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... Show moreThe 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.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.Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production. Show less
Elieh-Ali-Komi, D.; Bot, I.; Rodríguez-González, M.; Maurer, M. 2024
Mast cells (MCs) are commonly recognized for their crucial involvement in the pathogenesis of allergic diseases, but over time, it has come to light that they also play a role in the... Show moreMast cells (MCs) are commonly recognized for their crucial involvement in the pathogenesis of allergic diseases, but over time, it has come to light that they also play a role in the pathophysiology of non-allergic disorders including atherosclerosis. The involvement of MCs in the pathology of atherosclerosis is supported by their accumulation in atherosclerotic plaques upon their progression and the association of intraplaque MC numbers with acute cardiovascular events. MCs that accumulate within the atherosclerotic plaque release a cocktail of mediators through which they contribute to neovascularization, plaque progression, instability, erosion, rupture, and thrombosis. At a molecular level, MC-released proteases, especially cathepsin G, degrade low-density lipoproteins (LDL) and mediate LDL fusion and binding of LDL to proteoglycans (PGs). Through a complicated network of chemokines including CXCL1, MCs promote the recruitment of among others CXCR2+ neutrophils, therefore, aggravating the inflammation of the plaque environment. Additionally, MCs produce extracellular traps which worsen inflammation and contribute to atherothrombosis. Altogether, evidence suggests that MCs actively, via several underlying mechanisms, contribute to atherosclerotic plaque destabilization and acute cardiovascular syndromes, thus, making the study of interventions to modulate MC activation an interesting target for cardiovascular medicine. Show less
Delfos, L.; Depuydt, M.A.C.; Foks, A.C.; Bernabe Kleijn, M.N.A.; Kuiper, J.; Chemaly, M.; ... ; Bot, I. 2023
Circadian disturbance (CD) is the consequence of a mismatch between endogenous circadian rhythms, behaviour, and/or environmental cycles, and frequently occurs during shift work. Shift work has... Show moreCircadian disturbance (CD) is the consequence of a mismatch between endogenous circadian rhythms, behaviour, and/or environmental cycles, and frequently occurs during shift work. Shift work has been associated with elevated risk for atherosclerotic cardiovascular disease (asCVD) in humans, but evidence for the effectiveness of prevention strategies is lacking.\nHere, we applied time-restricted feeding (TRF) as a strategy to counteract atherosclerosis development during CD in female APOE∗3-Leiden.CETP mice, a well-established model for humanized lipoprotein metabolism. Control groups were subjected to a fixed 12:12 h light-dark cycle, while CD groups were subjected to 6-h phase advancement every 3 days. Groups had either ad libitum (AL) access to food or were subjected to TRF with restricted food access to the dark phase.\nTRF did not prevent the increase in the relative abundance of circulating inflammatory monocytes and elevation of (postprandial) plasma triglycerides during CD. Nonetheless, TRF reduced atherosclerotic lesion size and prevented an elevation in macrophage content of atherosclerotic lesions during CD, while it increased the relative abundance of anti-inflammatory monocytes, prevented activation of T cells, and lowered plasma total cholesterol levels and markers of hepatic cholesterol synthesis. These effects were independent of total food intake.\nWe propose that time restricted eating could be a promising strategy for the primary prevention of asCVD risk in shift workers, which warrants future study in humans.\nThis work was funded by the Novo Nordisk Foundation, the Netherlands Ministry of Social Affairs and Employment, Amsterdam Cardiovascular Sciences, and the Dutch Heart Foundation. Show less
Panhuis, W.I.H.; Schönke, M.; Modder, M.; Tom, H.E.; Lalai, R.A.; Pronk, A.C.M.; ... ; Kooijmana, S. 2023
