Megan Leask, Nicholas Sumpter, Ayona Roychowdhury, Karen L Gamble, Pariyaphon Lertprachakwong, Tony R Merriman, Jeffrey C Edberg and Richard Reynolds
University of Alabama at Birmingham
The gout flare is an immune response to monosodium urate crystals (MSUc) in the setting of hyperuricaemia. MSU crystals activate the NLRP3 inflammasome, with subsequent secretion of mature IL-1β from immune cells leading to an influx of phagocytes into the affected joints. This is the only known molecular mechanism that accounts for the gout flare phenotype, but does not explain why gout flares are episodic, unpredictable, and completely resolve on their own. One possibility is that activation of the NLRP3 inflammasome is contributed to via circadian changes in metabolites i.e. circadian changes in the ATP level in peripheral blood have been shown to activate the NLRP3 inflammasome. Because our genome-wide association study in gout indicates that the CLOCK transcription factor is enriched at genetic loci associated with gout we hypothesise that the circadian clock modifies the immune response to MSUc. To test this we utilized our ex vivo primary cell culture system that recapitulates NLRP3 activity and investigated whether timing of exposure to MSUc/LPS after plating PBMCs from healthy controls alters IL-1βproduction. Our experimental treatments were LPS/MSUc vs unstimulated and our stimulation times were 6, 12, 18 and 24 hrs post plating. Cells were collected for ELISA to measure IL-1β 2 hours post stimulation. Consistent with our previous assays we found that IL-1β is produced rapidly in response to MSUc/LPS stimulation. We also observed different levels of IL-1β for stimulation times. IL-1β production was greatest in the 12 hr post plating stimulation and was lowest in the 6 hr post plating stimulation. These data suggest that there is possibly a circadian mechanism underpinning the amount of IL-1β that is produced after stimulation. To investigate if MSUc exposure alters circadian gene expression we carried out LPS only, MSUc only and LPS/MSUc stimulations and assessed the expression of PER3, a key circadian rhythm gene. PER3 is expressed in a circadian manner in all three treatments however we did observe that the magnitude between treatments was altered and PER3 expression was lower in both the MSUc treatments (MSUc only and LPS/MSUc). Finally our single-cell sequencing data (Reynolds abstract) indicates that the major components (CLOCK, PER2, PER3, ARNTL, CRY1 and CRY2) of the circadian machinery are expressed in classical monocytes from people with gout. Our data supports the hypothesis that a circadian mechanism might be involved in the immune response to MSUc.