Rationale: Aggravated atherosclerosis in B lymphocyte-deficient chimeric mice and reduced atherosclerosis after transfer of unfractionated spleen B lymphocytes into splenectomized mice have led to the widely held notion that B lymphocytes are atheroprotective. However, B lymphocytes can be pathogenic, because their depletion by anti-CD20 antibody ameliorated atherosclerosis, and transfer of B2 lymphocytes aggravated atherosclerosis. These observations raise the question of the identity of the atheroprotective B-lymphocyte population.Objective: The purpose of the study was to identify an atheroprotective B-lymphocyte subset and mechanisms by which they confer atheroprotection. Methods and Results:
Atherosclerosis is a chronic inflammatory arterial disease characterized by focal accumulation of lipid and inflammatory cells. It is the number one cause of deaths in the Western world because of its complications of heart attacks and strokes. Statins are effective in only approximately one third of patients, underscoring the urgent need for additional therapies. B cells that accumulate in atherosclerotic lesions and the aortic adventitia of humans and mice are considered to protect against atherosclerosis development. Unexpectedly, we found that selective B cell depletion in apolipoprotein E-deficient (ApoE−/−) mice using a well-characterized mAb to mouse CD20 reduced atherosclerosis development and progression without affecting the hyperlipidemia imposed by a high-fat diet. Adoptive transfer of 5 × 106 or 5 × 107 conventional B2 B cells but not 5 × 106 B1 B cells to a lymphocyte-deficient ApoE−/− Rag-2−/− common cytokine receptor γ-chain–deficient mouse that was fed a high-fat diet augmented atherosclerosis by 72%. Transfer of 5 × 106 B2 B cells to an ApoE−/− mouse deficient only in B cells aggravated atherosclerosis by >300%. Our findings provide compelling evidence for the hitherto unrecognized proatherogenic role of conventional B2 cells. The data indicate that B2 cells can potently promote atherosclerosis development entirely on their own in the total absence of all other lymphocyte populations. Additionally, these B2 cells can also significantly augment atherosclerosis development in the presence of T cells and all other lymphocyte populations. Our findings raise the prospect of B cell depletion as a therapeutic approach to inhibit atherosclerosis development and progression in humans.
The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single cell RNA-Seq (scRNA-Seq) and 2 mass cytometry (CyTOF) studies. In a comprehensive meta-analysis, we demonstrate four macrophage subsets: resident, inflammatory, IFNIC and Trem2 foamy macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2 (ILC2) and CD8 T cells form prominent and separate populations. The CD4 T cells show a large population of Th17-like cells, which also contain γδ T cells. A small number of Tregs and Th1 cells is also identified. The present meta-analysis overcomes limitations of individual studies that, because of their experimental approach, overor under-represent certain cell populations. CyTOF identifies an even larger number of clusters, suggesting that surface markers provide more discriminatory information than transcriptomes. The present analysis provides evidence to further resolve some long-standing controversies in the field. First, Trem2 + foamy macrophages are not pro-inflammatory, but interferon-inducible cell (IFNIC) and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, Pf4, which had been considered specific for platelets and megakaryocytes, is also prominently expressed in resident vascular macrophages. Finally, the discovery of a prominent ILC2 cluster links the scRNA-Seq work to recent flow cytometry data suggesting a strong atheroprotective role of ILC2 cells. This resolves apparent discrepancies regarding the role of Th2 cells in atherosclerosis based on studies that pre-dated the discovery of ILC2 cells.
Background— Heart attacks and strokes, leading causes of deaths globally, arise from thrombotic occlusion of ruptured vulnerable atherosclerotic plaques characterized by abundant apoptosis, large necrotic cores derived from inefficient apoptotic cell clearance, thin fibrous caps, and focal inflammation. The genesis of apoptosis and necrotic cores in these vulnerable atherosclerotic plaques remains unknown. Cytotoxic CD8 + T lymphocytes represent up to 50% of leukocytes in advanced human plaques and dominate early immune responses in mouse lesions, yet their role in atherosclerosis also remains unresolved. Methods and Results— CD8 + T-lymphocyte depletion by CD8α or CD8β monoclonal antibody in apolipoprotein E-deficient mice fed a high-fat diet ameliorated atherosclerosis by reducing lipid and macrophage accumulation, apoptosis, necrotic cores, and monocyte chemoattractant protein 1, interleukin 1β, interferon γ, and vascular cell adhesion molecule 1. Transfer of CD8 + T cells into lymphocyte-deficient, apolipoprotein E-deficient mice partially reconstituted CD8 + T cells in lymphoid compartments and was associated with CD8 + T-cell infiltration in lesions, increased lipid and macrophage accumulation, apoptotic cells, necrotic cores, and interleukin 1β in atherosclerotic lesions. Transfer of CD8 + T cells deficient in perforin, granzyme B, or tumor necrosis factor α but not interferon γ failed to increase atherosclerotic lesions despite partial reconstitution in the lymphoid system and the presence in atherosclerotic lesions. Macrophages, smooth muscle cells, and endothelial cells were identified as apoptotic targets. Conclusions— We conclude that CD8 + T lymphocytes promote the development of vulnerable atherosclerotic plaques by perforin- and granzyme B–mediated apoptosis of macrophages, smooth muscle cells, and endothelial cells that, in turn, leads to necrotic core formation and further augments inflammation by tumor necrosis factor α secretion.
