Several studies have linked human endogenous retroviruses (HERVs) with autoimmune disease. We have previously demonstrated that the full-length polymorphic HERV-K113 provirus prevalence is increased in both Sjögren's syndrome (15.6%) and multiple sclerosis (11.9%) compared with the normal UK population (4%). Recent studies have shown that exogenous viral RNA can activate dendritic cells via the TLR7/8-mediated pathway. In this study we investigate the possibility that HERV-K113 RNA sequences are capable of activating both dendritic cells and macrophages. We examine the production of cytokines by both cell types in response to a series of different HERV-K113 RNA species to identify any differences in the profile produced.
A clone of the full-length HERV-K113 provirus was digested with restriction enzymes to generate different length fragments of the 5'-LTR and gag gene for in vitro transcription, producing single-stranded RNA species. The RNA was treated to remove any residual protein, DNA, lipopolysaccharide or other contaminants. After quantitation, the RNA was complexed with Lipofectamine 2000 (Invitrogen, Paisley, UK) or left uncomplexed before being used to treat cells. Cytokines levels in the cell supernatants were measured by ELISA. Dendritic cells were assayed for tumour necrosis factor (TNF), IL-6 and IP-10, while macrophages were assayed for TNF, IL-6, IL-8 and IL-10. Controls used were un-complexed RNA, poly-U synthetic RNA, and the chemical activator of TLR 7 and 8, Resiquimod (R-848).
Stimulation with R-848 produced TNF, IL-6, IL-8 and IL-10 from macrophages and TNF, IL-6 and IP-10 from dendritic cells (Table 1). Uncomplexed RNA did not stimulate cytokine production in any cell type demonstrating a lack of lipopolysacharide or other contaminant activation of cells. Stimulation of dendritic cells with each of the three complexed RNA species resulted in the production of IL-6, TNF and IP-10 (Table 1). Stimulation of macrophages only produced TNF. Although low levels of IL-6 were detected, there was no increase over control cells (Table 1). Neither IL-8 nor IL-10 was produced in macrophages in response to any of the RNA species. All three RNA species were more active than a poly-U synthetic RNA control.
Table 1. Cytokine release in response to one species of HERV-K113 RNA from different cells
The data demonstrate that RNA from HERVs is capable of inducing cytokine release by both macrophages and dendritic cells. The pattern of cytokine expression differs between the two cell types and may reflect different activation pathways in each case. This work demonstrates a possible mechanism of action for HERV induction of an inflammatory response leading to an autoimmune disease.