Expression and activation of human endogenous retroviruses of the W family in blood cells and astrocytes: implications for the pathogenesis of multiple sclerosis

Background Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system, with demyelination and gliosis. Proposed pathogenic co-factors triggering MS pathogenesis are the Epstein Barr virus (EBV), and two elements of the W family of human endogenous retroviruses (HERV-W): MSRV, that forms free virions, and syncytin-1, the ERVWE1env protein; both retroelements have neuropathogenic properties. In the past we studied MSRV in MS patients in various temporal and clinical stages; in all cases, striking parallelisms between MS behaviour and MSRV/HERV-W presence/ load were found. By simultaneous detection of MSRV and HHV-6, we found a direct correlation between MS and MSRV presence/load, but not for HHV-6. MS brains over-express MSRVenv and syncytin-1 transcripts, with respect to controls, while EBV presence was not detected. Materials and methods Since late EBV seroconversion is a strong risk factor for MS development, we performed in vitro experiments on PBMC from MS patients and MSRV+ volunteers, as well as on U87-MG astroglioma cells, that were studied as such or were exposed to EBV or to recombinant EBV glycoprotein350 (EBVgp350), or to proinflammatory cytokines. The levels of MSRVenv and syncytin-1 mRNAs were evaluated by discriminatory real time RTPCR assays. Flow cytometry was used to evaluate the HERV-Wenv protein on the plasmamembrane, as well the PBMC subsets. Results Basal expression of MSRVenv and syncytin-1 occurs in astrocytes and in NK, B and monocyte cells, but not in T cells. This uneven expression is amplified in naive MS patients. Astrocyte infection by EBV and exposure to EBVgp350 stimulate the expression of HERV-W/MSRV/ syncytin-1, with requirement of the NF-kB pathway. In EBVgp350-treated PBMC, MSRVenv and syncytin-1 are activated in B cells and monocytes, but not in T cells, nor in the highly expressing NK cells. The latter cells, but not the T cells, are activated by proinflammatory cytokines. Conclusions The study demonstrates that there are interactions among the above proposed MS-cofactors. In vivo, a pathogenic outcome would depend on activation in abnormal situations/tissues, as it may occur in delayed EBV infection, or in the presence of particular host genetic backgrounds, or both.</br

Expression and activation by Epstein Barr virus of human endogenous retroviruses-W in blood cells and astrocytes: inference for Multiple Sclerosis

Background: Proposed co-factors triggering the pathogenesis of multiple sclerosis (MS) are the Epstein Barr virus (EBV), and the potentially neuropathogenic MSRV (MS-associated retrovirus) and syncytin-1, of the W family of human endogenous retroviruses. Methodology/Principal Findings: In search of links, the expression of HERV-W/MSRV/syncytin-1, with/without exposure to EBV or to EBV glycoprotein350 (EBVgp350), was studied on peripheral blood mononuclear cells (PBMC) from healthy volunteers and MS patients, and on astrocytes, by discriminatory env-specific RT-PCR assays, and by flow cytometry. Basal expression of HERV-W/MSRV/syncytin-1 occurs in astrocytes and in monocytes, NK, and B, but not in T cells. This uneven expression is amplified in untreated MS patients, and dramatically reduced during therapy. In astrocytes, EBVgp350 stimulates the expression of HERV-W/MSRV/syncytin-1, with requirement of the NF-kB pathway. In EBVgp350-treated PBMC, MSRVenv and syncytin-1 transcription is activated in B cells and monocytes, but not in T cells, nor in the highly expressing NK cells. The latter cells, but not the T cells, are activated by proinflammatory cytokines. Conclusions/Significance: In vitro EBV activates the potentially immunopathogenic and neuropathogenic HERV-W/MSRV/ syncytin-1, in cells deriving from blood and brain. In vivo, pathogenic outcomes would depend on abnormal situations, as in late EBV primary infection, that is often symptomatic, or/and in the presence of particular host genetic backgrounds. In the blood, HERV-Wenv activation might induce immunopathogenic phenomena linked to its superantigenic properties. In the brain, toxic mechanisms against oligodendrocytes could be established, inducing inflammation, demyelination and axonal damage. Local stimulation by proinflammatory cytokines and other factors might activate further HERV-Ws, contributing to the neuropathogenity. In MS pathogenesis, a possible model could include EBV as initial trigger of future MS, years later, and HERV-W/MSRV/syncytin-1 as actual contributor to MS pathogenicity, in striking parallelism with disease behaviour

