Pathogenic variant c.61G > C in NLRP3 lowers NLRP3 inflammasome activation threshold in patients with keratitis Fugax Hereditaria

Abstract

AbstractPurpose: To elucidate the effect of NLRP3 variant c.61G > C on interleukin‐1β (IL‐1β) secretion in keratitis fugax hereditaria (KFH), a cryopyrin‐associated periodic syndrome limited to the eye, and to probe the potential modifying role of prednisolone.Methods: Peripheral blood mononuclear cells (PBMCs) isolated from whole blood of patients with KFH and healthy controls were grown under steady‐state conditions or primed with lipopolysaccharide (LPS) with or without prednisolone, and subsequently activated with adenosine triphosphate (ATP). Cell lysates and proteins precipitated from the cell culture media were separated by SDS‐PAGE. NLRP3, procaspase‐1, and IL‐1β were visualized by western blotting. The concentration of secreted IL‐1β in the culture media was quantified by ELISA.Results: A lower threshold for IL‐1β secretion following priming of the NLRP3 inflammasome with LPS was observed in patients‐derived PBMCs, compared to healthy controls (median, 124 vs. 16 pg/ml respectively). Interestingly, in PBMCs derived from patients with frequent KFH symptoms, LPS priming alone was able to trigger substantial IL‐1β secretion (median, 522 pg/ml), whereas those of patients experiencing occasional KFH attacks showed a subtler release of IL‐1β (median, 85 pg/ml). LPS and ATP treated PBMCs from patients with KFH showed significantly diminished IL‐1β secretion with prednisolone treatment (p = 0.04). NLRP3 expression was significantly enhanced with LPS stimulation (p = 0.03) whereas procaspase‐1 expression was not affected.Conclusions: PBMCs from patients with KFH are more prone to secrete IL‐1β, confirming the presumption that the c.61G > C is a gain‐of‐function variant. Furthermore, prednisolone is confirmed as a potent drug to reduce IL‐1β secretion in KFH.