BackgroundCircadian disturbance (CD) is the consequence of a mismatch between endogenous circadian rhythms, behaviour, and/or environmental cycles, and frequently occurs during shift work. Shift... Show moreBackgroundCircadian disturbance (CD) is the consequence of a mismatch between endogenous circadian rhythms, behaviour, and/or environmental cycles, and frequently occurs during shift work. Shift work has been associated with elevated risk for atherosclerotic cardiovascular disease (asCVD) in humans, but evidence for the effectiveness of prevention strategies is lacking.MethodsHere, we applied time-restricted feeding (TRF) as a strategy to counteract atherosclerosis development during CD in female APOE∗3-Leiden.CETP mice, a well-established model for humanized lipoprotein metabolism. Control groups were subjected to a fixed 12:12 h light–dark cycle, while CD groups were subjected to 6-h phase advancement every 3 days. Groups had either ad libitum (AL) access to food or were subjected to TRF with restricted food access to the dark phase.FindingsTRF did not prevent the increase in the relative abundance of circulating inflammatory monocytes and elevation of (postprandial) plasma triglycerides during CD. Nonetheless, TRF reduced atherosclerotic lesion size and prevented an elevation in macrophage content of atherosclerotic lesions during CD, while it increased the relative abundance of anti-inflammatory monocytes, prevented activation of T cells, and lowered plasma total cholesterol levels and markers of hepatic cholesterol synthesis. These effects were independent of total food intake.InterpretationWe propose that time restricted eating could be a promising strategy for the primary prevention of asCVD risk in shift workers, which warrants future study in humans.FundingThis work was funded by the Novo Nordisk Foundation, the Netherlands Ministry of Social Affairs and Employment, Amsterdam Cardiovascular Sciences, and the Dutch Heart Foundation. 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
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
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
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
(Ultra) Small superparamagnetic iron oxide nanoparticles, (U)SPIO, are widely used as magnetic resonance imaging contrast media and assumed to be safe for clinical applications in cardiovascular... Show more(Ultra) Small superparamagnetic iron oxide nanoparticles, (U)SPIO, are widely used as magnetic resonance imaging contrast media and assumed to be safe for clinical applications in cardiovascular disease. As safety tests largely relied on normolipidemic models, not fully representative of the clinical setting, we investigated the impact of (U)SPIOs on disease-relevant endpoints in hyperlipidemic models of atherosclerosis.RAW264.7 foam cells, exposed in vitro to Ferumoxide (dextran-coated SPIO), Ferumoxtran (dextran-coated USPIO), or Ferumoxytol (carboxymethyl dextran-coated USPIO) (all 1 mg Fe/ml) showed increased apoptosis and ROS accumulation for Ferumoxide and Ferumoxtran, whereas Ferumoxytol was tolerated well. Pro-apoptotic (TUNEL+) and pro-oxidant activity of Ferumoxide (0.3 mg Fe/kg) and Ferumoxtran (1 mg Fe/kg) were confirmed in plaque, spleen, and liver of hyperlipidemic ApoE-/- (n = 9/group) and LDLR-/- (n = 9-16/group) mice that had received single IV injections compared to saline-treated controls. Again, Ferumoxytol treatment (1 mg Fe/kg) failed to induce apoptosis or oxidative stress in these tissues. Concomitant antioxidant treatment (EUK-8/EUK-134) largely prevented these effects in vitro (-68%, P < 0.05) and in plaques from LDLR-/- mice (-60%, P < 0.001, n = 8/group). Repeated Ferumoxtran injections of LDLR-/- mice with pre-existing atherosclerosis enhanced plaque inflammation and apoptosis but did not alter plaque size. Strikingly, carotid artery plaques of endarterectomy patients who received Ferumoxtran (2.6 mg Fe/kg) before surgery (n = 9) also showed 5-fold increased apoptosis (18.2 vs. 3.7% respectively; P = 0.004) compared to controls who did not receive Ferumoxtran. Mechanistically, neither coating nor particle size seemed accountable for the observed cytotoxicity of Ferumoxide and Ferumoxtran.Ferumoxide and Ferumoxtran, but not Ferumoxytol, induced apoptosis of lipid-laden macrophages in human and murine atherosclerosis, potentially impacting disease progression in patients with advanced atherosclerosis. Show less
