Linda Beckers

Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis

CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells, is also expressed and functional on platelets. We investigated the thrombotic and inflammatory contributions of platelet CD40L in atherosclerosis. Although CD40L-deficient (Cd40l(-/-)) platelets exhibited impaired platelet aggregation and thrombus stability, the effects of platelet CD40L on inflammatory processes in atherosclerosis were more remarkable. Repeated injections of activated Cd40l(-/-) platelets into Apoe(-/-) mice strongly decreased both platelet and leukocyte adhesion to the endothelium and decreased plasma CCL2 levels compared with wild-type platelets. Moreover, Cd40l(-/-) platelets failed to form proinflammatory platelet-leukocyte aggregates. Expression of CD40L on platelets was required for platelet-induced atherosclerosis as injection of Cd40l(-/-) platelets in contrast to Cd40l(+/+) platelets did not promote lesion formation. Remarkably, injection of Cd40l(+/+), but not Cd40l(-/-), platelets transiently decreased the amount of regulatory T cells (Tregs) in blood and spleen. Depletion of Tregs in mice injected with activated Cd40l(-/-) platelets abrogated the athero-protective effect, indicating that CD40L on platelets mediates the reduction of Tregs leading to accelerated atherosclerosis. We conclude that platelet CD40L plays a pivotal role in atherosclerosis, not only by affecting platelet-platelet interactions but especially by activating leukocytes, thereby increasing platelet-leukocyte and leukocyte-endothelium interactions.

Deficient CD40-TRAF6 signaling in leukocytes prevents atherosclerosis by skewing the immune response toward an antiinflammatory profile

The CD40–CD40 ligand (CD40L) signaling axis plays an important role in immunological pathways. Consequently, this dyad is involved in chronic inflammatory diseases, including atherosclerosis. Inhibition of CD40L in apolipoprotein E (Apoe)–deficient (Apoe−/−) mice not only reduced atherosclerosis but also conferred a clinically favorable plaque phenotype that was low in inflammation and high in fibrosis. Blockade of CD40L may not be therapeutically feasible, as long-term inhibition will compromise systemic immune responses. Conceivably, more targeted intervention strategies in CD40 signaling will have less deleterious side effects. We report that deficiency in hematopoietic CD40 reduces atherosclerosis and induces features of plaque stability. To elucidate the role of CD40–tumor necrosis factor receptor-associated factor (TRAF) signaling in atherosclerosis, we examined disease progression in mice deficient in CD40 and its associated signaling intermediates. Absence of CD40-TRAF6 but not CD40-TRAF2/3/5 signaling abolishes atherosclerosis and confers plaque fibrosis in Apoe−/− mice. Mice with defective CD40-TRAF6 signaling display a reduced blood count of Ly6Chigh monocytes, an impaired recruitment of Ly6C+ monocytes to the arterial wall, and polarization of macrophages toward an antiinflammatory regulatory M2 signature. These data unveil a role for CD40–TRAF6, but not CD40–TRAF2/3/5, interactions in atherosclerosis and establish that targeting specific components of the CD40–CD40L pathway harbors the potential to achieve therapeutic effects in atherosclerosis.

Myeloid Type I Interferon Signaling Promotes Atherosclerosis by Stimulating Macrophage Recruitment to Lesions

Inflammatory cytokines are well-recognized mediators of atherosclerosis. Depending on the pathological context, type I interferons (IFNs; IFNalpha and IFNbeta) exert either pro- or anti-inflammatory immune functions, but their exact role in atherogenesis has not been clarified. Here, we demonstrate that IFNbeta enhances macrophage-endothelial cell adhesion and promotes leukocyte attraction to atherosclerosis-prone sites in mice in a chemokine-dependent manner. Moreover, IFNbeta treatment accelerates lesion formation in two different mouse models of atherosclerosis and increases macrophage accumulation in the plaques. Concomitantly, absence of endogenous type I IFN signaling in myeloid cells inhibits lesion development, protects against lesional accumulation of macrophages, and prevents necrotic core formation. Finally, we show that type I IFN signaling is upregulated in ruptured human atherosclerotic plaques. Hereby, we identify type I IFNs as proatherosclerotic cytokines that may serve as additional targets for prevention or treatment.

