Neuro-HIV-New insights into pathogenesis and emerging therapeutic targets.

HIV-associated neurocognitive disorders (HAND) is a term describing a complex set of cognitive impairments accompanying HIV infection. Successful antiretroviral therapy (ART) reduces the most severe forms of HAND, but milder forms affect over 50% of people living with HIV (PLWH). Pathogenesis of HAND in the ART era remains unknown. A variety of pathogenic factors, such as persistent HIV replication in the brain reservoir, HIV proteins released from infected brain cells, HIV-induced neuroinflammation, and some components of ART, have been implicated in driving HAND pathogenesis in ART-treated individuals. Here, we propose another factor-impairment of cholesterol homeostasis and lipid rafts by HIV-1 protein Nef-as a possible contributor to HAND pathogenesis. These effects of Nef on cholesterol may also underlie the effects of other pathogenic factors that constitute the multifactorial nature of HAND pathogenesis. The proposed Nef- and cholesterol-focused mechanism may provide a long-sought unified explanation of HAND pathogenesis that takes into account all contributing factors. Evidence for the impairment by Nef of cellular cholesterol balance, potential effects of this impairment on brain cells, and opportunities to therapeutically target this element of HAND pathogenesis are discussed.

Upregulated lncRNA-NEF predicts recurrence and poor treatment outcomes of ankylosing spondylitis.

INTRODUCTION: Osteoporosis is related to lncRNA-neighboring enhancer of FOXA2 (NEF) and inversely correlated to ankylosing spondylitis (AS), implying that lncRNA-NEF might also relate to AS. Thus, the study was carried out to investigate the involvement of lncRNA-NEF in AS. METHODS: The study included 60 AS patients and 60 healthy controls. LncRNA-NEF expression in synovial fluid samples was analyzed by reverse transcription quantitative real-time polymerase chain reaction. Disease activity of the 60 AS patients was determined using the Ankylosing Spondylitis Disease Activity Score (ASDAS) 1-4 and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Western blot was carried out to investigate the effects of lncRNA-NEF on inflammatory factors in human fibroblast-like synovial (HFLS) cells. A 3-year follow-up was performed to analyze the role of lncRNA-NEF in the prediction of the recurrence of AS. RESULTS: Our study observed that lncRNA-NEF expression was upregulated in synovial fluid of AS patients and significantly correlated with the ASDAS 1-4, BASDAI, erythrocyte sedimentation rate (ESR), and C-reactive protein level (p < .05). Treatment with nonsteroidal anti-inflammatory drugs significantly downregulated lncRNA-NEF expression (p < .01). A 3-year follow-up showed that patients with high lncRNA-NEF levels had a high recurrence rate (hazard ratio = 2.266). In addition, lncRNA-NEF was found to regulate the expression of inflammatory factors in HFLS cells. CONCLUSIONS: Therefore, lncRNA-NEF upregulation can predict recurrence and poor treatment outcomes of AS and has a great potential to serve as a predictive biomarker factor for the recurrent AS.

The HIV-1 proviral landscape reveals that Nef contributes to HIV-1 persistence in effector memory CD4+ T cells.

Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4+ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4+ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4+ T cells when compared with naive, central, and transitional memory CD4+ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.

HIV Nef and Antiretroviral Therapy Have an Inhibitory Effect on Autophagy in Human Astrocytes that May Contribute to HIV-Associated Neurocognitive Disorders.

A significant number of people living with HIV (PLWH) develop HIV-associated neurocognitive disorders (HAND) despite highly effective antiretroviral therapy (ART). Dysregulated macroautophagy (autophagy) is implicated in HAND pathogenesis. The viral protein Nef, expressed even with suppressive ART, and certain antiretrovirals affect autophagy in non-CNS cells. Astrocytes, vital for CNS microenvironment homeostasis and neuronal health, require autophagy for their own homeostasis. We hypothesized that extracellular Nef and/or ART impact astrocyte autophagy, thus contributing to HAND. We studied in-bulk and selective autophagic flux in primary human astrocytes treated with extracellular Nef and/or a combination of tenofovir+emtricitabine+raltegravir (ART) using Western blotting, a tandem fluorescent LC3 reporter, and transmission electron microscopy/morphometry. We show that after 24 h treatment, Nef and ART decrease autophagosomes through different mechanisms. While Nef accelerates autophagosome degradation without inducing autophagosome formation, ART inhibits autophagosome formation. Combination Nef+ART further depletes autophagosomes by inducing both abnormalities. Additionally, extracellular Nef and/or ART inhibit lysosomal degradation of p62, indicating Nef and/or ART affect in-bulk and selective autophagy differently. Dysregulation of both autophagic processes is maintained after 7 days of Nef and/or ART treatment. Persistent autophagy dysregulation due to chronic Nef and/or ART exposure may ultimately result in astrocyte and neuronal dysfunction, contributing to HAND.

