Modulation of the immune system for treatment of atherosclerosis

Cardiovascular diseases are the primary cause of death in the world with atherosclerosis as primary underlying cause. Atherosclerosis is characterized by cholesterol accumulation in the vessel wall and inflammation of the vessel wall of medium to large size arteries. Both cholesterol accumulation and inflammation are pathogenic in the context of atherosclerosis. Current treatment regimens are tailored to reduce cholesterol levels in the blood. However, even a successful lowering of cholesterol is in many patients not sufficient to prevent a major cardiovascular event due to unresolved inflammation. Therefore, the immune system provides an interesting therapeutic target for the treatment of atherosclerosis. In this thesis we have explored the effect on atherosclerosis of several immunomodulatory strategies in pre-clinical models.As cholesterol is not soluble in water, cholesterol is transported in the bloodstream in particles called lipoproteins. The low-density lipoprotein (LDL) carries the highest concentration of cholesterol and accumulates in the vessel wall where a pathogenic specific immune response against LDL is instigated. In this thesis we have used several strategies to modulate the specific immune response against LDL, inducing LDL-specific regulatory T cells, antibodies, and cytotoxic T cells. Through immunoproteasomal inhibition we assessed the effect of general immune inhibition on atherosclerosis.This research received support from the European Union’s Seventh Framework Programme (FP7/ 2007-2013) under grant agreement VIA no. 603131, which was also supported by financial contribution from Academic and SME/industrial partners. We further acknowledge the support from the Netherlands CardioVascular Research Initiative: the Dutch Heart Foundation, Dutch Federation of University Medical Centres, the Netherlands Organisation for Health Research and Development, and the Royal Netherlands Academy of Sciences for funding the the GENIUS II project (CVON2017-2020). Financial support by the Dutch Heart Foundation for the publication of this thesis is gratefully acknowledged. The realization of this thesis was also financially supported by the Leiden University.Biopharmaceutic

Inhibition of lysophosphatidic acid receptors 1 and 3 attenuates atherosclerosis development in LDL-receptor deficient mice

Lysophosphatidic acid (LPA) is a natural lysophospholipid present at high concentrations within lipid-rich atherosclerotic plaques. Upon local accumulation in the damaged vessels, LPA can act as a potent activator for various types of immune cells through its specific membrane receptors LPA1/3. LPA elicits chemotactic, pro-inflammatory and apoptotic effects that lead to atherosclerotic plaque progression. In this study we aimed to inhibit LPA signaling by means of LPA1/3 antagonism using the small molecule Ki16425. We show that LPA1/3 inhibition significantly impaired atherosclerosis progression. Treatment with Ki16425 also resulted in reduced CCL2 production and secretion, which led to less monocyte and neutrophil infiltration. Furthermore, we provide evidence that LPA1/3 blockade enhanced the percentage of non-inflammatory, Ly6Clow monocytes and CD4+ CD25+ FoxP3+ T-regulatory cells. Finally, we demonstrate that LPA1/3 antagonism mildly reduced plasma LDL cholesterol levels. Therefore, pharmacological inhibition of LPA1/3 receptors may prove a promising approach to diminish atherosclerosis development.Biopharmaceutic

Apolipoprotein A1 deficiency in mice primes bone marrow stem cells for T cell lymphopoiesis

The bone marrow has emerged as a potentially important target in cardiovascular disease as it generates all leukocytes involved in atherogenesis. In the current study, we evaluated whether a change in bone marrow functionality underlies the increased atherosclerosis susceptibility associated with high-density lipoprotein (HDL) deficiency. We found that HDL deficiency in mice due to the genetic lack of hepatocyte-derived apolipoprotein A1 (APOA1) was associated with an increase in the Lin-Sca-1+Kit+ (LSK) bone marrow stem cell population and lymphoid-primed multipotent progenitor numbers, which translated into a higher production and systemic flux of T cell subsets. In accordance with APOA1 deficiency-associated priming of stem cells to increase T lymphocyte production, atherogenic diet-fed low-density lipoprotein receptor knockout mice transplanted with bone marrow from APOA1-knockout mice displayed marked lymphocytosis as compared to wild-type bone marrow recipients. However, atherosclerotic lesion sizes and collagen contents were similar in the two groups of bone marrow recipients. In conclusion, systemic lack of APOA1 primes bone marrow stem cells for T cell lymphopoiesis. Our data provide novel evidence for a regulatory role of HDL in bone marrow functioning in normolipidemic mice.Biopharmaceutic

