoprotein (LDL), for binding to MHC-II. We found two peptides that bound to I-A^b, the MHC-II allele expressed in C57BL/6 and Apoe^−/− mice, with high affinity (Kd<10 nM). Immunizing mice with these peptides in a tolerogenic scheme (1× complete Freund's adjuvant, CFA, 4× incomplete Freund's adjuvant, IFA) was atheroprotective as evidenced by reduced lesion size (45% reduction by en face staining) and smaller aortic root lesions (40% reduction by histology). These data suggest that it is possible to elicit or amplify an atheroprotective autoimmune response in mice, possibly by a tolerogenic mechanism. The vaccinated mice produced more IL-10 in their aortas, but the numbers of regulatory T cells (Tregs) were unchanged. The vaccinated mice also produced IgG specific for the ApoB100 peptide they were immunized with. These antibodies cross-reacted with malondialdehyde (MDA)-modified LDL. Experiments are under way to identify the source of IL-10 and the relative role of Tregs and autoantibodies. Taken together, our data show that antigen presentation in atherosclerotic arteries is relevant. The atheroprotective peptide vaccines found in mice suggest that such vaccines can, in principle, be constructed for humans.
 

Regulation of Pro-atherogenic T Cell Responses
Andrew H. Lichtman, MD, PhD, Brigham and Women's Hospital, Boston, MA

T lymphocytes contribute to the development of atherosclerosis and to the destabilization of lesions that preceded acute coronary events. Most T cells within lesions are inflammatory helper T cells which produce interferon γ and/or IL-17. Experimental data from out laboratory has demonstrated that pro-atherogenic T cell responses can be diminished by changes in T cell subset differentiation toward Th1 or Th17 phenotypes, and by interfering with B7-CD28 family costimulatory pathways, Conversely, we have shown that blocking the PD-1/PD-L1 pathway, which is an emerging therapeutic target for cancer and chronic viral infections, signifincanlty enhances atherosclerotic lesion development and inflammation. We have also found that regulatory T cell suppression of pro-atherogenic T cell responses fails early during lesion development, and this correlates with loss of Treg in arterial walls due impaired migration, enhanced death, and phenotypic plasticity. Our work and the findings of other investigators support the rationale of treating atherosclerotic disease by blocking activation of atherosclerosis-antigen specific effector T cells and by sustaining Treg responses. The tools to accomplish these goals may include T cell costimulatory blockers, T cell inhibitory receptor agonists, and cytokine targeted drugs that will enhance Treg stability and diminish effector T cells mediated inflammation. The induction of long lasting T cell tolerance only to the disease-relevant antigens is also a goal of ongoing research, which would avoid the dangers of global immunosuppression.
 

Cholesterol Efflux Pathways Suppress Innate Immune Responses, Auto-immunity and Atherosclerosis
Alan Tall, Columbia University, New York

Leukocytosis is a risk factor for athero-thrombotic disease in humans, and develops in animal models of atherosclerosis in response to feeding high fat, high cholesterol diets. The ATP binding cassette transporters ABCA1 and ABCG1 promote cholesterol efflux to apoA-1 and HDL, respectively and are targets of LXR transcription factors. Mice lacking ABCA1/G1 develop a dramatic myeloproliferative phenotype with monocytosis and neutrophilia, associated with expansion and proliferation of hematopoietic stem and myeloid progenitor populations (HSPCs). The transporters are highly expressed in HSPCs where they act to control proliferative responses to growth factors (IL-3, GM-CSF) by regulating plasma membrane lipid rafts and cell surface expression of the common beta subunit of the IL-3/GM-CSF receptor. We have recently developed Abca1fl/flAbcg1fl/fl mice in order to assess the functions of these transporters in different populations of myeloid cells. Macrophage knockout of ABCA1/G1 (LysM-Cre) confirms a role of macrophage cholesterol efflux in suppressing inflammatory responses and atherosclerosis. Dendritic cell knockout of ABCA1/G1 (CD11c-Cre) produces a distinctive phenotype with T-cell activation and features of autoimmunity. ABCG4 is closely related to ABCG1 but is expressed primarily in the megakaryocyte progenitor (MkP) population of the bone marrow. ABCG4 deficient mice have MkP proliferation and expansion, thrombocytosis, increased platelet/leukocyte aggregates and accelerated atherosclerosis. ABCG4 promotes cholesterol efflux onto HDL, and thereby reduces the cell surface expression of the thrombopoietin (TPO) receptor. Overall results suggest that ATP binding cassette transporters promote cholesterol efflux, decrease membrane lipid raft formation and enhance the feedback down-regulation of growth factor receptors in response to growth factor binding, with anti-proliferative responses that may be beneficial in atherosclerosis and myeloproliferative neoplasms.
 

T Cell Fate in Atherosclerosis
Elena Galkina, PhD, Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School

Innate and adaptive immune responses both participate in atherogenesis. IL-17A⁺ T cell subsets have been shown to be protective in innate host defences against extracellular pathogens, but are highly pathogenic in several autoimmune diseases. The role of Th1, Th2 and T-regulatory subsets in atherogenesis is relatively well established; however, the involvement of Th17 cells remains unclear. While research from a number of laboratories suggested a pro-inflammatory role of IL-17A in atherogenesis, several laboratories found an opposite atheroprotective or no effects of IL-17A on plaque burden. Our laboratory generated IL-17A-deficient and IL-17 receptor A-deficient Apoe^−/− (Il17a^−/−Apoe^−/−and Il17ra^−/−Apoe^−/−) mice. Western diet fed Il17a^−/−Apoe^−/− and Il17ra^−/−Apoe^−/− mice had smaller atherosclerotic plaques in the aortic arch and aortic roots, but showed little difference in plaque burden in the thoracoabdominal aorta compared with Apoe^−/−controls. Our experiments demonstrated that the IL-17A/IL-17RA axis increases aortic arch inflammation during atherogenesis through the induction of aortic chemokines, and the acceleration of neutrophil and monocyte recruitment to this site underlining a pro-atherogenic role of IL-17A in this model. T cell subsets play diverse roles in inflammation and recent evidence suggests that T helper cells display limited plasticity under specific stimuli. The existence of a population of Foxp3⁺ T reg cells that co-expresses IL-17A or IFNγ has been demonstrated in several mouse models of inflammation. Our preliminary data demonstrate an elevation of Foxp3+IFNγ+ T reg cells in the aorta, and secondary lymphoid tissues of Apoe^−/− mice in comparison to age-matched C57Bl6 mice. Interestingly, all Foxp3+IFNγ+ T cells express both Th1 and Treg markers, including Tbet and GITR, suggesting that Foxp3+IFNγ+ T cells have attributes of both T regulatory and Th1 cells. Currently, we are exploring a potential role of T cell plasticity and mechanisms of the generation of Foxp3⁺IFNγ⁺ T cells in atherosclerosis.
 
Supported by NHLBI RO1 HL107522 (E.G.) and AHA Pre-doctoral Fellowship 11PRE7520041 (M.B.).