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NEF, HIV Protein Causes CVD, Metabolic Abnormalities,
Brain Disease - found in Reservoir
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NEF, HIV Protein - These considerations firstly apply to atherosclerosis, a pathology responsible for a range of cardiovascular co-morbidities of HIV disease.....Another co-morbidity likely related to the effects of Nef on cholesterol metabolism is HIV-associated neurocognitive disorder (HAND).....Nef-containing exosomes were found in plasma of patients receiving antiretroviral treatment and having no detectable viral load.....the origin of these exosomes is a small number of HIV infected cells residing in HIV reservoirs
PLOS July 25, 2019
In this study we show that HIV protein Nef released from infected cells in extracellular vesicles is taken up by un-infected ('bystander') cells impairing cholesterol metabolism in these cells. This impairment causes formation of excessive lipid rafts, re-localization of the inflammatory receptors into rafts, and triggers inflammation. These mechanisms may contribute to HIV-associated metabolic co-morbidities. Our work demonstrates how a single viral factor released from infected cells into circulation may cause a pleiotropy of pathogenic responses.
Author summary
HIV infects only a limited repertoire of cells expressing HIV receptors. Nevertheless, co-morbidities of HIV infection, such as atherosclerosis, dementia, renal impairment, myocardial pathology, abnormal haematopoiesis and others, involve dysfunction of cells that can not be infected by HIV. These co-morbidities persist even after successful application of antiretroviral therapy, when no virus is found in the blood. Many co-morbidities of HIV have a common element in their pathogenesis, impairment of cholesterol metabolism. In this study we show that HIV protein Nef released from infected cells in extracellular vesicles is taken up by un-infected ('bystander') cells impairing cholesterol metabolism in these cells. This impairment causes formation of excessive lipid rafts, re-localization of the inflammatory receptors into rafts, and triggers inflammation. These mechanisms may contribute to HIV-associated metabolic co-morbidities. Our work demonstrates how a single viral factor released from infected cells into circulation may cause a pleiotropy of pathogenic responses.
Another co-morbidity likely related to the effects of Nef on cholesterol metabolism is HIV-associated neurocognitive disorder (HAND).........These findings are consistent with a suggestion that Nef-containing exosomes constitute an important factor in pathogenesis of several metabolic co-morbidities of HIV disease. Nef is dispensable for HIV replication in vitro [39], but patients infected with Nef-deficient HIV strain develop very mild, if any, HIV disease [40], implying that the main pathogenic function of Nef is systemic rather than intracellular [16]. Recombinant Nef injected into apoe-/- mice fed a high-fat diet enhanced atherosclerosis and caused hypoalphalipoproteinemia and hypertriglyceridemia characteristic to HIV infection [41]. We propose that Nef creates a metabolic milieu that is favourable for HIV replication at the same time causes metabolic complications in the host. Cholesterol and lipid rafts play important role in HIV biology (for review see [10]), impairment of cholesterol metabolism and overabundance of rafts would benefit the virus, but at the same time may exacerbate co-morbidities that have impairment of cholesterol metabolism and inflammation as elements of their pathogenesis. These considerations firstly apply to atherosclerosis, a pathology responsible for a range of cardiovascular co-morbidities of HIV disease. Reduction in ABCA1 and consequently of cholesterol efflux, as well as increased abundance of TREM-1 in rafts, are recognized factors promoting accumulation of cholesterol in macrophages and inflammation [29, 42], two key elements in pathogenesis of atherosclerosis. Given that Nef-containing exosomes can be released from infected cells and have a considerable potency to affect cholesterol metabolism in uninfected cells, they can potentiate cholesterol accumulation and inflammatory response not only in HIV-infected, but in all macrophages, contributing significantly to the risk of atherosclerosis. Moreover, the effects of Nef on cholesterol metabolism in other cells involved in pathogenesis of atherosclerosis that cannot be infected with HIV, such as endothelial and smooth muscle cells, may also contribute to this co-morbidity.
