CD40L modulates CD4+ T-cell activation through receptor for activated C kinase 1.

Inhibition of the co-stimulatory ligand CD40L has shown beneficial effects in many experimental models of autoimmune disease and inflammation. Here, we show that CD40L deficiency in T cells in mice causes a reduction of CD4+ T-cell activation and specifically a strong reduction in IFN-γ-producing Th1 cells. In vitro, we could not reproduce this antigen presenting cell-dependent effects, but found that T-cell CD40L affects cell death and proliferation. We identified receptor of activated C kinase, the canonical PKC binding partner and known to drive proliferation and apoptosis, as a mediator of CD40L reverse signaling. Furthermore, we found that CD40L clustering stabilizes IFN-γ mediated Th1 polarization through STAT1, a known binding partner of receptor of activated C kinase. Together this highlights the importance of both CD40L forward and reverse signaling.

Adipocytes control hematopoiesis and inflammation through CD40 signaling.

The co-stimulatory CD40-CD40L dyad plays an important role in chronic inflammatory diseases associated with aging. Although CD40 is mainly expressed by immune cells, CD40 is also present on adipocytes. We aimed to delineate the role of adipocyte CD40 in the aging hematopoietic system and evaluated the effects of adipocyte CD40 deficiency on cardiometabolic diseases. Adult adipocyte CD40-deficient mice (AdiCD40KO) mice had a decrease in bone marrow hematopoietic stem cells (Lin-Sca+cKit+, LSK) and common lymphoid progenitors, which was associated with increased bone marrow adiposity and T-cell activation, along with elevated plasma corticosterone levels, a phenotype that became more pronounced with age. Atherosclerotic AdiCD40koApoE-/- (CD40AKO) mice also displayed changes in the LSK population, showing increased myeloid and lymphoid multipotent progenitors, and augmented corticosterone levels. Increased T-cell activation could be observed in bone marrow, spleen, and adipose tissue, while the numbers of B cells were decreased. Although atherosclerosis was reduced in CD40AKO mice, plaques contained more activated T cells and larger necrotic cores. Analysis of peripheral adipose tissue in a diet-induced model of obesity revealed that obese AdiCD40KO mice had increased T-cell activation in adipose tissue and lymphoid organs, but decreased weight gain and improved insulin sensitivity, along with increased fat oxidation. In conclusion, adipocyte CD40 plays an important role in maintaining immune cell homeostasis in bone marrow during aging and chronic inflammatory diseases, particularly of the lymphoid populations. Although adipocyte CD40 deficiency reduces atherosclerosis burden and ameliorates diet-induced obesity, the accompanying T-cell activation may eventually aggravate cardiometabolic diseases.

Macrophage CD40 signaling drives experimental autoimmune encephalomyelitis.

The costimulatory CD40L-CD40 dyad plays a major role in multiple sclerosis (MS). CD40 is highly expressed on MHCII+ B cells, dendritic cells and macrophages in human MS lesions. Here we investigated the role of the CD40 downstream signaling intermediates TNF receptor-associated factor 2 (TRAF2) and TRAF6 in MHCII+ cells in experimental autoimmune encephalomyelitis (EAE). Both MHCII-CD40-Traf2-/- and MHCII-CD40-Traf6-/- mice showed a reduction in clinical signs of EAE and prevented demyelination. However, only MHCII-CD40-Traf6-/- mice displayed a decrease in myeloid and lymphoid cell infiltration into the CNS that was accompanied by reduced levels of TNF-α, IL-6 and IFN-γ. As CD40-TRAF6 interactions predominantly occur in macrophages, we subjected CD40flfl LysMcre mice to EAE. This myeloid-specific deletion of CD40 resulted in a significant reduction in EAE severity, reduced CNS inflammation and demyelination. In conclusion, the CD40-TRAF6 signaling pathway in MHCII+ cells plays a key role in neuroinflammation and demyelination during EAE. Concomitant with the fact that CD40-TRAF6 interactions are predominant in macrophages, depletion of myeloid CD40 also reduces neuroinflammation. CD40-TRAF6 interactions thus represent a promising therapeutic target for MS. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

CD40L Deficiency Protects Against Aneurysm Formation.

