Atherosclerosis, the main underlying pathology of cardiovascular disease, is a chronic inflammatory disease characterized by lipid accumulation and immune cell responses in the vascular wall,... Show moreAtherosclerosis, the main underlying pathology of cardiovascular disease, is a chronic inflammatory disease characterized by lipid accumulation and immune cell responses in the vascular wall, resulting in plaque formation. It is well-known that atherosclerosis prevalence and manifestation vary by sex. However, sexual dimorphism in the immune landscape of atherosclerotic plaques has up to date not been studied at high-resolution. In this study, we investigated sex-specific differences in atherosclerosis development and the immunological landscape of aortas at single-cell level in aged Ldlr-/- mice.We compared plaque morphology between aged male and female chow diet-fed Ldlr-/- mice (22 months old) with histological analysis. Using single-cell RNA-sequencing and flow cytometry on CD45+ immune cells from aortas of aged Ldlr-/- mice, we explored the immune landscape in the atherosclerotic environment in males and females.We show that plaque volume is comparable in aged male and female mice, and that plaques in aged female mice contain more collagen and cholesterol crystals, but less necrotic core and macrophage content compared to males. We reveal increased immune cell infiltration in female aortas and found that expression of pro-atherogenic markers and inflammatory signaling pathways was enriched in plaque immune cells of female mice. Particularly, female aortas show enhanced activation of B cells (Egr1, Cd83, Cd180), including age-associated B cells, in addition to an increased M1/M2 macrophage ratio, where Il1b+ M1-like macrophages display a more pro-inflammatory phenotype (Nlrp3, Cxcl2, Mmp9) compared to males. In contrast, increased numbers of age-associated Gzmk+CD8+ T cells, dendritic cells, and Trem2+ macrophages were observed in male aortas.Altogether, our findings highlight that sex is a variable that contributes to immunological differences in the atherosclerotic plaque environment in mice and provide valuable insights for further preclinical studies into the impact of sex on the pathophysiology of atherosclerosis. Show less
Schaftenaar, F.H.; Dam, A.D. van; Bruin, G. de; Depuydt, M.A.C.; Mol, J.; Amersfoort, J.; ... ; Kuiper, J. 2024
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
Adaptive immunity plays a profound role in atherosclerosis pathogenesis by regulating antigen-specific responses, inflammatory signaling and antibody production. However, as we age, our immune... Show moreAdaptive immunity plays a profound role in atherosclerosis pathogenesis by regulating antigen-specific responses, inflammatory signaling and antibody production. However, as we age, our immune system undergoes a gradual functional decline, a phenomenon termed "immunosenescence". This decline is characterized by a reduction in proliferative naïve B- and T cells, decreased B- and T cell receptor repertoire and a pro-inflammatory senescence associated secretory profile. Furthermore, aging affects germinal center responses and deteriorates secondary lymphoid organ function and structure, leading to impaired T-B cell dynamics and increased autoantibody production. In this review, we will dissect the impact of aging on adaptive immunity and the role played by age-associated B- and T cells in atherosclerosis pathogenesis, emphasizing the need for interventions that target age-related immune dysfunction to reduce cardiovascular disease risk. Show less
Delfos, L.; Depuydt, M.A.C.; Foks, A.C.; Bernabe Kleijn, M.N.A.; Kuiper, J.; Chemaly, M.; ... ; Bot, I. 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
Mast cells have been associated with the progression and destabilization of advanced atherosclerotic plaques. Reducing intraplaque mast cell accumulation upon atherosclerosis progression could be a... Show moreMast cells have been associated with the progression and destabilization of advanced atherosclerotic plaques. Reducing intraplaque mast cell accumulation upon atherosclerosis progression could be a potent therapeutic strategy to limit plaque destabilization. Leukotriene B4 (LTB4) has been reported to induce mast cell chemotaxis in vitro. Here, we examined whether antagonism of the LTB4-receptor BLT1 could inhibit mast cell accumulation in advanced atherosclerosis. Expression of genes involved in LTB4 biosynthesis was determined