Pascal Kusters

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.

Immune Cell Crosstalk in Obesity: A Key Role for Costimulation?

In the past two decades, numerous experimental and clinical studies have established the importance of inflammation and immunity in the development of obesity and its metabolic complications, including insulin resistance and type 2 diabetes mellitus. In this context, T cells orchestrate inflammatory processes in metabolic organs, such as the adipose tissue (AT) and liver, thereby mediating obesity-related metabolic deterioration. Costimulatory molecules, which are present on antigen-presenting cells and naïve T cells in the AT, are known to mediate the crosstalk between the adaptive and innate immune system and to direct T-cell responses in inflammation. In this Perspectives in Diabetes article, we highlight the newest insights in immune cell interactions in obesity and discuss the role of costimulatory dyads in its pathogenesis. Moreover, the potential of therapeutic strategies that target costimulatory molecules in the metabolic syndrome is explored.

Blocking CD40-TRAF6 interactions by small-molecule inhibitor 6860766 ameliorates the complications of diet-induced obesity in mice

Background:Immune processes contribute to the development of obesity and its complications, such as insulin resistance, type 2 diabetes mellitus and cardiovascular disease. Approaches that target the inflammatory response are promising therapeutic strategies for obesity. In this context, we recently demonstrated that the interaction between the costimulatory protein CD40 and its downstream adaptor protein tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes adipose tissue inflammation, insulin resistance and hepatic steatosis in mice in the course of diet-induced obesity (DIO).Methods:Here we evaluated the effects of a small-molecule inhibitor (SMI) of the CD40-TRAF6 interaction, SMI 6860766, on the development of obesity and its complications in mice that were subjected to DIO.Results:Treatment with SMI 6860766 did not result in differences in weight gain, but improved glucose tolerance. Moreover, SMI 6860766 treatment reduced the amount of CD45+ leucocytes in the epididymal adipose tissue by 69%. Especially, the number of adipose tissue CD4+ and CD8+ T cells, as well as macrophages, was significantly decreased.Conclusions:Our results indicate that small-molecule-mediated inhibition of the CD40-TRAF6 interaction is a promising therapeutic strategy for the treatment of metabolic complications of obesity by improving glucose tolerance, by reducing the accumulation of immune cells to the adipose tissue and by skewing of the immune response towards a more anti-inflammatory profile.

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. FASEB J. 30, 1779–1788 (2016). www.fasebj.org

Targeting CD40-Induced TRAF6 Signaling in Macrophages Reduces Atherosclerosis

Background Disrupting the costimulatory CD40-CD40L dyad reduces atherosclerosis, but can result in immune suppression. The authors recently identified small molecule inhibitors that block the interaction between CD40 and tumor necrosis factor receptor-associated factor (TRAF) 6 (TRAF-STOPs), while leaving CD40-TRAF2/3/5 interactions intact, thereby preserving CD40-mediated immunity. Objectives This study evaluates the potential of TRAF-STOP treatment in atherosclerosis. Methods The effects of TRAF-STOPs on atherosclerosis were investigated in apolipoprotein E deficient (Apoe−/−) mice. Recombinant high-density lipoprotein (rHDL) nanoparticles were used to target TRAF-STOPs to macrophages. Results TRAF-STOP treatment of young Apoe−/− mice reduced atherosclerosis by reducing CD40 and integrin expression in classical monocytes, thereby hampering monocyte recruitment. When Apoe−/− mice with established atherosclerosis were treated with TRAF-STOPs, plaque progression was halted, and plaques contained an increase in collagen, developed small necrotic cores, and contained only a few immune cells. TRAF-STOP treatment did not impair “classical” immune pathways of CD40, including T-cell proliferation and costimulation, Ig isotype switching, or germinal center formation, but reduced CD40 and β2-integrin expression in inflammatory monocytes. In vitro testing and transcriptional profiling showed that TRAF-STOPs are effective in reducing macrophage migration and activation, which could be attributed to reduced phosphorylation of signaling intermediates of the canonical NF-κB pathway. To target TRAF-STOPs specifically to macrophages, TRAF-STOP 6877002 was incorporated into rHDL nanoparticles. Six weeks of rHDL-6877002 treatment attenuated the initiation of atherosclerosis in Apoe−/− mice. Conclusions TRAF-STOPs can overcome the current limitations of long-term CD40 inhibition in atherosclerosis and have the potential to become a future therapeutic for atherosclerosis.

Vascular CXCR4 Limits Atherosclerosis by Maintaining Arterial Integrity: Evidence from Mouse and Human Studies.

