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... Show moreAtherosclerosis 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.We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit β5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit β1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice.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.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. Show less
Smit, V.; Mol, J. de; Schaftenaar, F.H.; Depuydt, M.A.C.; Postel, R.J.; Smeets, D.: Verheijen, F.W.M.; ... ; Foks, A.C. 2023
Aging is a dominant driver of atherosclerosis and induces a series of immunological alterations, called immunosenescence. Given the demographic shift towards elderly, elucidating the unknown impact... Show moreAging is a dominant driver of atherosclerosis and induces a series of immunological alterations, called immunosenescence. Given the demographic shift towards elderly, elucidating the unknown impact of aging on the immunological landscape in atherosclerosis is highly relevant. While the young Western diet-fed Ldlr-deficient (Ldlr-/-) mouse is a widely used model to study atherosclerosis, it does not reflect the gradual plaque progression in the context of an aging immune system as occurs in humans.\nHere, we show that aging promotes advanced atherosclerosis in chow diet-fed Ldlr-/- mice, with increased incidence of calcification and cholesterol crystals. We observed systemic immunosenescence, including myeloid skewing and T-cells with more extreme effector phenotypes. Using a combination of single-cell RNA-sequencing and flow cytometry on aortic leukocytes of young versus aged Ldlr-/- mice, we show age-related shifts in expression of genes involved in atherogenic processes, such as cellular activation and cytokine production. We identified age-associated cells with pro-inflammatory features, including GzmK+CD8+ T-cells and previously in atherosclerosis undefined CD11b+CD11c+T-bet+ age-associated B-cells (ABCs). ABCs of Ldlr-/- mice showed high expression of genes involved in plasma cell differentiation, co-stimulation, and antigen presentation. In vitro studies supported that ABCs are highly potent antigen-presenting cells. In cardiovascular disease patients, we confirmed the presence of these age-associated T- and B-cells in atherosclerotic plaques and blood.\nCollectively, we are the first to provide comprehensive profiling of aged immunity in atherosclerotic mice and reveal the emergence of age-associated T- and B-cells in the atherosclerotic aorta. Further research into age-associated immunity may contribute to novel diagnostic and therapeutic tools to combat cardiovascular disease. Show less
Atherosclerosis is characterized by the accumulation of lipids and immune cells, including mast cells and B cells, in the arterial wall. Mast cells contribute to atherosclerotic plaque growth and... Show moreAtherosclerosis is characterized by the accumulation of lipids and immune cells, including mast cells and B cells, in the arterial wall. Mast cells contribute to atherosclerotic plaque growth and destabilization upon active degranulation. The FcεRI-IgE pathway is the most prominent mast cell activation route. Bruton's Tyrosine Kinase (BTK) is involved in FcεRI-signaling and may be a potential therapeutic target to limit mast cell activation in atherosclerosis. Additionally, BTK is crucial in B cell development and B-cell receptor signaling. In this project, we aimed to assess the effects of BTK inhibition on mast cell activation and B cell development in atherosclerosis. In human carotid artery plaques, we showed that BTK is primarily expressed on mast cells, B cells and myeloid cells. In vitro, BTK inhibitor Acalabrutinib dose-dependently inhibited IgE mediated activation of mouse bone marrow derived mast cells. In vivo, male Ldlr-/- mice were fed a high-fat diet for eight weeks, during which mice were treated with Acalabrutinib or control solvent. In Acalabrutinib treated mice, B cell maturation was reduced compared to control mice, showing a shift from follicular II towards follicular I B cells. Mast cell numbers and activation status were not affected. Acalabrutinib treatment did not affect atherosclerotic plaque size or morphology. In advanced atherosclerosis, where mice were first fed a high-fat diet for eight weeks before receiving treatment, similar effects were observed. Conclusively, BTK inhibition by Acalabrutinib alone did neither affect either mast cell activation nor early- and advanced atherosclerosis, despite the effects on follicular B cell maturation. Show less
