Endothelial cell (EC) sensing of disturbed blood flow triggers atherosclerosis, a disease of arteries that causes heart attack and stroke, through poorly defined mechanisms. The Notch pathway plays a central role in blood vessel growth and homeostasis, but its potential role in sensing of disturbed flow has not been previously studied. Here, we show using porcine and murine arteries and cultured human coronary artery EC that disturbed flow activates the JAG1-NOTCH4 signaling pathway. Light-sheet imaging revealed enrichment of JAG1 and NOTCH4 in EC of atherosclerotic plaques, and EC-specific genetic deletion of Jag1 ( Jag1 ECKO ) demonstrated that Jag1 promotes atherosclerosis at sites of disturbed flow. Mechanistically, single-cell RNA sequencing in Jag1 ECKO mice demonstrated that Jag1 suppresses subsets of ECs that proliferate and migrate. We conclude that JAG1-NOTCH4 sensing of disturbed flow enhances atherosclerosis susceptibility by regulating EC heterogeneity and that therapeutic targeting of this pathway may treat atherosclerosis.

Endothelial cell (EC) sensing of fluid shear stress regulates atherosclerosis, a disease of arteries that causes heart attack and stroke. Atherosclerosis preferentially develops at regions of arteries exposed to low oscillatory shear stress (LOSS), whereas high shear regions are protected. We show using inducible EC-specific genetic deletion in hyperlipidaemic mice that the Notch ligands JAG1 and DLL4 have opposing roles in atherosclerosis. While endothelial Jag1 promoted atherosclerosis at sites of LOSS, endothelial Dll4 was atheroprotective. Analysis of porcine and murine arteries and cultured human coronary artery EC exposed to experimental flow revealed that JAG1 and its receptor NOTCH4 are strongly upregulated by LOSS. Functional studies in cultured cells and in mice with EC-specific deletion of Jag1 show that JAG1-NOTCH4 signalling drives vascular dysfunction by repressing endothelial repair. These data demonstrate a fundamental role for JAG1-NOTCH4 in sensing LOSS during disease, and suggest therapeutic targeting of this pathway to treat atherosclerosis.

Neutrophils are implicated in the pathogenesis of atherosclerosis but are seldom detected in atherosclerotic plaques. We investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Here we report that levels of circulating neutrophil microvesicles are enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulate at disease-prone regions of arteries exposed to disturbed flow patterns, and promote vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, we demonstrate that neutrophil microvesicles promote inflammatory gene expression by delivering miR-155, enhancing NF-κB activation. Similarly, neutrophil microvesicles increase miR-155 and enhance NF-κB at disease-prone sites of disturbed flow in vivo. Enhancement of atherosclerotic plaque formation and increase in macrophage content by neutrophil microvesicles is dependent on miR-155. We conclude that neutrophils contribute to vascular inflammation and atherogenesis through delivery of microvesicles carrying miR-155 to disease-prone regions.

AbstractNeutrophils have been implicated in the pathogenesis of atherosclerosis, a lipid-driven disease of arteries, but they are seldom found in atherosclerotic plaques. To resolve this longstanding paradox, we investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Clinical and pre-clinical studies revealed that levels of circulating neutrophil microvesicles were enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulated at disease-prone regions of arteries that are exposed to complex flow patterns, and they promoted vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, it was demonstrated that neutrophil microvesicles promoted inflammatory gene expression by delivering a microRNA (miR-155) that enhanced NF-κB activation. Similary, neutrophil microvesicles increased miR-155 and enhanced NF-κB at disease-prone sites of disturbed flow in arteries of mice. We conclude that delivery of microvesicles carrying miR-155 to disease-prone regions of arteries provides a novel mechanism by which neutrophils contribute to vascular inflammation and atherogenesis.

Angiopoietins are a family of growth factors that are ligands for the tyrosine kinase receptor, Tie2. Angiopoietin 1 (Ang-1) is agonistic for Tie2, plays a key role in blood vessel maturation and stability and has been shown to possess anti-inflammatory properties. However, Tie2 expression has been demonstrated on human neutrophils and the observation that neutrophils migrate in response to Ang-1 in vitro has confounded research into its exact role in inflammation as well as its potential use as a therapeutic agent. We used a mouse model of peritoneal neutrophilic inflammation to determine if Ang-1 could stimulate neutrophil migration in vivo . Tie2 expression was demonstrated on mouse neutrophils. In addition, recombinant human Ang-1 induced significant chemotaxis of isolated mouse neutrophils in a Tie2- and CD18-dependent manner. Subsequently, co-immunoprecipitation of Ang-1 and CD18 demonstrated their interaction. Intraperitoneal injection of an engineered angiopoietin-1, MAT.Ang-1, induced significant neutrophil migration into the peritoneum and a significant increase in the levels of CCL4 in peritoneal lavage fluid. Depletion of resident peritoneal macrophages prior to, or concomitant injections of an anti-CCL4 antibody with MAT.Ang-1 resulted in a significant reduction in neutrophil recruitment. These data indicate a pro-inflammatory role for Ang-1 with respect to neutrophil recruitment.

