In a previous study, we established direct genetic evidence that HERV-Fc1 is associated with multiple sclerosis (42
). In 3 analyzed cohorts of MS patients and matching controls, a marker, rs391745, had the lowest combined P
value of 0.0003 for association with disease after Bonferroni correction (Fisher's formula). The identified locus on chromosome X harbored the HERV-Fc1 locus and no other coding capacities. No microRNA genes or small nucleolar RNA genes have been reported in the region, and the nearest known genes lie 141 kb upstream and 57 kb downstream, respectively, further substantiating the association.
In this study, anti-HERV-H/F Gag antibodies and flow cytometric analyses were used for the characterization of HERV-H/F Gag expression in PBMCs derived from MS patients. We showed, for the first time, significant differences in the expression of HERV-H/F Gag protein epitopes between PBMCs from healthy controls and those from MS patients. We found higher HERV-H/F Gag expression in monocytes and a tendency toward higher HERV-H/F Gag expression in CD19+
cells from patients with nonactive MS than in cells from healthy controls. In the group of patients with active MS, much greater differences in the HERV-H/F Gag expression relative to healthy controls were detected for CD4+
T cells. This is further evidence that the HERV-H/F family may play a role in the extensive activation of the adaptive immune responses in MS (15
). A previous study indicated that transcripts from HERV-H may play a biological role during T-cell activation. Transcription of HERV-H was restricted to the hematopoietic lineage and appeared to be maximal in T cells; therefore, its restriction could imply specificity (29
Strong cell-mediated immune responses to gammaretroviral HERV antigens (concomitantly with herpesvirus antigens) have been reported in MS (5
). Moreover, it has been shown that HERV peptides stimulate proliferation and immune response in MS patients with acute MS, but not in individuals with stable MS (15
). HERV peptide-specific proliferation and type 1 cytokine production by PBMCs was observed in individuals with acute MS, but not in those with stable MS, in whom a type 2 cytokine profile dominates (15
). Additionally, the authors observed that HERV-stimulated cytokine production is supported by CD4+
T helper cells. Different immune responses in patients with acute or chronic disease suggest that a cell-mediated and HERV-specific immune response is associated with MS (15
). In addition, the existence of a T-cell-mediated immune pathogenicity previously confirmed in vivo
with cell-free MSRV (HERV-W) virions injected into SCID mice humanized with human lymphoid grafts caused T-lymphocyte-dependent neuropathology (21
). This study suggests that MSRV retroviral particles from MS cultures have immunopathogenic properties mediated by T cells, which is compatible with the previously reported superantigen activity in vitro
We also found that in patients in the active phase of MS, the expression of HERV-H/F Gag by CD4+
T cells is significantly higher than that in CD8+
T cells. The importance of T cells in disease induction and further progression has been well documented (19
). Studies reported that brain-infiltrating CD8+
T cells persist as clonal expansions in the CSF and blood of MS patients (60
). Furthermore, selective enrichment of memory CD8+
T cells in the CSF of multiple sclerosis patients has been documented, suggesting a role for these CD8+
T cells in the pathogenesis of disease (26
). These data imply that many different T-cell populations can, in principle, be involved in the induction, propagation, and modulation of the disease. Both CD4+
T cells have been demonstrated in MS lesions, with CD4+
T cells predominating in acute lesions and CD8+
T cells being observed more frequently in chronic lesions (52
). Additionally, activated antigen-specific CD4+
T cells are found in all four described histopathological subtypes of MS (36
cells are the main effector T cells in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Furthermore, activated CD4+
Th1 cells contribute to inflammation in the CNS and to increased permeability of the blood brain barrier (4
). Recent data have established that interleukin 17 (IL-17)-producing CD4+
T cells, driven by IL-23 and referred to as TH17 cells, play a pivotal role in the pathogenesis of EAE (2
). In chronic TMEV disease (Theiler's murine encephalomyelitis virus [TMEV]-induced demyelinating disease)
, which is similar to the progressive forms of MS, recruitment of macrophages, TMEV-specific T cells, and antibodies induces apoptosis, inflammation, and demyelination in the CNS (46
). While early T-cell responses are directed to TMEV, the chronic T-cell-mediated disease involves the activation of myelin-reactive CD4+
The observed differences in HERV-H/F Gag expression for CD4+
T cells potentially substantiate the role of this HERV in regulating the activation of the adaptive immune responses in MS. The etiopathogenic mechanism and the importance of our findings are not yet known. The relatively normal levels of CD4+
T cells in patients with MS and the low CD4/CD8 ratio in active MS are in keeping with the apparently contradictory reports of the role of T helper cells in MS (34
) but are consistent with some earlier reports (41
A recently published flow cytometric study reported increased surface expression of specific gammaretroviral HERV-H and HERV-W Env epitopes on the surfaces of B cells and monocytes in patients with active MS. Furthermore, the higher expression of the HERV-H/W Env correlated with increased antibody reactivities to epitopes in sera from patients with active MS (6
). Together, the current and previous studies demonstrate that expression of these major structural components of the HERV H/F family is consistently increased in MS.