Another co-morbidity likely related to the effects of Nef on cholesterol metabolism is HIV-associated neurocognitive disorder (HAND). HIV infects macrophages, glial cells and astrocytes, but does not infect neurons, which play a key role in neurodegenerative disorders, nor endothelial cells, responsible for the integrity of blood-brain barrier [43]. Recently, Raymond and Khan [9, 17] in a series of elegant studies demonstrated the role of Nef-containing exosomes in pathogenesis of HAND, however, direct toxicity of Nef was suggested as a putative mechanism. The role of cholesterol metabolism and of lipid rafts in pathogenesis of neurodegenerative diseases is firmly established [25, 44], so the effects of Nef, through regulating lipid rafts directly and/or through potentiating inflammation, may contribute, if not drive, neurodegeneration caused by HIV infection.
Pathogenesis of several other co-morbidities of HIV infection may also involve impairment of cholesterol metabolism by Nef. Haematological co-morbidities of HIV infection were, on the one hand, attributed to the action of Nef [45], and, on the other hand, have cholesterol metabolism in the centre of their pathogenesis [46]. The role of ABCA1 and cholesterol metabolism in pathogenesis of diabetes, a frequent co-morbidity of HIV infection [47], is also well established [48]. The effects of Nef on these and other HIV co-morbidities may be direct, targeting cholesterol metabolism-related elements of their pathogenesis, but also indirect, through potentiating inflammation, which in turn has impairment of cholesterol metabolism and enhanced formation of lipid rafts among the key elements of pathogenesis [26].
Nef-containing exosomes were found in plasma of patients receiving antiretroviral treatment and having no detectable viral load [9, 17, 49]. Most likely, the origin of these exosomes is a small number of HIV infected cells residing in HIV reservoirs [5, 50]. Secretion of exNef from infected cells can occur despite effective HIV suppression: current antiretroviral drugs affect every step of the HIV life cycle except transcription and translation, thereby allowing production and release of viral components such as Nef [7, 8]. Interestingly, Nef itself is a potent activator of microvesicle exocytosis, and mechanism of release of Nef-containing exosomes from infected cells has been recently described [9, 51]. Delivery by exosomes, however, is not the only mechanism behind the bystander effects of Nef: transfer of Nef to uninfected cells by trogocytosis has been recently demonstrated [51].
It must, however, be recognized that the broad clinical relevance of our findings remains unclear. While the results of the experiments on the effects of exosomes from pooled plasma samples of few HIV-infected subjects are consistent with the proposed mechanisms, they provide only limited evidence for the importance of these mechanisms in clinical setting, especially in the context of ART treatment. A clinical study relating clinical endpoints to biological activity of exosomes isolated from plasma of HIV-infected subjects treated with different regimens of ART and with different levels of exNef in their blood is required to convincingly address this question.
In conclusion, Nef released from HIV-infected cells in exosomes may be responsible for systemic impairment of cholesterol metabolism in cells not infected by HIV, causing inflammation and contributing to multiple co-morbidities of HIV disease.
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1007907
DISCUSSION: In this study, we investigated how exosomes containing the HIV protein Nef alter cellular functions and cause inflammation associated with HIV infection. The main findings of this study can be summarized as follows: (i) Nef-containing exosomes (exNef) rapidly and effectively deliver Nef to macrophages; (ii) exNef affect cellular cholesterol metabolism (inhibit the abundance of ABCA1 and reduce cholesterol efflux) similar to the effect of HIV infection or recombinant Nef, but the effect of exNef was several orders of magnitude more potent than of recombinant Nef; (iii) exNef increase the abundance of lipid rafts through impairment of activation of Cdc42 followed by re-organization of actin cytoskeleton; (iv) exNef potentiate inflammatory responses in vitro and in vivo, likely through the effects on lipid rafts followed by activation of ERK1/2, activation of NLRP3 inflammasome and secretion of pro-inflammatory cytokines; (v) injection of exNef in vivo causes monocytosis, increases the abundance of rafts in monocytes, reduces ABCA1 abundance in several tissues, increases inflammatory cytokine production and causes hyperalphalipoproteinemia; (vi) ex vivo, exosomes isolated from HIV-infected cells or from plasma of HIV-infected subjects, but not from ΔNef-HIV infected cells or subjects, increase lipid rafts, TLR4 recruitment to rafts and inflammatory response in macrophages. The fact that Nef-containing exosomes produced by Nef-transfected HEK293 cells, HIV-infected MDMs and isolated from plasma of HIV-infected donors had similar effects on lipid rafts independently of the source, and exosomes not containing Nef did not show these effects, is a strong indication that the cause of these effects is in fact Nef, as opposed to other exosome constituents.
