Activation of MSRV-type endogenous retroviruses during infectious mononucleosis and Epstein-Barr virus latency: the missing link with multiple sclerosis?

Abstract

The etiology of multiple sclerosis (MS) is still unclear. The immuno-pathogenic phenomena leading to neurodegeneration are thought to be triggered by environmental (viral?) factors operating on predisposing genetic backgrounds. Among the proposed co-factors are the Epstein Barr virus (EBV), and the potentially neuropathogenic HERV-W/MSRV/Syncytin-1 endogenous retroviruses. The ascertained links between EBV and MS are history of late primary infection, possibly leading to infectious mononucleosis (IM), and high titers of pre-onset IgG against EBV nuclear antigens (anti-EBNA IgG). During MS, there is no evidence of MS-specific EBV expression, while a continuous expression of HERV-Ws occurs, paralleling disease behaviour. We found repeatedly extracellular HERV-W/MSRV and MSRV-specific mRNA sequences in MS patients (in blood, spinal fluid, and brain samples), and MRSV presence/load strikingly paralleled MS stages and active/remission phases. Aim of the study was to verify whether HERV-W might be activated in vivo, in hospitalized young adults with IM symptoms, that were analyzed with respect to expression of HERV-W/MSRV transcripts and proteins. Healthy controls were either EBV-negative or latently EBV-infected with/without high titers of anti-EBNA-1 IgG. The results show that activation of HERV-W/MSRV occurs in blood mononuclear cells of IM patients (2Log10 increase of MSRV-type env mRNA accumulation with respect to EBV-negative controls). When healthy controls are stratified for previous EBV infection (high and low, or no anti-EBNA-1 IgG titers), a direct correlation occurs with MSRV mRNA accumulation. Flow cytometry data show increased percentages of cells exposing surface HERV-Wenv protein, that occur differently in specific cell subsets, and in acute disease and past infection. Thus, the data indicate that the two main links between EBV and MS (IM and high anti-EBNA-1-IgG titers) are paralleled by activation of the potentially neuropathogenic HERV-W/MSRV. These novel findings suggest HERV-W/MSRV activation as the missing link between EBV and MS, and may open new avenues of intervention.

Figure 1. MSRV-type mRNA expression by PBMC from IM patients and healthy donors (HD).

The amounts of MSRV-type HERV-Wenv transcripts were evaluated by discriminatory real time RT-PCR, and normalized by the 2^−ΔCt method (see Methods for details). Data are expressed as medians (line), with maximum and minimum values (whiskers); boxes represent interquartile range of the samples. Statistical significance was evaluated by the Mann-Whitney U-test for comparison of two groups, and by the Kruskal-Wallis test for three or four groups. (A) Comparison of IM patients (N = 17) and all healthy donors (HD, N = 24). (B) Comparison of IM patients (N = 17) and HD donors stratified according to plasmatic anti-EBNA-1 IgG titers, as EBV-negative (EBV−, N = 9), <600 IU/ml (N = 7), and >600 IU/ml (N = 8). Kruskall-Wallis test gave p = 0.0005 for all four groups, and p = 0.014 for the three HD groups.

Figure 2. Presence of HERV-Wenv protein on the plasma membrane of PBMC subpopulations from IM patients and controls.

Flow cytometry evaluation of alive PBMC from IM patients (IM) and healthy donors (HD), either with anti-EBNA-1 IgG titers >600 IU/ml or EBV− negative (EBV−). Data were evaluated as percentage of cells with HERV-Wenv-specific staining, as specified in Methods, and are expressed as medians (line), with maximum and minimum values (whiskers); boxes represent interquartile range of the samples. Statistical significance was evaluated by the Mann-Whitney U test for comparison of two groups. When not specified, differences were not significant. All samples (four for each group) were evaluated in the same day. (A) Comparison of HERV-W-specific staining of whole PBMC. (B), (C), (D) HERV-W-specific staining of PBMC subpopulations sorted by specific staining with anti-CD19, anti-CD16 and anti-CD14 antibodies, respectively.