J774.1

Extracellular application of soluble UA enhanced production of pro-IL-1β, mature IL-1β and caspase-1 in LPS-primed and MSU-stimulated J774.1 cells

Soluble uric acid (UA) passes through UA transporters (UATs) into cells and activates the NLRP3 inflammasome, leading to increased secretion of IL-1β. ABCG2 exports UA into the extracellular space. It has been unclear if inhibiting ABCG2 activity increases cellular UA accumulation and production of IL-1β. Notsu et al. studied the effects of genetic and pharmacologic inhibition of ABCG2 on IL-1β production by macrophages under hyperuricemic conditions.

They had previously demonstrated that monosodium urate (MSU) crystal activates NLRP3 inflammasome in LPS-primed J774.1 cells to produce IL-1β and caspase-1. Treatment with MSU increased mature IL-1β (p17), caspase-1 (p20), and pro-IL-1β protein levels in LPS-primed cells. These data suggested MSU activated NLRP3 and increased transcription of pro-IL-1β. Treatment with soluble UA did not increase mature IL-1β, caspase-1, or pro-IL-1β protein levels in unprimed cells or LPS primed cells not stimulated with MSU. To study the effect of soluble UA on NLRP3 activity and IL-1β production, J774.1 cells were pretreated with UA (0-21 mg/dL) for 12h prior to stimulation with LPS and MSU. Western blot analysis revealed pretreatment with UA ≥14 mg/dL significantly increased pro-IL-1β, mature IL-1β, and caspase-1 in a concentration-dependent manner (Fig. 1A). Quantification of western blot data from 4 independent experiments confirmed significant protein expression increases at 21 mg/dL (Fig. 1B). Taken together, these data suggest soluble UA increases production of mature IL-1β by activating NLRP3 inflammasomes and stimulating transcription of pro-IL-1β in LPS-primed macrophages stimulated with MSU.