Interleukin-1 receptor accessory protein blockade limits the development of atherosclerosis and reduces plaque inflammation.

AIMS: The interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor required for signalling through the IL-1, IL-33, and IL-36 receptors. Using a novel anti-IL1RAP-blocking antibody, we investigated the role of IL1RAP in atherosclerosis. METHODS AND RESULTS: Single-cell RNA sequencing data from human atherosclerotic plaques revealed the expression of IL1RAP and several IL1RAP-related cytokines and receptors, including IL1B and IL33. Histological analysis showed the presence of IL1RAP in both the plaque and adventitia, and flow cytometry of murine atherosclerotic aortas revealed IL1RAP expression on plaque leucocytes, including neutrophils and macrophages. High-cholesterol diet fed apolipoprotein E-deficient (Apoe-/-) mice were treated with a novel non-depleting IL1RAP-blocking antibody or isotype control for the last 6 weeks of diet. IL1RAP blockade in mice resulted in a 20% reduction in subvalvular plaque size and limited the accumulation of neutrophils and monocytes/macrophages in plaques and of T cells in adventitia, compared with control mice. Indicative of reduced plaque inflammation, the expression of several genes related to leucocyte recruitment, including Cxcl1 and Cxcl2, was reduced in brachiocephalic arteries of anti-IL1RAP-treated mice, and the expression of these chemokines in human plaques was mainly restricted to CD68+ myeloid cells. Furthermore, in vitro studies demonstrated that IL-1, IL-33, and IL-36 induced CXCL1 release from both macrophages and fibroblasts, which could be mitigated by IL1RAP blockade. CONCLUSION: Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production.

Resident Memory T Cells in the Atherosclerotic Lesion Associate With Reduced Macrophage Content and Increased Lesion Stability.

BACKGROUND: Tissue resident memory T (TRM) cells are a T-cell subset that resides at the site of prior antigen recognition to protect the body against reoccurring encounters. Besides their protective function, TRM cells have also been implicated in inflammatory disorders. TRM cells are characterized by the expression of CD69 and transcription factors Hobit (homolog of Blimp-1 [B lymphocyte-induced maturation protein 1] in T cells) and Blimp-1. As the majority of T cells in the arterial intima expresses CD69, TRM cells may contribute to the pathogenesis of atherosclerosis as well. Here, we aimed to assess the presence and potential role of TRM cells in atherosclerosis. METHODS: To identify TRM cells in human atherosclerotic lesions, a single-cell RNA-sequencing data set was interrogated, and T-cell phenotypes were compared with that of integrated predefined TRM cells. The presence and phenotype of TRM in atherosclerotic lesions was corroborated using a mouse model that enabled tracking of Hobit-expressing TRM cells. To explore the function of TRM cells during atherogenesis, RAG1-/- (recombination activating gene 1 deficient) LDLr-/- (low-density lipoprotein receptor knockout) mice received a bone marrow transplant from HobitKO/CREBlimp-1flox/flox mice, which exhibit abrogated TRM cell formation, whereafter the mice were fed a Western-type diet for 10 weeks. RESULTS: Human atherosclerotic lesions contained T cells that exhibited a TRM cell-associated gene signature. Moreover, a fraction of these T cells clustered together with predefined TRM cells upon integration. The presence of Hobit-expressing TRM cells in the atherosclerotic lesion was confirmed in mice. These lesion-derived TRM cells were characterized by the expression of CD69 and CD49α. Moreover, we demonstrated that this small T-cell subset significantly affects lesion composition, by reducing the amount of intralesional macrophages and increasing collagen content. CONCLUSIONS: TRM cells, characterized by the expression of CD69 and CD49α, constitute a minor population in atherosclerotic lesions and are associated with increased lesion stability in a Hobit and Blimp-1 knockout mouse model.

Immunoproteasomal Inhibition With ONX-0914 Attenuates Atherosclerosis and Reduces White Adipose Tissue Mass and Metabolic Syndrome in Mice.

BACKGROUND: Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. METHODS: We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit ß5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit ß1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice. RESULTS: ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. CONCLUSIONS: We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.

Macrophage LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Is Required for the Effect of CD47 Blockade on Efferocytosis and Atherogenesis-Brief Report.

