Peter Stachon

Binding of CD40L to Mac-1’s I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis – but does not affect immunity and thrombosis in mice

Rationale: CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. Objective: Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. Methods and Results: CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the &agr;1 helix and the &bgr;-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr−/− mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. Conclusions: We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.

Tumor Necrosis Factor Receptor–Associated Factor 1 (TRAF1) Deficiency Attenuates Atherosclerosis in Mice by Impairing Monocyte Recruitment to the Vessel Wall

Background— Members of the tumor necrosis factor superfamily, such as tumor necrosis factor-&agr;, potently promote atherogenesis in mice and humans. Tumor necrosis factor receptor–associated factors (TRAFs) are cytoplasmic adaptor proteins for this group of cytokines. Methods and Results— This study tested the hypothesis that TRAF1 modulates atherogenesis in vivo. TRAF1−/−/LDLR−/− mice that consumed a high-cholesterol diet for 18 weeks developed significantly smaller atherosclerotic lesions than LDLR−/− (LDL receptor–deficient) control animals. As the most prominent change in histological composition, plaques of TRAF1-deficient animals contained significantly fewer macrophages. Bone marrow transplantations revealed that TRAF1 deficiency in both hematopoietic and vascular resident cells contributed to the reduction in atherogenesis observed. Mechanistic studies showed that deficiency of TRAF1 in endothelial cells and monocytes reduced adhesion of inflammatory cells to the endothelium in static and dynamic assays. Impaired adhesion coincided with reduced cell spreading, actin polymerization, and CD29 expression in macrophages, as well as decreased expression of the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in endothelial cells. Small interfering RNA studies in human cells verified these findings. Furthermore, TRAF1 messenger RNA levels were significantly elevated in the blood of patients with acute coronary syndrome. Conclusions— TRAF1 deficiency attenuates atherogenesis in mice, most likely owing to impaired monocyte recruitment to the vessel wall. These data identify TRAF1 as a potential treatment target for atherosclerosis.

The Oral Spleen Tyrosine Kinase Inhibitor Fostamatinib Attenuates Inflammation and Atherogenesis in Low-Density Lipoprotein Receptor–Deficient Mice

Objective— Spleen tyrosine kinase (SYK) has come into focus as a potential therapeutic target in chronic inflammatory diseases, such as rheumatoid arthritis and asthma, as well as in B-cell lymphomas. SYK has also been involved in the signaling of immunoreceptors, cytokine receptors, and integrins. We therefore hypothesized that inhibition of SYK attenuates the inflammatory process underlying atherosclerosis and reduces plaque development. Methods and Results— Low-density lipoprotein receptor–deficient mice consuming a high-cholesterol diet supplemented with 2 doses of the orally available SYK inhibitor fostamatinib for 16 weeks showed a dose-dependent reduction in atherosclerotic lesion size by up to 59±6% compared with the respective controls. Lesions of fostamatinib-treated animals contained fewer macrophages but more smooth muscle cells and collagen—characteristics associated with more stable plaques in humans. Mechanistically, fostamatinib attenuated adhesion and migration of inflammatory cells and limited macrophage survival. Furthermore, fostamatinib normalized high-cholesterol diet –induced monocytosis and inflammatory gene expression. Conclusion— We present the novel finding that the SYK inhibitor fostamatinib attenuates atherogenesis in mice. Our data identify SYK inhibition as a potentially fruitful antiinflammatory therapeutic strategy in atherosclerosis.

Coinhibitory Suppression of T Cell Activation by CD40 Protects Against Obesity and Adipose Tissue Inflammation in Mice