by single-cell RNA sequencing of human atherosclerotic plaques. Subsequently, Western-type diet fed LDLr-/- mice with pre-existing atherosclerosis were treated with the BLT1-antagonist CP105,696 or vehicle control three times per week by oral gavage. In the spleen, a significant reduction in CD11b+ myeloid cells was observed, including Ly6Clo and Ly6Chi monocytes as well as dendritic cells. However, atherosclerotic plaque size, collagen and macrophage content in the aortic root remained unaltered upon treatment. Finally, BLT1 antagonism did not affect mast cell numbers in the aortic root. Here, we show that human intraplaque leukocytes may be a source of locally produced LTB4. However, BLT1-antagonism during atherosclerosis progression does not affect either local mast cell accumulation or plaque size, suggesting that other mechanisms participate in mast cell accumulation during atherosclerosis progression. Show less
Liu, F.; Aulin, L.B.S.; Kossen, S.S.A.; Cathalina, J.E.J.; Bremmer, M.; Foks, A.C.; ... ; Hasselt, J.G.C. van 2022
Sepsis is a life-threatening condition driven by the dysregulation of the host immune response to an infection. The complex and interacting mechanisms underlying sepsis remain not fully understood.... Show moreSepsis is a life-threatening condition driven by the dysregulation of the host immune response to an infection. The complex and interacting mechanisms underlying sepsis remain not fully understood. By integrating prior knowledge from literature using mathematical modelling techniques, we aimed to obtain a deeper mechanistic insight into sepsis pathogenesis and to evaluate promising novel therapeutic targets, with a focus on Toll-like receptor 4 (TLR4)-mediated pathways. A Boolean network of regulatory relationships was developed for key immune components associated with sepsis pathogenesis after TLR4 activation. Perturbation analyses were conducted to identify therapeutic targets associated with organ dysfunction or antibacterial activity. The developed model consisted of 42 nodes and 183 interactions. Perturbation analyses suggest that over-expression of tumour necrosis factor alpha (TNF-α) or inhibition of soluble receptor sTNF-R, tissue factor, and inflammatory cytokines (IFN-γ, IL-12) may lead to a reduced activation of organ dysfunction related endpoints. Over-expression of complement factor C3b and C5b led to an increase in the bacterial clearance related endpoint. We identified that combinatory blockade of IFN-γ and IL-10 may reduce the risk of organ dysfunction. Finally, we found that combining antibiotic treatment with IL-1β targeted therapy may have the potential to decrease thrombosis. In summary, we demonstrate how existing biological knowledge can be effectively integrated using Boolean network analysis for hypothesis generation of potential treatment strategies and characterization of biomarker responses associated with the early inflammatory response in sepsis. Show less
Currently immunomodulatory compounds are under investigation for use in patients with cardiovascular disease, caused by atherosclerosis. These trials, using recurrent cardiovascular events as... Show moreCurrently immunomodulatory compounds are under investigation for use in patients with cardiovascular disease, caused by atherosclerosis. These trials, using recurrent cardiovascular events as endpoint, require enrollment of large patient groups. We investigated the effect of key risk factors for atherosclerosis development, ageing and smoking, on the immune system, with the objective to identify biomarkers differentiating between human populations, and potentially serving as endpoints for future phase 1B trials with immunomodulatory compounds. Blood was collected from young healthy volunteers (aged 18-25 years, n=30), young smokers (18-25 years, n=20), elderly healthy volunteers (>60 years, n=20), heavy smokers (>45 years, 15 packyears, n=11) and patients with stable coronary artery disease (CAD) (>60 years, n=27). Circulating immune cell subsets were characterized by flow cytometry, and collected plasma was evaluated by proteomics (Olink). Clear ageing effects were observed, mostly illustrated by a lower level in CD8+ and naïve CD4+ and CD8+ T cells, with an increase in CD4+ and CD8+ effector memory T cells in elderly healthy volunteers compared to young healthy volunteers. Heavy smokers showed a more inflammatory cellular phenotype, especially a shift in Th1/Th2 ratio: higher Th1 and lower Th2 percentages