In this study, we have shown that gut biopsies from 8 out of 12 individuals with ME displayed immunoreactivity consistent with the presence of HERV proteins. However, the same immunoreactivity was not observed in the biopsies of the controls. Additionally, we have shown that the immunoreactivity was observed in cells with a phenotype that is consistent with pDCs. These observations suggest that the presence of the HERV protein in pDCs may be associated with a pathological manifestation in at least a subset of individuals with ME.
Our detection of proteins that react with monoclonal antibodies to HERVs is consistent with HERV expression. Nevertheless, it could be argued that an exogenous retroviral infection may potentially account for the observed results. The present study was conducted using surplus clinical biopsies; therefore, the proper matched specimens were not available to conduct a rigorous transcriptional analysis on all cases. This issue was compounded by the de-identified nature of the specimens, thus preventing the collection of additional biological material. Nonetheless, cryopreserved lymphocytes and preserved RNA from a duodenal biopsy were available from two HERV-positive ME cases. Therefore, we performed unbiased next-generation sequencing (NGS) on both RNA derived from the duodenum and purified pDCs from these individuals. Multiple contigs of known HERV genes were observed; however, no open reading frames were identified that could account for an infectious retrovirus (unpublished data). We also attempted to identify an infectious murine leukemia virus (MLV)-related virus by co-culturing lymphocytes and purified pDCs using the DERSE indicator cell line (43
). The DERSE indicator cell line (a kind gift from Dr. Vineet KewalRamani) is derived from the prostate cancer cell line LNCaP and stably transfected with an MLV vector containing the green fluorescent protein (GFP) gene in reverse orientation. Only after rescue and transfer to new cells through reverse transcriptase and integrase enzymatic activity can the GFP be detected. In previous experiments, the sensitivity of this assay was established by detecting infectious MLV-related virus derived from two individual 22Rv1 cells per culture (unpublished data). In spite of using this sensitive detection method, we were unable to identify an infectious MLV-related retrovirus. Although we cannot definitively rule out the possibility that our observations were the result of an infectious retrovirus, our results using HERV-specific monoclonal and polyclonal antibodies, as well as NGS and co-culture methods, strongly argue against this.
A number of immunological observations have been described in relation to ME; however, the data presented here, to our knowledge, represent the first report of an association of pDCs with this disease. While the expression of endogenous retroviral proteins in the pDCs of ME cases does not intrinsically explain pathology, the observation that the immunoreactive proteins are only observed in pDCs is supportive of this concept. This supposition is further supported by our previous report of the dysregulation of inflammatory cytokines in a cohort of ME cases (44
). One subset of this cohort was characterized by elevated interleukin-8 (CXCL8), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interferon gamma-induced protein-10 (CXCL10), macrophage inflammatory proteins-alpha and beta (CCL3 and CCL4) and depressed interferon-alpha (IFN-α) production, all of which are produced in significant quantities by pDCs (45
). The production of type-I IFN by pDCs is a result of the engagement of Toll-like receptors (TLR) 7 and 9 in early endosomes (46
), whereas inflammatory cytokine production occurs as a result of their engagement in late endosomes (47
). A deviation in the spatiotemporal regulation of pDCs favoring TLR-activation in late endosomes over early endosomes would be consistent with our previous observations of cytokine dysregulation in those with ME. Similar abnormalities in the pDC function and distribution has also been reported in individuals with IBD. Brumgard et al
. observed that isolated pDCs from patients with flaring Crohn’s disease and ulcerative colitis overproduce inflammatory cytokines while underproducing type-I IFN in response to the synthetic TLR-9 agonist ODN 2216 (48
). Additionally, they reported on a significantly higher frequency of pDC in the inflamed colonic mucosa of patients with IBD. These data suggest that our observations in subjects with ME may not be unique to this disease but may, in fact, be common to diseases characterized by chronic inflammation.