OBJECTIVE: Antibody blockade of the "do not eat me" signal CD47 (cluster of differentiation 47) enhances efferocytosis and reduces lesion size and necrotic core formation in murine atherosclerosis. TNF (Tumor necrosis factor)-α expression directly enhances CD47 expression, and elevated TNF-α is observed in the absence of the proefferocytosis receptor LRP1 (low-density lipoprotein receptor-related protein 1), a regulator of atherogenesis and inflammation. Thus, we tested the hypothesis that CD47 blockade requires the presence of macrophage LRP1 to enhance efferocytosis, temper TNF-α-dependent inflammation, and limit atherosclerosis. Approach and Results: Mice lacking systemic apoE (apoE-/-), alone or in combination with the loss of macrophage LRP1 (double knockout), were fed a Western-type diet for 12 weeks while receiving anti-CD47 antibody (anti-CD47) or IgG every other day. In apoE-/- mice, treatment with anti-CD47 reduced lesion size by 25.4%, decreased necrotic core area by 34.5%, and decreased the ratio of free:macrophage-associated apoptotic bodies by 47.6% compared with IgG controls (P<0.05), confirming previous reports. Double knockout mice treated with anti-CD47 showed no differences in lesion size, necrotic core area, or the ratio of free:macrophage-associated apoptotic bodies compared with IgG controls. In vitro efferocytosis was 30% higher when apoE-/- phagocytes were incubated with anti-CD47 compared with IgG controls (P<0.05); however, anti-CD47 had no effect on efferocytosis in double knockout phagocytes. Analyses of mRNA and protein showed increased CD47 expression in anti-inflammatory IL (interleukin)-4 treated LRP1-/- macrophages compared with wild type, but no differences were observed in inflammatory lipopolysaccharide-treated macrophages. CONCLUSIONS: The proefferocytosis receptor LRP1 in macrophages is necessary for anti-CD47 blockade to enhance efferocytosis, limit atherogenesis, and decrease necrotic core formation in the apoE-/- model of atherosclerosis.

Krüppel-like factor 14 deletion in myeloid cells accelerates atherosclerotic lesion development.

AIMS: Atherosclerosis is the dominant pathologic basis of many cardiovascular diseases. Large genome-wide association studies have identified that single-nucleotide polymorphisms proximal to Krüppel-like factor 14 (KLF14), a member of the zinc finger family of transcription factors, are associated with higher cardiovascular risks. Macrophage dysfunction contributes to atherosclerosis development and has been recognized as a potential therapeutic target for treating many cardiovascular diseases. Herein, we address the biologic function of KLF14 in macrophages and its role during the development of atherosclerosis. METHODS AND RESULTS: KLF14 expression was markedly decreased in cholesterol loaded foam cells, and overexpression of KLF14 significantly increased cholesterol efflux and inhibited the inflammatory response in macrophages. We generated myeloid cell-selective Klf14 knockout (Klf14LysM) mice in the ApoE-/- background for the atherosclerosis study. Klf14LysMApoE-/- and litter-mate control mice (Klf14fl/flApoE-/-) were placed on the Western Diet for 12 weeks to induce atherosclerosis. Macrophage Klf14 deficiency resulted in increased atherosclerosis development without affecting the plasma lipid profiles. Klf14-deficient peritoneal macrophages showed significantly reduced cholesterol efflux resulting in increased lipid accumulation and exacerbated inflammatory response. Mechanistically, KLF14 upregulates the expression of a key cholesterol efflux transporter, ABCA1 (ATP-binding cassette transporter A1), while it suppresses the expression of several critical components of the inflammatory cascade. In macrophages, activation of KLF14 by its activator, perhexiline, a drug clinically used to treat angina, significantly inhibited the inflammatory response and increased cholesterol efflux in a KLF14-dependent manner in macrophages without triggering hepatic lipogenesis. CONCLUSIONS: This study provides insights into the anti-atherosclerotic effects of myeloid KLF14 through promoting cholesterol efflux and suppressing the inflammatory response. Activation of KLF14 may represent a potential new therapeutic approach to prevent or treat atherosclerosis.