Background— Costimulatory cascades such as the CD40L-CD40 dyad enhance immune cell activation and inflammation during atherosclerosis. Here, we tested the hypothesis that CD40 directly modulates traits of the metabolic syndrome in diet-induced obesity in mice. Methods and Results— To induce the metabolic syndrome, wild-type or CD40−/− mice consumed a high-fat diet for 20 weeks. Unexpectedly, CD40−/− mice exhibited increased weight gain, impaired insulin secretion, augmented accumulation of inflammatory cells in adipose tissue, and enhanced proinflammatory gene expression. This proinflammatory and adverse metabolic phenotype could be transplanted into wild-type mice by reconstitution with CD40-deficient lymphocytes, indicating a major role for CD40 in T or B cells in this context. Conversely, therapeutic activation of CD40 signaling by the stimulating antibody FGK45 abolished further weight gain during the study, lowered glucose levels, improved insulin sensitivity, and suppressed adipose tissue inflammation. Mechanistically, CD40 activation decreased the expression of proinflammatory cytokines in T cells but not in B cells or macrophages. Finally, repopulation of lymphocyte-free Rag1−/− mice with CD40−/− T cells provoked dysmetabolism and inflammation, corroborating a protective role of CD40 on T cells in the metabolic syndrome. Finally, levels of soluble CD40 showed a positive association with obesity in humans, suggesting clinical relevance of our findings. Conclusions— We present the surprising finding that CD40 deficiency on T cells aggravates whereas activation of CD40 signaling improves adipose tissue inflammation and its metabolic complications. Therefore, positive modulation of the CD40 pathway might describe a novel therapeutic concept against cardiometabolic disease.

TRAF5 Deficiency Accelerates Atherogenesis in Mice by Increasing Inflammatory Cell Recruitment and Foam Cell Formation

Rationale: Tumor necrosis factor receptor–associated factors (TRAFs) are cytoplasmic adaptor proteins for the TNF/interleukin-1/Toll-like receptor superfamily. Ligands of this family comprise multiple important cytokines such as TNF&agr;, CD40L, and interleukin-1&bgr; that promote chronic inflammatory diseases such as atherosclerosis. We recently reported overexpression of TRAF5 in murine and human atheromata and that TRAF5 promotes inflammatory functions of cultured endothelial cells and macrophages. Objective: This study tested the hypothesis that TRAF5 modulates atherogenesis in vivo. Methods and Results: Surprisingly, TRAF5−/−/LDLR−/− mice consuming a high-cholesterol diet for 18 weeks developed significantly larger atherosclerotic lesions than did TRAF5+/+/LDLR−/− controls. Plaques of TRAF5-deficient animals contained more lipids and macrophages, whereas smooth muscle cells and collagen remained unchanged. Deficiency of TRAF5 in endothelial cells or in leukocytes enhanced adhesion of inflammatory cells to the endothelium in dynamic adhesion assays in vitro and in murine vessels imaged by intravital microscopy in vivo. TRAF5 deficiency also increased expression of adhesion molecules and chemokines and potentiated macrophage lipid uptake and foam cell formation. These findings coincided with increased activation of JNK and appeared to be independent of TRAF2. Finally, patients with stable or acute coronary heart disease had significantly lower amounts of TRAF5 mRNA in blood compared with healthy controls. Conclusions: Unexpectedly, TRAF5 deficiency accelerates atherogenesis in mice, an effect likely mediated by increased inflammatory cell recruitment to the vessel wall and enhanced foam cell formation.

CD40L Deficiency Attenuates Diet-Induced Adipose Tissue Inflammation by Impairing Immune Cell Accumulation and Production of Pathogenic IgG-Antibodies

Background Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L – an established marker and mediator of cardiovascular disease – induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo. Methodology/Principal Findings WT or CD40L−/− mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L−/− mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L−/− mice. However, CD40L−/− mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L−/− mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels. Conclusion We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.

