June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Lens epithelial cell Prdx6 deficiency activates ROS-Inflammasome pathway-driven cell death
Author Affiliations & Notes
  • Bhavana Chhunchha
    Ophthalmology and Visual Sciences, University of Nebraska Medical Center College of Medicine, Omaha, Nebraska, United States
  • Eri Kubo
    Department of Ophthalmology, Kanazawa Medical University, Kanazawa, Ishikawa, Japan
  • Dhirendra P Singh
    Ophthalmology and Visual Sciences, University of Nebraska Medical Center College of Medicine, Omaha, Nebraska, United States
  • Footnotes
    Commercial Relationships   Bhavana Chhunchha None; Eri Kubo None; Dhirendra Singh None
  • Footnotes
    Support  EY024589
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 663 – F0018. doi:
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    • Get Citation

      Bhavana Chhunchha, Eri Kubo, Dhirendra P Singh; Lens epithelial cell Prdx6 deficiency activates ROS-Inflammasome pathway-driven cell death. Invest. Ophthalmol. Vis. Sci. 2022;63(7):663 – F0018.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Augmentation of oxidative stress-induced inflammation due to deterioration of antioxidant defense with advancing age is suggested to be associated with aging-related blinding diseases. The multifunctional antioxidant protein Peroxiredoxin 6 (Prdx6) provides cytoprotection and delays lens opacity by regulating reactive oxygen species (ROS) homeostasis. Herein, we showed, for the first time, that Prdx6-deficiency in mouse (m) lens epithelial cells (mLECs) causes ROS-induced Nlrp3 inflammasome activation-mediated inflammatory cell death.

Methods : Prdx6-/- (a model for aging) and Prdx6+/+ LECs-derived from Prdx6-deficient and wild-type of 12 months old mice lenses were treated or untreated with N-acetylcysteine (NAC) or Prdx6, and then exposed to H2O2 (0-100µM) for different periods. ROS levels were quantified by H2-DCF-DA dye and cell death was evaluated by LDH (a marker for pyroptotic cell death) and MTS assays. Western blot or ELISA and real-time PCR were used to monitor expression or activation of Prdx6, NF-κB, IκBα, Nlrp3 inflammasome (Nlrp3, ASC, caspase-1) activation, IL-1β, and gasdermin-D in Prdx6+/+ and Prdx6-/- LECs lysates or cell culture supernatant. SN50, a NF-κB inhibitor was used to block NF-κB activation. Two-tailed Student’s t-test and one–way ANOVA were used for statistical analysis.

Results : Compared to Prdx6+/+, Prdx6-/- LECs bearing elevated ROS showed markedly increased expression and activation of Nlrp3 and inflammatory proteins; caspase-1, IL-1β, ASC, NF-κB and gasdermin-D with elevated caspase-1 activity, IL-1β secretion and gasdermin-D cleavage involved in cell death/pyroptosis. These factors and cell death were further increased in Prdx6-/- LECs in response to H2O2. Under these conditions, a supply of Prdx6 or NAC or SN50 to Prdx6-/- LECs attenuated ROS-driven aberrant activation of Nlrp3, the inflammatory factors, and therapeutically reduced caspase-1 activation, release of IL-1β and LDH, and suppressed cell death (p<0.001). Coupled with a significantly the lower vulnerability of Prdx6+/+ LECs, these results revealed that Prdx6-deficiency caused ROS-dependent activation of Nlrp3 inflammasome assembly and pyroptosis in LECs.

Conclusions : Our findings underscore the importance of Prdx6 in the redox-signaling network of Nlrp3 inflammasome activation regulating LECs homeostasis as its deficiency is associated with ROS/Nlrp3 inflammasome activation-dependent pyroptotic cell death.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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