Impaired macrophage trafficking and increased helper T-cell recruitment with loss of cadherin-11 in atherosclerotic immune response.

Inflammation caused by infiltrating macrophages and T cells promotes plaque growth in atherosclerosis. Cadherin-11 (CDH11) is a cell-cell adhesion protein implicated in several fibrotic and inflammatory diseases. Much of the research on CDH11 concerns its role in fibroblasts, although its expression in immune cells has been noted as well. The objective of this study was to assess the effect of CDH11 on the atherosclerotic immune response. In vivo studies of atherosclerosis indicated an increase in Cdh11 in plaque tissue. However, global loss of Cdh11 resulted in increased atherosclerosis and inflammation. It also altered the immune response in circulating leukocytes, decreasing myeloid cell populations and increasing T-cell populations, suggesting possible impaired myeloid migration. Bone marrow transplants from Cdh11-deficient mice resulted in similar immune cell profiles. In vitro examination of Cdh11-/- macrophages revealed reduced migration, despite upregulation of a number of genes related to locomotion. Flow cytometry revealed an increase in CD3+ and CD4+ helper T-cell populations in the blood of both the global Cdh11 loss and the bone marrow transplant animals, possibly resulting from increased expression by Cdh11-/- macrophages of major histocompatibility complex class II molecule genes, which bind to CD4+ T cells for coordinated activation. CDH11 fundamentally alters the immune response in atherosclerosis, resulting in part from impaired macrophage migration and altered macrophage-induced T-cell activation.NEW & NOTEWORTHY Cadherin-11 is well known to contribute to inflammatory and fibrotic disease. Here, we examined its role in atherosclerosis progression, which is predominantly an inflammatory process. We found that while cadherin-11 is associated with plaque progression, global loss of cadherin-11 exacerbated the disease phenotype. Moreover, loss of cadherin-11 in bone marrow-derived immune cells resulted in impaired macrophage migration and an unexplained increase in circulating helper T cells, presumably due to altered macrophage function without cadherin-11.

Hypercholesterolemia Impairs Clearance of Neutrophil Extracellular Traps and Promotes Inflammation and Atherosclerotic Plaque Progression.

Objective: Hypercholesterolemia-induced NETosis and accumulation of neutrophil extracellular traps (NETs) in the atherosclerotic lesion exacerbates inflammation and is causally implicated in plaque progression. We investigated whether hypercholesterolemia additionally impairs the clearance of NETs mediated by endonucleases such as DNase1 and DNase1L3 and its implication in advanced atherosclerotic plaque progression. Approach and Results: Using a mouse model, we demonstrate that an experimental increase in the systemic level of NETs leads to a rapid increase in serum DNase activity, which is critical for the prompt clearance of NETs and achieving inflammation resolution. Importantly, hypercholesterolemic mice demonstrate an impairment in this critical NET-induced DNase response with consequent delay in the clearance of NETs and defective inflammation resolution. Administration of tauroursodeoxycholic acid, a chemical chaperone that relieves endoplasmic reticulum stress, rescued the hypercholesterolemia-induced impairment in the NET-induced DNase response suggesting a causal role for endoplasmic reticulum stress in this phenomenon. Correction of the defective DNase response with exogenous supplementation of DNase1 in Apoe-/- mice with advanced atherosclerosis resulted in a decrease in plaque NET content and significant plaque remodeling with decreased area of plaque necrosis and increased collagen content. From a translational standpoint, we demonstrate that humans with hypercholesterolemia have elevated systemic extracellular DNA levels and decreased plasma DNase activity. Conclusions: These data suggest that hypercholesterolemia impairs the NET-induced DNase response resulting in defective clearance and accumulation of NETs in the atherosclerotic plaque. Therefore, strategies aimed at rescuing this defect could be of potential therapeutic benefit in promoting inflammation resolution and atherosclerotic plaque stabilization.

CD200 Limits Monopoiesis and Monocyte Recruitment in Atherosclerosis.

[Figure: see text].

HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation.

[Figure: see text].

Deficiency of Endothelial CD40 Induces a Stable Plaque Phenotype and Limits Inflammatory Cell Recruitment to Atherosclerotic Lesions in Mice.

