Dr Mihir Wechalekar

Recipient: Dr Mihir Wechalekar
Intended department: Bruce Miller Fellowship & ARA funded- Department of Rheumatology- Flinders University


Improving Outcomes in Rheumatoid Arthritis


Rheumatoid Arthritis (RA) is an inflammatory, destructive and debilitating autoimmune disease, where by the immune system infiltrates and attacks self-tissues. In RA patients these tissues are the synovial membrane, a specialised connective tissue encasing the fluid lubricant in joints. Although significant progress has been made in treating RA, current therapies are ineffective in over 50% of patients and long term disease remission is rare. In these patients with ongoing active disease, societal impact in terms of disability, quality of life and impact on work-capacity are profound. Strong evidence suggests that the immune cell types that invade the synovial membrane heavily influence disease prognosis as well as treatment success. However, the mechanisms regulating such responses remain poorly understood. In part because of this, current treatment approaches are mostly empirical, with very variable responses.
Background and research significance: The research in its wider context Current therapeutic intervention in RA costs the Australian health system approximately 500M/year, and better allocation of treatments presents a significant opportunity to improve patient outcomes while reducing the economic burden to the Australian taxpayer. Importantly the research proposed in this application, and now well under way, will result in greater prediction of treatment responses, eliminating ineffective therapy and potentially reduce the time taken to achieve disease remission. This is particularly important as it is now known that effective early treatment of RA is critical to preventing irreversible bone loss and life-long disability. Moreover, approximately 50% of patients with RA are no longer in full-time work 4 years after diagnosis of disease; indeed, good treatment response translates to less deformity and disability and our previous work1 has shown that those who respond well to treatment stay in the work force longer. The research proposed in the original study is set out with the aim to characterise cellular subsets and tissue phenotypes in early RA synovial tissue (ST) before and after treatments, in order to investigate different disease outcomes in response to traditional or biologic therapies. We gratefully acknowledge the Bruce Miller-ARA Post-Doctoral Fellowship for 2018, which enabled us to significantly advance our research by employing a research associate and establishment of novel techniques of analysis of synovial tissue (e.g. the Vectra microscopy) which would not have been possible without the support of this grant. The research methodologies we proposed to employ in this project can be separated in to 4 broad techniques: flow cytometry (perfect for assessing new cellular subsets), microscopy (ideal for assessing tissue phenotype), transcriptomic analysis (to assess the complete set of RNA transcripts that are produced by genes of inflammatory cells) and serum cytokine analysis (immune system messenger and inflammatory molecules), which together can be used for the identification of new biomarkers that predict effective responses in different patients. The 2018 Bruce Miller-ARA call for funding was submitted as a 2-year project, where our first step was to perform optimisation and analysis of flow cytometry to identify lymphocyte infiltrates in the ST and to perform routine Immunohistochemistry (IHC) for these early RA ST samples (see below). As the project progresses into the second year, upon which we will have recruited both pre- and post-treatment samples from the joints of RA patients (as well as matching blood samples from these patients) we plan to correlate clinical disease activity, with the presence or absence of the types of cells identified via flow cytometry and IHC, but also with the serum cytokine analysis (ELISA) and transcriptomic analysis (real-time PCR) biomarkers that might confer response or relapse to treatment. In 2018, we have made significant progress towards achieving the aims of this grant. These achievement progress milestones include: (1) establishment and implementation of multi-colour flow cytometry to investigate the cellular infiltrates in the tissue from early RA joints; during 2018, we expanded our panel to include novel T cell subsets; (2) we developed- for the first time in synovial tissue- a multi-colourmicroscopy method with automated computational assessment of tissue phenotype. This is a major milestone for severa reasons including (1) RA ST is a finite resource and extracting the most amount of data from each sample is important; (2) Preservation of structural information and identifying cells that express more than one marker is difficult with traditional IHC, but possible with multi-colour microscopy, and; (3) The automation of sample analysis will remove human analysis bias and reliability issues .In this application, we aimed to fully characterise the cellular composition of the synovial tissue (ST)infiltrates as well as clinical responses to routinely prescribed treatments in order to develop biomarkers that can predict the most effective treatment for each individual patient. Furthermore, in collaboration with a leading