OBJECTIVE: The mechanisms underlying formation of arterial aneurysms remain incompletely understood. Because inflammation is a common feature during the progressive degeneration of the aortic wall, we studied the role of the costimulatory molecule CD40L, a major driver of inflammation, in aneurysm formation. APPROACH AND RESULTS: Transcriptomics data obtained from human abdominal aortic aneurysms and normal aortas revealed increased abundance of both CD40L and CD40 in media of thrombus-free and thrombus-covered human abdominal aortic aneurysms samples. To further unravel the role of CD40L in aneurysm formation, apolipoprotein E-deficient (Apoe-/-) and Cd40l-/-Apoe-/- mice were infused with angiotensin II for 7 and 28 days. Only a minority of Cd40l-/-Apoe-/- mice (33% and 17%) developed (dissecting) aneurysms compared with 75% and 67% of Apoe-/- littermates after 7 and 28 days of infusion, respectively. Total vessel area of the aorta at the suprarenal level was 52% smaller in angiotensin II-infused Cd40l-/-Apoe-/- mice compared with that in angiotensin II-infused Apoe-/- mice. Chimeric Apoe-/- mice repopulated with Cd40l-/-Apoe-/- bone marrow afforded a similar protection against dissecting aneurysm formation. Moreover, lack of CD40L protected mice from fatal aneurysm rupture. T helper cell and macrophage accumulation in aneurysmal tissue was reduced in Cd40l-/-Apoe-/- mice with a concomitant decrease in expression of proinflammatory chemo- and cytokines. In addition, aneurysms of Cd40l-/-Apoe-/- mice displayed reduced abundance of matrix metalloproteinase-13 and an increase in tissue inhibitor of metalloproteinase-3 while activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 was diminished. CONCLUSIONS: Deficiency of (hematopoietic) CD40L protects against dissecting aneurysm formation and reduces the incidence of fatal rupture. This is associated with a decreased accumulation and activation of inflammatory cells and a dampened protease activity in the arterial wall.

Constitutive CD40 Signaling in Dendritic Cells Limits Atherosclerosis by Provoking Inflammatory Bowel Disease and Ensuing Cholesterol Malabsorption.

The costimulatory molecule CD40 is a major driver of atherosclerosis. It is expressed on a wide variety of cell types, including mature dendritic cells (DCs), and is required for optimal T-cell activation and expansion. It remains undetermined whether and how CD40 on DCs impacts the pathogenesis of atherosclerosis. Here, the effects of constitutively active CD40 in DCs on atherosclerosis were examined using low-density lipoprotein-deficient (Ldlr-/-) bone marrow chimeras that express a transgene containing an engineered latent membrane protein 1 (LMP)/CD40 fusion protein conferring constitutive CD40 signaling under control of the DC-specific CD11c promoter (DC-LMP1/CD40). As expected, DC-LMP1/CD40/Ldlr-/- chimeras (DC-LMP1/CD40) showed increased antigen-presenting capacity of DCs and increased T-cell numbers. However, the mice developed extensive neutrophilia compared to CD40wt/Ldlr-/- (CD40wt) chimeras. Despite overt T-cell expansion and neutrophilia, a reduction in conventional DC frequency and a dramatic (approximately 80%) reduction in atherosclerosis was observed. Further analyses revealed that cholesterol and triglyceride levels had decreased by 37% and 60%, respectively, in DC-LMP1/CD40 chimeras. Moreover, DC-LMP1/CD40 chimeras developed inflammatory bowel disease characterized by massive transmural influx of leukocytes and lymphocytes, resulting in villous degeneration and lipid malabsorption. Constitutive activation of CD40 in DCs results in inflammation of the gastrointestinal tract, thereby impairing lipid uptake, which consequently results in attenuated atherosclerosis.

CD27 co-stimulation increases the abundance of regulatory T cells and reduces atherosclerosis in hyperlipidaemic mice.