IFNγ-stimulated B cells inhibit T follicular helper cells and protect against atherosclerosis

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 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.Biopharmaceutic

Diet-induced dyslipidemia induces metabolic and migratory adaptations in regulatory T cells

A hallmark of advanced atherosclerosis is inadequate immunosuppression by regulatory T(Treg) cells inside atherosclerotic lesions. Dyslipidemia has been suggested to alter Treg cellmigration by affecting the expression of specific membrane proteins, thereby decreasing Treg cellmigration towards atherosclerotic lesions. Besides membrane proteins, cellular metabolism has beenshown to be a crucial factor in Treg cell migration. We aimed to determine whether dyslipidemiacontributes to altered migration of Treg cells, in part, by affecting cellular metabolism.Dyslipidemia was induced by feeding Ldlr-/- mice a Western-type diet for 16-20weeks and intrinsic changes in Treg cells affecting their migration and metabolism were examined.Dyslipidemia was associated with altered mTORC2 signaling in Treg cells, decreased expression ofmembrane proteins involved in migration, including CD62L, CCR7 and S1Pr1, and decreased Tregcell migration towards lymph nodes. Furthermore, we discovered that diet-induced dyslipidemiainhibited mTORC1 signaling, induced PPARδ activation and increased fatty acid (FA) oxidation inTreg cells. Moreover, mass-spectrometry analysis of serum from Ldlr-/- mice with normolipidemia ordyslipidemia showed increases in multiple PPARδ ligands during dyslipidemia. Treatment with asynthetic PPARδ agonist increased the migratory capacity of Treg cells in vitro and in vivo in an FAoxidation dependent manner. Furthermore, diet-induced dyslipidemia actually enhanced Treg cellmigration into the inflamed peritoneum and into atherosclerotic lesions in vitro.Altogether, our findings implicate that dyslipidemia does not contribute toatherosclerosis by impairing Treg cell migration as dyslipidemia associated with an effector-likemigratory phenotype in Treg cells.Analytical BioScience

CD8+ T-cells contribute to lesion stabilization in advanced atherosclerosis by limiting macrophage content and CD4+ T-cell responses

Human endarterectomy samples analyzed by flow cytometry showed a negative correlation between the percentage of CD8+ T-cells and macrophages, suggesting a possible protective role for these cells in lesion development. To further test this hypothesis, LDLr-/- mice were fed a Western-type diet (WTD) for 10 weeks to induce atherosclerosis, after which they received CD8α-depleting or isotype control antibody for six weeks. Depletion of CD8+ T-cells in advanced atherosclerosis resulted in less stable lesions, with significantly reduced collagen content in the trivalve area, increased macrophage content and increased necrotic core area compared to controls. Mechanistically, we observed that CD8 depletion specifically increased the fraction of Th1 CD4+ T-cells in the lesions. Treatment of WTD-fed LDLr-/- mice with a FasL-neutralizing antibody resulted in similar changes in macrophages and CD4+ T-cell skewing as CD8+ T-cell depletion. T lymphocytes play an important role in atherosclerosis development, but the role of the CD8+ T-cell remains debated, especially in the clinically relevant advanced stages of atherosclerosis development. Here, we set out to determine the role of CD8+ T-cells in advanced atherosclerosis. These findings demonstrate for the first time a local, protective role for CD8+ T-cells in advanced atherosclerosis, through limiting accumulation of Th1 cells and macrophages, identifying a novel regulatory mechanism for these cells in atherosclerosis. Methods and Results Aims ConclusionDrug Delivery Technolog

