Dhirendra P. Singh

The abnormal activation of the Nlrp3 (Nod-like receptor pyrin 3) inflammasome, in response to oxidative stress or impaired antioxidant defense, is linked to aging-related diseases. Previously, we have shown that Peroxiredoxin (Prdx)6 deficiency triggers reactive oxygen species (ROS)-dependent activation of Kruppel-like factor (Klf)9/Nlrp3 inflammasome in aging lens epithelial cells (LECs). Herein, we test the therapeutic efficacy of Prdx6 delivery in abating the oxidative stress-induced aberrant activation of the Klf9/NF-ĸB/Nlrp3 pathway and subsequent pyroptotic cell death in LECs and Prdx6-deficient (Prdx6^−/−) LECs. Similar to aged LECs, Prdx6-depleted LECs exhibited activation of Nlrp3 inflammasome components—including ASC, Caspase-1, IL-1β, IL-18, GSDMD—and displayed heightened sensitivity to H₂O₂/UVB-induced oxidative damage. The delivery of TAT-HA-Prdx6 or the overexpression of Prdx6 in Prdx6^−/− mLECs significantly suppressed the aberrant activation of these inflammatory components and restored redox balance by eliminating ROS levels during oxidative stress. Similarly, TAT-HA-Prdx6 effectively internalized into SRA-hLECs and suppressed the H₂O₂- and/or UVB-induced upregulation of Nlrp3 and its components. Furthermore, the oxidative stress or Prdx6 deficiency led to increased Nlrp3 promoter activity and NF-ĸB activation, accompanied by decreased cytosolic IĸBα and increased phosphorylation of IĸBα; these alterations were reversed by Prdx6 overexpression. The elevated Klf9 transcription observed in aging and Prdx6^−/− mLECs or under oxidative stress was also inhibited by Prdx6 delivery. Additionally, Prdx6^−/− mLECs and aging LECs displayed increased TXNIP and reduced TRX levels, which were normalized by Prdx6 restoration. Collectively, this study provides the first evidence that the loss of Prdx6 drives aberrant activation of Klf9/NF-ĸB/Nlrp3 inflammasome axis, leading to pyroptotic cell death. Prdx6 delivery represents a promising therapeutic strategy to rescue cells from pyroptosis (oxidative stress-induced inflammatory cell death).

Deregulated Nlrp3 (NOD-like receptor pyrin 3) inflammasome activation is strongly associated with age-related blinding diseases, including cataract. Previously, we demonstrated that loss of peroxiredoxin6 (Prdx6) promotes reactive oxygen species (ROS) amplification and aberrant activation of Klf9 and Nlrp3 inflammasome activity–driven pyroptosis. In this study, using aging mouse(m)/human(h) lenses and lens epithelial cells (LECs), we reveal a critical link between Nlrp3 and thioredoxin (TRX)-interacting protein (TXNIP), which increases during aging and oxidative stress conditions. We found that aging lenses exhibiting opacity showed elevated ROS levels, increased TXNIP expression, along with upregulation of Nlrp3 inflammasome components, including caspase-1, ASC, IL-1β, IL-18, and gasderminD (GSDMD), with significantly reduced TRX1. mLECs overexpressing TXNIP were more susceptible to hydrogen peroxide (H₂O₂), Lipopolysaccharide (LPS), ultraviolet B (UVB)-induced oxidative stress, displaying increased ROS accumulation, reduced cell viability, and enhanced activation of Nlrp3 inflammasome and its downstream inflammatory mediators, hallmarks of pyroptotic cell death. Conversely, TXNIP knockdown suppressed Nlrp3 inflammasome activation, decreased ROS production, and significantly improved cell survival, indicating a protective effect against oxidative injury. Ex vivo, TAT-HA-Prdx6 delivery inhibited H₂O₂-induced Nlrp3 activation and preserved lens transparency, demonstrating its potent antioxidant and anti-inflammatory effects. Collectively, these findings identify TXNIP as a key regulator of Nlrp3 inflammasome signaling and thereby highlight the therapeutic potential of TXNIP silencing (ShTXNIP) or TAT-HA-Prdx6 delivery to halt Nlrp3-mediated pyroptosis during aging or oxidative stress conditions.