OBJECTIVES: The co-stimulatory CD40L-CD40 dyad exerts a critical role in atherosclerosis by modulating leukocyte accumulation into developing atherosclerotic plaques. The requirement for cell-type specific expression of both molecules, however, remains elusive. Here, we evaluate the contribution of CD40 expressed on endothelial cells (ECs) in a mouse model of atherosclerosis. METHODS AND RESULTS: Atherosclerotic plaques of apolipoprotein E-deficient (Apoe -/- ) mice and humans displayed increased expression of CD40 on ECs compared with controls. To interrogate the role of CD40 on ECs in atherosclerosis, we induced EC-specific (BmxCreERT2-driven) deficiency of CD40 in Apoe -/- mice. After feeding a chow diet for 25 weeks, EC-specific deletion of CD40 (iEC-CD40) ameliorated plaque lipid deposition and lesional macrophage accumulation but increased intimal smooth muscle cell and collagen content, while atherosclerotic lesion size did not change. Leukocyte adhesion to the vessel wall was impaired in iEC-CD40-deficient mice as demonstrated by intravital microscopy. In accord, expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in the vascular endothelium declined after deletion of CD40. In vitro, antibody-mediated inhibition of human endothelial CD40 significantly abated monocyte adhesion on ECs. CONCLUSION: Endothelial deficiency of CD40 in mice promotes structural features associated with a stable plaque phenotype in humans and decreases leukocyte adhesion. These results suggest that endothelial-expressed CD40 contributes to inflammatory cell migration and consecutive plaque formation in atherogenesis.

What is the meaning of ANCA positivity in IgG4-related disease?

OBJECTIVES: To evaluate the prevalence and meaning of antineutrophil cytoplasmic antibodies (ANCA) positivity in a cohort of IgG4-related disease (IgG4-RD). METHODS: We identified patients with ANCA determination from a retrospective cohort of 69 patients with IgG4-RD. ANCA were measured by indirect immunofluorescence microscopy (IIF) and/or proteinase 3 (PR3)-ANCA and MPO-ANCA by ELISA. IIF patterns were classified as perinuclear (P-ANCA), cytoplasmic (C-ANCA) and atypical (X-ANCA). We compared the ANCA-positive vs the ANCA-negative IgG4-RD group. RESULTS: Out of 69 patients, 31 IgG4-RD patients had an ANCA determination. Four patients with concomitant systemic autoimmune diseases were excluded. We found positive ANCA by IIF in 14 (56%) of 25 patients tested. The most common IIF pattern was C-ANCA in eight (57.1%), followed by dual C-ANCA/X-ANCA in four (28.6%) and P-ANCA and dual C-ANCA/P-ANCA in one each (7.1%). Of the 20 patients with ANCA determination by both IIF and ELISA, four have positive ANCA by ELISA (three for MPO-ANCA and one for PR3-ANCA). Of the two patients with only ELISA determination, one was positive for MPO-ANCA. The prevalence of ANCA positivity by ELISA was 22.7% (5 out of 22 patients). ANCA was more frequent in the Mikulizc/systemic phenotype (42.9%) compared with other phenotypes (P = 0.04). ANCA-positive IgG4-RD patients had more frequently lymph node and kidney involvement, high IgG1 levels and erythrocyte sedimentation rate, and positive antinuclear antibodies. CONCLUSION: ANCA are found in a significant number of patients with IgG4-RD and differed from the ANCA-negative group in terms of clinical and serological features.

Follicular regulatory helper T cells control the response of regulatory B cells to a high-cholesterol diet.

AIMS: B cell functions in the process of atherogenesis have been investigated but several aspects remain to be clarified. METHODS AND RESULTS: In this study, we show that follicular regulatory helper T cells (TFR) control regulatory B cell (BREG) populations in Apoe-/- mice models on a high-cholesterol diet (HCD). Feeding mice with HCD resulted in up-regulation of TFR and BREG cell populations, causing the suppression of proatherogenic follicular helper T cell (TFH) response. TFH cell modulation is correlated with the growth of atherosclerotic plaque size in thoracoabdominal aortas and aortic root plaques, suggesting that TFR cells are atheroprotective. During adoptive transfer experiments, TFR cells transferred into HCD mice decreased TFH cell populations, atherosclerotic plaque size, while BREG cell population and lymphangiogenesis are significantly increased. CONCLUSION: Our results demonstrate that, through different strategies, both TFR and TFH cells modulate anti- and pro-atherosclerotic immune processes in an Apoe-/- mice model since TFR cells are able to regulate both TFH and BREG cell populations as well as lymphangiogenesis and lipoprotein metabolism.