Atherosclerosis is a lipid-driven chronic infammatory disease; however, whether it can be classifed as an autoimmune disease remains unclear. In this study, we applied single-cell T cell receptor... Show moreAtherosclerosis is a lipid-driven chronic infammatory disease; however, whether it can be classifed as an autoimmune disease remains unclear. In this study, we applied single-cell T cell receptor seqencing (scTCR-seq) on human carotid artery plaques and matched peripheral blood mononuclear cell samples to assess the extent of TCR clonality and antigen-specifc activation within the various T cell subsets. We observed the highest degree of plaque-specifc clonal expansion in efector CD4+ T cells, and these clonally expanded T cells expressed genes such as CD69, FOS and FOSB, indicative of recent TCR engagement, suggesting antigen-specifc stimulation. CellChat analysis suggested multiple potential interactions of these efector CD4+ T cells with foam cells. Finally, we integrated a published scTCR-seq dataset of the autoimmune disease psoriatic arthritis, and we report various commonalities between the two diseases. In conclusion, our data suggest that atherosclerosis has an autoimmune compondent driven by autoreactive CD4+ T cells. Show less
Postel, R.J.; Smit, V.; De Mol, J.; Bernabé Kleijn, M.N.A.; De Jong, M.J.M.; Delfos, L.; ... ; Foks, A.C. 2022
Aging is associated with changes in the composition and function of the immune system, such as decreased B cell numbers and reduced antibody responses (1). In addition, aging is related to an... Show moreAging is associated with changes in the composition and function of the immune system, such as decreased B cell numbers and reduced antibody responses (1). In addition, aging is related to an increased incidence of inflammatory diseases, such as the lipid-driven chronic inflammatory disease atherosclerosis, the main underlying cause of cardiovascular disease (2). B cells play a major role in atherosclerosis progression by antibody secretion, antigen presentation and T cell regulation. Different B cell subsets, with either proatherogenic or antiatherogenic properties, have been identified in atherosclerosis, but the impact of aging on B cells during atherosclerosis remains to be elucidated. In contrast to young atherosclerotic mice where few B cells are found within the plaque, single cell RNA sequencing and flow cytometry data from our group revealed that B cells are one of the most dominant leukocytes present in atherosclerotic aortas from naturally aged LDLr-/- mice. In this study, we aimed to gain further insights into the role of these aged B cells on T cell immunity in atherosclerosis by depleting B cells in aged atherosclerotic mice. Aged (85-95 weeks) atherosclerotic male LDLr-/- mice were kept on a chow diet for six weeks, during which the mice received 250 μg B cell-depleting anti-CD20 antibody (Genentech) (n=12) or 250 μg rat IgG2a isotype control (n=12) once a week intraperitoneally. Mice that received the anti-CD20 antibody showed effective B cell depletion in the blood and lymphoid organs, such as the spleen and lymph nodes, but also locally in the atherosclerotic plaque. In addition, B cell depleted mice showed a significant increase in the percentage of T-bet expressing CD4+ T cells in aortic plaques compared to control mice (αCD20: 25.05±2.88% vs. ctrl: 15.94±2.14%, p<0.05). Furthermore, the percentage of RORyt+ and IL-17+ CD4+ T cells in the spleen and heart draining lymph nodes was increased after B cell depletion, indicating a shift towards Th17 differentiation.Collectively, we show that CD20+ B cell depletion in aged LDLr-/- mice skews CD4+ T cells towards a Th1 phenotype in the atherosclerotic plaque and towards a Th17 phenotype in lymphoid organs, suggesting a protective role for aged B cells in atherosclerosis. However, B cells are a heterogeneous population and further research should elucidate whether aged B cells exert a pro- or anti-atherogenic role. In conclusion, our results indicate that aged B cells have an important function in CD4+ T cell differentiation and could be attractive targets to combat age-related cardiovascular disease. Show less