IL-12 and GM-CSF in DNA/MVA immunizations against HIV-1 CRF12_BF Nef induced T-cell responses with an enhanced magnitude, breadth and quality.

In Argentina, the HIV epidemic is characterized by the co-circulation of subtype B and BF recombinant viral variants. Nef is an HIV protein highly variable among subtypes, making it a good tool to study the impact of HIV variability in the vaccine design setting. We have previously reported a specific cellular response against NefBF with low cross-reactivity to NefB in mice. The aim of this work was to analyze whether the co-administration of IL-12 and GM-CSF, using DNA and MVA vaccine vectors, could improve the final cellular response induced. Mice received three DNA priming doses of a plasmid that express NefBF plus DNAs expressing IL-12 and/or GM-CSF. Afterwards, all the groups were boosted with a MVAnefBF dose. The highest increase in the magnitude of the NefBF response, compared to that induced in the control was found in the IL-12 group. Importantly, a response with higher breadth was detected in groups which received IL-12 or GM-CSF, evidenced as an increased frequency of recognition of homologous (BF) and heterologous (B) Nef peptides, as well as a higher number of other Nef peptide pools representing different viral subtypes. However, these improvements were lost when both DNA cytokines were simultaneously administered, as the response was focused against the immunodominant peptide with a detrimental response towards subdominant epitopes. The pattern of cytokines secreted and the specific-T-cell proliferative capacity were improved in IL-12 and IL-12+GM-CSF groups. Importantly IL-12 generated a significant higher T-cell avidity against a B heterologous peptide.This study indicates that the incorporation of DNA expressing IL-12 in DNA/MVA schemes produced the best results in terms of improvements of T-cell-response key properties such as breadth, cross-reactivity and quality (avidity and pattern of cytokines secreted). These relevant results contribute to the design of strategies aimed to induce T-cell responses against HIV antigens with higher quality.

Strong ability of Nef-specific CD4+ cytotoxic T cells to suppress human immunodeficiency virus type 1 (HIV-1) replication in HIV-1-infected CD4+ T cells and macrophages.

A restricted number of studies have shown that human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic CD4+ T cells are present in HIV-1-infected individuals. However, the roles of this type of CD4+ T cell in the immune responses against an HIV-1 infection remain unclear. In this study, we identified novel Nef epitope-specific HLA-DRB1*0803-restricted cytotoxic CD4+ T cells. The CD4+ T-cell clones specific for Nef187-203 showed strong gamma interferon production after having been stimulated with autologous B-lymphoblastoid cells infected with recombinant vaccinia virus expressing Nef or pulsed with heat-inactivated virus particles, indicating the presentation of the epitope antigen through both exogenous and endogenous major histocompatibility complex class II processing pathways. Nef187-203-specific CD4+ T-cell clones exhibited strong cytotoxic activity against both HIV-1-infected macrophages and CD4+ T cells from an HLA-DRB1*0803+ donor. In addition, these Nef-specific cytotoxic CD4+ T-cell clones exhibited strong ability to suppress HIV-1 replication in both macrophages and CD4+ T cells in vitro. Nef187-203-specific cytotoxic CD4+ T cells were detected in cultures of peptide-stimulated peripheral blood mononuclear cells (PBMCs) and in ex vivo PBMCs from 40% and 20% of DRB1*0803+ donors, respectively. These results suggest that HIV-1-specific CD4+ T cells may directly control HIV-1 infection in vivo by suppressing virus replication in HIV-1 natural host cells.

From viral infection to pulmonary arterial hypertension: a role for viral proteins?

The vascular pathology seen in severe pulmonary arterial hypertension (PAH) is remarkably similar despite the fact that it arises in diverse conditions including idiopathic cases, those associated with collagen vascular diseases, with abnormal blood flow, such as patients with Eisenmenger physiology, and with the use of anorexigen drugs. The pathogenesis of severe PAH is clearly complex, and probably results from the interaction of multiple modulating genes with environmental factors. HIV is evidently a risk factor for the development of PAH, and the increased prevalence of the disease in HIV-infected patients compared with the general population has been noted for several years. The mechanism by which infection leads to full-blown PAH is, however, unknown. Attempts to localize the virus in the vascular lesions or endothelial cells of affected patients have been unsuccessful, suggesting that a direct role of the virus is unlikely, and indicating that the underlying mechanism in pulmonary arterial hypertension associated with HIV (HIV-PAH) is related to the indirect action of infection, possibly through the action of pleiotropic viral proteins. One such candidate HIV protein is one of the first to be detected after invasion of the host cell, Nef. In this article we discuss recent studies on a potential role for Nef in HIV-PAH, with special reference to the knowledge gained from the SIV model of HIV infection.