Follicular B Cells Promote Atherosclerosis via T Cell-Mediated Differentiation Into Plasma Cells and Secreting Pathogenic Immunoglobulin G.

OBJECTIVE: B cells promote or protect development of atherosclerosis. In this study, we examined the role of MHCII (major histocompatibility II), CD40 (cluster of differentiation 40), and Blimp-1 (B-lymphocyte-induced maturation protein) expression by follicular B (FO B) cells in development of atherosclerosis together with the effects of IgG purified from atherosclerotic mice. APPROACH AND RESULTS: Using mixed chimeric Ldlr-/- mice whose B cells are deficient in MHCII or CD40, we demonstrate that these molecules are critical for the proatherogenic actions of FO B cells. During development of atherosclerosis, these deficiencies affected T-B cell interactions, germinal center B cells, plasma cells, and IgG. As FO B cells differentiating into plasma cells require Blimp-1, we also assessed its role in the development of atherosclerosis. Blimp-1-deficient B cells greatly attenuated atherosclerosis and immunoglobulin-including IgG production, preventing IgG accumulation in atherosclerotic lesions; Blimp-1 deletion also attenuated lesion proinflammatory cytokines, apoptotic cell numbers, and necrotic core. To determine the importance of IgG for atherosclerosis, we purified IgG from atherosclerotic mice. Their transfer but not IgG from nonatherosclerotic mice into Ldlr-/- mice whose B cells are Blimp-1-deficient increased atherosclerosis; transfer was associated with IgG accumulating in atherosclerotic lesions, increased lesion inflammatory cytokines, apoptotic cell numbers, and necrotic core size. CONCLUSIONS: The mechanism by which FO B cells promote atherosclerosis is highly dependent on their expression of MHCII, CD40, and Blimp-1. FO B cell differentiation into IgG-producing plasma cells also is critical for their proatherogenic actions. Targeting B-T cell interactions and pathogenic IgG may provide novel therapeutic strategies to prevent atherosclerosis and its adverse cardiovascular complications.

Cytoplasmic domain of tissue factor promotes liver fibrosis in mice.

AIM: To evaluate the role of tissue factor (TF) and protease activated receptor (PAR)-2 in liver fibrosis. METHODS: Using CCl4 administration for eight weeks, we induced hepatic fibrosis in wild-type C57BL/6 mice and in mice with deletion of the cytoplasmic signalling domain of TF (TF§CT/§CT), deletion of PAR-2 (PAR-2-/-) and combined deletion of TF signalling domain and PAR-2 (TF§CT/§CT/PAR-2-/-). Hepatic fibrosis area was assessed by quantitative imaging of picrosirius red staining. Hepatic collagen content was assessed by hydroxyproline levels. Hepatic stellate cells (αSMA positive) and hepatic macrophages (CD68 positive) were identified by immunohistochemistry. Hepatic gene expression was determined by PCR and liver TGFß1 content by ELISA. RESULTS: CCl4 treated mice with deletion of the PAR-2 gene (PAR-2-/-) and the cytoplasmic domain of TF (TF§CT/§CT) developed significantly less hepatic fibrosis, characterised by reduced liver fibrosis area and hydroxyproline content, compared to control wildtype mice treated with CCl4. The observed reduction in histological fibrosis was accompanied by a significant decrease in the hepatic content of TGFß, the prototypic fibrogenic cytokine, as well as fewer activated hepatic stellate cells and hepatic macrophages. Deletion of the TF cytoplasmic signalling domain reduced hepatic fibrosis to levels similar to those observed in mice lacking PAR-2 signalling but combined deletion provided no added protection against fibrosis indicating a lack of mutual modulating effects that have been observed in other contexts such as angiogenic responses. CONCLUSION: Tissue factor cytoplasmic domain is involved in TF-PAR-2 signalling initiating hepatic fibrosis and is a potential therapeutic target, as its deletion would not impact coagulation.

Cytotoxic lymphocytes and atherosclerosis: significance, mechanisms and therapeutic challenges.

