The effect of the circadian clock on the mechanism of gouty inflammation.

Abstract

Background: Circadian oscillators are present in every tissue of the body and synchronized by both external and endogenous signals to regulate physiological functions of the body and maintain tissue homeostasis. Multiple studies suggested that proteins encoded by circadian clock genes affect the development and/or the severity of chronic inflammation that causes a variety of diseases such as rheumatoid arthritis, atherosclerosis and colitis. However, no studies yet demonstrated involvement of circadian clocks of immune cells in development of gout attacks, despite the fact that gout attacks are known to be time-of-day dependent. Aims: To determine whether MSU (monosodium urate crystals) crystal exposure resulted in a change in expression of circadian clock components in the THP-1 monocyte/macrophage-like cell model and whether specific components of the clock (REV-ERBa and BMAL1) influenced MSU crystal-induced NLRP3 (nucleotide-binding domain (NOD)-like receptor protein 3) inflammasome activity in THP-1 cells. Methods: THP-1 and PMA-differentiated THP-1 macrophage-like cells were treated +/-MSU crystals over 24h time-course and the expression of the full range of core circadian genes (CLOCK (circadian locomotor output cycles kaput), BMAL1 (bran and muscle Arnt-like protein-1), REV-ERBa, CRY1/2 (cryptochrome 1/2), PER1/2/3) was measured by RT-qPCR. The protein levels of core circadian clock component REV-ERBα and TLR4 (Toll-like receptor 4) were measured by Western blot. The activity of NLRP3 inflammasome complex was measured by Caspase-1 Glo assay in THP-1 cells treated with heme (a REV-ERBa agonist), SR-8278 (REV-ERBa antagonist) and prednisolone in the presence/absence of MSU crystals. The assessment of cell numbers was done by using Cyquant GR dye. Results: Time had a significant effect on the expression of BMAL, REV-ERBa, CRY1 and PER1 in both cell types. The treatment with MSU crystals resulted in a time-dependent reduction of REV-ERBα in monocytes and altered expression of REV-ERBα and CRY1 in macrophage-like cells in a time-dependent manner. Overexpression of BMAL1 led to reduction in NLRP3 inflammasome activity in both cell types in the presence of MSU crystals, yet in the absence of MSU crystals BMAL1-overexpresson increased NLRP3 activity. Knockdown of BMAL1 induced the expression of NLRP3 inflammasome in both THP-1 cell lines in the presence of MSU crystals, however, NLRP3 activation was decreased in the absence of MSU crystals. Heme and prednisolone treatment demonstrated cell- and time- dependent regulation of NLRP3 activity. The effects of heme were mediated in part via alteration in REV-ERBα activity as well as other regulatory mechanisms. Conclusions: Our findings suggest that alternations in the expression of a core circadian components such as REV-ERBα produce an effect on MSU-induced inflammatory response in THP-1 monocytes/macrophages-like cells. Furthermore, the result from this study demonstrated that both REV-ERBα and BMAL1 regulate the activity of MSU-induced NLRP3 inflammasome, however, the exact mechanism of their ant-inflammatory activity is yet to be identified.