Stimulation of the co-inhibitory BTLA pathway protects against atherosclerosis by selectively reducing atherogenic B cells

B-and T-lymphocyte attenuator (BTLA) is a co-inhibitory molecule which was identified as a marker for Th1 cells. However, nowadays it is known that BTLA is most abundantly expressed on B cells. Also, we showed earlier that BTLA was specifically expressed on atherogenic follicular B2 cells with low expression on other B cell subtypes. Since it has been shown that stimulating co-inhibitory pathways can greatly influence atherosclerosis, we aimed to induce BTLA signaling in diet-induced atherosclerosis.For the initiaton study, LDLR-/- mice were fed a western type diet (WTD) for 6 weeks and received twice a week i.p. injections with an agonistic BTLA antibody. Mice in the control groups either received PBS or an isotype control antibody. For the progression study, mice were first fed a WTD for 10 weeks after which they received the same treatment as in the initiation experiment.Lesion development in mice receiving the agonistic BTLA antibody was significantly lower than in mice treated with PBS (PBS:2.6±0.8x105 μm2 vs. BTLA:1.5±0.8x105 μm2; p5 μm2 vs. BTLA; p=0.07). This corresponded with a major decrease in circulating (PBS: 44.2±4.9%, Isotype: 50.6±5.8% vs. BTLA: 24.5±12.0%; p+ B cells (PBS:29.1±4.2%, Isotype:30.8.±3.6% vs. BTLA:21.1±7.4%; pIn conclusion, stimulation of the BTLA pathway in experimental atherosclerosis in LDLR-/- mice reduces lesion development and increases stability of established lesions, presumably by specifically inhibiting atherogenic B cells, while leaving atheroprotective B cell subsets unaffected.Biopharmaceutic

Bidirectional effects of IL-10+ regulatory B cells in Ldlr−/− mice

Limiting the pro-inflammatory immune response is critical for the treatment of atherosclerosis. Regulatory B cells (Bregs) can modulate the immune response through interleukin-10 (IL-10). Current data regarding Bregs and atherosclerosis is scarce and conflicting.\n B cells on atherosclerosis.\n B cells on atherosclerosis.\n B cells in atherosclerosis.Biopharmaceutic

Apolipoprotein A1 modulates the ability of bone marrow to facilitate lymphocyte production after transplantation into Ldl receptor knockout mice

Apolipoprotein A1 (APOA1) deficiency is associated with severe reductions in plasma HDL cholesterol and therefore an increased atherosclerosis susceptibility. Although APOA1 can directly impact on inflammation and atherosclerosis, it can also suppress proliferation of hematopoietic stem cells and skew hematopoiesis, and thus alter immune cell numbers and function. In this study we aimed to dissociate the direct effects of APOA1 deficiency on immune cells in the context of atherosclerosis from effects that are generated through changes in bone marrow (BM) immune cell production.BM from wild-type and APOA1 knockout mice was transplanted into hypercholesterolemic LDL receptor knockout mice.Plasma cholesterol levels and atherosclerotic plaque size and collagen content were unaltered upon reconstitution with BM from APOA1 knockout mice. However, APOA1 knockout BM recipients displayed lymphocytosis, and a strong increase in circulating CD4 T-cell numbers. This coincided with a strong increase in splenocyte numbers and a relative increase in splenic and circulating naïve T-cells. An increase in thymus size and thymic CCR9 expression suggested an increase in T-cell production, driven by an increase in hematopoietic stem cells and T lineage differentiation in the donor BM from APOA1 knockout mice.In conclusion, our studies show that a genetic lack of APOA1 translated into long term changes in immune cell production from BM progenitors, leading to the development of naïve T-cells from the thymus, resulting in lymphocytosis. Furthermore, our studies suggest that the effect of APOA1 deficiency on BM does not underlie the diet-induced increase in atherosclerosis susceptibility associated with global APOA1 deficiency.Biopharmaceutic