compared to young healthy volunteers. A significant decrease in circulating atheroprotective oxLDL-specific IgM was found in patients with CAD compared to young healthy volunteers. Elevated pro-inflammatory and chemotactic proteins TREM1 and CCL11 were observed in elderly volunteers compared to young volunteers. In addition, heavy smokers had an increase in pro-inflammatory cytokine IL-6 and lysosomal protein LAMP3. These data show that ageing and smoking are associated with an inflammatory immunophenotype, and that heavy smokers or aged individuals may serve as potential populations for future clinical trials investigating immunomodulatory drugs targeted for cardiovascular disease. 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
Increasing evidence has shown that immune checkpoint molecules of the T-cell immunoglobulin and mucin domain (Tim) family are associated with diverse physiologic and pathologic processes. Previous... Show moreIncreasing evidence has shown that immune checkpoint molecules of the T-cell immunoglobulin and mucin domain (Tim) family are associated with diverse physiologic and pathologic processes. Previous studies of the role of Tim-1 in atherosclerosis using anti-Tim-1 antibodies have yielded contradictory results. We thus aimed to investigate atherosclerosis development in Tim-1 deficient mice.Mice with a specific loss of the Tim-1 mucin-domain (Tim-1Δmucin) and C57BL/6 (WT) mice received a single injection of a recombinant adeno-associated virus encoding murine Pcsk9 (rAAV2/8-D377Y-mPcsk9) and were fed a Western type diet for 13 weeks to introduce atherosclerosis.Tim-1Δmucin mice developed significantly larger lesions in the aortic root compared to WT mice, with significantly more macrophages and a trend towards a larger necrotic core. Furthermore, Tim-1Δmucin mice showed a significant loss of IL-10+ B cells and regulatory B cell subsets and increased pro-atherogenic splenic follicular B cells compared to WT mice. Moreover, Tim-1Δmucin mice displayed a dramatic reduction in Th2-associated immune response compared to controls but we did not observe any changes in humoral immunity.In summary, Tim-1Δmucin mice displayed a profound impairment in IL-10+ B cells and an imbalance in the Th1/Th2 ratio, which associated with exacerbated atherosclerosis. Show less
Mol, J. de; Postel, R.J.; Smit, V.; Bernabé Kleijn, M.N.A.; Bot, I.; Kuiper, J.; 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
Douna, H.; Mol, J. de; Amersfoort, J.; Schaftenaar, F.H.; Kiss, M.G.; Suur, B.E.; ... ; Foks, A.C. 2022
B and T cells are interconnected in the T follicular helper-germinal center B cell (TFH-GC B cell) axis, which is hyperactive during atherosclerosis development and loss of control along this axis... Show moreB and T cells are interconnected in the T follicular helper-germinal center B cell (TFH-GC B cell) axis, which is hyperactive during atherosclerosis development and loss of control along this axis results in exacerbated atherosclerosis. Inhibition of the TFH-GC B cell axis can be achieved by providing negative co-stimulation to TFH cells through the PD-1/PD-L1 pathway. Therefore, we investigated a novel therapeutic strategy using PD-L1-expressing B cells to inhibit atherosclerosis. We found that IFNγ-stimulated B cells significantly enhanced PD-L1 expression and limited TFH cell development. To determine whether IFNγ-B cells can reduce collar-induced atherosclerosis, apoE -/- mice fed a Western-type diet were treated with PBS, B cells or IFNγ-B cells for a total of 5 weeks following collar placement. IFNγ-B cells significantly increased PD-L1hi GC B cells and reduced plasmablasts. Interestingly, IFNγ-B cells-treated mice show increased atheroprotective Tregs and T cell-derived IL-10. In line with these findings, we observed a significant reduction in total lesion volume in carotid arteries of IFNγ-B cells-treated mice compared to PBS-treated mice and a similar trend was observed compared to B cell-treated mice. In conclusion, our data show that IFNγ-stimulated B cells strongly upregulate PD-L1, inhibit TFH cell responses and protect against 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
Ingen, E. van; Foks, A.C.; Woudenberg, T.; Bent, M.L. van der; Jong, A. de; Hohensinner, P.J.; ... ; Nossent, A.Y. 2021