Aims: The co-stimulatory receptor CD27 modulates responses of T cells, B cells, and NK cells. Various T cell subsets participate in atherogenesis. However, the role of CD27 in atherosclerosis remains unexplored. Methods and results: Here we investigated the effect of bone marrow-derived and systemic CD27 deficiency in Apolipoprotein E-deficient (Apoe-/-) mice in early and advanced stages of atherosclerosis. Lethally-irradiated Apoe-/- mice reconstituted with Cd27-/-Apoe-/- bone marrow and consuming an atherogenic diet displayed a markedly increased plaque size and lesional inflammation compared to mice receiving Cd27+/+Apoe-/- bone marrow. Accordingly, chow diet-fed Cd27-/-Apoe-/- mice showed exacerbated lesion development and increased inflammation at the age of 18 weeks. At a more advanced stage of atherosclerosis (28 weeks), lesion size and phenotype did not differ between the two groups. Systemic and bone marrow-derived CD27 deficiency reduced the abundance of regulatory T cells (Treg) in blood, lymphoid organs, and the aorta. Numbers of other immune cells were not affected while expression of inflammatory cytokine genes (e.g. IL-1ß and IL-6) was increased in the aorta when haematopoietic CD27 was lacking. In vitro, Tregs of CD27-deficient mice showed similar suppressive capacity compared with their wild-type controls and migrated equally towards CCL19 and CCL21. However, thymic Cd27-/- Tregs underwent increased apoptosis and expressed fewer markers of proliferation in vivo. Reconstitution of Cd27-/-Apoe-/- mice with Cd27+/+Apoe-/- Tregs reversed the increase in atherosclerosis. Conclusion: We demonstrate that CD27 co-stimulation increases the number of Tregs and limits lesion development and inflammation in experimental atherosclerosis, particularly during early stages of disease. Thus, our study suggests that promotion of CD27 function may mitigate atherosclerosis.

Inhibition of CD40-TRAF6 interactions by the small molecule inhibitor 6877002 reduces neuroinflammation.

BACKGROUND: The influx of leukocytes into the central nervous system (CNS) is a key hallmark of the chronic neuro-inflammatory disease multiple sclerosis (MS). Strategies that aim to inhibit leukocyte migration across the blood-brain barrier (BBB) are therefore regarded as promising therapeutic approaches to combat MS. As the CD40L-CD40 dyad signals via TNF receptor-associated factor 6 (TRAF6) in myeloid cells to induce inflammation and leukocyte trafficking, we explored the hypothesis that specific inhibition of CD40-TRAF6 interactions can ameliorate neuro-inflammation. METHODS: Human monocytes were treated with a small molecule inhibitor (SMI) of CD40-TRAF6 interactions (6877002), and migration capacity across human brain endothelial cells was measured. To test the therapeutic potential of the CD40-TRAF6-blocking SMI under neuro-inflammatory conditions in vivo, Lewis rats and C57BL/6J mice were subjected to acute experimental autoimmune encephalomyelitis (EAE) and treated with SMI 6877002 for 6 days (rats) or 3 weeks (mice). RESULTS: We here show that a SMI of CD40-TRAF6 interactions (6877002) strongly and dose-dependently reduces trans-endothelial migration of human monocytes. Moreover, upon SMI treatment, monocytes displayed a decreased production of ROS, tumor necrosis factor (TNF), and interleukin (IL)-6, whereas the production of the anti-inflammatory cytokine IL-10 was increased. Disease severity of EAE was reduced upon SMI treatment in rats, but not in mice. However, a significant reduction in monocyte-derived macrophages, but not in T cells, that had infiltrated the CNS was eminent in both models. CONCLUSIONS: Together, our results indicate that SMI-mediated inhibition of the CD40-TRAF6 pathway skews human monocytes towards anti-inflammatory cells with reduced trans-endothelial migration capacity, and is able to reduce CNS-infiltrated monocyte-derived macrophages during neuro-inflammation, but minimally ameliorates EAE disease severity. We therefore conclude that SMI-mediated inhibition of the CD40-TRAF6 pathway may represent a beneficial treatment strategy to reduce monocyte recruitment and macrophage activation in the CNS and has the potential to be used as a co-treatment to combat MS.