A potential dysregulation of pDCs may suggest an explanation for a number of other clinical observations associated with ME. As stated previously, pDCs are most remarkable for their ability to produce copious amounts of type-I IFN. In 1978, Trinchieri and colleagues indirectly identified pDCs by their ability to activate natural killer (NK) cell-mediated cytotoxicity through the production of IFN-α (49
) and several subsequent studies have expanded upon the importance of type-I IFN in modulating NK cell function (50
). Decreased NK cell activity is a commonly reported observation associated with ME (51
); therefore, an aberrant pDC response leading to a decrease in IFN-α would be consistent with dysregulation of NK cells. Inflammatory cytokine and chemokine abnormalities have also been reported in association with ME by other researchers. For instance, Natelson et al
. reported elevated levels of CXCL8 and IL-10 in the spinal fluid of individuals with influenzalike onset of ME (52
). Moreover, Vernon et al
. and others reported that CXCL8 gene transcription was elevated in those with ME (52
). Finally, Chao et al
. reported IL-6 to be up-regulated in subsets of ME cases (54
In our previous report, the expression of inflammatory cytokines was more prevalent in a subset of individuals characterized by a gamma-delta T-cell clonality (44
). A similar clonality has been observed in autoimmune diseases characterized by the expression of HERVs, such as rheumatoid arthritis and MS (55
). Additionally, the expansion of gamma-delta T-cells in response to the expression of endogenous gamma retroviruses has been reported in animal models (57
). Some HERV proteins act as superantigens (34
), promoting the expansion of T-cell populations and, therefore, the observation of gamma-delta T-cell clonality associated with ME, as well as other diseases, may be the result of HERV superantigen stimulation.
Although the Canadian consensus criteria for ME and the Fukuda criteria for CFS do not include symptoms of autoimmunity, the recent study by Fluge et al
. supports the notion that at least a subset of individuals with ME may have an autoimmune element associated with their disease (11
). Autoimmune diseases, such as SLE, MS and rheumatoid arthritis, have several symptoms that overlap with those of ME and all have been associated with the pDC dysfunction. Moreover, the same autoimmune diseases are also reported to be associated with the expression of HERVs, although a physical connection between HERVs and pDCs has not yet been reported. As Stoye points out in his recent review (58
), the role of HERVs in autoimmunity remains an unproven hypothesis; however, an increasing number of studies suggest that HERVs may have the capacity to contribute to disease pathology (59
). It is noteworthy that work conducted in the laboratory of Dr. Bridget Huber showed that HERV-K18 expression could be induced by herpes viruses such as Epstein-Barr virus and human herpes virus 6 (HHV-6) (34
). Consistent with that work and with the data presented here, both of these viruses have been observed in the duodenum of individuals with ME (62
The expression of HERV proteins in autoimmune diseases such as SLE, MS and Sjögren’s syndrome, is also evident by reports of antibodies to retroviral proteins in those who are not found to be infected with an exogenous retrovirus (26
). If the expression of HERV proteins in pDCs is found to be associated with these autoimmune diseases, it may help explain the presence of such antibodies. Inflammation is known to increase HERV expression (63
); therefore, if pDC-associated inflammation drives the expression of endogenous retroviruses, it is also conceivable that dysregulated expression of other proteins in pDCs may occur. Consequently, the antigen-presenting abilities of pDCs may contribute to the production of auto-reactive antibodies, as is observed in ME (65
). The etiopathology associated with autoimmune diseases, HERV expression, and pDCs is clearly not a simple relationship. Nevertheless, the prospect of an involvement of HERVs and pDCs in ME pathology provides an opportunity for more directed studies into the immune dysregulation associated with this disease.
The expression of HERV proteins in pDCs may lead to ME-related pathology. Conversely, their expression might merely be the result of the inflammation associated with the disease, or perhaps a combination of both. Nevertheless, the presence of these proteins in the pDCs of individuals with ME but not in controls does support an involvement of pDCs in ME. The results presented in this report, if confirmed and extended by other studies, may provide a greater understanding over the etiopathology of ME.