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Introduction Genome wide association studies have identified Tribbles-1 (TRIB1) to be significantly associated with all major plasma lipid traits and as a risk factor for ischaemic heart disease and myocardial infarction. Studies in mice using Trib1 full body KO and liver-specific over-expression and KO models have shown that hepatic expression of TRIB1 reduces circulating lipids. Additionally, Trib1 has been implicated as a regulator of alternatively activated macrophages. However the potential interplay between hepatocytes, macrophages and Trib1 remain unexplored. This study aimed to assess whether myeloid Trib1 regulates tissue macrophage polarisation and investigate its consequences on plasma lipid homeostasis. Methods We developed myeloid specific Trib1 conditional knockout (Trib1 fl/fl x Lyz2Cre; Trib1KO) and over-expressor mice (ROSA26Trib1.Tg x Lyz2Cre; Trib1Tg), thereby deleting or over-expressing Trib1 in myeloid cells. Plasma lipid levels were directly measured by ion mobility. Macrophage phenotype was characterised in the liver (Kupffer cells, KCs), adipose (ATMs) and BMDMs by qPCR and semi-quantitative immunofluorescence analysis. Western blotting was used to assess regulators of macrophage polarisation. Furthermore, microarray analysis of human monocyte derived macrophages (MDMs) was employed to identify potential TRIB1-regulated cytokines. Results Loss of myeloid Trib1 increased levels of plasma triglyceride, VLDL-C (p<0.05) and promoted pro-inflammatory polarisation in KCs (p<0.01), ATMs (p<0.01) and BMDMs (p<0.05), while Trib1Tg mice revealed opposing changes in all parameters assessed. Western blotting showed TRIB1 modulates protein levels of C/EBP-β2 and –β² (p<0.05), both key regulators of macrophage polarisation, via the control of COP1 activity and miR-155 expression. Microarray analysis of MDMs indicated TRIB1 may regulate production of a number of pro-inflammatory cytokines that are implicated in fatty liver disease and adipocyte lipolysis. Reduced expression of these was confirmed in in Trib1Tg BMDMs (p<0.05). Conclusions Myeloid Trib1 is a potent regulator of lipid homeostasis, the loss of which promotes inflammation in metabolic tissues. Our observations uncover a novel mechanism of KC-hepatocyte cross talk mediated through Trib1.

Rationale: Blood flow-induced shear stress controls endothelial cell (EC) physiology during atherosclerosis via transcriptional mechanisms that are incompletely understood. The mechanosensitive transcription factor TWIST is expressed during embryogenesis, but its role in EC responses to shear stress and focal atherosclerosis is unknown. Objective: To investigate whether TWIST regulates endothelial responses to shear stress during vascular dysfunction and atherosclerosis and compare TWIST function in vascular development and disease. Methods and Results: The expression and function of TWIST1 was studied in EC in both developing vasculature and during the initiation of atherosclerosis. In zebrafish, twist was expressed in early embryonic vasculature where it promoted angiogenesis by inducing EC proliferation and migration. In adult porcine and murine arteries, TWIST1 was expressed preferentially at low shear stress regions as evidenced by quantitative polymerase chain reaction and en face staining. Moreover, studies of experimental murine carotid arteries and cultured EC revealed that TWIST1 was induced by low shear stress via a GATA4-dependent transcriptional mechanism. Gene silencing in cultured EC and EC-specific genetic deletion in mice demonstrated that TWIST1 promoted atherosclerosis by inducing inflammation and enhancing EC proliferation associated with vascular leakiness. Conclusions: TWIST expression promotes developmental angiogenesis by inducing EC proliferation and migration. In addition to its role in development, TWIST is expressed preferentially at low shear stress regions of adult arteries where it promotes atherosclerosis by inducing EC proliferation and inflammation. Thus, pleiotropic functions of TWIST control vascular disease and development.

Aim: TRIB1 is a pseudokinase and recent GWAS analyses have linked this gene to the control of lipid homeostasis. Analysis of full body TRIB1 knockout and a liver specific TRIB1 overexpressing mouse have demonstrated that hepatic expression of TRIB1 reduces VLDL production, plasma triglycerides and cholesterol. Additionally, TRIB1 has been shown to be necessary for the development of M2 tissue macrophages. However, the interplay between TRIB1, macrophages and hepatocytes is unexplored.

