Klf9 amplifies Nlrp3 inflammasome-mediated inflammatory death signaling in lens epithelial cells via transactivation of Nlrp3 gene

Abstract

Purpose : Oxidative stress-induced Nlrp3 (Nod Like Receptor Pyrin 3) inflammasome-mediated inflammatory response is a critical mechanism for aging diseases. Previously, we demonstrated that increased KLF9 (Kruppel-like factor 9) expression in aging lens epithelial cells (LECs) leads to reactive oxygen species (ROS) amplification and cell death. Here we show that Nlrp3 is a new target gene for Klf9-mediated transcription, and aberrant expression of KLF9 is a cause of Nlrp3 inflammasome-mediated inflammatory form of LECs death.

Methods : C57BL/6 mice (m) LEC from different ages (2 months (M), 8M and 19M old) were exposed to H₂O₂ (0-100μM) or LPS (0-5μM) for different periods. Levels of Klf9, Nlrp3, ASC, caspase-1 (Cas-1), IL-1β, GSDMD and LDH, ROS and cell viability were determined by Western blot, qPCR, ELISA, H2DCF-DA dye and MTS, respectively. In silico analysis identified Klf9 sites in Nlrp3 promoter (-1866/+166). Effect of H₂O₂ or LPS was done by transfecting LECs with Nlrp3-LUC promoter or its mutants with Klf9-siRNA or Klf9 plasmid. ChIP assay with the treated transfectants revealed the levels of DNA-Klf9 binding. A two-tailed Student’s t-test and one–way ANOVA were used for statistical analysis.

Results : Compared to younger mice, mLECs from the older mice containing elevated ROS showed that aberrant Klf9 mRNA and protein expression was associated with increased expression and activation of Nlrp3 and inflammatory proteins; Cas-1, IL-1β and ASC with elevated Cas-1, IL-1β, LDH secretion and GSDMD cleavage involved in cell death (p<0.001). This inflammatory process and cell death were further increased in response to LPS or H₂O₂ or LECs overexpressing Klf9. Conversely, the LECs-Klf9-SiRNA showed reduced levels of ROS and inflammatory molecules with enhanced cell viability (p<0.001). ChIP and promoter assays in LECs revealed that Klf9 upregulated Nlrp3 transcription by binding to repeated GC boxes (nC^A/GCCCn) in the Nlrp3 promoter.

Conclusions : Klf9 is a regulator of Nlrp3 transcription and its aberrant expression in response to aging/oxidative stress is a main event in Nlrp3 inflammasome-driven inflammatory pathway. Since Klf9 depletion represses Nlrp3 inflammasome inflammatory pathway, this may be a promising target to abate oxidative-/aging-related diseases.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.