JAG1-NOTCH4 mechanosensing drives atherosclerosis.

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 (Jag1ECKO) demonstrated that Jag1 promotes atherosclerosis at sites of disturbed flow. Mechanistically, single-cell RNA sequencing in Jag1ECKO 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.

Neutrophil microvesicles drive atherosclerosis by delivering miR-155 to atheroprone endothelium.

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.

Angiopoietin-1 enhances neutrophil chemotaxis in vitro and migration in vivo through interaction with CD18 and release of CCL4.

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.

Temporal interleukin-1beta secretion from primary human peripheral blood monocytes by P2X7-independent and P2X7-dependent mechanisms.

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.

Constriction of rat extra-splenic veins to lipopolysaccharide involves endothelin-1.

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 microg 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 ET(A) and ET(B) antagonists ABT-627 (atrasentan, 6.3 x 10(-6) M) and A-192621(1.45 x 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 induces acute interferon-gamma production through polyclonal stimulation of chicken leukocytes.

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.

Course of infection and immune responses in the respiratory tract of IBV infected broilers after superinfection with E. coli.

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.

The role of phagocytic cells in enhanced susceptibility of broilers to colibacillosis after Infectious Bronchitis Virus infection.

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 4dpi, 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 4dpi, 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 7dpi 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-1beta, IL-6, IL-8, IL-10, IL-18 and IFN-gamma mRNA was found 1dpi. 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.

Fast track selection of immunogens for novel vaccines through visualisation of the early onset of the B-cell response.

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.