The functional consequence of the pathogenic variant in the inflammasome component NLRP3 as the underlying cause of keratitis fugax hereditaria

Abstract

PurposeKeratitis fugax hereditaria (KFH) is a rare periodic autoinflammatory corneal disease caused by a heterozygous pathogenic variant c.61G>C in the NLRP3 gene. Other NLRP3 variants cause cryopyrin‐associated periodic syndromes (CAPS), characterized by enhanced production of IL‐1β. While more than 50% of KFH patients use prednisolone to relieve pain, there are no data on how the c.61G>C NLRP3 variant or prednisolone treatment affects IL‐1β production in KFH patients. We carried out a functional study to elucidate these questions.MethodsPeripheral blood mononuclear cells (PBMCs) isolated from whole blood of patients with KFH (n = 8) and healthy controls (n = 4) were grown under steady‐state conditions or exposed to NLRP3 inflammasome priming with LPS (500 ng/ml) together with or without prednisolone (100 µM) and subsequently activating inflammasome with ATP(1 mM). Proteins precipitated from the cell culture media were separated by SDS‐PAGE and transferred to a membrane by western blotting. IL‐1β was visualized from the membranes by immunodetection. Alternatively, the concentration of secreted IL‐1β in the media was quantified by ELISA.ResultsFollowing NLRP3 inflammasome activating stimuli, a reduced threshold for IL‐1β secretion was observed in severely affected KFH patients compared to mildly affected patients or healthy controls. In these patients, LPS priming alone was able to trigger IL‐1β secretion while in the mildly affected patients and healthy controls IL‐1β secretion required a canonical two‐hit activation of the NLRP3 inflammasome by both LPS and ATP. In both KFH and healthy control‐derived PBMCs, IL‐1β secretion declined after prednisolone treatment.ConclusionsOur study demonstrated that patients with severe KFH are more prone to IL‐1β secretion, thus indicating that the c.61G>C is a gain‐of‐function variant. Furthermore, prednisolone is a potent treatment for reducing IL‐1β secretion in KFH.