Increasing evidence reveals that the deregulation of cellular antioxidant response with advancing age, resulting in the continuing amplification of oxidative stress-induced inflammatory response, is a pre-eminent cause for the onset of aging-related disease states, including blinding diseases. However, several safeguards, like an antioxidant defense system, are genetically in place to maintain redox homeostasis. Nonetheless, if the homeostatic capacity of such systems fails (like in aging), an inflammatory pathway elicited by excessive oxidative stress-evoked aberrant NLRP3 (NOD, LRR- and pyrin domain-containing protein 3) inflammasome activation can become pathogenic and lead to disease states. Among all known inflammasomes, NLRP3 is the most studied and acts as an intracellular sensor to detect danger(s). Upon activation, NLRP3 recruits apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization and facilitates the recruitment of activated Caspase-1 (Cas-1), which results in the release of inflammatory cytokines, IL-1β and IL-18 and the activation of GasderminD, an executor of pyroptosis. NLRP3 inflammasome is tightly regulated in favor of cell health. However, when and how the activation of NLRP3 and its inflammatory components goes awry, leading to cellular derangement, and what regulatory factors are involved in the normal physiological and aging/oxidative conditions will be included in this review. Also, we address the latest findings to highlight the connection between oxidative stress, antioxidants, and NLRP3 activation as this begets aging diseases and explore the cellular pathways that are in place to regulate oxidative-induced inflammations and the pathobiological consequences of dysregulated inflammatory responses and vice versa.

Cataracts, the leading cause of blindness globally, are caused by oxidative stress and inflammation, which disrupt lens transparency due to increased accumulation of reactive oxygen species (ROS) as well as protein and DNA damage during aging. Using in vitro, ex vivo, and in vivo models, we determined the protective efficacy of N-acetylcysteine amide (NACA) against oxidative stress-induced and aging-induced cataractogenesis. We found that lens epithelial cells exposed to the oxidative stress inducers hydrogen peroxide (H₂O₂) or tert-butyl hydroperoxide showed significant ROS accumulation and reduced cellular viability. These effects were inhibited by NACA via the suppression of ROS and thioredoxin-interacting protein (Txnip) expression, a regulator of oxidative stress-related cellular damage and inflammation. In ex vivo lens experiments, NACA significantly reduced H₂O₂-induced lens opacity and preserved lens integrity. Similarly to NACA-treated lenses ex vivo, the integrity and opacity of aged mouse lenses, when topically instilled with NACA, were preserved and reduced, respectively, and are directly related to reduced Txnip and increased thioredoxin (Trx) expression levels. Overall, our findings demonstrated the protective ability of NACA to abate aberrant redox-active pathways, particularly the ROS/TRX/TXNIP axis, thereby preventing cataractogenesis and preserving eye lens integrity and ultimately impeding aging-related cataracts.