Cytotoxic lymphocytes encompass natural killer lymphocytes (cells) and cytotoxic T cells that include CD8+ T cells, natural killer (NK) T cells, γ, δ (γδ)-T cells and human CD4 + CD28- T cells. These cells play critical roles in inflammatory diseases and in controlling cancers and infections. Cytotoxic lymphocytes can be activated via a number of mechanisms that may involve dendritic cells, macrophages, cytokines or surface proteins on stressed cells. Upon activation, they secrete pro-inflammatory cytokines as well as anti-inflammatory cytokines, chemokines and cytotoxins to promote inflammation and the development of atherosclerotic lesions including vulnerable lesions, which are strongly implicated in myocardial infarctions and strokes. Here, we review the mechanisms that activate and regulate cytotoxic lymphocyte activity, including activating and inhibitory receptors, cytokines, chemokine receptors-chemokine systems utilized to home to inflamed lesions and cytotoxins and cytokines through which they affect other cells within lesions. We also examine their roles in human and mouse models of atherosclerosis and the mechanisms by which they exert their pathogenic effects. Finally, we discuss strategies for therapeutically targeting these cells to prevent the development of atherosclerotic lesions and vulnerable plaques and the challenge of developing highly targeted therapies that only minimally affect the body's immune system, avoiding the complications, such as increased susceptibility to infections, which are currently associated with many immunotherapies for autoimmune diseases. LINKED ARTICLES: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc.

Killer cells in atherosclerosis.

Cytotoxic lymphocytes (killer cells) play a critical role in host defence mechanisms, protecting against infections and in tumour surveillance. They can also exert detrimental effects in chronic inflammatory disorders and in autoimmune diseases. Tissue cell death and necrosis are prominent features of advanced atherosclerotic lesions including vulnerable/unstable lesions which are largely responsible for most heart attacks and strokes. Evidence for accumulation of killer cells in both human and mouse lesions together with their cytotoxic potential strongly suggest that these cells contribute to cell death and necrosis in lesions leading to vulnerable plaque development and potentially plaque rupture. Killer cells can be divided into two groups, adaptive and innate immune cells depending on whether they require antigen presentation for activation. Activated killer cells detect damaged or stressed cells and kill by cytotoxic mechanisms that include perforin, granzymes, TRAIL or FasL and in some cases TNF-α. In this review, we examine current knowledge on killer cells in atherosclerosis, including CD8 T cells, CD28- CD4 T cells, natural killer cells and γδ-T cells, mechanisms responsible for their activation, their migration to developing lesions and effector functions. We also discuss pharmacological strategies to prevent their deleterious vascular effects by preventing/limiting their cytotoxic effects within atherosclerotic lesions as well as potential immunomodulatory therapies that might better target lesion-resident killer cells, to minimise any compromise of the immune system, which could result in increased susceptibility to infections and reductions in tumour surveillance.

Opposing roles of B lymphocyte subsets in atherosclerosis.

Atherosclerosis is initiated by cholesterol entry into arteries that triggers chronic immune-inflammatory lesions in the vessels. Early lesions are clinically insignificant but advanced complex lesions and vulnerable rupture prone lesions impact on quality of life and can be life threatening. Rupture of vulnerable atherosclerotic lesions initiates thrombotic occlusion of vital arteries precipitating heart attacks and strokes that remain major killers globally despite therapeutic use of statins to lower blood cholesterol levels. Conventional B2 cells are proatherogenic whereas peritoneal Bla cells are atheroprotective. Depletion of B2 cells by administration of mAb to CD20 or to BAFF receptor or in BAFF receptor-deficient mice ameliorates atherosclerosis. B2 cells may promote atherosclerosis by production of IgG, secretion of proinflammatory cytokine TNFα and activation of CD4 T cells. Together these B2 cell mechanisms contribute to generation of rupture-prone vulnerable atherosclerotic plaques characterised by large necrotic cores. In contrast, peritoneal Bla cells protect against atherosclerosis by secretion of natural IgM that scavenges apoptotic cells and oxidised LDL and reduces necrotic cores in atherosclerotic lesions. These atheroprotective effects can be further increased by stimulating Bla cells by administration of apoptotic cells, liposomes of phosphatidylserine abundant on surfaces of apoptotic cell, by mAb to TIM1, a phosphatidylserine receptor expressed by B1a cells and by TLR4-MyD88 activation. Experimental studies of atherosclerosis in mouse models indicate that reductions in atherogenic B2 cells and/or activation of atheroprotective B1a cells protects against atherosclerosis development, findings which have potential for clinical translation to reduce risks of deaths from heart attacks and strokes.