pharmaceutical partner we aim to identify new targets for potential therapies for the personalised treatment of RA. This is important as current therapies are ineffective in over 50% of patients and long-term disease remission is rare and it is known that effective early treatment can prevent lifelong bone loss and disability in RA. As detailed above, there are several research methodologies required to complete this 2-year project, and we have been successful in implementing and improving some of these methods in order to extract the most amount of data out of the limited samples. Furthermore, new patient recruitment is still ongoing. Our main discovery since starting this project in 2018, is that early RA ST samples contain a novel immunecell subset called T peripheral helper cells. This is the first time anyone has characterised these cells in early RA and their identification is of critical importance to the pathophysiology of the disease as they have been proposed to be involved in the mechanisms perpetuating inflammation (but until now had not been identified in early RA). Furthermore, we identify more of these type of cells in patients that have more severe disease. We need to collect more data to confirm this second observation, however, it indicates to us that our original hypothesis, that the type of cells in the ST can influence patient prognosis, is indeed true. Additionally, we have recently used transcriptomic profiling by RNAseq on a subset of the early RA patients to identify unique immune cell signatures that are associated with disease and response to treatment. In particular we have identified alterations in gene expression of gene involved in both T cell and plasma cell differentiation after standard therapy in RA. Importantly, as per the aims of this project, we are now in the process of extending this transcriptional profiling to the remaining patients treated with standard therapies, but also those patients in the study that have been treated with biologic therapies. Finally, by the end of 2018, we mostly established a multi-colour microscopy technique for the histopathological assessment of ST. We now have the ability to use computational analysis to count the number of different cell subsets in the ST, and to distinguish between samples with different phenotypes. This is a step change in the way that histopathological analysis is performed, and development of this technique will translate into high impact data for this project as well as all future projects in all   rheumatology laboratories. The research proposed in this project has the potential to provide stratified treatment options for patient with RA. The promise of personalised medicine for RA has resulted in a recent surge of interest in ST biology with the aim of translating a comprehensive understanding of the biological processes that governlymphocyte infiltration and maintenance in ST into better disease prognosis and treatment responses. The methods employed in this project, along with the unique cohort of patient samples from ST and blood, will allow for identification of cellular and molecular biomarkers that will help to provide personalised medicine for RA. Furthermore, the ST bio-bank at the Flinders Medical Centre is exclusive in Australia and the collection of treatment naive ST samples with matching post-treatment (either DMARD or biologic therapy) is a unique resource, unparalleled worldwide. This bio-bank provides the unique opportunity to identify ST and peripheral biomarkers that will allow for the application of personalised medicine in RA. The proposed project will facilitate a more comprehensive understanding of ST “pathotypes” to elucidate mechanisms of non-response. Critically, the data generated from this proposal will provide preliminary data for larger research grants enabling the joint development and testing of new-targeted therapies for RA. The long-term benefit of this work will be that this application will be greater prediction of treatment response, eliminating ineffective therapy to potentially reduce time to achieve disease remission and minimize exposure to treatments with potentially serious side effects.With the gratefully acknowledged support of the Bruce Miller-ARA Post-Doctoral Fellowship for 2019, we aim to continue this work. This additional funding, we believe, will be critical for the successful completion of this project. In 2019, we are confident we will make additional progress by facilitating a more comprehensive understanding of ST “pathotypes” to elucidate mechanisms of non-response; by building on the data generated from this proposal will provide preliminary data for larger research grants, accelerate research outputs, increase our international standing further and competitiveness for obtaining future funding. Given that it appears probable that ST biology has the potential to dictate treatment efficacy and that comprehensive analysis of ST infiltrates will yield new cellular targets for therapy, in 2019, we propose to continue this work by (1) Characterising novel and established lymphoid cell subsets in early RA ST tissue; (2) Investigating disease outcomes in a large cohort of early RA patients in the context of therapeutic intervention and cellular ST “pathotypes”; and (3) Identifying peripheral cellular and molecular biomarkers of treatment efficacy.