Adoptive transfer of CX3CR1 transduced-T regulatory cells improves homing to the atherosclerotic plaques and dampens atherosclerosis progression.

AIM: Loss of immunosuppressive response supports inflammation during atherosclerosis. We tested whether adoptive cell therapy (ACT) with Tregulatory cells (Tregs), engineered to selectively migrate in the atherosclerotic plaque, would dampen the immune-inflammatory response in the arterial wall in animal models of familial hypercholesterolaemia (FH). METHODS AND RESULTS: FH patients presented a decreased Treg suppressive function associated to an increased inflammatory burden. A similar phenotype was observed in Ldlr -/- mice accompanied by a selective increased expression of the chemokine CX3CL1 in the aorta but not in other districts (lymph nodes, spleen, and liver). Treg overexpressing CX3CR1 were thus generated (CX3CR1+-Tregs) to drive Tregs selectively to the plaque. CX3CR1+-Tregs were injected (i.v.) in Ldlr -/- fed high-cholesterol diet (western type diet, WTD) for 8 weeks. CX3CR1+-Tregs were detected in the aorta, but not in other tissues, of Ldlr -/- mice 24 h after ACT, corroborating the efficacy of this approach. After 4 additional weeks of WTD, ACT with CX3CR1+-Tregs resulted in reduced plaque progression and lipid deposition, ameliorated plaque stability by increasing collagen and smooth muscle cells content, while decreasing the number of pro-inflammatory macrophages. Shotgun proteomics of the aorta showed a metabolic rewiring in CX3CR1+-Tregs treated Ldlr -/- mice compared to controls that was associated with the improvement of inflammation-resolving pathways and disease progression. CONCLUSION: ACT with vasculotropic Tregs appears as a promising strategy to selectively target immune activation in the atherosclerotic plaque.

Positive disease-specific autoantibodies have limited clinical significance in diagnosing IgG4-related disease in daily clinical practice.

OBJECTIVES: The 2019 ACR/EULAR classification criteria for IgG4-related disease (IgG4-RD) have exclusion criteria including positive disease-specific autoantibodies, and these have been documented to have a high specificity. This study aimed to further validate these criteria as well as identify characteristics of patients showing false-negative results. METHODS: We retrospectively analysed 162 IgG4-RD patients and 130 mimickers. The sensitivity, specificity and fulfilment rates for each criterion were calculated, and intergroup comparisons were performed to characterize the false-negative cases. RESULTS: Both the IgG4-RD patients and mimickers were aged ≥65 years with male predominance. The final diagnoses of mimickers were mainly malignancy, vasculitis, sarcoidosis and aneurysm. The classification criteria had a sensitivity of 72.8% and specificity of 100%. Of the 44 false-negative cases, one did not fulfil the entry criteria, 20 fulfilled one exclusion criterion and 27 did not achieve sufficient inclusion criteria scores. The false-negative cases had fewer affected organs, lower serum IgG4 levels, and were less likely to have received biopsies than the true-positive cases. Notably, positive disease-specific autoantibodies were the most common exclusion criterion fulfilled in 18 patients, only two of whom were diagnosed with a specific autoimmune disease complicated by IgG4-RD. In addition, compared with the true-positive cases, the 18 had comparable serum IgG4 levels, number of affected organs, and histopathology and immunostaining scores despite higher serum IgG and CRP levels. CONCLUSIONS: The ACR/EULAR classification criteria for IgG4-RD have an excellent diagnostic specificity in daily clinical practice. Positive disease-specific autoantibodies may have limited clinical significance for the diagnosis of IgG4-RD.

Tc17 CD8+ T cells accumulate in murine atherosclerotic lesions, but do not contribute to early atherosclerosis development.

