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Principal Investigator: TONY R MERRIMAN
Organization: UNIVERSITY OF ALABAMA AT BIRMINGHAM
Fiscal Year: 2024
Award: $317,476
Funding agency: National Institute of Arthritis and Musculoskeletal and Skin Diseases
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.
Terms: <Affect><Agonist><Ancillary Study><Beta Proprotein Interleukin 1><Blood><Blood Cells><Blood Reticuloendothelial System><Blood Sample><Blood Serum><Blood monocyte><Blood specimen><Body Tissues><Candidate Disease Gene><Candidate Gene><Cartilage><Cartilaginous Tissue><Cell Body><Cell Communication and Signaling><Cell Culture System><Cell Culture Techniques><Cell Signaling><Cells><Clinical><Clinical Research><Clinical Study><Clinical Trials><Collection><Consent><Data><Deposit><Deposition><Differential Gene Expression><Effector Cell><Enrollment><Environment><Expression Signature><Fatty Acid Desaturases><Fatty Acid Desaturating Enzymes><Fatty Acids><Flare><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Frequencies><GWA study><GWAS><Gene Expression><Gene Expression Monitoring><Gene Expression Pattern Analysis><Gene Expression Profile><Gene Expression Profiling><Gene Transcription><Genes><Genetic><Genetic Transcription><Genomics><Gout><Homolog of Drosophila TOLL><Hour><IL-1 beta><IL-1 β><IL-1-b><IL-1β><IL1-Beta><IL1-β><IL1B Protein><IL1F2><IL1β><Immune><Immunes><Individual><Inflammasome><Inflammation><Inflammatory><Inflammatory Arthritis><Innate Immune Response><Interleukin 1beta><Interleukin-1 beta><Interleukin-1β><Intracellular Communication and Signaling><Investigation><Joint Diseases><Joints><Knowledge><Marrow monocyte><Measurement><Measures><Mediating><Molecular><Molecular Genetics><Mononuclear><Myelogenous><Myeloid><Myeloid Cells><Names><Organ><PBMC><Parents><Participant><Pathogenesis><Pathway interactions><Patients><Peripheral Blood Cell><Peripheral Blood Mononuclear Cell><Persons><Phagocytes><Phagocytic Cell><Phagocytosis><Phenotype><Preinterleukin 1 Beta><Production><Protocol><Protocols documentation><Publishing><RNA Expression><RNA Seq><RNA sequencing><RNAseq><Randomized, Controlled Trials><Regulation><Research><Research Resources><Resources><Role><Sampling><Serum><Signal Transduction><Signal Transduction Systems><Signaling><Site><Source><Stimulant><Stimulus><Symptoms><Synovia><Synovial Cell><Synovial Fluid><System><TIL4><TLR protein><TLR2><TLR2 gene><TLR2 receptor><TLR4><TLR4 gene><Testing><Time><Tissue-Specific Differential Gene Expression><Tissue-Specific Gene Expression><Tissues><Toll Homologue><Toll-Like Receptor 2><Toll-Like Receptor Family Gene><Toll-like receptors><Toll/Interleukin 1 Receptor-Like 4><Toll/Interleukin 1 Receptor-Like 4 Gene><Toll/Interleukin 1 Receptor-Like Protein 4><Transcript Expression Analyses><Transcript Expression Analysis><Transcription><Urate><Validation><Visit><Work><amebocyte><analyze gene expression><arm><arthropathic><arthropathies><arthropathy><biological signal transduction><cell culture><cell cultures><cell type><design><designing><differential expression><differentially expressed><enroll><flow cytophotometry><gene expression analysis><gene expression assay><gene expression pattern><gene expression signature><genome wide association><genome wide association scan><genome wide association studies><genome wide association study><genomewide association scan><genomewide association studies><genomewide association study><global gene expression><global transcription profile><innovate><innovation><innovative><insight><joint disorder><knock-down><knockdown><molecular targeted therapeutics><molecular targeted therapies><molecular targeted treatment><monocyte><name><named><naming><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><novel><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><parent><pathway><peripheral blood><prevent><preventing><primary outcome><randomized control trial><response><scRNA-seq><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><social role><toll-like receptor 4><transcriptional differences><transcriptional profile><transcriptional profiling><transcriptional signature><transcriptome><transcriptome profiling><transcriptome sequencing><transcriptomic profiling><transcriptomic sequencing><transcriptomics><validations><whole genome association analysis><whole genome association studies><whole genome association study>