Toll-Like Receptor (TLR)4 and MyD88 are Essential for Atheroprotection by Peritoneal B1a B Cells.

BACKGROUND: We previously identified peritoneal B1a cells that secrete natural IgM as a key atheroprotective B cell subset. However, the molecules that activate atheroprotective B1a cells are unknown. Here, we investigated whether Toll-like receptors (TLRs) TLR2, TLR4, and TLR9 expressed by B1a cells are required for IgM-mediated atheroprotection. METHODS AND RESULTS: We adoptively transferred B1a cells from wild-type mice or from mice deficient in TLR2, TLR4, TLR9, or myeloid differentiation primary response 88 (MyD88) into ApoE-/- mice depleted of peritoneal B1a cells by splenectomy and fed a high-fat diet for 8 weeks. Elevations in plasma total, anti-oxLDL (oxidized low-density lipoprotein), anti-leukocyte, anti-CD3, anti-CD8, and anti-CD4 IgMs in atherosclerotic mice required B1a cells expressing TLR4 and MyD88, indicating a critical role for TLR4-MyD88 signaling for IgM secretion. Suppression of atherosclerosis was also critically dependent on B1a cells expressing TLR4-MyD88. Atherosclerosis suppression was associated not only with reductions in lesion apoptotic cells, necrotic cores, and oxLDL, but also with reduced lesion CD4+ and CD8+ T cells. Transforming growth factor beta 1 (TGF-ß1) expression, including macrophages expressing TGF-ß1, was increased, consistent with increased IgM-mediated phagocytosis of apoptotic cells by macrophages. Reductions in lesion inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin (IL) 1ß, and IL-18 were consistent with augmented TGF-ß1 expression. CONCLUSIONS: TLR4-MyD88 expression on B1a cells is critical for their IgM-dependent atheroprotection that not only reduced lesion apoptotic cells and necrotic cores, but also decreased CD4 and CD8 T-cell infiltrates and augmented TGF-ß1 expression accompanied by reduced lesion inflammatory cytokines TNF-α, IL-1ß, and IL-18.

B-cell-specific depletion of tumour necrosis factor alpha inhibits atherosclerosis development and plaque vulnerability to rupture by reducing cell death and inflammation.

AIMS: B2 lymphocytes promote atherosclerosis development but their mechanisms of action are unknown. Here, we investigated the role of tumour necrosis factor alpha (TNF-α) produced by B2 cells in atherogenesis. METHODS AND RESULTS: We found that 50% of TNF-α-producing spleen lymphocytes were B2 cells and ∼20% of spleen and aortic B cells produced TNF-α in hyperlipidemic ApoE(-/-) mice. We generated mixed bone marrow (80% µMT/20% TNF-α(-/-)) chimeric LDLR(-/-) mice where only B cells did not express TNF-α. Atherosclerosis was reduced in chimeric LDLR(-/-) mice with TNF-α-deficient B cells. TNF-α expression in atherosclerotic lesions and in macrophages were also reduced accompanied by fewer apoptotic cells, reduced necrotic cores, and reduced lesion Fas, interleukin-1ß and MCP-1 in mice with TNF-α-deficient B cells compared to mice with TNF-α-sufficient B cells. To confirm that the reduced atherosclerosis is attributable to B2 cells, we transferred wild-type and TNF-α-deficient B2 cells into ApoE(-/-) mice deficient in B cells or in lymphocytes. After 8 weeks of high fat diet, we found that atherosclerosis was increased by wild-type but not TNF-α-deficient B2 cells. Lesions of mice with wild-type B2 cells but not TNF-α-deficient B2 cells also had increased apoptotic cells and necrotic cores. Transferred B2 cells were found in lesions of recipient mice, suggesting that TNF-α-producing B2 cells promote atherosclerosis within lesions. CONCLUSION: We conclude that TNF-α produced by B2 cells is a key mechanism by which B2 cells promote atherogenesis through augmenting macrophage TNF-α production to induce cell death and inflammation that promote plaque vulnerability.

A CD1d-dependent lipid antagonist to NKT cells ameliorates atherosclerosis in ApoE-/- mice by reducing lesion necrosis and inflammation.