Diet-induced obesity in mice diminishes hematopoietic stem and progenitor cells in the bone marrow.

Obesity is associated with chronic low-grade inflammation, characterized by leukocytosis and inflammation in the adipose tissue. Continuous activation of the immune system is a stressor for hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). Here we studied how diet-induced obesity (DIO) affects HSPC population dynamics in the BM. Eight groups of age-matched C57Bl/6 mice received a high-fat diet (45% kilocalories from fat) ranging from 1 d up to 18 wk. The obesogenic diet caused decreased proliferation of lineage(-)Sca-1(+)c-Kit(+) (LSK) cells in the BM and a general suppression of progenitor cell populations including common lymphoid progenitors and common myeloid progenitors. Within the LSK population, DIO induced a shift in stem cells that are capable of self-renewal toward maturing multipotent progenitor cells. The higher differentiation potential resulted in increased lymphoid and myeloid ex vivo colony-forming capacity. In a competitive BM transplantation, BM from obese animals showed impaired multilineage reconstitution when transplanted into chow-fed mice. Our data demonstrate that obesity stimulates the differentiation and reduces proliferation of HSPCs in the BM, leading to a decreased HSPC population. This implies that the effects of obesity on HSPCs hampers proper functioning of the immune system.-Van den Berg, S. M., Seijkens, T. T. P., Kusters, P. J. H., Beckers, L., den Toom, M., Smeets, E., Levels, J., de Winther, M. P. J., Lutgens, E. Diet-induced obesity in mice diminishes hematopoietic stem and progenitor cells in the bone marrow.

Constitutive GITR Activation Reduces Atherosclerosis by Promoting Regulatory CD4+ T-Cell Responses-Brief Report.

OBJECTIVE: Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is expressed on CD4(+) effector memory T cells and regulatory T cells; however, its role on these functionally opposing cell types in atherosclerosis is not fully understood. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice (Ldlr(-/-)) were lethally irradiated and reconstituted with either bone marrow from B-cell-restricted Gitrl transgenic mice or from wild-type controls and fed a high-cholesterol diet for 11 weeks. Chimeric Ldlr(-/-) Gitrl(tg) mice showed a profound increase in both CD4(+) effector memory T cells and regulatory T cells in secondary lymphoid organs. Additionally, the number of regulatory T cells was significantly enhanced in the thymus and aorta of these mice along with increased Gitrl and Il-2 transcript levels. Atherosclerotic lesions of Ldlr(-/-) Gitrl(tg) chimeras contained more total CD3(+) T cells as well as Foxp3(+) regulatory T cells overall, leading to significantly less severe atherosclerosis. CONCLUSIONS: These data indicate that continuous GITR stimulation through B cell Gitrl acts protective in a mouse model of atherosclerosis by regulating the balance between regulatory and effector memory CD4(+) T cells.

Platelet CD40 Exacerbates Atherosclerosis by Transcellular Activation of Endothelial Cells and Leukocytes.