Aims (Ultra) Small superparamagnetic iron oxide nanoparticles, (U)SPIO, are widely used as magnetic resonance imaging contrast media and assumed to be safe for clinical applications in... Show moreAims (Ultra) Small superparamagnetic iron oxide nanoparticles, (U)SPIO, are widely used as magnetic resonance imaging contrast media and assumed to be safe for clinical applications in cardiovascular disease. As safety tests largely relied on normolipidaemic models, not fully representative of the clinical setting, we investigated the impact of (U)SPIOs on disease-relevant endpoints in hyperlipidaemic models of atherosclerosis. Methods and results RAW264.7 foam cells, exposed in vitro to ferumoxide (dextran-coated SPIO), ferumoxtran (dextran-coated USPIO), or ferumoxytol [carboxymethyl (CM) dextran-coated USPIO] (all 1 mg Fe/mL) showed increased apoptosis and reactive oxygen species accumulation for ferumoxide and ferumoxtran, whereas ferumoxytol was tolerated well. Pro-apoptotic (TUNEL+) and pro-oxidant activity of ferumoxide (0.3 mg Fe/kg) and ferumoxtran (1 mg Fe/kg) were confirmed in plaque, spleen, and liver of hyperlipidaemic ApoE(-/-) (n = 9/group) and LDLR-/- (n = 9-16/group) mice that had received single IV injections compared with saline-treated controls. Again, ferumoxytol treatment (1 mg Fe/kg) failed to induce apoptosis or oxidative stress in these tissues. Concomitant antioxidant treatment (EUK-8/EUK-134) largely prevented these effects in vitro (-68%, P < 0.05) and in plaques from LDLR-/- mice (-60%, P < 0.001, n = 8/group). Repeated ferumoxtran injections of LDLR-/- mice with pre-existing atherosclerosis enhanced plaque inflammation and apoptosis but did not alter plaque size. Strikingly, carotid artery plaques of endarterectomy patients who received ferumoxtran (2.6 mg Fe/kg) before surgery (n = 9) also showed five-fold increased apoptosis (18.2 vs. 3.7%, respectively; P = 0.004) compared with controls who did not receive ferumoxtran. Mechanistically, neither coating nor particle size seemed accountable for the observed cytotoxicity of ferumoxide and ferumoxtran. Conclusions Ferumoxide and ferumoxtran, but not ferumoxytol, induced apoptosis of lipid-laden macrophages in human and murine atherosclerosis, potentially impacting disease progression in patients with 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
Velden, J. van der; Asselbergs, F.W.; Bakkers, J.; Batkai, S.; Bertrand, L.; Bezzina, C.R.; ... ; Thum, T. 2022
Cardiovascular diseases represent a major cause of morbidity and mortality, necessitating research to improve diagnostics, and to discover and test novel preventive and curative therapies. All of... Show moreCardiovascular diseases represent a major cause of morbidity and mortality, necessitating research to improve diagnostics, and to discover and test novel preventive and curative therapies. All of which warrant experimental models that recapitulate human disease. The translation of basic science results to clinical practice is a challenging task. In particular for complex conditions such as cardiovascular diseases, which often result from multiple risk factors and co-morbidities. This difficulty might lead some individuals to question the value of animal research, citing the translational 'valley of death', which largely reflects the fact that studies in rodents are difficult to translate to humans. This is also influenced by the fact that new, human-derived in vitro models can recapitulate aspects of disease processes. However, it would be a mistake to think that animal models cannot provide a vital step in the translational pathway as they do provide important pathophysiological insights into disease mechanisms particularly on a organ and systemic level. While stem cell-derived human models have the potential to become key in testing toxicity and effectiveness of new drugs, we need to be realistic, and carefully validate all new human-like disease models. In this position paper, we highlight recent advances in trying to reduce the number of animals for cardiovascular research ranging from stem cell-derived models to in situ modelling of heart properties, bioinformatic models based on large datasets, and improved current animal models, which show clinically relevant characteristics observed in patients with a cardiovascular disease. We aim to provide a guide to help researchers in their experimental design to translate bench findings to clinical routine taking the replacement, reduction and refinement (3R) as a guiding concept. 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