The physiological quantum of stress-inducible transcriptional protein, Lens Epithelium-Derived Growth Factor (LEDGF), is vital for the maintenance of cellular physiology. Erratic epigenetic reprogramming in response to oxidative stress or with advancing age is found to be a major cause in the gene silencing, leading to pathobiologies. Using aging human (h) eye lens/lens epithelial cells (LECs) coupled with redox-active Peroxiredoxin 6 (Prdx6)-deficient (Prdx6^−/−) mLECs as model systems, herein, we showed that in aging/oxidative stress, the human LEDGF gene was regulated by unique methylation patterns of CGs nucleotides within and around the Sp1 binding site(s) of CpG island of the LEDGF promoter (−170 to −27nts). The process caused the repression of LEDGF and its target, Hsp27, resulting in reactive oxygen species (ROS) amplification and cellular insults. This phenomenon was opposed to the unmethylated promoter in LECs. Clinically, we observed that the loss of LEDGF in the Prdx6^−/− mLECs or aging lenses/LECs, correlating with increased expression of DNMT1, DNMT3a, and DNMT3b along with the methyl CpG binding protein 2 (MeCP2). Upon oxidative stress, the expression of these molecules was increased with the dramatic reduction in LEDGF expression. While demethylating agent, 5-Aza deoxycytidine (5-AzaC) transposed the aberrant methylation status, and revived LEDGF and Hsp27 expression. Mechanistically, the chloramphenicol acetyltransferase (CAT) reporter gene driven by the LEDGF promoter (−170/ + 35) and ChIP assays uncovered that 5-AzaC acted on GC/Sp1 sites to release LEDGF transcription. The data argued, for the first time, that de novo methylation of CGs around and within Sp1 sites of the CpG island directly disrupted Sp1 activity, which ensued in LEDGF repression and its biological functions. The findings should improve our understanding of cellular insults-associated with aberrant DNMTs-mediated LEDGF’s activity, and can offer strategies for therapeutic intervention to halt aging/oxidative stress-induced abnormalities.

The continuum of antioxidant response dysregulation in aging/oxidative stress-driven Nlrp3 inflammasome activation-mediated inflammatory response is associated with age-related diseases. Peroxiredoxin (Prdx) 6 is a key antioxidant that provides cytoprotection by regulating redox homeostasis. Herein, using lens epithelial cells (LECs) derived from the targeted inactivation of Prdx6 gene and aging lenses, we present molecular evidence that Prdx6-deficiency causes oxidative-driven Nlrp3 inflammasome activation, resulting in pyroptosis in aging/redox active cells wherein Prdx6 availability offsets the inflammatory process. We observed that Prdx6^−/− and aging LECs harboring accumulated reactive oxygen species (ROS) showed augmented activation of Nlrp3 and bioactive inflammatory components, like Caspase-1, IL-1β, ASC and Gasdermin-D. Similar to lipopolysaccharide treatment, oxidative exposure led to further ROS amplification with increased activation of the Nlrp3 inflammasome pathway. Mechanistically, we found that oxidative stress enhanced Kruppel-like factor 9 (Klf9) expression in aging/Prdx6^−/− mLECs, leading to a Klf9-dependent increase in Nlrp3 transcription, while the elimination of ROS by the delivery of Prdx6 or by silencing Klf9 prevented the inflammatory response. Altogether, our data identify the biological significance of Prdx6 as an intrinsic checkpoint for regulating the cellular health of aging or redox active LECs and provide opportunities to develop antioxidant-based therapeutic(s) to prevent oxidative/aging-related diseases linked to aberrant Nlrp3 inflammasome activation.

A major hallmark of aging-associated diseases is the inability to evoke cellular defense responses. Transcriptional protein Nrf2 (nuclear factor erythroid-derived 2-related factor) plays a pivotal role in the oxidative stress response, cellular homeostasis, and health span. Nrf2’s activation has been identified as a therapeutic target to restore antioxidant defense in aging. Here, we demonstrated that FDA-approved drug, hydralazine (Hyd), was a reactivator of the Nrf2/ARE (antioxidant response element) pathway in various ages and types of mouse (m) or human (h) lens epithelial cells (LECs) and mice lenses in-vitro/in-vivo. This led to Hyd-driven abatement of carbonyls, reduced reactive oxygen species (ROS), and reduced 4-HNE/MDA-adducts with cytoprotection, and extended lens healthspan by delaying/preventing lens opacity against aging/oxidative stress. We elucidated that Hyd activated the protective signaling by inducing Nrf2 to traverse from the cytoplasm to the nucleus and potentiated the ARE response by direct interaction of Nrf2 and ARE sequences of the promoter. Loss-of-function study and cotreatment of Hyd and antioxidant, N-acetyl cysteine (NAC) or Peroxiredoxin (Prdx)6, specified that Nrf2/ARE-driven increase in the promoter activity was Hyd-dependent. Our study provides proof-of concept evidence and, thereby, paves the way to repurposing Hyd as a therapeutic agent to delay/prevent aging and oxidative-related disorders.

