International Journal of Molecular Sciences · 5 authors, 2 centres
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This study demonstrates that deficiency of the antioxidant enzyme Prdx6 in lens epithelial cells leads to reactive oxygen species accumulation and activation of the Nlrp3 inflammasome, resulting in pyroptosis. The mechanism involves Klf9-dependent suppression of antioxidant genes and upregulation of Nlrp3 transcription. These findings suggest that Prdx6 delivery or Klf9/Nlrp3 inhibition could be therapeutic strategies for age-related inflammatory diseases.
**Background:** Age-related diseases are linked to chronic inflammation driven by oxidative stress and dysregulation of antioxidant defenses. The Nlrp3 inflammasome is a key mediator of inflammatory signaling, and its activation is triggered by reactive oxygen species (ROS). Peroxiredoxin 6 (Prdx6) is a multifunctional antioxidant enzyme that maintains redox homeostasis. This study investigates whether Prdx6 deficiency leads to Nlrp3 inflammasome activation in lens epithelial cells (LECs) and explores the underlying molecular mechanisms involving the transcription factor Klf9.
**Methods:** The study used LECs from Prdx6 knockout (Prdx6−/−) and wild-type (Prdx6+/+) mice, primary LECs from aging C57BL/6 mice (2 months, 8 months, 19 months), primary human LECs from donors aged 16 and 65 years, and the SRA-hLEC cell line. ROS levels were measured using H2-DCF-DA and CellROX deep red reagent. Protein expression of Nlrp3, ASC, Caspase-1, IL-1β, IL-18, GSDMD, Prdx6, and Klf9 was assessed by Western blot. Secreted IL-1β and IL-18 were quantified by ELISA. mRNA levels were measured by RT-qPCR. Nlrp3 promoter activity was assessed using luciferase reporter assays. Gain- and loss-of-function experiments were performed by overexpressing Klf9 or Prdx6 via plasmid transfection or lentiviral infection, and by knocking down Klf9 using lentiviral shRNA.
**Key Results:** Prdx6−/− mLECs showed significantly higher ROS levels and spontaneous cell death compared to wild-type cells. They also exhibited increased expression and activation of Nlrp3 inflammasome components, including cleaved Caspase-1, ASC oligomers, and elevated secretion of IL-1β and IL-18. Similarly, aging mLECs and human LECs (65 y) displayed progressive ROS accumulation, reduced Prdx6 expression, and increased Nlrp3 inflammasome activation. H2O2 or LPS treatment further amplified these effects in Prdx6−/− cells. Klf9 expression was elevated in aging and Prdx6−/− LECs, correlating with suppression of antioxidant genes Prdx6 and Txnrd2. Overexpression of Klf9 in LECs increased ROS and Nlrp3 expression while reducing Prdx6 and Txnrd2. Luciferase reporter assays demonstrated that Klf9 upregulates Nlrp3 transcription, identifying Nlrp3 as a novel target gene of Klf9. Knockdown of Klf9 reduced ROS and protected cells from H2O2-induced death. Restoration of Prdx6 expression in Prdx6−/− mLECs via lentiviral infection significantly reduced ROS levels and suppressed Nlrp3 inflammasome activation.
**Clinical Implications:** This study identifies Prdx6 as a critical regulator of Nlrp3 inflammasome activation in LECs, with deficiency leading to ROS-driven inflammatory cell death (pyroptosis) via a Klf9-dependent mechanism. These findings suggest that Prdx6 loss contributes to age-related lens pathologies such as cataract. The authors propose that therapeutic strategies combining Prdx6 delivery with Klf9 or Nlrp3 inhibitors could prevent or delay age-related inflammatory diseases. The study provides a mechanistic basis for developing antioxidant-based therapies targeting the Nlrp3 inflammasome pathway.