HIV productively infects CD4+ T-cells, macrophages and related cells expressing CD4 receptor and CCR5 or CXCR4 co-receptors, but not other cell types that lack these molecules, and cannot replicate in tissues where susceptible cells are underrepresented. Nevertheless, clinical manifestations of HIV infection often involve dysfunction of cells and tissues which are not, and could not be, infected by HIV. HIV disease is associated with numerous co-morbidities, such as atherosclerosis, metabolic syndrome, myocardial pathology, abnormal adipose tissue, dementia, respiratory complications, abnormal haematopoiesis, and many others [1]. Paradoxically, many co-morbidities persist, albeit with reduced severity, even after successful application of antiretroviral therapy (ART), when no virus is detected in the blood and immunodeficiency is mitigated. One example is atherosclerosis and dyslipidaemia associated with HIV infection [2]. Pathogenesis of these co-morbidities involves vascular endothelial and smooth muscle cells as well as hepatic cells, none of which susceptible to HIV infection. Macrophages, which are also involved in pathogenesis of atherosclerosis, can be infected by HIV, however, the proportion of infected monocytes in blood and macrophages in tissues of ART-treated patients is too low to be a major driver of systemic atherosclerosis. One explanation of the systemic pathology in treated HIV infection is "bystander" effects. The effect of HIV infection on bystander cells has been described (for review see [3]) and was attributed to individual HIV proteins released from infected cells [4, 5] and taken up by uninfected cells. Nef (Negative Regulatory Factor), for example, is known to affect tissues through cytotoxicity, and other HIV proteins released from infected host cells may contribute to the systemic effects of the infection in various ways [6]. These effects can take place even in the context of effective anti-retroviral treatment due to ongoing expression of HIV proteins in long-living infected cells and HIV replication in viral reservoirs [5, 7]. Nef was found in blood of HIV-infected patients receiving ART [8, 9].
Many of the diverse co-morbidities of HIV disease have a common element that plays a prominent role in their pathogenesis, impairment of cholesterol metabolism. Cholesterol also plays a key role in the lifecycle of HIV, and HIV interacts with host cholesterol metabolism machinery [10]. We have previously identified the molecular mechanism by which HIV infection affects cholesterol metabolism [11]. HIV targets a pathway responsible for removal of excessive cholesterol from peripheral cells, reverse cholesterol transport pathway, and the key element of this pathway, lipid transporter ATP binding cassette transporter type A1 (ABCA1).
We demonstrated the central role of the viral protein Nef in this activity: Nef inactivates host cell's ABCA1 leading to reduction of cholesterol efflux and accumulation of intracellular cholesterol [11-13]. The concentration of free Nef in plasma of HIV-infected patients is, however, very low, and most Nef is secreted by HIV-infected cells in exosomes [9]. Exosomes were implicated in pathogenesis of HIV disease [14] and are increasingly considered an important way of cell-to-cell communication ensuring rapid and targeted delivery of the molecules from one cell to another [15]. The contribution of Nef-containing exosomes to systemic effects of HIV infection [16] and to pathogenesis of HIV-associated dementia [17] has been proposed. In this study, we investigated the effect of Nef-containing exosomes on uninfected macrophages and identified the mechanism that may play a key role in pathogenesis of several metabolic co-morbidities of HIV infection.
Nef-containing exosomes reorganize lipid rafts
One consequence of inactivation of ABCA1 and suppression of cholesterol efflux is an increase in the abundance of lipid rafts [23, 24]. Lipid rafts play a prominent role in pathogenesis of many diseases, from cardiovascular disease and inflammation to dementia [25, 26]. We previously demonstrated that intracellular expression of Nef results in increased abundance of rafts in macrophages [12].........The abundance of rafts was also increased in CD4+ T-cells (p<0.05, n = 3), although the effects were smaller, presumably because T-cells, compared to MDM, have lower abundance of ABCA1 [12], the main target of exNef. Collectively, these findings indicate that exNef increases the abundance of lipid rafts, changes lipid raft composition and reduces the abundance of ABCA1 in rafts.
These experiments indicate that exNef potentiates a signalling cascade originating from TLR4 and resulting in NLRP3 inflammasome activation and release of IL-1β........Thus, it appears that exNef, likely through its effects on ABCA1, impairs activation of Cdc42 leading to reduction of actin polymerization, increased abundance or lipid rafts and potentiated inflammatory response.
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