Self-DNA (eg, released from dying cells or in neutrophil extracellular traps) and an increased expression of the antimicrobial peptide Cramp/LL37 in atherosclerotic lesions may thus stimulate a pDC-driven pathway of autoimmune activation and the generation of anti-double-stranded-DNA antibodies, critically aggravating atherosclerosis lesion formation. These key factors may thus represent novel therapeutic targets.

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.

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 wildtype platelets. Moreover, Cd40l ؊/؊ platelets failed to form proinflammatory plateletleukocyte aggregates. Expression of CD40L on platelets was required for plateletinduced 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

We used a novel approach of cytostatically induced leucocyte depletion and subsequent reconstitution with leucocytes deprived of classical (inflammatory/Gr1hi) or non-classical (resident/Gr1lo) monocytes to dissect their differential role in atheroprogression under high-fat diet (HFD). Apolipoprotein E-deficient (Apoe−/−) mice lacking classical but not non-classical monocytes displayed reduced lesion size and macrophage and apoptotic cell content. Conversely, HFD induced a selective expansion of classical monocytes in blood and bone marrow. Increased CXCL1 levels accompanied by higher expression of its receptor CXCR2 on classical monocytes and inhibition of monocytosis by CXCL1-neutralization indicated a preferential role for the CXCL1/CXCR2 axis in mobilizing classical monocytes during hypercholesterolemia. Studies correlating circulating and lesional classical monocytes in gene-deficient Apoe−/− mice, adoptive transfer of gene-deficient cells and pharmacological modulation during intravital microscopy of the carotid artery revealed a crucial function of CCR1 and CCR5 but not CCR2 or CX3CR1 in classical monocyte recruitment to atherosclerotic vessels. Collectively, these data establish the impact of classical monocytes on atheroprogression, identify a sequential role of CXCL1 in their mobilization and CCR1/CCR5 in their recruitment.

Methods and Results: Compared to Apoe؊/؊ mice, Cramp ؊/؊ Apoe ؊/؊ mice exhibit reduced lesion sizes with lower macrophage numbers. In atherosclerotic aortas, we could detect CRAMP specifically in neutrophils, but not in monocytes or macrophages. By use of intravital microscopy, CRAMP was found to be deposited by activated neutrophils on inflamed endothelium of large arteries. In this location cathelicidins promote adhesion of classical monocytes and neutrophils, but not nonclassical monocytes in a formyl-peptide receptor-dependent manner. Key Words: atherosclerosis Ⅲ monocyte recruitment Ⅲ neutrophil A therosclerosis is a chronic inflammation of the arterial vessel wall with relatively well-defined roles for leukocytes such as macrophages and lymphocytes. 1,2 Recent studies, however, have revealed that neutrophils infiltrate atherosclerotic lesions at various time points, 3-5 and depletion studies provide evidence for a proatherogenic role of neutrophils. 5,6 Nevertheless, mechanistic insights into how neutrophils promote early atherosclerotic lesion formation remain elusive. Neutrophils contain granules with more than 300 different proteins that undergo limited exocytosis on neutrophil extravasation. 7 Some of these proteins are able to activate and recruit immune cells and thus have been coined alarmins. 8 Cathelicidins (CRAMP in mouse, LL37 in humans) residing in neutrophil secondary granules were shown to potently activate and recruit monocytes and macrophages, 9,10 thus fulfilling alarmin criteria. Because cathelicidins were identified in atherosclerotic lesions, 11 we investigated their role in a mouse model of atherosclerosis. Conclusions: Editorial, see p 1036 In This Issue, see p 1035 MethodsDetailed Methods are provided in the Online Supplement. Plaque StudiesCramp Ϫ/Ϫ mice 12 were crossed with Apoe Ϫ/Ϫ mice. Atherosclerotic lesion size as well as lesional neutrophil and macrophage content were assessed by histology and immunohistochemistry. Intravital MicroscopyLeukocyte adhesion to the carotid artery was studied by intravital fluorescence microscopy as described previously. 5 ResultsTo investigate the role of CRAMP in early atherosclerotic lesion formation, we fed Apoe Ϫ/Ϫ and CrampOriginal received January 29, 2012; revision received February 15, 2012; accepted February 27, 2012. In January 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.88 days.Brief UltraRapid Communications are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive observations, but have a relatively narrow scope.

The transforming and tumor growthpromoting properties of Axl, a member of the Tyro3, Axl, and Mer (TAM) family of receptor tyrosine kinases (TAMRs), are well recognized. In contrast, little is known about the role of the TAMR ligand growth arrest-specific gene 6 (Gas6) in tumor biology. By using Gas6-deficient (Gas6 ؊/؊ ) mice, we show that bone marrow-derived Gas6 promotes growth and metastasis in different experimental cancer models, including one resistant to vascular endothelial growth factor inhibitors. Mechanistic studies reveal that circulating leukocytes produce minimal Gas6. However, once infiltrated in the tumor, leukocytes upregulate Gas6, which is mitogenic for tumor cells. Consistent herewith, impaired tumor growth in Gas6 ؊/؊ mice is rescued by transplantation of wild-type bone marrow and, conversely, mimicked by transplantation of Gas6 ؊/؊ bone marrow into wild-type hosts. These findings highlight a novel role for Gas6 in a positive amplification loop, whereby tumors promote their growth by educating infiltrating leukocytes to up-regulate the production of the mitogen Gas6. Hence, inhibition of Gas6 might offer novel opportunities for the treatment of cancer. IntroductionGrowth arrest-specific gene 6 (Gas6) was discovered because its expression is up-regulated in fibroblasts under growth-arrest conditions. 1,2 This factor exerts pleiotropic functions in health and disease: it amplifies platelet aggregation during thrombus formation, 3,4 enhances erythropoiesis, 5 and increases leukocyte extravasation in inflammatory conditions, 6 among other functions. 7,8 Gas6 binds to the Tyro3, Axl, and Mer (TAM) family of receptor tyrosine kinases (TAMRs), which consists of Tyro3 (Sky/Rse), Axl (Ufo/ Ark), and Mer (Eyk), although the binding affinity of this ligand differs for each receptor (AxlϾTyro3Ͼ ϾMer). 9,10 TAMRs, in particular Axl, have transforming properties. Indeed, overexpression of a truncated version of Axl in premalignant cells is sufficient to induce tumors in mice. 11 Axl is also highly expressed in human tumor cells in vitro, [12][13][14][15] as well as in a large variety of primary human cancers, including leukemia, 16 gastric cancer, 17 colon cancer, 18 breast cancer, 19 ovarian cancer, 20 and glioblastoma, 21 among others. In gastric cancer, Axl expression is associated with lymph node metastasis, an adverse prognostic factor. 17 However, little is known about the role of Gas6 in cancer. Gas6 is overexpressed in human ovarian, endometrial, gastric, thyroid, and glioblastoma tumors. 17,[20][21][22][23] In those studies, expression of Gas6 was detected in tumor cells, endothelial cells, and astrocytes, 20-24 but expression of Gas6 in tumor-infiltrated leukocytes has not been studied. Gas6 promotes proliferation and survival of different cancer cell lines, including prostate and melanoma tumor cells. 13,14 Interestingly, when glioma tumors coexpress high levels of Axl and Gas6, the survival of cancer patients is shortened. 21 Inhibition of Gas6 by a soluble Axl trap in vitro or a dominantneg...

Note: The editorial process for this article was fully handled by Prof. G. Y. H. Lip, Editor-in-Chief.

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.