Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.

HIV infection has a profound effect on "bystander" cells causing metabolic co-morbidities. This may be mediated by exosomes secreted by HIV-infected cells and containing viral factors. Here we show that exosomes containing HIV-1 protein Nef (exNef) are rapidly taken up by macrophages releasing Nef into the cell interior. This caused down-regulation of ABCA1, reduction of cholesterol efflux and sharp elevation of the abundance of lipid rafts through reduced activation of small GTPase Cdc42 and decreased actin polymerization. Changes in rafts led to re-localization of TLR4 and TREM-1 to rafts, phosphorylation of ERK1/2, activation of NLRP3 inflammasome, and increased secretion of pro-inflammatory cytokines. The effects of exNef on lipid rafts and on inflammation were reversed by overexpression of a constitutively active mutant of Cdc42. Similar effects were observed in macrophages treated with exosomes produced by HIV-infected cells or isolated from plasma of HIV-infected subjects, but not with exosomes from cells and subjects infected with ΔNef-HIV or uninfected subjects. Mice injected with exNef exhibited monocytosis, reduced ABCA1 in macrophages, increased raft abundance in monocytes and augmented inflammation. Thus, Nef-containing exosomes potentiated pro-inflammatory response by inducing changes in cholesterol metabolism and reorganizing lipid rafts. These mechanisms may contribute to HIV-associated metabolic co-morbidities

Inhibition of extracellular cyclophilins with cyclosporine analog and development of atherosclerosis in apolipoprotein E-deficient mice.

Cyclophilins exert both intracellular and extracellular activi-ties related to immune responses and inflammation, which have been implicated in pathogenesis of atherosclerosis. Pan-inhibition of cyclophilins has both pro- and antiatherosclerotic properties, but specific contributions of extracellular and intracellular cyclophilins to these effects have not been characterized. Here, using selective inhibitor of extracellular cyclophilins, we investigated the role of these molecules in atherosclerosis. Apolipoprotein E–null mice fed a high-fat diet received intraperitoneal injections every second day of either vehicle or two analogs of cyclosporine A (CsA): [Melle]4-CsA (NIM811), a nonimmunosupressive cell-permeable inhibitor of both intracellular and extracellular cyclophilins; and [(4R)-4-[(6-carboxy-1H-benzo[d]imidazol-2-yl)-methyl]-4-methyl-L

HIV protein Nef causes dyslipidemia and formation of foam cells in mouse models of atherosclerosis

Patients with HIV are at an increased risk of cardiovascular disease. In this study we investigated the effect of Nef, a secreted HIV protein responsible for the impairment of cholesterol efflux, on the development of atherosclerosis in two animal models. ApoE_/_ mice fed a high-fat diet and C57BL/6 mice fed a high-fat, high-cholesterol diet were injected with recombinant Nef (40 ng/injection) or vehicle, and the effects of Nef on development of atherosclerosis, inflammation, and dyslipidemia were assessed. In apoE_/_ mice, Nef significantly increased the size of atherosclerotic lesions and caused vessel remodeling. Nef caused elevation of total cholesterol and triglyceride levels in the plasma while reducing high-density lipoprotein cholesterol levels. These changes were accompanied by a reduction of ABCA1 abundance in the liver, but not in the vessels. In C57BL/6 mice, Nef caused a significant number of lipid-laden macrophages presented in adventitia of the vessels; these cells were absent from the vessels of control mice. Nef caused sharp elevations of plasma triglyceride levels and body weight. Taken together, our findings suggest that Nef causes dyslipidemia and accumulation of cholesterol in macrophages within the vessel wall, supporting the role of Nef in pathogenesis of atherosclerosis in HIV-infected patients

Modification of lipid rafts by extracellular vesicles carrying HIV-1 protein Nef induces redistribution of amyloid precursor protein and Tau, causing neuronal dysfunction

HIV-associated neurocognitive disorders (HANDs) are a frequent outcome of HIV infection. Effective treatment of HIV infection has reduced the rate of progression and severity but not the overall prevalence of HANDs, suggesting ongoing pathological process even when viral replication is suppressed. In this study, we investigated how HIV-1 protein Nef secreted in extracellular vesicles (exNef) impairs neuronal functionality. ExNef were rapidly taken up by neural cells in vitro, reducing the abundance of ABC transporter A1 (ABCA1) and thus cholesterol efflux and increasing the abundance and modifying lipid rafts in neuronal plasma membranes. ExNef caused a redistribution of amyloid precursor protein (APP) and Tau to lipid rafts and increased the abundance of these proteins, as well as of Ab . ExNef further potentiated phosphorylation of Tau and activation of inflammatory pathways. These changes were accompanied by neuronal functional impairment. Disruption of lipid rafts with cyclodextrin reversed the phenotype. Short-term treatment of C57BL/6 mice with either purified recombinant Nef or exNef similarly resulted in reduced abundance of ABCA1 and elevated abundance of APP in brain tissue. The abundance of ABCA1 in brain tissue of HIV-infected human subjects diagnosed with HAND was lower, and the abundance of lipid rafts was higher compared with HIV-negative individuals. Levels of APP and Tau in brain tissue correlated with the abundance of Nef. Thus, modification of neuronal cholesterol trafficking and of lipid rafts by Nef may contribute to early stages of neurodegeneration and pathogenesis in HAND. 4

Effect of ELK-2A2K2E on plasma lipids and lipoproteins.

<p><b>A -</b> Total cholesterol; <b>B -</b> non-HDL cholesterol; <b>C</b> - Triglycerides; <b>D -</b> HDL cholesterol; <b>E </b><b>-</b> apoA-I. *p<0.05, **p<0.01 <i>versus</i> vehicle; n = 8 for each point.</p

Effect of ELK-2A2K2E treatment on inflammation and oxidation in aorta.

<p><b>A -</b> Analysis of macrophage infiltration within the aortic sinus region after staining with anti CD68; *p<0.001 <i>versus</i> vehicle; n = 8 for each bar. <b>B -</b> Representative section of an aortic sinus from a control mouse stained with anti CD68. <b>C -</b> Representative section of an aortic sinus from a mouse treated with ELK-2A2K2E stained with anti CD68. <b>D </b><b>-</b> Analysis of the abundance of VCAM-1 in sections from the aortic sinus region; *p<0.001 <i>versus</i> vehicle; n = 8 for each bar. <b>E -</b> Representative section of an aortic sinus from a control mouse stained with anti VCAM-1. <b>F -</b> Representative section of an aortic sinus from a mouse treated with ELK-2A2K2E stained with anti VCAM-1. <b>G -</b> Analysis of the abundance of nitrotyrosine (NT) in sections from the aortic sinus region; *p<0.001 <i>versus</i> vehicle; n = 8 for each bar. <b>H -</b> Representative section of an aortic sinus from a control mouse stained with anti- nitrotyrosine. <b>I -</b> Representative section of an aortic sinus from a mouse treated with ELK-2A2K2E stained with anti-nitrotyrosine. Values represent positive staining with the corresponding antibody as a percentage of lesion area.</p