AIMS: Atherosclerosis-related deaths from heart attacks and strokes remain leading causes of global mortality, despite the use of lipid-lowering statins. Thus, there is an urgent need to develop additional therapies. METHODS AND RESULTS: Reports that NKT cells promote atherosclerosis and an NKT cell CD1d-dependent lipid antagonist (DPPE-PEG350, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethyleneglycol)-350]) reduces allergen-induced inflammation led us to investigate its therapeutic potential in preventing the development and progression of experimental atherosclerosis. DPPE-PEG350 was administered to hyperlipidaemic ApoE(-/-) mice with/without established atherosclerosis. Atherosclerosis and immune cells were assessed in the aortic sinus lesions. Lesion expression of monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion protein-1 (VCAM-1) responsible for inflammatory immune cell recruitment as well as mRNA expression of IFNγ and its plasma levels were investigated. Necrotic cores and lesion smooth muscle and collagen contents important in plaque stability were determined as were plasma lipid levels. DPPE-PEG350 reduced atherosclerosis development and delayed progression of established atherosclerosis without affecting plasma lipids. CD4 and CD8 T cells and B cells in atherosclerotic lesions were decreased in DPPE-PEG350-treated mice. Lesion MCP-1 and VCAM-1 protein expression and necrotic core size were reduced without affecting lesion smooth muscle and collagen content. IFNγ and lymphocytes were unaffected by the treatment. CONCLUSION: The attenuation of progression of established atherosclerosis together with reduced development of atherosclerosis in hyperlipidaemic mice by the NKT antagonist, without affecting NKT cell or other lymphocyte numbers, suggests that targeting lesion inflammation via CD1d-dependent activation of NKT cells using DPPE-PEG350 has a therapeutic potential in treating atherosclerosis.

Obligatory Role for B Cells in the Development of Angiotensin II-Dependent Hypertension.

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-ß 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.

Phosphatidylserine liposomes mimic apoptotic cells to attenuate atherosclerosis by expanding polyreactive IgM producing B1a lymphocytes.

AIMS: To investigate whether activation of atheroprotective peritoneal B1a cells by apoptotic cells or phosphatidylserine liposomes (PSLs) can enhance their protective actions during atherosclerosis development. METHODS AND RESULTS: Male apolipoprotein E-knockout (ApoE-/-) mice were treated with apoptotic cells or PSLs at the beginning of 8-week high-fat diet. Intraperitoneally administered apoptotic cells attenuated atherosclerosis in hypercholesterolemic ApoE-/- mice by 53% and macrophage accumulation by 52%, effects mimicked by administering PSLs and abolished by B1a cell depletion by splenectomy. These effects were associated with reduced lesion CD4+ and CD8+ T cells, mRNAs of MCP-1, VCAM-1, TNF-α, IL-1ß, IL-12, and IL-18 while anti-inflammatory TGF-ß mRNA levels doubled. Apoptotic cells or PSLs increased B1a lymphocytes including TIM-1+ B1a cells in vivo and in vitro while other lymphocyte populations were unaffected. Total plasma IgM, anti-leucocyte, anti-CD3, anti-CD4, and anti-oxLDL IgM were elevated. IgM in atherosclerotic lesions was also elevated and this was associated with reduced lesion MDA-LDL (oxLDL), apoptotic cells and necrotic core size. These effects of activating B1a cells could be attributed to B1a-derived polyreactive IgM deposited in lesions that reduce inflammatory cytokines by lowering lesion ox-LDL via anti-oxLDL IgM, T-cells via anti-leucocyte, anti-CD3, and anti-CD4 IgM, apoptotic cells and necrotic core size via IgM binding to apoptotic cells and enhancing phagocytosis, which also elevates anti-inflammatory cytokines. CONCLUSION: Targeting B1a cell activation by PSLs may be a potentially potent therapeutic strategy to attenuate atherosclerosis and reduce the incidence of atherosclerosis-dependent myocardial infarction and stroke.

CD4+ natural killer T cells potently augment aortic root atherosclerosis by perforin- and granzyme B-dependent cytotoxicity.