AIMS: CD8+ T cells can differentiate into subpopulations that are characterized by a specific cytokine profile, such as the Tc17 population that produces interleukin-17. The role of this CD8+ T-cell subset in atherosclerosis remains elusive. In this study, we therefore investigated the contribution of Tc17 cells to the development of atherosclerosis. METHODS AND RESULTS: Flow cytometry analysis of atherosclerotic lesions from apolipoprotein E-deficient mice revealed a pronounced increase in RORγt+CD8+ T cells compared to the spleen, indicating a lesion-specific increase in Tc17 cells. To study whether and how the Tc17 subset affects atherosclerosis, we performed an adoptive transfer of Tc17 cells or undifferentiated Tc0 cells into CD8-/- low-density lipoprotein receptor-deficient mice fed a Western-type diet. Using flow cytometry, we showed that Tc17 cells retained a high level of interleukin-17A production in vivo. Moreover, Tc17 cells produced lower levels of interferon-γ than their Tc0 counterparts. Analysis of the aortic root revealed that the transfer of Tc17 cells did not increase atherosclerotic lesion size, in contrast to Tc0-treated mice. CONCLUSION: These findings demonstrate a lesion-localized increase in Tc17 cells in an atherosclerotic mouse model. Tc17 cells appeared to be non-atherogenic, in contrast to their Tc0 counterpart.

The Role of T Cells Reactive to the Cathelicidin Antimicrobial Peptide LL-37 in Acute Coronary Syndrome and Plaque Calcification.

The human cationic anti-microbial peptide LL-37 is a T cell self-antigen in patients with psoriasis, who have increased risk of cardiovascular events. However, the role of LL-37 as a T cell self-antigen in the context of atherosclerosis remains unclear. The objective of this study was to test for the presence of T cells reactive to LL-37 in patients with acute coronary syndrome (ACS). Furthermore, the role of T cells reactive to LL-37 in atherosclerosis was assessed using apoE-/- mice immunized with the LL-37 mouse ortholog, mCRAMP. Peripheral blood mononuclear cells (PBMCs) from patients with ACS were stimulated with LL-37. PBMCs from stable coronary artery disease (CAD) patients or self-reported subjects served as controls. T cell memory responses were analyzed with flow cytometry. Stimulation of PBMCs with LL-37 reduced CD8+ effector T cell responses in controls and patients with stable CAD but not in ACS and was associated with reduced programmed cell death protein 1 (PDCD1) mRNA expression. For the mouse studies, donor apoE-/- mice were immunized with mCRAMP or adjuvant as controls, then T cells were isolated and adoptively transferred into recipient apoE-/- mice fed a Western diet. Recipient mice were euthanized after 5 weeks. Whole aortas and hearts were collected for analysis of atherosclerotic plaques. Spleens were collected for flow cytometric and mRNA expression analysis. Adoptive transfer experiments in apoE-/- mice showed a 28% reduction in aortic plaque area in mCRAMP T cell recipient mice (P < 0.05). Fifty six percent of adjuvant T cell recipient mice showed calcification in atherosclerotic plaques, compared to none in the mCRAMP T cell recipient mice (Fisher's exact test P = 0.003). Recipients of T cells from mice immunized with mCRAMP had increased IL-10 and IFN-γ expression in CD8+ T cells compared to controls. In conclusion, the persistence of CD8+ effector T cell response in PBMCs from patients with ACS stimulated with LL-37 suggests that LL-37-reactive T cells may be involved in the acute event. Furthermore, studies in apoE-/- mice suggest that T cells reactive to mCRAMP are functionally active in atherosclerosis and may be involved in modulating plaque calcification.

Naive CD8+ T Cells Expressing CD95 Increase Human Cardiovascular Disease Severity.

