Richard J Reynolds, Megan Leask, Nicholas Sumpter, Tony R Merriman, Pariyaphon Lertprachakwong, Jeffrey C Edberg.
Affiliation(s):
University of Alabama at Birmingham
Gene expression studies in gout represent a powerful approach for understanding the mechanistic basis of flares because case and non-case differences in expression could reveal the biology of activation and response of immune cells to relevant, gout-specific stimuli. We have undertaken and integrated several gene expression studies in samples from participants of the UAB Gout Registry with validation in an ex vivo system where fresh peripheral blood mononuclear cells are stimulated with monosodium urate (MSU) crystals and toll-like receptor agonists. Using RNA collected from whole blood followed by RNAseq, we found 48 statistically significant (FDR < 0.10) differentially expressed genes between gout cases (N = 13) and non-gout controls (N = 6). In a separate cross-sectional RNAseq study of whole blood, 36 genes were differentially expressed (p < 0.05) between participants with gout in active flare (N = 5) and intercritical gout (N=13). Several of the genes indicated from the two studies are known to be associated with gout from GWAS (e.g., TMEM176A/B, FADS2), suggesting that the underlying genetic associations may contribute to gene expression differences with risk for gout, including risk for active flares. TMEM176A/B and FADS2 had increased expression in classical, intermediate and nonclassical monocytes, relative to all other subsets, as inferred by scRNAseq of samples from people (N = 5) with intercritical gout or gout in active. The percent of intermediate monocyte cells expressing TMEM176B appeared to be lower in people in active flare compared to intercritical, but consistent trends were not observed among the monocyte subsets. Time course studies, utilizing the ex-vivo sytem (N = 5 non-gout donors) and qPCR, showed reduced TMEM176B gene expression by orders of magnitude post stimulation with LPS and/or MSU crystals (P-value = 0.005), however the lowered expression was most durable in the crystal-stimulated replicates (time X treatment interaction effect; P-value = 0.001). As expected, MSU crystals and lipopolysaccharide (LPS) synergistically stimulate secretion of IL1β, as measured by ELISA, indicating NLRP3 inflammasome activation (MSU X LPS interaction effect (P-value = 1.6E-5). We have implicated a number of differentially expressed genes between gout cases and controls, and between intercritical gout cases and those in active flare, and we have developed an ex vivo system to validate their expression signatures. TMEM176B has been reported to negatively regulate the NLRP3 inflammasome. The question to pursue in future studies is whether the altered expression of genes, such as TMEM176B, is ancillary to the activated NLRP3 inflammasome or whether it is causal. Nevertheless our experiment suggests the NLRP3 inflammasome, when activated with MSU crystals, negatively regulates TMEM176B expression.