We have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in... Show moreWe have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in early and established plaques, with reduced macro-phage content in established plaques. Within the plaque, different subtypes of macrophages are present. Here, we aimed to investigate whether miR-494-3p directly influences macrophage polarization and activation. Human macrophages were polarized into either proinflammatory M1 or anti-inflammatory M2 macrophages and simultaneously treated with 3GA-494 or a control antisense (3GA-ctrl). We show that 3GA-494 treatment inhibited miR-494-3p in M1 macrophages and dampened Ml polarization, while in M2 macrophages miR-494-3p expression was induced and M2 polarization enhanced. The proinflammatory marker CCR2 was reduced in 3GA-494-treated atherosclerosis-prone mice. Pathway enrichment analysis predicted an overlap between miR-494-3p target genes in macrophage polarization and Wnt signaling. We demonstrate that miR-494-3p regulates expression levels of multiple Wnt signaling components, such as LRP6 and TBL1X. Wnt signaling appears activated upon treatment with 3GA-494, both in cultured M1 macrophages and in plaques of hypercholesterolemic mice. Taken together, 3GA-494 treatment dampened M1 polarization, at least in part via activated Wnt signaling, while M2 polarization was enhanced, which is both favorable in reducing atherosclerotic plaque formation and increasing plaque stability. Show less
Mol, J. de; Kuiper, J.; Tsiantoulas, D.; Foks, A.C. 2021
Aging is considered to be an important risk factor for several inflammatory diseases. B cells play a major role in chronic inflammatory diseases by antibody secretion, antigen presentation and T... Show moreAging is considered to be an important risk factor for several inflammatory diseases. B cells play a major role in chronic inflammatory diseases by antibody secretion, antigen presentation and T cell regulation. Different B cell subsets have been implicated in infections and multiple autoimmune diseases. Since aging decreases B cell numbers, affects B cell subsets and impairs antibody responses, the aged B cell is expected to have major impacts on the development and progression of these diseases. In this review, we summarize the role of B cells in health and disease settings, such as atherosclerotic disease. Furthermore, we provide an overview of age-related changes in B cell development and function with respect to their impact in chronic inflammatory diseases. Show less
Brown fat activation attenuates atherosclerosis development by accelerating triglyceride-rich lipoprotein turnover and/or stimulation of reverse cholesterol transport via the SRB1 (scavenger... Show moreBrown fat activation attenuates atherosclerosis development by accelerating triglyceride-rich lipoprotein turnover and/or stimulation of reverse cholesterol transport via the SRB1 (scavenger receptor class B type 1). The aim of this study was to investigate the specific role of hepatic SRB1 in the atheroprotective properties of brown fat activation.APOE*3-Leiden.CETP mice, a well-established model of human-like lipoprotein metabolism andatherosclerosis, were treated with vehicle or adenoassociated virus serotype 8-short hairpin RNA, which decreased hepatic SRB1 protein levels by 40% to 55%. After 2 weeks, mice without or with hepatic SRB1 knockdown were treated with vehicle or the β3-adrenergic receptor agonist CL316 243 to activate brown fat for 4 weeks to determine HDL (highdensity lipoprotein) catabolism and for 9 weeks to evaluate atherosclerosis. Surprisingly, hepatic SRB1 knockdown additively improved the beneficial effects of β3-adrenergic receptor agonism on atherosclerosis development. In fact, hepatic SRB1 knockdown per se not only increased HDL-cholesterol levels but also reduced plasma triglyceride and non-HDL-cholesterol levels, thus explaining the reduction in atherosclerosis development. Mechanistic studies indicated that this is due to increased lipolytic processing and hepatic uptake of VLDL (very low density lipoprotein) by facilitating VLDL-surface transfer to HDL.Hepatic SRB1 knockdown in a mouse model with an intact ApoE (apolipoprotein E)-LDLR (low density lipoprotein receptor) clearance pathway, relevant to human lipoprotein metabolism, reduced atherosclerosis and improved the beneficial effect of brown fat activation on atherosclerosis development, explained by pleiotropic effects of hepatic SRB1 knockdown on lipolytic processing and hepatic uptake of VLDL. Brown fat activation could thus be an effective strategy to treat cardiovascular disease also in subjects with impaired SRB1 function. Show less