Two-Photon Microscopy for Imaging of the (Atherosclerotic) Vascular Wall: A Proof of Concept Study

Background: Understanding atherogenesis will benefit significantly from simultaneous imaging, both ex vivo and in vivo, of structural and functional information at the (sub)cellular level within intact arteries. Due to limited penetration depth and loss of resolution with depth, intravital and confocal fluorescence microscopy are not suitable to study (sub)cellular details in arteries with wall thicknesses above 50 µm. Methods: Using two-photon laser scanning microscopy (TPLSM), which combines 3D resolution and large penetration depth, we imaged mouse carotid arteries. Results: In thin slices, (sub)cellular structures identified using histochemical techniques could also be identified using TPLSM. Ex vivo, structural experiments on intact atherosclerotic arteries of Apo-E–/– mice demonstrated that in contrast to confocal or wide-field microscopy, TPLSM can be used to visualize (sub) cellular structural details of atherosclerotic plaques. In vivo, pilot experiments were carried out on healthy arteries of wild-type C57BL6 and atherosclerotic arteries of Apo-E–/– mice. As an example of functional measurements, we visualized fluorescently labeled leukocytes in vivo in the lumen. Additionally, detailed morphological information of vessel wall and atherosclerotic plaque was obtained after topical staining. Conclusions: Thus, TPLSM potentially allows combined functional and structural studies and can therefore be eminently suitable for investigating structure-function relationships at the cellular level in atherogenesis in the mouse.

Gene Profiling in Atherosclerosis Reveals a Key Role for Small Inducible Cytokines Validation Using a Novel Monocyte Chemoattractant Protein Monoclonal Antibody

Background—Pathological aspects of atherosclerosis are well described, but gene profiles during atherosclerotic plaque progression are largely unidentified. Methods and Results—Microarray analysis was performed on mRNA of aortic arches of ApoE−/− mice fed normal chow (NC group) or Western-type diet (WD group) for 3, 4.5, and 6 months. Of 10 176 reporters, 387 were differentially (>2×) expressed in at least 1 group compared with a common reference (ApoE−/−, 3- month NC group). The number of differentially expressed genes increased during plaque progression. Time-related expression clustering and functional grouping of differentially expressed genes suggested important functions for genes involved in inflammation (especially the small inducible cytokines monocyte chemoattractant protein [MCP]-1, MCP-5, macrophage inflammatory protein [MIP]-1&agr;, MIP-1&bgr;, MIP-2, and fractalkine) and matrix degradation (cathepsin-S, matrix metalloproteinase-2/12). Validation experiments focused on the gene cluster of small inducible cytokines. Real-time polymerase chain reaction revealed a plaque progression–dependent increase in mRNA levels of MCP-1, MCP-5, MIP-1&agr;, and MIP-1&bgr;. ELISA for MCP-1 and MCP-5 showed similar results. Immunohistochemistry for MCP-1, MCP-5, and MIP-1&agr; located their expression to plaque macrophages. An inhibiting antibody for MCP-1 and MCP-5 (11K2) was designed and administered to ApoE−/− mice for 12 weeks starting at the age of 5 or 17 weeks. 11K2 treatment reduced plaque area and macrophage and CD45+ cell content and increased collagen content, thereby inducing a stable plaque phenotype. Conclusions—Gene profiling of atherosclerotic plaque progression in ApoE−/− mice revealed upregulation of the gene cluster of small inducible cytokines. Further expression and in vivo validation studies showed that this gene cluster mediates plaque progression and stability.

The CD40-TRAF6 axis is the key regulator of the CD40/CD40L system in neointima formation and arterial remodeling

We investigated the role of CD40 and CD40L in neointima formation and identified the downstream CD40-signaling intermediates (tumor necrosis factor [TNF]-receptor associated factors [TRAF]) involved. Neointima formation was induced in wild-type, CD40(-/-), CD40L(-/-), and in CD40(-/-) mice that contained a CD40 transgene with or without mutations at the CD40-TRAF2,3&5, TRAF6, or TRAF2,3,5&6 binding sites. Compared with wild-type mice, CD40(-/-) mice showed a significant decrease in neointima formation with increased collagen deposition and decreased inflammatory cell infiltration. Neointima formation was also impaired in wild-type mice reconstituted with CD40(-/-) bone marrow. In vitro, the capacity of CD40(-/-) leukocytes to adhere to the endothelium was reduced. Ligated carotid arteries of CD40(-/-) mice showed a smaller total vessel volume and an impaired remodeling capacity, reflected by decreased gelatinolytic/collagenolytic activity. Comparable results were found in mice with defects in CD40-TRAF6 and CD40-TRAF 2/3/5&6 binding, but not in mice with defects in CD40-TRAF2/3&5 binding. Neointima formation and vascular remodeling in CD40-receptor-deficient mice is impaired, due to a decreased inflammatory cell infiltration and matrix-degrading protease activity, with CD40-TRAF6 signaling as the key regulator. This identifies the CD40-TRAF6 axis as a potential therapeutic target in vascular disease.