OBJECTIVE: Beyond their eminent role in hemostasis and thrombosis, platelets are recognized as mediators of inflammation. Platelet cluster of differentiation 40 (CD40) ligand (CD40L and CD154) plays a key role in mediating platelet-induced inflammation in atherosclerosis. CD40, the receptor for CD40L, is present on platelets; however, the role of CD40 on this cell type is until now undefined. APPROACH AND RESULTS: We found that in both mice and humans, platelet CD40 mediates the formation of platelet-leukocyte aggregates and the release of chemokine (C-X-C motif) ligand 4. Leukocytes were also less prone to adhere to CD40-deficient thrombi. However, platelet CD40 was not involved in platelet aggregation. Activated platelets isolated from Cd40(-/-)Apoe(-/-) mice adhered less to the endothelium upon injection into Apoe(-/-) mice when compared with CD40-sufficient platelets. Furthermore, lack of CD40 on injected platelets led to reduced leukocyte recruitment to the carotid artery as assayed by intravital microscopy. This was accompanied by a decrease in endothelial vascular cell adhesion molecule-1, platelet endothelial cell adhesion molecule, VE-cadherin, and P-selectin expression. To investigate the effect of platelet CD40 in atherosclerosis, Apoe(-/-) mice received thrombin-activated Apoe(-/-) or Cd40(-/-)Apoe(-/-) platelets every 5 days for 12 weeks, starting at the age of 17 weeks, when atherosclerotic plaques had already formed. When compared with mice that received Apoe(-/-) platelets, those receiving Cd40(-/-)Apoe(-/-) platelets exhibited a >2-fold reduction in atherosclerosis. Plaques of mice receiving CD40-deficient platelets were less advanced, contained less macrophages, neutrophils, and collagen, and displayed smaller lipid cores. CONCLUSIONS: Platelet CD40 plays a crucial role in inflammation by stimulating leukocyte activation and recruitment and activation of endothelial cells, thereby promoting atherosclerosis.

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

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.

Hypercholesterolemia-induced priming of hematopoietic stem and progenitor cells aggravates atherosclerosis.

Modulation of hematopoietic stem and progenitor cells (HSPCs) determines immune cell function. In this study, we investigated how hypercholesterolemia affects HSPC biology and atherosclerosis. Hypercholesterolemia induced loss of HSPC quiescence, characterized by increased proliferation and expression of cyclin B1, C1, and D1, and a decreased expression of Rb, resulting in a 3.6- fold increase in the number of HSPCs in hypercholesterolemic Ldlr(-/-) mice. Competitive bone marrow (BM) transplantations showed that a hypercholesterolemic BM microenvironment activates HSPCs and skews their development toward myeloid lineages. Conversely, hypercholesterolemia-primed HSPCs acquired an enhanced propensity to generate myeloid cells, especially granulocytes and Ly6C(high) monocytes, even in a normocholesterolemic BM microenvironment. In conformity, macrophages differentiated from hypercholesterolemia-primed HSPCs produced 17.0% more TNF-α, 21.3% more IL-6, and 10.5% more MCP1 than did their normocholesterolemic counterparts. Hypercholesterolemia-induced priming of HSPCs generated leukocytes that more readily migrated into the artery, which resulted in a 2.1-fold increase in atherosclerotic plaque size. In addition, these plaques had a more advanced phenotype and exhibited a 1.2-fold increase in macrophages and 1.8-fold increase in granulocytes. These results identify hypercholesterolemia-induced activation and priming of HSPCs as a novel pathway in the development of atherosclerosis. Inhibition of this proinflammatory differentiation pathway on the HSPC level has the potential to reduce atherosclerosis.

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 Ly6C(high) 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.

Hematopoietic G-protein-coupled receptor kinase 2 deficiency decreases atherosclerotic lesion formation in LDL receptor-knockout mice.

Leukocyte chemotaxis is deemed instrumental in initiation and progression of atherosclerosis. It is mediated by G-protein-coupled receptors (e.g., CCR2 and CCR5), the activity of which is controlled by G-protein-coupled receptor kinases (GRKs). In this study, we analyzed the effect of hematopoietic deficiency of a potent regulator kinase of chemotaxis (GRK2) on atherogenesis. LDL receptor-deficient (LDLr(-/-)) mice with heterozygous hematopoietic GRK2 deficiency, generated by bone marrow transplantation (n=15), displayed a dramatic attenuation of plaque development, with 79% reduction in necrotic core and increased macrophage content. Circulating monocytes decreased and granulocytes increased in GRK2(+/-) chimeras, which could be attributed to diminished granulocyte colony-forming units in bone marrow. Collectively, these data pointed to myeloid cells as major mediators of the impaired atherogenic response in GRK2(+/-) chimeras. LDLr(-/-) mice with macrophage/granulocyte-specific GRK2 deficiency (LysM-Cre GRK2(flox/flox); n=8) failed to mimic the aforementioned phenotype, acquitting these cells as major responsible subsets for GRK2 deficiency-associated atheroprotection. To conclude, even partial hematopoietic GRK2 deficiency prevents atherosclerotic lesion progression beyond the fatty streak stage, identifying hematopoietic GRK2 as a potential target for intervention in atherosclerosis.