These findings are in accordance with previous reports indicating significantly higher antibody reactivity to HERV-H-derived Env and Gag antigens in sera (10
) and CSF (7
) from MS patients than in healthy controls. The increased expression of gammaretroviral HERV Env and Gag epitopes in PBMCs and increased anti-HERV antibody reactivities in the active phase of MS also substantiate a role for HERVs as potential biomarkers in the disease.
In the present study, the expression of HERV-H/F Gag protein epitopes in PBMCs was positively correlated with the levels of extracellular HERV-Fc1 RNA molecules detected in plasma, suggesting that the detected Gag proteins are Fc1 related. However, further studies with HERV-Fc1-specific antibodies are necessary to assess the levels of specific HERV-Fc1-encoded proteins in MS patients. Finally, flow cytometry analyses of HERV-H/F Gag expression in PBMCs could be useful in follow-up studies of MS patients to assess disease activity and response to the treatment. The importance of the observed expression has yet to be elucidated.
Extracellular HERV-Fc1 RNAs were detected in plasma samples from both healthy controls and MS patients. A 4-fold increase in the amount of extracellular HERV-Fc1 RNA was found in plasma samples from patients with active MS compared with healthy controls. The above data, obtained by HERV-Fc1 gag-specific absolute Q-PCR assays, show that HERV-Fc1-like RNA sequences are expressed and released to a greater extent by cells from MS patients and that the HERV-Fc1 gag gene is at least partially transcribed. Most importantly, our results show a statistically significant association between the extracellular HERV-Fc1 RNA titer in plasma and disease activity as assessed by comparing the clinically defined active/nonactive disease. The association may reflect the increased cellular activation observed during active disease periods, which may augment the activation and perhaps replication of HERV-Fc1. We are aware that the detected HERV-Fc1 RNA transcripts, containing only gag sequences, do not comprise full-length proviral sequences but may represent, e.g., terminated transcripts. However, we have observed a linear relationship between extracellular RNA for HERV-Fc1 gag transcript and HERV-H/F Gag protein levels, which suggests that most of the detected transcripts were full length. As observed, a 4-fold increase in levels of extracellular HERV-Fc1 gag RNA in patients with active MS strengthens the link between HERV-Fc1 and MS and at least indicates that HERV-Fc1 expression in plasma can be used as a marker of disease activity in MS.
At the DNA level, we have not seen any change in HERV-Fc1 gag copy numbers in lymphocytes of MS patients. There was no significant change in HERV-Fc1 gag DNA copies in MS patients compared with healthy controls. However, we have to take into account that certain individuals may have HERV-Fc1 copies on their X chromosomes without the in-frame stop codon between the gag pol genes, which could readily express longer HERV-Fc1 viral transcripts following transactivation by cofactors, as yet undefined.
An important caveat for this study is that we have not controlled for possible effects of the treatment of MS patients on the transcriptional activation of HERV-Fc1 elements. However, over half of the patients registered as having active MS in this study were not in treatment at the time of sampling. Nevertheless, a significant decrease in anti-Env antibody reactivity for HERV-H and HERV-W as a consequence of IFN-β therapy, closely linked to the efficacy of therapy, has been reported (51
). Furthermore, there are also suggestions that IFN therapy caused prompt and complete inhibition of MSRV circulation (38
The pathogenic potential of HERV-Fc1 has not yet been fully explored. The proviral DNA contains two stop codons and a frameshift in the pol
gene. The reading frames of the gag
genes appear to be complete (3
). Possible HERV-Fc1 infectivity can result from the contributions of other endogenous viruses through recombination or pseudotyping. Furthermore, the production of pathogenic molecules could be related to HERV-Fc1 retroviral expression. We suggest that there may be a pathogenic cascade in MS involving several specific pathogens interacting with particular genetic elements, leading to enhanced HERV-Fc1 protein expression.
In this paper, we show for the first time the quantification of HERV-Fc1 gag
transcripts in plasma from patients with active or nonactive MS. We found the highest levels of extracellular HERV-Fc1 RNAs in the patients with active disease. Despite the diversity of findings related to HERV expression, with several conflicting and negative reports (40
), numerous studies in the past have reported expression of HERVs in MS (13
). This applies to particles, viral proteins, and viral RNA (22
). However, causality has been notoriously difficult to establish from these experiments. Even if a significant association was established, the direction of causality was uncertain: did the virus induce disease, or did the disease induce virus? However, this is fundamentally different for HERV-Fc1, where genetic evidence also points to an association. Nevertheless, it remains to be directly demonstrated whether HERV-Fc1 is responsible for pathogenesis in MS.