Background: The CXCL12/CXCR4 chemokine ligand/receptor axis controls (progenitor) cell homeostasis and trafficking. So far, an atheroprotective role of CXCL12/CXCR4 has only been implied through pharmacological intervention, in particular, because the somatic deletion of the CXCR4 gene in mice is embryonically lethal. Moreover, cell-specific effects of CXCR4 in the arterial wall and underlying mechanisms remain elusive, prompting us to investigate the relevance of CXCR4 in vascular cell types for atheroprotection. Methods: We examined the role of vascular CXCR4 in atherosclerosis and plaque composition by inducing an endothelial cell (BmxCreERT2-driven)–specific or smooth muscle cell (SMC, SmmhcCreERT2- or TaglnCre-driven)–specific deficiency of CXCR4 in an apolipoprotein E–deficient mouse model. To identify underlying mechanisms for effects of CXCR4, we studied endothelial permeability, intravital leukocyte adhesion, involvement of the Akt/WNT/&bgr;-catenin signaling pathway and relevant phosphatases in VE-cadherin expression and function, vascular tone in aortic rings, cholesterol efflux from macrophages, and expression of SMC phenotypic markers. Finally, we analyzed associations of common genetic variants at the CXCR4 locus with the risk for coronary heart disease, along with CXCR4 transcript expression in human atherosclerotic plaques. Results: The cell-specific deletion of CXCR4 in arterial endothelial cells (n=12–15) or SMCs (n=13–24) markedly increased atherosclerotic lesion formation in hyperlipidemic mice. Endothelial barrier function was promoted by CXCL12/CXCR4, which triggered Akt/WNT/&bgr;-catenin signaling to drive VE-cadherin expression and stabilized junctional VE-cadherin complexes through associated phosphatases. Conversely, endothelial CXCR4 deficiency caused arterial leakage and inflammatory leukocyte recruitment during atherogenesis. In arterial SMCs, CXCR4 sustained normal vascular reactivity and contractile responses, whereas CXCR4 deficiency favored a synthetic phenotype, the occurrence of macrophage-like SMCs in the lesions, and impaired cholesterol efflux. Regression analyses in humans (n=259 796) identified the C-allele at rs2322864 within the CXCR4 locus to be associated with increased risk for coronary heart disease. In line, C/C risk genotype carriers showed reduced CXCR4 expression in carotid artery plaques (n=188), which was furthermore associated with symptomatic disease. Conclusions: Our data clearly establish that vascular CXCR4 limits atherosclerosis by maintaining arterial integrity, preserving endothelial barrier function, and a normal contractile SMC phenotype. Enhancing these beneficial functions of arterial CXCR4 by selective modulators might open novel therapeutic options in atherosclerosis.

Cathelicidin regulates myeloid cell accumulation in adipose tissue and promotes insulin resistance during obesity

Note: The review process for this manuscript was fully handled by Gregory Y. H. Lip, Editor in Chief.

Helminth antigens counteract a rapid high-fat diet-induced decrease in adipose tissue eosinophils

Brown adipose tissue (BAT) activation and white adipose tissue (WAT) beiging can increase energy expenditure and have the potential to reduce obesity and associated diseases. The immune system is a potential target in mediating brown and beige adipocyte activation. Type 2 and anti-inflammatory immune cells contribute to metabolic homeostasis within lean WAT, with a prominent role for eosinophils and interleukin (IL)-4-induced anti-inflammatory macrophages. We determined eosinophil numbers in epididymal WAT (EpAT), subcutaneous WAT (ScAT) and BAT after 1 day, 3 days or 1 week of high-fat diet (HFD) feeding in C57Bl/6 mice. One day of HFD resulted in a rapid drop in eosinophil numbers in EpAT and BAT, and after 3 days, in ScAT. In an attempt to restore this HFD-induced drop in adipose tissue eosinophils, we treated 1-week HFD-fed mice with helminth antigens from Schistosoma mansoni or Trichuris suis and evaluated whether the well-known protective metabolic effects of helminth antigens involves BAT activation or beiging. Indeed, antigens of both helminth species induced high numbers of eosinophils in EpAT, but failed to induce beiging. In ScAT, Schistosoma mansoni antigens induced mild eosinophilia, which was accompanied by slightly more beiging. No effects were observed in BAT. To study type 2 responses on brown adipocytes directly, T37i cells were stimulated with IL-4. This increased Ucp1 expression and strongly induced the production of eosinophil chemoattractant CCL11 (+26-fold), revealing that brown adipocytes themselves can attract eosinophils. Our findings indicate that helminth antigen-induced eosinophilia fails to induce profound beiging of white adipocytes.

Exploring immune checkpoints as potential therapeutic targets in atherosclerosis

In the past decades, the inflammatory nature of atherosclerosis has been well-recognized and despite the development of therapeutic strategies targeted at its classical risk factors such as dyslipidemia and hypertension, atherosclerosis remains a major cause of morbidity and mortality. Additional strategies targeting the chronic inflammatory pathways underlying the development of atherosclerosis are therefore required. Interactions between different immune cells result in the secretion of inflammatory mediators, such as cytokines and chemokines, and fuel atherogenesis. Immune checkpoint proteins have a critical role in facilitating immune cell interactions and play an essential role in the development of atherosclerosis. Although the therapeutic potential of these molecules is well-recognized in clinical oncology, the use of immune checkpoint modulators in atherosclerosis is still limited to experimental models. Here, we review recent insights on the role of immune checkpoint proteins in atherosclerosis. Additionally, we explore the therapeutic potential and challenges of immune checkpoint modulating strategies in cardiovascular medicine and we discuss novel therapeutic approaches to target these proteins in atherosclerosis.

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.