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

AIMS: The importance of transforming growth factor beta (TGFß) as an immune regulatory cytokine in atherosclerosis has been established. However, the role of TGFß 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 TGFß signalling in DCs on atherosclerosis by using mice carrying a transgene resulting in functional inactivation of TGFß receptor II (TGFßRII) 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 TGFßRII 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.

Peritoneal Dialysis Aggravates and Accelerates Atherosclerosis in Uremic ApoE-/- Mice.

BACKGROUND: Atherosclerosis is highly prevalent in people with chronic kidney disease (CKD), including those receiving peritoneal dialysis (PD). Although it is lifesaving, PD induces profound systemic inflammation, which may aggravate atherosclerosis. Therefore, the hypothesis is that this PD-induced inflammation aggravates atherosclerosis via immune cell activation. METHODS AND RESULTS: ApoE-/- mice were subjected to a 5/6 nephrectomy to induce CKD. Three weeks later, mice were fed a high-cholesterol diet. Half of the nephrectomized mice then received daily peritoneal infusions of 3.86% Physioneal for 67 further days (CKD+PD) until the end of the experiment, and were compared with mice without CKD. Sham operated and PD-only mice were additional controls. CKD+PD mice displayed more severe atherosclerotic disease than control mice. Plaque area increased, and plaques were more advanced with a vulnerable phenotype typified by decreased collagen content and decreased fibrous cap thickness. Increased CD3+ T-cell numbers were present in plaques and perivascular adipose tissue of CKD and CKD+PD mice. Plaques of CKD+PD mice contained more iNOS+ immune cells. Spleens of CKD+PD mice showed more CD4+ central memory, terminally differentiated type 1 T-helper (Th1), Th17, and CX3C motif chemokine receptor 1+ (CX3CR1) CD4+ T-cells with less regulatory and effector T-cells. CONCLUSIONS: PD-fluid exposure in uremic mice potentiates systemic and vascular T-cell-driven inflammation and aggravates atherosclerosis. PD polarized CD4+ T-cells toward an inflammatory Th1/Th17 phenotype, and increased CX3CR1+ CD4+ T-cells, which are associated with vascular homing in CKD-associated atherosclerosis. Targeting CD4+ T-cell activation and CX3CR1+ polarization has the potential to attenuate atherosclerosis in PD patients.

T cell specific deletion of Casitas B lineage lymphoma-b reduces atherosclerosis, but increases plaque T cell infiltration and systemic T cell activation.

Introduction: Atherosclerosis is a lipid-driven inflammatory disease of the arterial wall, and the underlying cause of the majority of cardiovascular diseases. Recent advances in high-parametric immunophenotyping of immune cells indicate that T cells constitute the major leukocyte population in the atherosclerotic plaque. The E3 ubiquitin ligase Casitas B-lymphoma proto-oncogene-B (CBL-B) is a critical intracellular regulator that sets the threshold for T cell activation, making CBL-B a potential therapeutic target to modulate inflammation in atherosclerosis. We previously demonstrated that complete knock-out of CBL-B aggravated atherosclerosis in Apoe-/- mice, which was attributed to increased macrophage recruitment and increased CD8+ T cell activation in the plaque. Methods: To further study the T cell specific role of CBL-B in atherosclerosis, Apoe-/- CD4cre Cblb fl/fl (Cbl-bcKO) mice and Apoe-/-CD4WTCblbfl/fl littermates (Cbl-bfl/fl) were fed a high cholesterol diet for ten weeks. Results: Cbl-bcKO mice had smaller atherosclerotic lesions in the aortic arch and root compared to Cbl-bfl/fl, and a substantial increase in CD3+ T cells in the plaque. Collagen content in the plaque was decreased, while other plaque characteristics including plaque necrotic core, macrophage content, and smooth muscle cell content, remained unchanged. Mice lacking T cell CBL-B had a 1.4-fold increase in CD8+ T cells and a 1.8-fold increase in regulatory T cells in the spleen. Splenic CD4+ and CD8+ T cells had increased expression of C-X-C Motif Chemokine Receptor 3 (CXCR3) and interferon-γ (IFN-γ), indicating a T helper 1 (Th1)-like/effector CD8+ T cell-like phenotype. Conclusion: In conclusion, Cbl-bcKO mice have reduced atherosclerosis but show increased T cell accumulation in the plaque accompanied by systemic T cell activation.