Blocking CD40-TRAF6 signaling is a therapeutic target in obesity-associated insulin resistance.

Significance Inflammation is a critical contributor to the pathogenesis of metabolic disorders associated with obesity. A group of molecules crucial in regulating the immune system are costimulatory molecules, including CD40. Our current study shows that CD40 acts as a double-edged sword in the metabolic syndrome through the initiation of differential signaling cascades. The CD40-TNF receptor-associated factor (TRAF) 2/3/5 signaling pathway protects against metabolic dysfunction and inflammation associated with obesity; conversely, the CD40-TRAF6 pathway contributes to the detrimental consequences of obesity. In the present study, we therefore designed, validated, and used a small-molecule inhibitor that blocks CD40-TRAF6 interactions. The improvement of insulin resistance by this specific CD40-TRAF6 inhibitor could represent a therapeutic breakthrough in the field of immunometabolism. The immune system plays an instrumental role in obesity and insulin resistance. Here, we unravel the role of the costimulatory molecule CD40 and its signaling intermediates, TNF receptor-associated factors (TRAFs), in diet-induced obesity (DIO). Although not exhibiting increased weight gain, male CD40−/− mice in DIO displayed worsened insulin resistance, compared with wild-type mice. This worsening was associated with excessive inflammation of adipose tissue (AT), characterized by increased accumulation of CD8+ T cells and M1 macrophages, and enhanced hepatosteatosis. Mice with deficient CD40-TRAF2/3/5 signaling in MHCII+ cells exhibited a similar phenotype in DIO as CD40−/− mice. In contrast, mice with deficient CD40-TRAF6 signaling in MHCII+ cells displayed no insulin resistance and showed a reduction in both AT inflammation and hepatosteatosis in DIO. To prove the therapeutic potential of inhibition of CD40-TRAF6 in obesity, DIO mice were treated with a small-molecule inhibitor that we designed to specifically block CD40-TRAF6 interactions; this compound improved insulin sensitivity, reduced AT inflammation, and decreased hepatosteatosis. Our study reveals that the CD40-TRAF2/3/5 signaling pathway in MHCII+ cells protects against AT inflammation and metabolic complications associated with obesity whereas CD40-TRAF6 interactions in MHCII+ cells aggravate these complications. Inhibition of CD40-TRAF6 signaling by our compound may provide a therapeutic option in obesity-associated insulin resistance.

CD40L Deficiency Ameliorates Adipose Tissue Inflammation and Metabolic Manifestations of Obesity in Mice

Objective—Obese adipose tissue shows hallmarks of chronic inflammation, which promotes the development of metabolic disorders. The mechanisms by which immune cells interact with each other or with metabolism-associated cell types, and the players involved, are still unclear. The CD40-CD40L costimulatory dyad plays a pivotal role in immune responses and in diseases such as atherosclerosis and may therefore be a mediator of obesity. Here we investigated whether CD40L is involved in adipose tissue inflammation and its associated metabolic changes. Methods and Results—To assess a putative role of CD40L in obesity in vivo, we evaluated metabolic and inflammatory consequences of 18 weeks of high-fat feeding in CD40L+/+ and CD40L−/− mice. In addition, C57Bl6 mice were injected with neutralizing anti-CD40L (&agr;CD40L) antibody for 12 weeks while being fed a high-fat diet. Genetic deficiency of CD40L attenuated the development of diet-induced obesity, hepatic steatosis, and increased systemic insulin sensitivity. In adipose tissue, it impaired obesity-induced immune cell infiltration and the associated deterioration of glucose and lipid metabolism. Accordingly, &agr;CD40L treatment improved systemic insulin sensitivity, glucose tolerance, and CD4+ T-cell infiltration in adipose tissue with limited effects on adipose tissue weight. Conclusion—CD40L plays a crucial role in the development of obesity-induced inflammation and metabolic complications.

