The role of miR-23a~24~27a cluster in the pathogenesis of treatment resistant rheumatoid arthritis

Background: Rheumatoid arthritis (RA) is a symmetric polyarthritis arising from autoimmune dysregulation leading to severe disability and increased risk of co- morbidities and death. A chronic disproportionate inflammatory process lies at the heart of disease pathogenesis. Breach of self-tolerance, subsequent immune effector cell activation in the context of abundant expression of effector cytokines all contribute to uncontrolled inflammation. Molecular safeguards that normally operate to promote immune regulation appear defective in RA. Intensive basic and translational research over the last 30 years have contributed the emergence of an array of new therapeutics for the treatment of RA, which has transformed patient outcomes. The identity of the cytokine targeting treatments that have been most successful elucidates a functional hierarchy that implicates elements of both innate and adaptive immunity. In particular, dysregulation of TNFα and IL-6 biology are at the core of effector pathways and as such unravelling their detailed regulation is of critical importance. Moreover their primary synthesis places myeloid cells, and, in particular, blood-derived monocytes at the heart of pathogenic circuitry. Best current clinical practice is to treat early disease and deploy aggressive treatments directed towards restoration of immune balance in virtually all patients. However only a proportion of such patients will actually have poor prognosis disease and in reality merit such aggressive interventions - the identification of such clinical endotypes is a major challenge for the next decade. The field of epigenetics and consequent regulatory control of inflammatory cells offers rich potential in this regard. Examples of such regulatory elements are small RNA species – microRNAs (miRs), which serve as negative regulators of cellular transcription and thereby repress protein translation. Importantly they do so across functionally integrated pathways, operating beyond individual moieties. A growing body of evidence implicates a significant role of miRs in the regulation of inflammatory processes in the context of RA. Objectives: To identify miR species that are differentially regulated in patients with poor response to therapeutic intervention, compared to patients with well- controlled disease and healthy controls. Thereafter, to characterise candidate 2 miRs arising from these investigations to thereby determine their functional significance. Together these studies will shed light on a substantially ignored area of RA biology, namely the underlying mechanisms that subserve drug resistance in RA. Key Results: Microarray profiling of CD14+ monocytes derived from patients with drug resistance upon receipt of DMARDs or biologic treatments, compared with good responders or matched healthy controls identified the miR-23a~24-2~27a cluster to be significantly repressed in monocytes from resistant RA. Further analysis identified that two members of the cluster, miR-23a and miR-27a are implicated in a feedback loop regulating the IL-6 pathway. Thus IL-6 stimulation of primary monocytes suppresses the expression of this miR cluster, permitting expression of their direct molecular target, namely IL-6R, thus sensitising cells to further IL-6 signalling. I also observed that cells lacking miR-23a and miR-27a express higher levels of the pro-inflammatory cytokines TNFα and IL-6 when stimulated with LPS, further confirming that lack of these miRs has direct implications for chronic inflammatory processes. The remaining member of this miR cluster, miR-24, was shown to directly target methylene tetrahydrofolate reductase but not dihydrofolate reductase enzymes, implicating it in the target pathway of methotrexate (MTX), the most commonly used anchor DMARD. Although this is unlikely to confer disease resistance, this interaction suggests that miR-24 levels could be predictive of tolerability of methotrexate use. The potential biomarker capabilities of miR-24 in relation to MTX use, or miR-23a and miR-27 with regards to responsiveness to anti-IL-6 or JAK signalling inhibition therapeutics will be evaluated in my future work. Conclusion: This series of studies has elucidated highly novel pathways that mediate amplification of inflammatory responses in blood-derived monocytes through feedback pathways operating via regulatory miRs. Furthermore, analysis of a distinct cohort of RA patients allowed identification of miR species that have the potential to be utilized as clinical biomarkers for treatment efficacy or tolerability evaluation. Although a separate validation study is required, the detailed investigation of the role of these miRs performed here provides a clear mechanistic insight into their function and will certainly support future discovery .