Regulation of human endogenous retroviruses of the W family by type III interferon λ2 and HIV in astrocytes

Astrocytes play a key role in important neurological diseases such as multiple sclerosis (MS) and neuroAIDS. The multiple sclerosis-associated retrovirus (MSRV) is an active member of the W family of human endogenous retroviruses (HERV), able to produce extracellular particles; another HERV-W element is ERVWE1, producing only an env protein, named syncytin-1, that can be found intracellularly and on the plasma-membrane, but not extracellularly. MSRVenv and syncytin-1 proteins have pro-inflammatory properties that might be pathogenic, as observed in vitro and in animal models: they may cause neuroinflammation, neurodegeneration, alterations of the immune system and stress responses; both have been suggested as co-factors triggering the immunopathogenesis of MS.λ2 has been shown to favour the replication of exogenous retroviruses, such as HIV, we designed studies to detect possible effects of IFNλ2 on the above HERV-W retroelements in U87 MG astrocytes, by using real time RT-PCR assays that can selectively identify either MSRVenv or syncytin-1 transcripts.. Treatment of astrocytes with IFNλ2 was found to enhance the expression of both MSRVenv and syncytin-1, dose-dependently. If cells are infected by HIV, the expression of both elements is modified. Surprisingly, HIV caused opposite effects on the two HERV-W retroelements, since MSRVenv transcription was up-regulated, whilst that of syncytin-1 was down-regulated. Cell pre-treatment with IFNλ2 did not alter the effects of subsequent HIV infection on MSRVenv and syncytin-1. behaves as a pro-inflammatory cytokine, that can facilitate the expression of both exogenous and endogenous human retroviruses. Since Type III IFN. This cell line has the IL-28R receptor, and is sensitive to IFNλ2. It is totally unknown the role played by Type III IFN in neuropathologies like neuro-AIDS and MS, but it could be hypothesized that IFNλ

Novel reliable real-time PCR for differential detection of MSRV<i>env</i> and syncytin-1 in RNA and DNA from patients with multiple sclerosis

Two components of the HERV-W family of human endogenous retroviruses are activated during multiple sclerosis (MS) and proposed immunopathogenic co-factors: MSRV (MS-associated retrovirus), and ERVWE1 (whose env protein, syncytin-1, reaches the plasma membrane). MSRVenv and syncytin-1 are closely related, and difficult to distinguish each other. The sequences of extracellular MSRVenv and of syncytin-1 available in GenBank were compared with those found in MS patients and controls of the cohort under study. With respect to syncytin-1, MSRVenv sequences have a 12-nucleotide insertion in the trans-membrane moiety. Based on this insertion, discriminatory real-time PCR assays were developed, that can amplify selectively either MSRVenv or syncytin-1. The data of MS patients and controls indicated that MSRV and ERVWE1 are both expressed in the brain of MS patients, while only MSRV is present in the blood; MSRV was released in culture by PBMCs of MSRV-producer individuals. These cells expressed the complete MSRVenv gene in the absence of syncytin-1 expression, up to the final, fully glycosylated envelope protein product, since western blot staining with anti-HERV-Wenv antibody detected two bands of the same molecular weight (73 and 61 kDa) of the fully glycosylated and partially glycosylated HERV-Wenv uncleaved proteins. Beyond MSRVenv DNA copy numbers were more abundant in MS patients than in healthy humans, while syncytin-1 were unchanged. These findings reinforce the link between MSRV and MS

Clinical and therapy features of MS patients.

*<p>: n.a.: not applicable.</p

Chromosomal representation of multiple sites of integration in human DNA of HERV-W<i>env</i> loci covering ≥80% of the HERV-Wenv gene.

