Dr Mihir Dilip Wechalekar 

Recipient: Dr Mihir Dilip Wechalekar
Intended department: Jointly funded by the BB  & A Miller Foundation and the ARA- Flinders University
Project:

 

Improving outcomes in Rheumatoid Arthritis

 

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 into 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-colour microscopy method with automated computational assessment of tissue phenotype. This is a major milestone for several 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.

Additionally, we have recently used transcriptomic profiling by RNA seq 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.