Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death.

The perpetuation of inflammation is an important pathophysiological contributor to the global medical burden. Chronic inflammation is promoted by non-programmed cell death1,2; however, how inflammation is instigated, its cellular and molecular mediators, and its therapeutic value are poorly defined. Here we use mouse models of atherosclerosis-a major underlying cause of mortality worldwide-to demonstrate that extracellular histone H4-mediated membrane lysis of smooth muscle cells (SMCs) triggers arterial tissue damage and inflammation. We show that activated lesional SMCs attract neutrophils, triggering the ejection of neutrophil extracellular traps that contain nuclear proteins. Among them, histone H4 binds to and lyses SMCs, leading to the destabilization of plaques; conversely, the neutralization of histone H4 prevents cell death of SMCs and stabilizes atherosclerotic lesions. Our data identify a form of cell death found at the core of chronic vascular disease that is instigated by leukocytes and can be targeted therapeutically.

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 NET Release Fails to Reduce Adipose Tissue Inflammation in Mice.

Obesity-associated diseases such as Type 2 diabetes, liver disease and cardiovascular diseases are profoundly mediated by low-grade chronic inflammation of the adipose tissue. Recently, the importance of neutrophils and neutrophil-derived myeloperoxidase and neutrophil elastase on the induction of insulin resistance has been established. Since neutrophil elastase and myeloperoxidase are critically involved in the release of neutrophil extracellular traps (NETs), we here hypothesized that NETs may be relevant to early adipose tissue inflammation. Thus, we tested the effect of the Peptidyl Arginine Deiminase 4 inhibitor Cl-amidine, a compound preventing histone citrullination and subsequent NET release, in a mouse model of adipose tissue inflammation. C57BL6 mice received a 60% high fat diet for 10 weeks and were treated with either Cl-amidine or vehicle. Flow cytometry of adipose tissue and liver, immunohistological analysis and glucose and insulin tolerance tests were performed to determine the effect of the treatment and diet. Although high fat diet feeding induced insulin resistance no significant effect was observed between the treatment groups. In addition no effect was found in leukocyte infiltration and activation in the adipose tissue and liver. Therefore we concluded that inhibition of neutrophil extracellular trap formation may have no clinical relevance for early obesity-mediated pathogenesis of the adipose tissue and liver.

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.

Atherosclerotic Plaque Destabilization in Mice: A Comparative Study.

Atherosclerosis-associated diseases are the main cause of mortality and morbidity in western societies. The progression of atherosclerosis is a dynamic process evolving from early to advanced lesions that may become rupture-prone vulnerable plaques. Acute coronary syndromes are the clinical manifestation of life-threatening thrombotic events associated with high-risk vulnerable plaques. Hyperlipidemic mouse models have been extensively used in studying the mechanisms controlling initiation and progression of atherosclerosis. However, the understanding of mechanisms leading to atherosclerotic plaque destabilization has been hampered by the lack of proper animal models mimicking this process. Although various mouse models generate atherosclerotic plaques with histological features of human advanced lesions, a consensus model to study atherosclerotic plaque destabilization is still lacking. Hence, we studied the degree and features of plaque vulnerability in different mouse models of atherosclerotic plaque destabilization and find that the model based on the placement of a shear stress modifier in combination with hypercholesterolemia represent with high incidence the most human like lesions compared to the other models.

Deficiency of the sialyltransferase St3Gal4 reduces Ccl5-mediated myeloid cell recruitment and arrest: short communication.