<p>The <i>in silico</i> analysis of the current version of the human genome was performed by NCBI Basic Local Alignment Search Tool (BLAST) program, using as queries the MSRV<i>env</i> and syncytin-1 sequences (PV14 MSRV clone, GenBank accession number AF331500, and ERVW-1<i>env</i> coding sequence NM_014590.3, respectively). From the initial BLAST-identified regions, only the sequences covering ≥80% of the HERV-Wenv gene were selected as target and are reported. Red dashes: target:score ≥200; mauves dashes: target:score 80–200. Hits: number of loci; Hit GIs: Locus number all matches; MT: mitochondrial chromosome.</p

Basal expression of MSRVenv/ syncytin-1/HERV-Wenv in U87-MG astrocytes and PBMC subsets. A.

<p>Detection of MSRVenv and syncytin-1 mRNAs of U87-MG cells, by real time RT-PCR (means of three experiments run in duplicate, calculated by the 2^−ΔCt method; bars indicate standard deviation. <b>B</b>. Flow cytometry evaluation of the HERV-Wenv protein on U87-MG plasma membrane. Shaded histograms: HERV-Wenv-specific staining; open histograms: isotype control. <b>C.</b> MSRV<i>env</i> and Syncytin-1 mRNA expression on PBMC from MSRV(+) donors as such, and after immunobeads separation in CD3⁺T, CD19⁺ B, CD56⁺/CD19⁻/CD3⁻ NK and CD19⁻/CD3–/CD56⁻ monocyte subsets, and subsequent monocyte differentiation into MDM (bars indicate standard deviation). <b>D</b>. Flow cytometry evaluation of the HERV-Wenv protein on the membrane of PBMC from a representative MSRV(+) donor <i>in toto</i> and after sorting of the CD3⁺T, CD19⁺ B, CD56⁺/CD19⁻/CD3⁻ NK and CD19⁻/CD3⁻/CD56⁻ monocyte subsets. Shaded histograms: HERV-Wenv-specific staining; open histograms: isotype control. <b>E.</b> Cell populations distribution of PBMC from a representative MSRV(+) donor before, and after capture by magnetic beads charged with anti-HERV-Wenv or with an unrelated isotype antibody. The unprocessed PBMC and the cells retained by the immunobeads were sorted for T, B, NK and monocyte markers. The unretained cells were also analysed (not shown). <b>F</b>. Presence of surface HERV-Wenv protein in blood cells from five MSRV(+) HD, five untreated MS patients and three MS patients under effective therapy, evaluated by flow cytometry in PBMC <i>in toto</i> and after sorting of the CD3⁺T, CD19⁺ B, CD56⁺/CD19⁻/CD3⁻ NK and CD19⁻/CD3−/CD56⁻ monocyte subsets. Each dot represents an individual; horizontal bars represent the means. HERV-Wenv positivity of CD3⁺T cells was <2% for all samples (not shown).</p

Expression of MSRVenv and syncytin-1 by PBMC subsets exposed to EBVgp350 or proinflammatory cytokines.

<p><b>A.</b> Levels of MSRV<i>env</i> and syncytin-1 mRNAs of PBMC from MSRV(+) HD treated overnight with 1–100 ng/ml of EBVgp350, either as such or separated in T, B, NK and monocyte subsets. <b>B.</b> Comparison of the MSRVenv and syncytin-1 mRNA levels of monocytes and MDM after overnight EBVgp350 treatment. A and B: Data are the means of three experiments run in duplicate, calculated by the 2^−ΔΔCt method; <b>C.</b> Expression of the HERV-Wenv protein on the plasma membrane, evaluated by flow cytometry as present env-specific positivity of PBMC treated for 24 h with TNFα (1 ng/ml), IFNγ (1000 IU/ml), or PMA (50 NM); the bars indicate standard deviations.</p

Env genes integrated in the human genome that are recognized by MSRV<i>env</i>- and syncytin-1-specific real-time PCR assays.

a<p>BLAST program, using as query the MSRV<i>env</i> amplicon; * amino acids; § nucleotides; ° Accession number; ^b BLAST program, using as query the syncytin-1 amplicon.</p