OBJECTIVE: Cardiovascular disease (CVD) remains a significant global health concern with a high degree of mortality. While CD4+ T cells have been extensively studied in CVD, the importance of CD8+ T cells in this disease, despite their abundance and increased activation in human atherosclerotic plaques, remains largely unknown. Thus, the objective of this study was to compare peripheral T-cell signatures between humans with a high (severe) risk of CVD (including myocardial infarction or stroke) and those with a low risk of CVD. Approach and Results: Using mass cytometry, we uncovered a naive CD8+ T (TN) cell population expressing CD95 (termed CD95+CD8+ stem cell memory T [CD8 TSCM] cells) that was enriched in patients with high compared with low CVD. This T-cell subset enrichment within individuals with high CVD was a relative increase and resulted from the loss of CD95lo cells within the TN compartment. We found that CD8 TSCM cells positively correlated with CVD risk in humans, while CD8+ TN cells were inversely correlated. Atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice also displayed respective 7- and 2-fold increases in CD8+ TSCM frequencies within the peripheral blood and aorta-draining paraaortic lymph nodes compared with C57BL/6J mice. CD8+ TSCM cells were 1.7-fold increased in aortas from western diet fed ApoE-/- mice compared with normal laboratory diet-fed ApoE-/- mice. Importantly, transfer of TSCM cells into immune-deficient Rag.Ldlr recipient mice that lacked T cells increased atherosclerosis, illustrating the importance of these cells in atherogenesis. CONCLUSIONS: CD8+ TSCM cells are increased in humans with high CVD. As these TSCM cells promote atherosclerosis, targeting them may attenuate atherosclerotic plaque progression.

Bee Venom Phospholipase A2 Ameliorates Atherosclerosis by Modulating Regulatory T Cells.

Atherosclerosis is a chronic inflammatory disease caused by lipids and calcareous accumulations in the vascular wall due to an inflammatory reaction. Recent reports have demonstrated that regulatory T (Treg) cells have an important role as a new treatment for atherosclerosis. This study suggests that bee venom phospholipase A2 (bvPLA2) may be a potential therapeutic agent in atherosclerosis by inducing Treg cells. We examined the effects of bvPLA2 on atherosclerosis using ApoE-/- and ApoE-/-/Foxp3DTR mice. In this study, bvPLA2 increased Treg cells, followed by a decrease in lipid accumulation in the aorta and aortic valve and the formation of foam cells. Importantly, the effect of bvPLA2 was found to depend on Treg cells. This study suggests that bvPLA2 can be a potential therapeutic agent for atherosclerosis.

Protective Role of C3aR (C3a Anaphylatoxin Receptor) Against Atherosclerosis in Atherosclerosis-Prone Mice.

OBJECTIVE: Emerging evidence suggests that C3aR (C3a anaphylatoxin receptor) signaling has protective roles in various inflammatory-related diseases. However, its role in atherosclerosis has been unknown. The purpose of the study was to investigate the possible protective role of C3aR in aortic atherosclerosis and explore molecular and cellular mechanisms involved in the protection. Approach and Results: C3ar-/-/Apoe-/- mice were generated by cross-breeding of atherosclerosis-prone Apoe-/- mice and C3ar-/- mice. C3ar-/-/Apoe-/- mice and Apoe-/- mice (as a control) underwent high-fat diet for 16 weeks were assessed for (1) atherosclerotic plaque burden, (2) aortic tissue inflammation, (3) recruitment of CD11b+ leukocytes into atherosclerotic lesions, and (4) systemic inflammatory responses. Compared with Apoe-/- mice, C3ar-/-/Apoe-/- mice developed more severe atherosclerosis. In addition, C3ar-/-/Apoe-/- mice have increased local production of proinflammatory mediators (eg, CCL2 [chemokine (C-C motif) ligand 2], TNF [tumor necrosis factor]-α) and infiltration of monocyte/macrophage in aortic tissue, and their lesional macrophages displayed an M1-like phenotype. Local pathological changes were associated with enhanced systemic inflammatory responses (ie, elevated plasma levels of CCL2 and TNF-α, increased circulating inflammatory cells). In vitro analyses using peritoneal macrophages showed that C3a stimulation resulted in upregulation of M2-associated signaling and molecules, but suppression of M1-associated signaling and molecules, supporting the roles of C3a/C3aR axis in mediating anti-inflammatory response and promoting M2 macrophage polarization. CONCLUSIONS: Our findings demonstrate a protective role for C3aR in the development of atherosclerosis and suggest that C3aR confers the protection through C3a/C3aR axis-mediated negative regulation of proinflammatory responses and modulation of macrophage toward the anti-inflammatory phenotype.