Acute cardiovascular diseases, such as myocardial infarction or stroke, are still a major cause of death in Western Society. The main underlying pathology of cardiovascular diseases is... Show moreAcute cardiovascular diseases, such as myocardial infarction or stroke, are still a major cause of death in Western Society. The main underlying pathology of cardiovascular diseases is atherosclerosis, which is caused by the accumulation of lipids and inflammatory cells in the vessel wall, in so-called atherosclerotic plaques. Mast cells accumulate within these atherosclerotic plaques and activation of mast cells leads to the progression and destabilization of advanced plaques via the secretion of pro-inflammatory mediators and cytokines. Mast cells can be activated by various stimuli, of which crosslinking of the Fce receptor I (FceRI) with IgE-antigen complexes is best known. Bruton’s tyrosine kinase (BTK), a cytoplasmic nonreceptor tyrosine kinase, is involved in the downstream signaling of FceRI-mediated mast cell activation and degranulation. Therefore, BTK might be an attractive target to interfere in the FceRI-mediated mast cell activation pathway. In this study, we thus aimed to assess the effects of the BTK inhibitor ACP-196 on FceRI-mediated mast cell activation, plaque progression and destabilization in an atherosclerotic mouse model.Male LDLr knockout mice, 7-11 weeks old, were treated with ACP-196 (25 mg/kg p.o., n=15) or control solvent (n=14) three times per week for eight weeks. During treatment, mice were fed a Western-type diet (WTD) to induce atherosclerotic plaque formation. During the experiment, plasma total cholesterol levels and body weight did not differ between the control and treatment group. After eight weeks, mice were sacrificed and hearts were isolated to determine atherosclerotic plaque size and stability in the aortic root by histology. Other immunological relevant tissues, such as aorta, spleen and mediastinal lymph nodes were harvested to examine mast cell activation status and other immune cells by flow cytometry. After eight weeks of ACP-196 treatment in LDLr knockout mice, a significant 59% reduction in the frequency of CD117+ FceRI+ mast cells was observed in aortic plaques of ACP-196 treated mice (0.24±0.06%) compared to control mice (0.57 ±0.08%, p<0.05), while relative mast cell activation status was not affected. Additionally, ACP-196 treatment inhibited B cell maturation in the circulation, spleen, mediastinal lymph nodes and peritoneal cavity of LDLr knockout mice compared to control mice. However, these effects on immune cells did not translate into effects on atherosclerosis, as ACP-196 treatment (size:12.3±2%; collagen:14.5±1.9%) did not significantly affect atherosclerotic plaque size and collagen content when compared to control mice (size:11.5±1.4%; collagen: 13.6±1.5%).Conclusively, these findings suggest that ACP-196 treatment leads to reduced migration of mast cells to the atherosclerotic plaques of LDLr knockout mice, but does not directly affect mast cell activation and initial atherosclerotic lesion development. Show less
Cytotoxic CD4+ T cells have previously been found in peripheral blood of patients with coronary artery disease (1), however their occurrence in atherosclerotic plaques and their association with... Show moreCytotoxic CD4+ T cells have previously been found in peripheral blood of patients with coronary artery disease (1), however their occurrence in atherosclerotic plaques and their association with the pathophysiology of atherosclerosis has not been established. Single-cell RNA sequencing was performed on human carotid atherosclerotic plaques of 18 patients to identify specific T cell populations (2). Next, human femoral and carotid atherosclerotic plaques (n=95) and matched blood samples (n=49) were analyzed by flow cytometry for the presence of CD4+GZMB+ T cells. Plaque morphology was assessed by Movat’s Pentachrome staining. A distinct cytotoxic GZMB+ PRF1+ CD28- CD4+ T cell cluster was identified using single-cell RNA sequencing. Furthermore, flow cytometry analysis showed that the percentage of GZMB+CD4+ T cells was significantly elevated in plaque compared to blood (Blood: 12.17±2.0 vs. Plaque: 17.40±1.0; P=0.0002). Moreover, a significant positive correlation was observed between the percentage of GZMB+CD4+ T cells in blood versus plaque (P=0.031). In line with the proinflammatory character of these cells, we found a positive association of GZMB+CD4+ T cells (P=0.036) with necrotic core size, whereas no correlation was found with this subtype in the circulation. In this study we have shown an enrichment of cytotoxic CD4+ T cells in atherosclerotic lesions, which positively correlate with necrotic core size. Future studies are aimed at elucidating the role of these cells in advanced atherosclerosis. Show less
