Abstract
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by continuous inflammation and synovial hyperplasia. Currently, there is no cure, and treatment consists of early intervention with disease-modifying antirheumatic drugs (DMARDs) to slow disease progression and control symptoms. Some people with RA have suboptimal or no response to current treatments, thus there is an urgent need to understand the underlying disease mechanisms that could contribute to treatment resistance. Recent research has highlighted the role of elevated type I interferon (IFN-I) levels in treatment response. Persistent viral infection with the Epstein-Barr virus (EBV) has been linked to overactivation of the IFN-I response and to autoimmune disease. However, the three-way interaction between EBV, elevated IFN-I and treatment response is understudied. Understanding the contribution of the IFN-I pathway in this context may guide more effective and individualised treatment outcomes.
To establish an approach to this issue, peripheral blood mononuclear cells from 6 healthy participants were stimulated for 24 hours with an optimised concentration of a major IFN-I protein, IFNα, at 1000U/mL either alone or in combination with three selected DMARDs: Methotrexate (MTX), Infliximab (IFX), and Tocilizumab (TCZ). DMARD stimulation occurred at two timepoints: 30 minutes pre-IFNα or 30 minutes post-IFNα. Total RNA was extracted from cells, reverse transcribed into cDNA, and analysed for the expression of eight interferon-stimulated genes (ISGs): IRF1, IRF7, IFI6, IFITM3, ISG15, TRIM21, MxA, and PKR, using quantitative real-time polymerase chain reaction.
Downregulation of all ISGs was observed following treatment with DMARDs, irrespective of administration order. Significantly, IFI6 was decreased by all three treatments. An IFN score, calculated as the median fold change of all ISG expression, showed substantial inter-individual variability across conditions. Individuals with high baseline IFN scores experienced a decrease in their scores following DMARD treatment, whereas the inverse was observed for participants with low baseline IFN activity. Normalisation to IFNα-stimulated controls revealed greater post-treatment decreases with IFX and TCZ, whereas the inverse was observed with MTX. Qualitative measurement of antibodies against EBV nuclear antigen (EBNA) and viral capsid antigen were analysed using Enzyme-Linked Immunosorbent Assay on the participants' plasma. Preliminary data suggest that higher IFN scores occurred in participants who were positive for anti-EBNA-IgG.
The results in this thesis demonstrate direct modulation of ISG expression by DMARDs under physiologically relevant conditions of the IFN-I pathway. Furthermore, the heterogeneity in individual immune responses to DMARDs in this experimental set-up is of sufficient magnitude to justify further exploration. A biologically plausible explanation is that DMARD-induced reduction in antiviral ISG expression that keeps EBV in a latent state may lead to the reactivation of the virus, and the viral reactivation may, in turn, reduce DMARD efficacy. These data suggest that the IFN-I signature should not be viewed as a universal prognostic marker across all RA treatments, but rather as a potential tool to stratify individuals who might preferentially benefit from different classes of DMARDs.