Introduction Mitogen activated protein kinase (MAPK) cascades are involved in the regulation of several cellular responses including migration or proliferation. We have shown previously that tribbles-1 (trb-1), a member of the tribbles family of adaptor/scaffold proteins controls vascular smooth muscle proliferation and chemotaxis via a direct interaction with the JNK activator kinase, MKK4. However, a regulatory role in monocyte/macrophage function for members of the tribbles family has also been reported. With the current work, we aimed to gain a better understanding of the early steps of monocyte recruitment under inflammatory conditions, and in particular, the contribution of tribbles-1 to these processes. Methods We induced experimental peritonitis in trb-1 KO and trb-1 transgenic mice, where the expression of this gene was specifically modulated in the monocyte/macrophage lineage, and their littermate controls by injecting 1ml 4% thioglycollate to the peritoneal cavity. The composition of the cells infiltrating the peritoneum was analysed at 0, 6 and 24 hours post-injection by multi-colour flow cytometry. We also measured the cytokine content of the lavage fluids via the BDTM Cytometric Bead Array (CBA). Results At 6 and 24 hours post-injection we found a significant increase in the infiltration of monocytes to the peritoneal cavity in trb-1 KO animals compared to littermate controls, while the trb-1 over-expressing monocytes refrained from entering the peritoneum. We quantified the amount of IL-1a/b, TNFa, MIP1a/b and KC in the samples and found no significant difference in the production of any of these cytokines. Conclusions Trb-1 is involved in the regulation of monocyte migration under inflammatory conditions and therefore may play a significant role in the early steps of monocyte recruitment. Via this mechanism, trb-1 may contribute to the development of chronic inflammation and atherosclerosis. Further investigations will determine whether differences in the expression of leukocyte cell adhesion molecules (CAM) or other signalling abnormalities are responsible for the altered migration pattern of trb-1 KO and trb-1 transgenic monocytes.

The processing and regulated secretion of IL-1beta are critical points of control of the biological activity of this important pro-inflammatory cytokine. IL-1beta is produced by both monocytes and macrophages, but the rate and mechanism of release differ according to the differentiation status and the origin of these cells. We aimed to study the control of processing and release in human blood monocytes and human monocyte-derived macrophages. Toll-like receptor (TLR)-induced IL-1beta production and release were investigated for dependence upon caspase-1, P2X7 receptor activation, and loss of membrane asymmetry associated with microvesicle shedding. TLR agonists induced P2X7 receptor-dependent IL-1beta release in both monocytes and macrophages; however, only monocytes also showed P2X7 receptor-independent release of mature IL-1beta. Furthermore, in monocytes ATP-mediated PS exposure could be activated independently of IL-1beta production. Release of IL-1beta from monocytes showed selectivity for specific TLR agonists and was accelerated by P2X7 receptor activation. Human monocytes released more IL-1beta/cell than macrophages. These data have important implications for inflammatory diseases that involve monocyte activation and IL-1 release.

The spleen has an important role in blood volume regulation and increased resistance of post-capillary hilar veins (in mesentery adjoining the spleen) can regulate this. This study investigated whether venular constriction to lipopolysaccharide (LPS) involved endothelin-1 (ET-1). Pressure myography was used to study isolated extra-splenic (hilar) vessels from male Wistar rats (n = 111). Arteries and veins were treated with LPS (50 µg ml−1) for 4 h. Extra-splenic veins constricted to LPS (p < 0.05), but there was no effect on arteries. Denudation did not abolish venular constriction to LPS, indicating an endothelial independent mechanism. However, the dual ET-1 receptor antagonist bosentan (10−5 M) and specific ETA and ETB antagonists ABT-627 (atrasentan, 6.3 × 10−6 M) and A-192621(1.45 × 10−6 M) completely abolished constriction of LPS-treated veins. ET-1 alone also constricted the extra-splenic arteries and veins (p < 0.05), with a greater response observed in veins (p < 0.05). ELISA also confirmed that serum and spleen levels of ET-1 increased in response to LPS (p < 0.05). That LPS-induced constriction of extra-splenic veins is mediated by ET-1. Greater constriction of post- versus pre-capillary extra-splenic vessels to LPS would result in increased intra-splenic fluid extravasation and hypovolaemia in vivo.

Infectious Bronchitis Virus, a member of the Coronaviridae, is a respiratory pathogen in poultry. We found that in vitro stimulation with IBV resulted in ChIFN-gamma production in splenocytes of both infected birds and uninfected birds. The non-specific stimulation did not occur when other avian viruses or other coronaviruses were used or when mammalian cells were stimulated with IBV. Inactivation of IBV reduced ChIFN-gamma production, but ChIFN-gamma remained elevated compared to unstimulated cells. An increase in ChIFN-gamma mRNA was detected in splenocytes from IBV-infected and uninfected chickens as early as 1 h after stimulation with IBV. These results indicate that IBV acts as a polyclonal stimulus, inducing a rapid production of IFN-gamma even without previous exposure to the virus. (c) 2008 Elsevier Inc. All rights reserved.

Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. The aim of this study was to investigate how IBV affects the course of events upon infection with E. coli. Broilers were inoculated with IBV H120 vaccine virus or virulent M41 and challenged 5 days later with E. coli 506. A PBS and E. coli group without previous virus inoculation were included. Sections of trachea, lung and airsacs were stained for CD4, CD8, gammadelta-TCR, alphabeta1-TCR, and for macrophages (KUL-01) and both pathogens. Changes in the mucociliary barrier of trachea, lung and airsacs did not predispose for bacterial superinfection. The disease in the lungs of the E. coli group and both IBV/E. coli groups was similar. Lesions in the airsacs were more pronounced and of longer duration in the IBV/E. coli groups. The immunocytological changes differed substantially between the E. coli group and both IBV/E. coli groups. In trachea, lungs and airsacs the CD4+ and CD8+ populations were significantly larger than in the E. coli and PBS groups. In the lungs and the airsacs the macrophages were more numerous in the IBV/E. coli and the E. coli groups than in the PBS group. The presence of high numbers of T cells and macrophages in IBV infected birds most likely induced an altered immune response, which is responsible for the enhanced clinical signs of colibacillosis.

Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. We investigated whether IBV affects recruitment and function of phagocytic cells and examined NO production, phagocytic and bactericidal activity, and kinetics of peripheral blood mononuclear cells (PBMC) and splenocytes. Moreover, we measured cytokine mRNA expression in lung and spleen samples. Broilers were inoculated with IBV H120 vaccine or virulent M41 and challenged 5 days later with E. coli 506. A PBS control and E. coli group without previous virus inoculation were also included. Birds were sacrificed at various time points after inoculation (h/dpi). Inoculation with IBV induced extended and more severe colibacillosis than with E. coli alone. At 4 dpi, the number of KUL-01+ PBMC in all E. coli-inoculated groups was significantly higher than in PBS-inoculated birds, which correlated with lesion scores. From 1 to 4 dpi, NO production by PBMC from all E. coli-inoculated animals was elevated compared to PBS birds. Bactericidal activity of PBMC in IBV-inoculated animals at 7 dpi was lower than in PBS- and E. coli-inoculated birds, but phagocytic capacity and recruitment were not severely impaired. In spleen samples of IBV-infected animals reduced expression of IL-1β, IL-6, IL-8, IL-10, IL-18 and IFN-γ mRNA was found 1 dpi. Our results suggest that enhanced colibacillosis after IBV infection or vaccination is caused at least by altered innate immunity and less by impairment of phagocytic cell function.

The measurement of T cell responses in chickens, not only for quantitative aspects but also for the qualitative nature of the responses, becomes increasingly important. However, there are very few assays available to measure T cell function. Therefore, we have developed enzyme-linked immunosorbent spot assay (ELISPOT) and an intracellular cytokine staining (ICCS) assay. ELISPOT assay for the detection of chicken interferon-gamma (ChIFN-γ) production was set up and shown to be reproducible for both polyclonal and antigen-specific stimuli such as Newcastle disease virus (NDV). However, the ELISPOT assay lacks the ability to identify individual cytokine-producing cells. Separation of CD4+ and CD8+ T cell populations gave additional information, but appeared to have the disadvantage of a loss of cell interactions during stimulation. In a further refinement, individual cells were identifiable by ICCS, which gives the possibility to characterize for multiple characteristics, such as cytokine production and phenotype of the cell. Using ICCS, ChIFN-γ production was evaluated. Although cells were detected at only low frequencies, polyclonal stimulation of peripheral blood mononuclear cell (PBMC) or spleen cells resulted in a significant increase in ChIFN-γ production by CD4+ and CD8+ cells.

A most essential step in vaccine research and development, ie vaccine studies in animals, seriously suffer from long timespans needed to arrive at effective immunogens. In this report we show how almost immediately after vaccination the antibody inducing potential of low immunogenic 'self' antigens can be accurately assessed. (We expect that this timespan can be reduced even more when 'non self' antigens are used, since such responses should be stronger.) The method takes advantage of the immediate onset after vaccination of the immune response in the spleen. This novel method allows detection of antigen-specific B cells of the spleen as early as 7 days after immunization and at frequencies as low as 10 in 1,000,000 cells. The method depends on sequential staining with PE- and APC-conjugated tetramers, made with the same biotinylated peptide. The antigenic peptides are biotinylated and tetramerized with either PE neutravidin or APC streptavidin. We expect that this method can be generally applied to visualize B cell responses, irrespective of the way they are induced. In addition to the fast selection and development of novel immunogens, this procedure can be used to delineate the kinetics of the B cell response, to phenotypically characterize and to isolate antigen-specific B cells, and, perhaps most importantly, to count them at the clonal level before any circulating antibodies can be detected.