P2Y6 Deficiency Limits Vascular Inflammation and Atherosclerosis in Mice

Objective— Nucleotides such as ATP, ADP, UTP, and UDP serve as proinflammatory danger signals via purinergic receptors on their release to the extracellular space by activated or dying cells. UDP binds to the purinergic receptor Y6 (P2Y6) and propagates vascular inflammation by inducing the expression of chemokines such as monocyte chemoattractant protein 1, interleukin-8, or its mouse homologsCCL1 (chemokine [C-C motif] ligand 1)/keratinocyte chemokine, CXCL2 (chemokine [C-X-C motif] ligand 2)/macrophage inflammatory protein 2, and CXCL5 (chemokine [C-X-C motif] ligand 5)/LIX, and adhesion molecules such as vascular cell adhesion molecule 1 and intercellular cell adhesion molecule 1. Thus, P2Y6 contributes to leukocyte recruitment and inflammation in conditions such as allergic asthma or sepsis. Because atherosclerosis is a chronic inflammatory disease driven by leukocyte recruitment to the vessel wall, we hypothesized a role of P2Y6 in atherogenesis. Approach and Results— Intraperitoneal stimulation of wild-type mice with UDP induced rolling and adhesion of leukocytes to the vessel wall as assessed by intravital microscopy. This effect was not present in P2Y6-deficient mice. Atherosclerotic aortas of low-density lipoprotein receptor–deficient mice consuming high-cholesterol diet for 16 weeks expressed significantly more transcripts and protein of P2Y6 than respective controls. Finally, P2Y6 −/−/low-density lipoprotein receptor–deficient mice consuming high-cholesterol diet for 16 weeks developed significantly smaller atherosclerotic lesions compared with P2Y6 +/+/low-density lipoprotein receptor–deficient mice. Bone marrow transplantation identified a crucial role of P2Y6 on vascular resident cells, most likely endothelial cells, on leukocyte recruitment and atherogenesis. Atherosclerotic lesions of P2Y6-deficient mice contained fewer macrophages and fewer lipids as determined by immunohistochemistry. Mechanistically, RNA expression of vascular cell adhesion molecule 1 and interleukin-6 was decreased in these lesions and P2Y6-deficient macrophages took up less modified low-density lipoprotein cholesterol. Conclusions— We show for the first time that P2Y6 deficiency limits atherosclerosis and plaque inflammation in mice.

Purinergic Receptor Y6 Deficiency Limits Vascular Inflammation and Atherosclerosis in Mice

Objective— Nucleotides such as ATP, ADP, UTP, and UDP serve as proinflammatory danger signals via purinergic receptors on their release to the extracellular space by activated or dying cells. UDP binds to the purinergic receptor Y6 (P2Y6) and propagates vascular inflammation by inducing the expression of chemokines such as monocyte chemoattractant protein 1, interleukin-8, or its mouse homologsCCL1 (chemokine [C-C motif] ligand 1)/keratinocyte chemokine, CXCL2 (chemokine [C-X-C motif] ligand 2)/macrophage inflammatory protein 2, and CXCL5 (chemokine [C-X-C motif] ligand 5)/LIX, and adhesion molecules such as vascular cell adhesion molecule 1 and intercellular cell adhesion molecule 1. Thus, P2Y6 contributes to leukocyte recruitment and inflammation in conditions such as allergic asthma or sepsis. Because atherosclerosis is a chronic inflammatory disease driven by leukocyte recruitment to the vessel wall, we hypothesized a role of P2Y6 in atherogenesis. Approach and Results— Intraperitoneal stimulation of wild-type mice with UDP induced rolling and adhesion of leukocytes to the vessel wall as assessed by intravital microscopy. This effect was not present in P2Y6-deficient mice. Atherosclerotic aortas of low-density lipoprotein receptor–deficient mice consuming high-cholesterol diet for 16 weeks expressed significantly more transcripts and protein of P2Y6 than respective controls. Finally, P2Y6 −/−/low-density lipoprotein receptor–deficient mice consuming high-cholesterol diet for 16 weeks developed significantly smaller atherosclerotic lesions compared with P2Y6 +/+/low-density lipoprotein receptor–deficient mice. Bone marrow transplantation identified a crucial role of P2Y6 on vascular resident cells, most likely endothelial cells, on leukocyte recruitment and atherogenesis. Atherosclerotic lesions of P2Y6-deficient mice contained fewer macrophages and fewer lipids as determined by immunohistochemistry. Mechanistically, RNA expression of vascular cell adhesion molecule 1 and interleukin-6 was decreased in these lesions and P2Y6-deficient macrophages took up less modified low-density lipoprotein cholesterol. Conclusions— We show for the first time that P2Y6 deficiency limits atherosclerosis and plaque inflammation in mice.