Clinical hypertension is associated with raised serum IgG antibodies. However, whether antibodies are causative agents in hypertension remains unknown. We investigated whether hypertension in mice is associated with B-cell activation and IgG production and moreover whether B-cell/IgG deficiency affords protection against hypertension and vascular remodeling. Angiotensin II (Ang II) infusion (0.7 mg/kg per day; 28 days) was associated with (1) a 25% increase in the proportion of splenic B cells expressing the activation marker CD86, (2) an 80% increase in splenic plasma cell numbers, (3) a 500% increase in circulating IgG, and (4) marked IgG accumulation in the aortic adventitia. In B-cell-activating factor receptor-deficient (BAFF-R(-/-)) mice, which lack mature B cells, there was no evidence of Ang II-induced increases in serum IgG. Furthermore, the hypertensive response to Ang II was attenuated in BAFF-R(-/-) (Δ30±4 mm Hg) relative to wild-type (Δ41±5 mm Hg) mice, and this response was rescued by B-cell transfer. BAFF-R(-/-) mice displayed reduced IgG accumulation in the aorta, which was associated with 80% fewer aortic macrophages and a 70% reduction in transforming growth factor-β expression. BAFF-R(-/-) mice were also protected from Ang II-induced collagen deposition and aortic stiffening (assessed by pulse wave velocity analysis). Finally, like BAFF-R deficiency, pharmacological depletion of B cells with an anti-CD20 antibody attenuated Ang II-induced hypertension by ≈35%. Hence, these studies demonstrate that B cells/IgGs are crucial for the development of Ang II-induced hypertension and vessel remodeling in mice. Thus, B-cell-targeted therapies-currently used for autoimmune diseases-may hold promise as future treatments for hypertension.
We have recently identified conventional B2 cells as atherogenic and B1a cells as atheroprotective in hypercholesterolemic ApoE−/− mice. Here, we examined the development of atherosclerosis in BAFF-R deficient ApoE−/− mice because B2 cells but not B1a cells are selectively depleted in BAFF-R deficient mice. We fed BAFF-R−/− ApoE−/− (BaffR.ApoE DKO) and BAFF-R+/+ApoE−/− (ApoE KO) mice a high fat diet (HFD) for 8-weeks. B2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while B1a cells and non-B lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. B cells were absent in atherosclerotic lesions of BaffR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by B2 cells, despite low but measurable numbers of B2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. BAFF-R deficiency in ApoE−/− mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, IL1-β and proinflammatory chemokine MCP-1 was also reduced. Body weight and plasma cholesterols were unaffected in BaffR.ApoE DKO mice. Our data indicate that B2 cells are important contributors to the development of atherosclerosis and that targeting the BAFF-R to specifically reduce atherogenic B2 cell numbers while preserving atheroprotective B1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation.
Background-CD4ϩCD25ϩFoxp3ϩ regulatory T cells (Tregs) attenuate atherosclerosis, but their therapeutic applicationby adoptive transfer is limited by the need for their expansion in vitro and limited purity. Recently, an interleukin (IL)-2/anti-IL-2 neutralizing monoclonal antibody (IL-2/anti-IL-2 mAb) complex has been shown to expand these Tregs. We examined the capacity of a modified IL-2/anti-IL-2 mAb treatment to expand Tregs and inhibit both the progression and development of developed atherosclerosis. Methods and Results-Six-week old apolipoprotein E-deficient mice fed a high-fat diet for 8 weeks were administered IL-2/anti-IL-2 mAb commencing 2 weeks after starting the diet. Tregs in the spleen, lymph node, and liver were selectively expanded without affecting CD4ϩ, CD8ϩ, or natural killer cells. Tregs were increased in lesions and lesion size reduced. CD4ϩ T-cells, macrophages, mature dendritic cells, proliferating cell nuclear antigenϩ cells, and monocyte chemoattractant protein-1 and vascular cell adhesion molecule-1 were reduced. In anti-CD3-stimulated splenocytes, proliferation and secretion of Th1, Th2, and Th17 (IL-17) cytokines and IL-1␤ were reduced. To determine whether treatment attenuated progression of developed atherosclerosis, 6-week-old apolipoprotein E-deficient mice were fed a high-fat diet for 6 weeks, followed by IL-2/anti-IL-2 mAb treatment for 6 weeks while continuing the high-fat diet. Treatment also increased Tregs without affecting CD4ϩ, CD8ϩ, or natural killer cells, suppressed inflammation, and greatly attenuated progression of atherosclerosis. Conclusions-IL-
Objective-High-mobility group box protein 1 (HMGB1) is a DNA-binding protein and cytokine highly expressed in atherosclerotic lesions, but its pathophysiological role in atherosclerosis is unknown. We investigated its role in the development of atherosclerosis in ApoEϪ/Ϫ mice. Methods and Results-Apolipoprotein E-deficient (ApoEϪ/Ϫ) mice fed a high-fat diet were administered a monoclonal anti-HMGB1 neutralizing antibody, and the effects on lesion size, immune cell accumulation, and proinflammatory mediators were assessed using Oil Red O, immunohistochemistry, and real-time polymerase chain reaction. As with human atherosclerotic lesions, lesions in ApoEϪ/Ϫ mice expressed HMGB1. Treatment with the neutralizing antibody attenuated atherosclerosis by 55%. Macrophage accumulation was reduced by 43%, and vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression was attenuated by 48% and 72%, respectively. CD11cϩ dendritic cells were reduced by 65%, and the mature (CD83ϩ) population was reduced by 60%. Treatment also reduced CD4ϩ cells by nearly 50%. mRNAs in lesions encoding tumor necrosis factor-␣ and interleukin-1␤ tended to be reduced. Mechanistically, HMGB1 stimulated macrophage migration in vitro and in vivo; in vivo, it markedly augmented the accumulation of F4/80ϩGr-1(Ly-6C)ϩ macrophages and also increased F4/80ϩCD11bϩ macrophage numbers. Conclusion-HMGB1
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