RATIONALE: CD4(+) natural killer T (NKT) cells augment atherosclerosis in apolipoprotein E-deficient (ApoE)(-/-) mice but their mechanisms of action are unknown. OBJECTIVES: We investigated the roles of bystander T, B, and NK cells; NKT cell-derived interferon-γ, interleukin (IL)-4, and IL-21 cytokines; and NKT cell-derived perforin and granzyme B cytotoxins in promoting CD4(+) NKT cell atherogenicity. METHODS AND RESULTS: Transfer of CD4(+) NKT cells into T- and B-cell-deficient ApoE(-/-)Rag2(-/-) mice augmented aortic root atherosclerosis by ≈75% that was ≈30% of lesions in ApoE(-/-) mice; macrophage accumulation similarly increased. Transferred NKT cells were identified in the liver and atherosclerotic lesions of recipient mice. Transfer of CD4(+) NKT cells into T-, B-cell-deficient, and NK cell-deficient ApoE(-/-)Rag2(-/-)γC(-/-) mice also augmented atherosclerosis. These data indicate that CD4(+) NKT cells can exert proatherogenic effects independent of other lymphocytes. To investigate the role of NKT cell-derived interferon-γ, IL-4, and IL-21 cytokines and perforin and granzyme B cytotoxins, CD4(+) NKT cells from mice deficient in these molecules were transferred into NKT cell-deficient ApoE(-/-)Jα18(-/-) mice. CD4(+) NKT cells deficient in IL-4, interferon-γ, or IL-21 augmented atherosclerosis in ApoE(-/-)Jα18(-/-) mice by ≈95%, ≈80%, and ≈70%, respectively. Transfer of CD4(+) NKT cells deficient in perforin or granzyme B failed to augment atherosclerosis. Apoptotic cells, necrotic cores, and proinflammatory VCAM-1 (vascular cell adhesion molecule) and MCP-1 (monocyte chemotactic protein) were reduced in mice receiving perforin-deficient NKT cells. CD4(+) NKT cells are twice as potent as CD4(+) T cells in promoting atherosclerosis. CONCLUSIONS: CD4(+) NKT cells potently promote atherosclerosis by perforin and granzyme B-dependent apoptosis that increases postapoptotic necrosis and inflammation.

Natural killer (NK) cells augment atherosclerosis by cytotoxic-dependent mechanisms.

AIM: Although natural killer (NK) cells, a key component of the innate immune system, have been identified in human and mouse atherosclerotic lesions, their role in atherosclerosis development remains unclear. To determine their role in atherosclerosis, we used both loss- and gain-of-function experiments in ApoE(-/-) mice fed a high-fat diet. METHODS AND RESULTS: Treatment of ApoE(-/-) mice with anti-Asialo-GM1 antibodies depleted NK cells without affecting other lymphocytes, including natural killer T cells, and greatly attenuated atherosclerosis. These effects were independent of plasma lipids. To confirm the atherogenicity of NK cells, these cells were isolated from mouse spleens for adoptive transfer into lymphocyte-deficient ApoE(-/-)Rag2(-/-)IL2rg(-/-) mice. Transfer of NK cells from wild-type mice into ApoE(-/-)Rag2(-/-)IL2rg(-/-) mice doubled lesion size, confirming a pro-atherogenic role for NK cells. To determine whether their atherogenicity was dependent on production of interferon-γ (IFN-γ) or cytotoxins, we compared the transfer of NK cells deficient in IFN-γ, perforin, and granzyme B with the transfer of wild-type NK cells. Transfer of IFN-γ-deficient NK cells increased lesion size in the lymphocyte-deficient ApoE(-/-) mice as wild-type NK cells. However, granzyme B- and perforin-deficient NK cells did not affect lesion size. Only wild-type NK cells increased necrotic core size, whereas perforin- and granzyme B-deficient NK cells did not. Plasma lipid levels were largely unaffected by the cell transfer. CONCLUSION: Our loss- and gain-of-function findings provide definitive evidence that NK cells are atherogenic and their production of perforin and granzyme B contributes to atherosclerosis and the expansion of necrotic cores.

Immune regulation by CD52-expressing CD4 T cells.

T-cell regulation by CD52-expressing CD4 T cells appears to operate by two different and possibly synergistic mechanisms. The first is by its release from the cell surface of CD4 T cells that express high levels of CD52 that then binds to the inhibitory sialic acid-binding immunoglobulin-like lectins-10 (Siglec-10) receptor to attenuate effector T-cell activation by impairing phosphorylation of T-cell receptor associated lck and zap-70. The second mechanism appears to be by crosslinkage of the CD52 molecules by an as yet unidentified endogenous ligand that is mimicked by a bivalent anti-CD52 antibody that results in their expansion.

BAFF receptor mAb treatment ameliorates development and progression of atherosclerosis in hyperlipidemic ApoE(-/-) mice.

