Revealing novel mechanisms of gout flares by transcriptional phenotyping synovial and peripheral blood mononuclear cells. - Gout is the most common inflammatory arthritis, but beyond the well described role of the NLRP3 inflammasome, the molecular genetic causes of gout flares is least well known among the arthropathies. Published and preliminary results from our group have implicated genes associated with inflammation in gout which do (e.g., TMEM176B) or may not (e.g, FADS2) relate directly to inflammasome activation. A powerful approach for understanding the mechanistic basis of gout flares is through transcriptomic profiling of mononuclear cells from the target organ of inflammation, the synovial fluid, but these studies have not been rigorously carried out. We have designed a first of its kind, time-sensitive ancillary study, through the parent clinical trial, Treat-to-Target vs Treat-to-Avoid-Symptoms (TRUST), which will allow novel comparisons of candidate gene expression in mononuclear cells of synovial fluid to peripheral blood cells prior to and during active gout flares. These studies will require consenting TRUST participants for arthrocentesis during active flares. This additional, fresh biospecimen collection, facilitated by the TRUST trial, is highly time-sensitive and crucial for thorough investigation of molecular mechanisms of gout flares. We will use these samples to carry out Aim 1 - differential gene expression analysis of myeloid subsets before and during a flare in peripheral blood and synovial fluid samples during active flare. Flow cytometry on samples from 20 individuals will be used to isolate non-classical and classical monocytes followed by bulk RNA sequencing. Single cell RNAseq will be done on peripheral blood mononuclear cell (PBMC) samples from five of the 20 individuals to infer specific cell-types. In Aim 2, using our existing ex vivo cell culture paradigm we will stimulate PBMCs with LPS and C18.0 fatty acid (FA) in the presence/absence of MSU crystals, followed by scRNAseq 24 hours post stimulation. Cells will be isolated from fresh samples of blood collected from people with gout (intercritical) enrolled at UAB. scRNAseq transcriptomes will be compared between unstimulated, C18.0 FA and LPS stimulated cells. Readouts will allow mechanistic insight into gout flares that are TLR-agonist mediated and related to potential gout flare triggers. Finally, we will knockdown candidate genes, by attenuating their expression and then measure NLRP3 inflammasome activation. Altered expression of secreted IL-1β would confirm their mechanistic role in gout flares. The proposed research is highly innovative by sampling, with never before collections of matched samples of peripheral blood and synovial fluid, by analyses, novel transcriptomic analyses of myeloid cells in samples and in an ex vivo system, and by design, leveraging resources of the TRUST parent clinical design for this time-sensitive ancillary study. Our work will provide new knowledge of genetically-based mechanisms of gout flares and aid in identification of new therapeutic targets.