Reprogramming macrophages to an anti-inflammatory phenotype by helminth antigens reduces murine atherosclerosis

Atherosclerosis is a lipid‐driven inflammatory disease of the vessel wall, characterized by the chronic activation of macrophages. We investigated whether the helminth‐derived antigens [soluble egg antigens (SEAs)] could modulate macrophage inflammatory responses and protect against atherosclerosis in mice. In bone marrow‐derived macrophages, SEAs induce anti‐inflammatory macrophages, typified by high levels of IL‐10 and reduced secretion of proinflammatory mediators. In hyperlipidemic LDLR‐/‐ mice, SEA treatment reduced plaque size by 44%, and plaques were less advanced compared with PBS‐injected littermate controls. The atheroprotective effect of SEAs was found to be mainly independent of cholesterol lowering and T‐lymphocyte responses but instead could be attributed to diminished myeloid cell activation. SEAs reduced circulating neutrophils and inflammatory Ly6Chigh monocytes, and macrophages showed high IL‐10 production. In line with the observed systemic effects, atherosclerotic lesions of SEA‐treated mice showed reduced intraplaque inflammation as inflammatory markers [TNF‐α, monocyte chemotactic protein 1 (MCP‐1), intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and CD68], neutrophil content, and newly recruited macrophages were decreased. We show that SEA treatment protects against atherosclerosis development by dampening inflammatory responses. In the future, helminth‐derived components may provide novel opportunities to treat chronic inflammatory diseases, as they diminish systemic inflammation and reduce the activation of immune cells.—Wolfs, I. M. J., Stöger, J. L., Goossens, P., Pöttgens, C., Gijbels, M. J. J., Wijnands, E., van der Vorst, E. P. C., van Gorp, P., Beckers, L., Engel, D., Biessen, E. A. L., Kraal, G., van Die, I., Donners, M. M. P. C., de Winther, M. P. J. Reprogramming macrophages to an anti‐inflammatory phenotype by helminth antigens reduces murine atherosclerosis. FASEB J. 28, 288–299 (2014). www.fasebj.org

Abrogated transforming growth factor beta receptor II (TGFβRII) signalling in dendritic cells promotes immune reactivity of T cells resulting in enhanced atherosclerosis

AIMS The importance of transforming growth factor beta (TGFβ) as an immune regulatory cytokine in atherosclerosis has been established. However, the role of TGFβ signalling in dendritic cells (DCs) and in DC-mediated T cell proliferation and differentiation in atherosclerosis is unknown. METHODS AND RESULTS Here, we investigated the effect of disrupted TGFβ signalling in DCs on atherosclerosis by using mice carrying a transgene resulting in functional inactivation of TGFβ receptor II (TGFβRII) signalling in CD11c(+) cells (Apoe(-/-)CD11cDNR). Apoe(-/-)CD11cDNR mice exhibited an over two-fold increase in the plaque area compared with Apoe(-/-) mice. Plaques of Apoe(-/-)CD11cDNR mice showed an increase in CD45(+) leucocyte content, and specifically in CD3(+), CD4(+) and CD8(+) cells, whereas macrophage content was not affected. In lymphoid organs, Apoe(-/-)CD11cDNR mice had equal amounts of CD11c(+) cells, and CD11c(+)CD8(+) and CD11c(+)CD8(-) subsets, but showed a subtle shift in the CD11c(+)CD8(-) population towards the more inflammatory CD11c(+)CD8(-)CD4(-) DC subset. In addition, the number of plasmacytoid-DCs decreased. Maturation markers such as MHCII, CD86 and CD40 on CD11c(hi) cells did not change, but the CD11cDNR DCs produced more TNFα and IL-12. CD11c(+) cells from CD11cDNR mice strongly induced T-cell proliferation and activation, resulting in increased amounts of effector T cells producing high amounts of Th1 (IFN-γ), Th2 (IL-4, IL-10), Th17 (IL-17), and Treg (IL-10) cytokines. CONCLUSION Here, we show that loss of TGFβRII signalling in CD11c(+) cells induces subtle changes in DC subsets, which provoke uncontrolled T cell activation and maturation. This results in increased atherosclerosis and an inflammatory plaque phenotype during hypercholesterolaemia.