AIMS: Option to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE(-/-) mouse. METHODS AND RESULTS: To determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE(-/-) mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93(-) CD19(+) B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19(+) B cells, CD4(+) and CD8(+) T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1ß, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE(-/-) mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93(-) CD19(+) B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment. CONCLUSION: Anti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE(-/-) mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases.

Cytotoxic and proinflammatory CD8+ T lymphocytes promote development of vulnerable atherosclerotic plaques in apoE-deficient mice.

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 CD8ß 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 1ß, 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 1ß 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.

Cytokine therapy with interleukin-2/anti-interleukin-2 monoclonal antibody complexes expands CD4+CD25+Foxp3+ regulatory T cells and attenuates development and progression of atherosclerosis.

BACKGROUND: CD4+CD25+Foxp3+ regulatory T cells (Tregs) attenuate atherosclerosis, but their therapeutic application by 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 CD4+, CD8+, or natural killer cells. Tregs were increased in lesions and lesion size reduced. CD4+ T-cells, macrophages, mature dendritic cells, proliferating cell nuclear antigen+ 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-1ß 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 CD4+, CD8+, or natural killer cells, suppressed inflammation, and greatly attenuated progression of atherosclerosis. CONCLUSIONS: IL-2/anti-IL-2 mAb treatment in vivo attenuates atherosclerosis via selective Tregs expansion. The findings suggest that cytokine-based IL-2/anti-IL-2 mAb complex therapy could represent an attractive approach for treating atherosclerosis, because it markedly attenuates progression as well as development, by modulating its immunoinflammatory component.

Protective role of natural IgM-producing B1a cells in atherosclerosis.

Atherosclerosis initiated by hyperlipidemia is modulated by immune cells in its development, progression, and rupture that results in thrombotic arterial occlusion leading to strokes and myocardial infarction. B cells initially thought to be atheroprotective provide opposing roles by their different subsets. Unlike B2 cells that are atherogenic, serosal B1a cells are atheroprotective by producing natural IgM antibodies that clear modified low-density lipoprotein and apoptotic and necrotic debris. In addition to natural IgM antibodies, B1a cells may act as regulatory B cells by producing the anti-inflammatory cytokine interleukin-10, which inhibits proinflammatory cytokines secreted by activated macrophages and T cells in atherosclerotic lesions. These findings suggest in vivo expansion of atheroprotective B1a cells as a potential therapeutic strategy to augment the benefits of lipid-lowering statin therapy.

Depletion of B2 but not B1a B cells in BAFF receptor-deficient ApoE mice attenuates atherosclerosis by potently ameliorating arterial inflammation.

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-ß 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.

Protease-activated receptor 2 promotes experimental liver fibrosis in mice and activates human hepatic stellate cells.

UNLABELLED: Protease-activated receptor (PAR) 2 is a G-protein-coupled receptor that is activated after proteolytic cleavage by serine proteases, including mast cell tryptase and activated coagulation factors. PAR-2 activation augments inflammatory and profibrotic pathways through the induction of genes encoding proinflammatory cytokines and extracellular matrix proteins. Thus, PAR-2 represents an important interface linking coagulation and inflammation. PAR-2 is widely expressed in cells of the gastrointestinal tract, including hepatic stellate cells (HSCs), endothelial cells, and hepatic macrophages; however, its role in liver fibrosis has not been previously examined. We studied the development of CCl(4) -induced liver fibrosis in PAR-2 knockout mice, and showed that PAR-2 deficiency reduced the progression of liver fibrosis, hepatic collagen gene expression, and hydroxyproline content. Reduced fibrosis was associated with decreased transforming growth factor beta (TGFß) gene and protein expression and decreased matrix metalloproteinase 2 and tissue inhibitor of matrix metalloproteinase 1 gene expression. In addition, PAR-2 stimulated activation, proliferation, collagen production, and TGFß protein production by human stellate cells, indicating that hepatic PAR-2 activation increases profibrogenic cytokines and collagen production both in vivo and in vitro. CONCLUSION: Our findings demonstrate the capacity of PAR-2 activation to augment TGFß production and promote hepatic fibrosis in mice and to induce a profibrogenic phenotype in human HSCs. PAR-2 antagonists have recently been developed and may represent a novel therapeutic approach in preventing fibrosis in patients with chronic liver disease.