RATIONALE: Sialylation by α2,3-sialyltransferases has been shown to be a crucial glycosylation step in the generation of functional selectin ligands. Recent evidence suggests that sialylation also affects the binding of chemokines to their corresponding receptor. OBJECTIVE: Because the chemokine receptors for Ccl5 and Ccl2 are important in atherogenic recruitment of neutrophils and monocytes, we here investigated the role of α2,3-sialyltransferase IV (ST3Gal-IV) in Ccl5- and Ccl2-mediated myeloid cell arrest and further studied its relevance in a mouse model of atherosclerosis. METHODS AND RESULTS: St3Gal4-deficient myeloid cells showed a reduced binding of Ccl5 and an impaired Ccl5-triggered integrin activation. Correspondingly, Ccl5-induced arrest on tumor necrosis factor-α-stimulated endothelium was almost completely abrogated, as observed in flow chamber adhesion assays and during ex vivo perfusion or intravital microscopy of carotid arteries. Moreover, Ccl5-triggered neutrophil and monocyte extravasation into the peritoneal cavity was severely reduced in St3Gal4(-/-) mice. In contrast, St3Gal4 deficiency did not significantly affect Ccl2 binding and only marginally decreased Ccl2-induced flow arrest of myeloid cells. In agreement with the crucial role of leukocyte accumulation in atherogenesis, and the importance of Ccl5 chemokine receptors mediating myeloid cell recruitment to atherosclerotic vessels, St3Gal4 deficiency drastically reduced the size, stage, and inflammatory cell content of atherosclerotic lesions in Apoe(-/-) mice on high-fat diet. CONCLUSIONS: In summary, these findings identify ST3Gal-IV as a promising target to reduce inflammatory leukocyte recruitment and arrest.

Distinct functions of chemokine receptor axes in the atherogenic mobilization and recruitment of classical monocytes.

We used a novel approach of cytostatically induced leucocyte depletion and subsequent reconstitution with leucocytes deprived of classical (inflammatory/Gr1(hi) ) or non-classical (resident/Gr1(lo) ) 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 CX3 CR1 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.

Simvastatin reduces endotoxin-induced acute lung injury by decreasing neutrophil recruitment and radical formation.

INTRODUCTION: Treatment of acute lung injury (ALI) remains an unsolved problem in intensive care medicine. As simvastatin exerts protective effects in inflammatory diseases we explored its effects on development of ALI and due to the importance of neutrophils in ALI also on neutrophil effector functions. METHODS: C57Bl/6 mice were exposed to aerosolized LPS (500 µg/ml) for 30 min. The count of alveolar, interstitial, and intravasal neutrophils were assessed 4 h later by flow cytometry. Lung permeability changes were assessed by FITC-dextran clearance and albumin content in the BAL fluid. In vitro, we analyzed the effect of simvastatin on neutrophil adhesion, degranulation, apoptosis, and formation of reactive oxygen species. To monitor effects of simvastatin on bacterial clearance we performed phagocytosis and bacterial killing studies in vitro as well as sepsis experiments in mice. RESULTS: Simvastatin treatment before and after onset of ALI reduces neutrophil influx into the lung as well as lung permeability indicating the protective role of simvastatin in ALI. Moreover, simvastatin reduces the formation of ROS species and adhesion of neutrophils without affecting apoptosis, bacterial phagocytosis and bacterial clearance. CONCLUSION: Simvastatin reduces recruitment and activation of neutrophils hereby protecting from LPS-induced ALI. Our results imply a potential role for statins in the management of ALI.

Hematopoietic interferon regulatory factor 8-deficiency accelerates atherosclerosis in mice.

OBJECTIVE: Inflammatory leukocyte accumulation drives atherosclerosis. Although monocytes/macrophages and polymorphonuclear neutrophilic leukocytes (PMN) contribute to lesion formation, sequelae of myeloproliferative disease remain to be elucidated. METHODS AND RESULTS: We used mice deficient in interferon regulatory factor 8 (IRF8(-/-)) in hematopoietic cells that develop a chronic myelogenous leukemia-like phenotype. Apolipoprotein E-deficient mice reconstituted with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow displayed an exacerbated atherosclerotic lesion formation compared with controls. The chronic myelogenous leukemia-like phenotype in mice with IRF8(-/-) bone marrow, reflected by an expansion of PMN in the circulation, was associated with an increased lesional accumulation and apoptosis of PMN, and enlarged necrotic cores. IRF8(-/-) compared with IRF8(+/+) PMN displayed unaffected reactive oxygen species formation and discharge of PMN granule components. In contrast, accumulating in equal numbers at sites of inflammation, IRF8(-/-) macrophages were defective in efferocytosis, lipid uptake, and interleukin-10 cytokine production. Importantly, depletion of PMN in low-density lipoprotein receptor or apolipoprotein E-deficient mice with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow abrogated increased lesion formation. CONCLUSIONS: These findings indicate that a chronic myelogenous leukemia-like phenotype contributes to accelerated atherosclerosis in mice. Among proatherosclerotic effects of other cell types, this, in part, is linked to an expansion of functionally intact PMN.