Signaling through the coinhibitory programmed death (PD)-1/PD-L1 pathway regulates T cell responses and can inhibit ongoing immune responses. Inflammation is a key process in the development of... Show moreSignaling through the coinhibitory programmed death (PD)-1/PD-L1 pathway regulates T cell responses and can inhibit ongoing immune responses. Inflammation is a key process in the development of atherosclerosis, the underlying cause for the majority of cardiovascular diseases. Dampening the excessive immune response that occurs during atherosclerosis progression by promoting PD-1/PD-L1 signaling may have a high therapeutic potential to limit disease burden. In this study we therefore aimed to assess whether an agonistic PD-1 antibody can diminish atherosclerosis development.Ldlr-/- mice were fed a western-type diet (WTD) while receiving 100 μg of an agonistic PD-1 antibody or control vehicle twice a week. Stimulation of the PD-1 pathway delayed the WTD-induced monocyte increase in the circulation up to 3 weeks and reduced T cell activation and proliferation. CD4+ T cell numbers in the atherosclerotic plaque were reduced upon PD-1 treatment. More specifically, we observed a 23% decrease in atherogenic IFNγ-producing splenic CD4+ T cells and a 20% decrease in cytotoxic CD8+ T cells, whereas atheroprotective IL-10 producing CD4+ T cells were increased with 47%. Furthermore, we found an increase in regulatory B cells, B1 cells and associated atheroprotective circulating oxLDL-specific IgM levels in agonistic PD-1-treated mice. This dampened immune activation following agonistic PD-1 treatment resulted in reduced atherosclerosis development (p < 0.05).Our data show that stimulation of the coinhibitory PD-1 pathway inhibits atherosclerosis development by modulation of T- and B cell responses. These data support stimulation of coinhibitory pathways as a potential therapeutic strategy to combat atherosclerosis. Show less
The immune system is strongly involved in atherosclerosis and immune regulation generally leads to attenuated atherosclerosis. B and T lymphocyte attenuator (BTLA) is a novel co-receptor that... Show moreThe immune system is strongly involved in atherosclerosis and immune regulation generally leads to attenuated atherosclerosis. B and T lymphocyte attenuator (BTLA) is a novel co-receptor that negatively regulates the activation of B and T cells, however, there have been no reports of BTLA and its function in atherosclerosis or cardiovascular disease (CVD). We aimed to assess the dominant BTLA expressing leukocyte in CVD patients and to investigate whether BTLA has a functional role in experimental atherosclerosis. Stimulation of the BTLA pathway in Ldlr-/-mice reduces initial lesion development and increases collagen content of established lesions, presumably by shifting the balance between atherogenic follicular B cells and atheroprotective B cells and directing CD4+ T cells towards regulatory T cells. We provide the first evidence that BTLA is a very promising target for the treatment of atherosclerosis.We show that BTLA is primarily expressed on B cells in CVD patients and follicular B2 cells in low-density lipoprotein receptor-deficient (Ldlr-/-) mice. We treated Ldlr-/- mice that were fed a Western-type diet (WTD) with PBS, an isotype antibody or an agonistic BTLA antibody (3C10) for 6 weeks. We report here that the agonistic BTLA antibody significantly attenuated atherosclerosis. This was associated with a strong reduction in follicular B2 cells, while regulatory B and T cells were increased. The BTLA antibody showed similar immunomodulating effects in a progression study in which Ldlr-/- mice were fed a WTD for 10 weeks before receiving antibody treatment. Most importantly, BTLA stimulation enhanced collagen content, a feature of stable lesions, in preexisting lesions. Show less
