Expression of HERV-Fc1, a human endogenous retrovirus, is increased in patients with active multiple sclerosis

Abstract

Multiple sclerosis (MS) is considered to be an autoimmune disease with an unknown cause and with immune system dysregulation. Among environmental factors, viruses are most often connected with the etiology of MS. Human endogenous retroviruses (HERVs) constitute 5 to 8% of human genomic DNA and have been detected as transcripts and proteins in the central nervous system (CNS) and peripheral blood, frequently in the context of neuroinflammation. HERV-Fc1, which belongs to the HERV-H/F family, has received our attention largely because of the genetic association with MS. We studied the expression of a capsid (Gag) protein of HERV-H/F origin by flow cytometry in peripheral blood mononuclear cells (PBMCs) from healthy controls and from MS patients with nonactive or active disease. There was a significant increase in HERV-H/F Gag expression in CD4(+) (P < 0.001) and CD8(+) (P < 0.001) T lymphocytes and in monocytes (P = 0.0356) in PBMCs from MS patients with active disease. Furthermore, we have undertaken the first rigorous SYBR green-based absolute quantitative PCR (Q-PCR) evaluation approach to quantify extracellular HERV-Fc1 RNA viral loads in plasma from MS patients and healthy controls. We found a 4-fold increase in extracellular HERV-Fc1 RNA titers in patients with active MS compared with healthy controls (P < 0.001). These findings strengthen the link between HERV-Fc1 and the pathology of MS. The cause and biological consequences of these differential expression levels will be the subject of further investigation. HERV-Fc1 biology could be a compelling area for understanding the pathology of MS and possibly other autoimmune disorders.

Fig 1

HERV-Fc1 gag DNA copy number calculations in the control group for the two genders. Shown are SYBR green absolute Q-PCR assay analyses of HERV-Fc1-positive healthy controls; HERV-Fc1 gag gene copy numbers are calculated per human genome equivalent (30 subjects for each gender). The data are expressed as median copy numbers/reaction plus standard deviation (SD), quantified on the basis of an external DNA calibration curve obtained from serial dilutions of plasmids containing the amplicon of interest, as described in Materials and Methods. The P value was calculated using the nonparametric Mann-Whitney U test.

Fig 2

Absolute quantification of extracellular HERV-Fc1 gag RNA molecule copy numbers in plasma samples from healthy controls and patients with MS. The data are expressed as a median RNA copy number/reaction (1 ml) plus SD. Experimental values were quantified on the basis of the external RNA calibration curve obtained from serial dilutions of plasmid containing the amplicon of interest transcribed in vitro (see Materials and Methods). P values were calculated using the nonparametric Mann-Whitney U test.

Fig 3

Flow cytometry analysis of intracellular HERV-H/F Gag expression in PBMCs from healthy controls and patients with nonactive and active MS. The bar graphs show comparative analysis of HERV-H/F Gag expression in CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, and monocytes in peripheral blood of healthy controls and patients with nonactive and active MS. The y axis represents FI. The bars show the median values and SD. The P values show statistical differences (<0.05) between the different groups. P values were calculated using the nonparametric Mann-Whitney U test. *, P ≤ 0.05; ***, P ≤ 0.001.

Fig 4

Correlation between HERV-H/F Gag expression and levels of extracellular HERV-Fc1 gag RNA in patients with nonactive MS. The graphs show a positive correlation between levels of extracellular HERV-Fc1 gag RNA and HERV-H/F Gag expression in CD4⁺, CD8⁺, and CD19⁺ cells and monocytes in patients with nonactive MS. The x axes represent FI for CD4⁺, CD8⁺, and CD19⁺ cells and monocytes These relationships were evaluated using the Spearman correlation test; r, Spearman rank correlation. *, P ≤ 0.05; **, P ≤ 0.01.

Fig 5

Correlation between HERV-H/F Gag expression and levels of extracellular HERV-Fc1 gag RNA in patients with active MS. The graphs show a positive correlation between levels of extracellular HERV-Fc1 gag RNA and HERV-H/F Gag expression in CD4⁺, CD8⁺, and CD19⁺ cells and monocytes in patients with active MS. The x axes represent FI for CD4⁺, CD8⁺, and CD19⁺ cells and monocytes. These relationships were evaluated using the Spearman correlation test; r, Spearman rank correlation. The orange dots indicate patients without drug treatment in the course of blood sampling. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.