Targeting the ACOD1-itaconate axis stabilizes atherosclerotic plaques.

Inflammatory macrophages are key drivers of atherosclerosis that can induce rupture-prone vulnerable plaques. Skewing the plaque macrophage population towards a more protective phenotype and reducing the occurrence of clinical events is thought to be a promising method of treating atherosclerotic patients. In the current study, we investigate the immunomodulatory properties of itaconate, an immunometabolite derived from the TCA cycle intermediate cis-aconitate and synthesised by the enzyme Aconitate Decarboxylase 1 (ACOD1, also known as IRG1), in the context of atherosclerosis. Ldlr-/- atherogenic mice transplanted with Acod1-/- bone marrow displayed a more stable plaque phenotype with smaller necrotic cores and showed increased recruitment of monocytes to the vessel intima. Macrophages from Acod1-/- mice contained more lipids whilst also displaying reduced induction of apoptosis. Using multi-omics approaches, we identify a metabolic shift towards purine metabolism, in addition to an altered glycolytic flux towards production of glycerol for triglyceride synthesis. Overall, our data highlight the potential of therapeutically blocking ACOD1 with the aim of stabilizing atherosclerotic plaques.

The immunobiology of CD154-CD40-TRAF interactions in atherosclerosis.

Atherosclerosis is a chronic disease of the large arteries that is responsible for the majority of cardiovascular events. In its pathogenesis, the immune system plays a pivotal role. The effectuation of the immune response through interactions between immune cells that is mediated by co-stimulatory molecules, determine atherosclerosis severity. This review will highlight the role of one of the most powerful co-stimulatory dyads, the CD154 (also known as CD40 ligand, CD40L)-CD40 dyad, in atherosclerosis. Its cell-type specific actions, signal transduction cascades and its therapeutic potentials will be discussed.

Deficiency of germinal center kinase TRAF2 and NCK-interacting kinase (TNIK) in B cells does not affect atherosclerosis.

Background: Atherosclerosis is the underlying cause of many cardiovascular diseases, such as myocardial infarction or stroke. B cells, and their production of pro- and anti-atherogenic antibodies, play an important role in atherosclerosis. In B cells, TRAF2 and NCK-interacting Kinase (TNIK), a germinal center kinase, was shown to bind to TNF-receptor associated factor 6 (TRAF6), and to be involved in JNK and NF-κB signaling in human B cells, a pathway associated with antibody production. Objective: We here investigate the role of TNIK-deficient B cells in atherosclerosis. Results: ApoE-/-TNIKfl/fl (TNIKBWT) and ApoE-/-TNIKfl/flCD19-cre (TNIKBKO) mice received a high cholesterol diet for 10 weeks. Atherosclerotic plaque area did not differ between TNIKBKO and TNIKBWT mice, nor was there any difference in plaque necrotic core, macrophage, T cell, α-SMA and collagen content. B1 and B2 cell numbers did not change in TNIKBKO mice, and marginal zone, follicular or germinal center B cells were unaffected. Total IgM and IgG levels, as well as oxidation specific epitope (OSE) IgM and IgG levels, did not change in absence of B cell TNIK. In contrast, plasma IgA levels were decreased in TNIKBKO mice, whereas the number of IgA+ B cells in intestinal Peyer's patches increased. No effects could be detected on T cell or myeloid cell numbers or subsets. Conclusion: We here conclude that in hyperlipidemic ApoE-/- mice, B cell specific TNIK deficiency does not affect atherosclerosis.