Increasing levels of oxidative-stress due to deterioration of the Nrf2 (NFE2-related factor)/ARE (antioxidant response element) pathway is found to be a primary cause of aging pathobiology. Metformin having anti-aging effects can delay/halt aging-related diseases. Herein, using lens epithelial cell lines (LECs) of human (h) or mouse (m) and aging h/m primary LECs along with lenses as model systems, we demonstrated that Metformin could correct deteriorated Bmal1/Nrf2/ARE pathway by reviving AMPK-activation, and transcriptional activities of Bmal1/Nrf2, resulting in increased antioxidants enzymatic activity and expression of Phase II enzymes. This ensued reactive oxygen species (ROS) mitigation with cytoprotection and prevention of lens opacity in response to aging/oxidative stress. It was intriguing to observe that Metformin internalized lens/LECs and upregulated OCTs (Organic Cation Transporters). Mechanistically, we found that Metformin evoked AMPK activation-dependent increase of Bmal1, Nrf2, and antioxidants transcription by promoting direct E-Box and ARE binding of Bmal1 and Nrf2 to the promoters. Loss-of-function and disruption of E-Box/ARE identified that Metformin acted by increasing Bmal1/Nrf2-mediated antioxidant expression. Data showed that AMPK-activation was a requisite for Bmal1/Nrf2-antioxidants-mediated defense, as pharmacologically inactivating AMPK impeded the Metformin’s effect. Collectively, the results for the first-time shed light on the hitherto incompletely uncovered crosstalk between the AMPK and Bmal1/Nrf2/antioxidants mediated by Metformin for blunting oxidative/aging-linked pathobiology.

Aberrant Sumoylation-mediated protein dysfunction is involved in a variety of oxidative and aging pathologies. We previously reported that Sumoylation-deficient Prdx6K^{(lysine)122/142R(Arginine)} linked to the TAT-transduction domain gained stability and protective efficacy. In the present study, we formulated wild-type TAT-HA-Prdx6^WT and Sumoylation-deficient Prdx6-loaded poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) to further enhance stability, protective activities, and sustained delivery. We found that in vitro and subconjuctival delivery of Sumoylation-deficient Prdx6-NPs provided a greater protection of lens epithelial cells (LECs) derived from human and Prdx6^−/−-deficient mouse lenses against oxidative stress, and it also delayed the lens opacity in Shumiya cataract rats (SCRs) than TAT-HA-Prdx6^WT-NPs. The encapsulation efficiencies of TAT-HA-Prdx6-NPs were ≈56%–62%. Dynamic light scattering (DLS) and atomic force microscopy (AFM) analyses showed that the NPs were spherical, with a size of 50–250 nm and a negative zeta potential (≈23 mV). TAT-HA-Prdx6 analog-NPs released bioactive TAT-HA-Prdx6 (6%–7%) within 24 h. Sumoylation-deficient TAT-HA-Prdx6-NPs provided 35% more protection by reducing the oxidative load of LECs exposed to H₂O₂ compared to TAT-HA-Prdx6^WT-NPs. A subconjuctival delivery of TAT-HA-Prdx6 analog-NPs demonstrated that released TAT-HA-Prdx6^K122/142R could reduce lens opacity by ≈60% in SCRs. Collectively, our results demonstrate for the first time that the subconjuctival delivery of Sumoylation-deficient Prdx6-NPs is efficiently cytoprotective and provide a proof of concept for potential use to delay cataract and oxidative-related pathobiology in general.