Tumor Necrosis Factor Receptor Associated Factor 6 Is Not Required for Atherogenesis in Mice and Does Not Associate with Atherosclerosis in Humans

Background Tumor necrosis factor receptor-associated factors (TRAFs) are important signaling molecules for a variety of pro-atherogenic cytokines including CD40L, TNF α, and IL1β. Several lines of evidence identified TRAF6 as a pro-inflammatory signaling molecule in vitro and we previously demonstrated overexpression of TRAF6 in human and Murine atherosclerotic plaques. This study investigated the role of TRAF6-deficiency in mice developing atherosclerosis, a chronic inflammatory disease. Methodology/Principal Findings Lethally irradiated low density lipoprotein receptor-deficient mice (TRAF6+/+/LDLR−/−) were reconstituted with TRAF6-deficient fetal liver cells (FLC) and consumed high cholesterol diet for 18 weeks to assess the relevance of TRAF6 in hematopoietic cells for atherogenesis. Additionally, TRAF6+/−/LDLR−/− mice received TRAF6-deficient FLC to gain insight into the role of TRAF6 deficiency in resident cells. Surprisingly, atherosclerotic lesion size did not differ between the three groups in both aortic roots and abdominal aortas. Similarly, no significant differences in plaque composition could be observed as assessed by immunohistochemistry for macrophages, lipids, smooth muscle cells, T-cells, and collagen. In accord, in a small clinical study TRAF6/GAPDH total blood RNA ratios did not differ between groups of patients with stable coronary heart disease (0.034±0.0021, N = 178), acute coronary heart disease (0.029±0.0027, N = 70), and those without coronary heart disease (0.032±0.0016, N = 77) as assessed by angiography. Conclusion Our study demonstrates that TRAF6 is not required for atherogenesis in mice and does not associate with clinical disease in humans. These data suggest that pro- and anti-inflammatory features of TRAF6 signaling outweigh each other in the context of atherosclerosis.

Extracellular ATP Induces Vascular Inflammation and Atherosclerosis via Purinergic Receptor Y2 in Mice

Objective—A solid body of evidence supports a role of extracellular ATP and its P2 receptors in innate and adaptive immunity. It promotes inflammation as a danger signal in various chronic inflammatory diseases. Thus, we hypothesize contribution of extracellular ATP and its receptor P2Y2 in vascular inflammation and atherosclerosis. Approach and Results—Extracellular ATP induced leukocyte rolling, adhesion, and migration in vivo as assessed by intravital microscopy and in sterile peritonitis. To test the role of extracellular ATP in atherosclerosis, ATP or saline as control was injected intraperitoneally 3× a week in low-density lipoprotein receptor−/− mice consuming high cholesterol diet. Atherosclerosis significantly increased after 16 weeks in ATP-treated mice (n=13; control group, 0.26 mm2; ATP group, 0.33 mm2; P=0.01). To gain into the role of ATP-receptor P2Y2 in ATP-induced leukocyte recruitment, ATP was administered systemically in P2Y2-deficient or P2Y2-competent mice. In P2Y2-deficient mice, the ATP-induced leukocyte adhesion was significantly reduced as assessed by intravital microscopy. P2Y2 expression in atherosclerosis was measured by real-time polymerase chain reaction and immunohistochemistry and demonstrates an increased expression mainly caused by influx of P2Y2-expressing macrophages. To investigate the functional role of P2Y2 in atherogenesis, P2Y2-deficient low-density lipoprotein receptor−/− mice consumed high cholesterol diet. After 16 weeks, P2Y2-deficient mice showed significantly reduced atherosclerotic lesions with decreased macrophages compared with P2Y2-competent mice (n=11; aortic arch: control group, 0.25 mm2; P2Y2-deficient, 0.14 mm2; P=0.04). Mechanistically, atherosclerotic lesions from P2Y2-deficient mice expressed less vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 RNA. Conclusions—We show that extracellular ATP induces vascular inflammation and atherosclerosis via activation of P2Y2.