A role for human endogenous retrovirus-K (HML-2) in rheumatoid arthritis: investigating mechanisms of pathogenesis

Abstract

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections within the human genome. These molecular fossils draw parallels with present-day exogenous retroviruses and have been linked previously with immunopathology within rheumatoid arthritis (RA). Mechanisms of pathogenesis for HERV-K in RA such as molecular mimicry were investigated. To clarify a role for HERVs in RA, potential autoantigens implicated in autoimmunity were scanned for sequence identity with retroviral epitopes. Short retroviral peptides modelling shared epitopes were synthesized, to survey anti-serum of RA patients and disease controls. A novel real-time polymerase chain reaction (PCR) assay was also developed to quantify accurately levels of HERV-K (HML-2) gag expression, relative to normalized housekeeping gene expression. Both serological and molecular assays showed significant increases in HERV-K (HML-2) gag activity in RA patients, compared to disease controls. The real-time PCR assay identified significant up-regulation in HERV-K mRNA levels in RA patients compared to inflammatory and healthy controls. Exogenous viral protein expression and proinflammatory cytokines were also shown to exert modulatory effects over HERV-K (HML-2) transcription. From our data, it can be concluded that RA patients exhibited significantly elevated levels of HERV-K (HML-2) gag activity compared to controls. Additional factors influencing HERV activity within the synovium were also identified. The significant variation in RA patients, both serologically and transcriptionally, may be an indication that RA is an umbrella term for a number of separate disease entities, of which particular HERV polymorphisms may play a role in development.

Fig. 2

Degree of inhibition shown by two patient bleeds (1 and 2) after incubation with non-biotinylated peptide and negative control peptide.

Fig. 1

(a) Levels of antibody reactivity to PLSK [human endogenous retrovirus (HERV)-K] peptide in RA patients and disease controls. (b) Levels of antibody reactivity in RA patients and disease controls to KPR [HERV-K (HML-2)] peptide. (c) Levels of antibodies specific for biotinylated KPR peptide in RA patient anti-sera and disease controls. RA, rheumatoid arthritis (n = 48); SLE, systemic lupus erythematosus (n = 23); OA, osteoarthritis (n = 27); IBD, inflammatory bowel disease (n = 17); NHD, normal healthy donors (n = 37).

Fig. 5

Correlation of all samples tested by both biotinylated enzyme-linked immunosorbent assay with KPR peptide and quantitative reverse transcription–polymerase chain reaction (n = 30). The R² value for the gradient of the trendline is shown on the graph (0·953).

Fig. 4

Comparison of human endogenous retrovirus (HERV)-K (HML-2) gag1 expression in different cell lines. Cell lines surveyed include human fibroblast-like synoviocytes (HFLS) derived from a rheumatoid arthritis patient (RA), osteoarthritis patient (OA), normal healthy donor (NHD) and mouse-derived fibroblasts (MF). A human non-synovial fibroblast cell line (HEK-293) was also tested.

Fig. 3

Levels of human endogenous retrovirus (HERV)-K (HML-2) transcription in patient samples as determined by real-time polymerase chain reaction to the HERV-K (HML-2) gag1 gene. Rheumatoid arthritis (RA) patient (n = 34), osteoarthritis patient (OA) (n = 17), non-RA inflammatory controls – irritable bowel disease (IC) (n = 5) and normal healthy donors (NHD) (n = 5).

Fig. 6

(a) Levels of human endogenous retrovirus (HERV)-K (HML2) gag1 transcriptional activity after incubation with proinflammatory cytokines [interleukin (IL)-1β, IL-6 and tumour necrosis factor-α]. Cell lines incubated included fibroblast-like synoviocytes taken from rheumatoid arthritis (RA) patient, osteoarthritis patient (OA) and normal healthy door (NHD). (b) Levels of HERV-K (HML-2) gag1 after transfection of plasmids encoding Epstein–Barr virus gene products EBNA1 (Epstein–Barr nuclear antigen), LMP1 (late membrane protein 1) and LMP2A (late membrane protein 2a). Cell lines transfected included human fibroblast-like synoviocytes (HFLS) derived from an RA patient, HFLS taken from an OA patient and a healthy donor and a Hodgkin's lymphoma cell line (KMH2).