TXNIP-Deficiency and Prdx6 Delivery Inhibit Aging/Oxidative Stress–Driven TXNIP-Nlrp3 Inflammasome Activation and Mitigate Pyroptosis in Lens Epithelial Cells

TL;DR

This study shows that reducing TXNIP or delivering Prdx6 can prevent harmful inflammation and cell death in aging lenses, offering potential treatments for age-related eye diseases.

Contribution

The study identifies TXNIP as a key regulator of Nlrp3 inflammasome activation and proposes therapeutic strategies to mitigate pyroptosis in aging lenses.

Findings

TXNIP overexpression increases oxidative stress and Nlrp3 inflammasome activation in lens epithelial cells.

TXNIP knockdown reduces ROS and protects cells from pyroptosis during oxidative stress.

TAT-HA-Prdx6 delivery inhibits Nlrp3 activation and preserves lens transparency in ex vivo models.

Abstract

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.

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Full text

1. Introduction

Aging or age-associated diseases are the progressive loss of organ and tissue function due to increasing oxidative load and loss of antioxidant responses. Increased productivity of reactive oxygen species (ROS) is found to be the main cause for the development of age-related diseases [1,2,3,4,5,6]. The cellular antioxidant defense system normalizes/controls ROS formation and reduces cellular damage. The oxidative damage often arises from defects in redox-regulating antioxidant enzymes. Thus, understanding how antioxidants act at the molecular level is essential for preventing age-related diseases [5,7,8]. Among the antioxidant enzymes, peroxiredoxin (Prdxs) and thioredoxin system (TRX) play a crucial role in response to oxidative stress and protect the cells by maintaining cellular redox homeostasis by acting as H_2_O_2_ or ROS optimizers [6,7,8,9,10,11,12,13,14,15]. In mammals, Prdxs are classified based on Cysteine (Cys) residues in the active core and their catalytic mechanism. Prdx1-4 are typical (2-Cys), Prdx5 is atypical (2-Cys), while Prdx6 has only one Cys (1-Cys). Prdx6 is a unique member of the Prdx family, crucially having glutathione (GSH) peroxidase, calcium-independent phospholipase A2 (aiPLA2), and lysophosphatidylcholine acyl transferase (LPCAT) activities [9,16,17,18]. In the Prdx family members, Prdx6 is the only member that has the capability to protect the cells by reducing phospholipid hydroperoxides [19]. Prdx6 is predominantly localized in the cytoplasm. However, recently it has also been detected in lysosomal-type organelles, mitochondria, plasma membrane, and endoplasmic reticulum [9,17,19,20,21,22]. Notably, Prdx6 repairs peroxidized cellular membranes by directly reducing oxidized phospholipids and by removing and replacing oxidized sn-2 fatty acyl chains via its aiPLA2 and LPCAT activities [22,23,24,25,26]. Recently, Prdx6 has gained attention as it has remarkable antioxidant activity, along with anti-apoptotic as well as anti-inflammatory properties, including facilitation of selenium (Se) for Gpx4 expression and activity [9,15,20,23,27,28,29,30,31,32,33,34]. Nonetheless, reduced expression of Prdx6 has been observed during aging and oxidative stress conditions in many types of cells and tissues, including lens epithelial cells (LECs), neuronal cells, trabecular meshwork (TM) cells, and retinal pigment epithelium (RPE) cells [8,9,18,22,35]. This protein protects the cells against various kinds of endogenous, exogenous, or environmental stressors, such as ROS-producing agents, including ultraviolet (UVB), paraquat, glutamate, cobalt chloride, endoplasmic reticulum (ER) stress, tBOOH, and lipopolysaccharide (LPS). Prdx6 mitigates oxidative damage and suppresses different types of cell death, such as apoptosis, necroptosis, pyroptosis, and ferroptosis [7,8,9,10,20,21,23,29,31,36,37]. Another major endogenous antioxidant, TRX, is a 12 kDa protein with redox activity that plays a crucial role in aging by maintaining redox homeostasis. During aging and oxidative stress, TRX expression or activity is significantly declined. In mammals, TRX1 is found mainly in the cytoplasm, plasma membrane, and nucleus, while TRX2 is found only in mitochondria. TRX function and expression are regulated by thioredoxin-interacting protein (TXNIP) [6,14,38,39,40,41,42,43,44,45,46,47].

A multifunctional protein, TXNIP, also known as vitamin D3-upregulated protein 1 (VDUP1) or thioredoxin-binding protein 2 (TBP-2), is expressed in a wide range of tissues and cell types and negatively regulates the activity of TRX [38,40,42,47,48,49,50,51,52,53,54,55,56]. Recently, TXNIP has emerged as a critical molecular link between aging, oxidative stress, and inflammation. However, TXNIP has been found to be involved in multiple biological and pathological processes, including inflammatory responses and cell death. It does so by binding to TRX-TXNIP and TXNIP-Nlrp3. This results in excessive ROS production–induced inflammatory response. Growing evidence dictates that TXNIP plays an aberrant role in the development of metabolic, neurological, and inflammatory disorders [40,48,49,57,58,59,60,61,62,63]. It has been found that TXNIP is aberrantly expressed in aging tissues and senescent cells, as well as cells facing oxidative or metabolic stresses [50,51,53,64,65]. TXNIP was initially known to localize in the cytoplasm; it was later found that under oxidative stress, it could move to diverse intracellular positions [38,66]. TXNIP is a critical regulator of redox balance and has also been identified as an important contributor to the development of cellular senescence [42]. TXNIP has been found to interact with Nlrp3 and activate the Nlrp3 inflammasome pathway. Inflammation is closely linked to the activation of Nlrp3 inflammasome [67]. Nlrp3-TXNIP can be activated by diverse stimuli and regulates inflammatory responses, while also being implicated in numerous age-related chronic diseases [62,67,68]. Moreover, inflammatory signaling is closely linked to the activation of Nlrp3 inflammasome, which consists of the sensor Nlrp3, the adaptor apoptosis-associated speck-like protein (ASC), and the effector caspase-1. Inflammatory diseases mostly involve a multiprotein complex that plays a central role in inflammation, with the Nlrp3 inflammasome being the most extensively studied [9,20,67,69,70,71,72,73]. This multiprotein complex is activated through a two-step process. In the first priming step, cells detect pathogen- or damage-associated molecular patterns (PAMPs or DAMPs) via Toll-like receptor 4 (TLR4), leading to increased expression and activity of inflammasome components. In the second activation step, cellular stress signals promote the Nlrp3 inflammasome complex. Activated Nlrp3 is deubiquitinated and recruits the adaptor protein ASC, which in turn binds to pro-caspase-1, forming a large multimeric complex. Activated caspase-1 within this complex cleaves pro-interleukin-1β (IL-1β) and pro-IL-18 into mature IL-1β and IL-18, triggering a robust inflammatory response [9,20,67,74,75,76,77].

During aging/oxidative conditions, TXNIP plays as an essential factor in exacerbating Nlrp3 inflammasome activation, and this results in inflammatory cell death, pyroptosis, leading to inflammatory disorders [48,49,51,56,78,79,80]. Furthermore, TXNIP is an endogenous inhibitor of the antioxidant TRX. TRX is a major thiol-reducing and ROS-scavenger molecule. The binding of TXNIP to TRX blocks TRX activity and enhances oxidative stress. The TXNIP-TRX complex–mediated redox-sensitive signaling triggers Nlrp3 inflammasome activation. In this scenario, TXNIP is a key driver of oxidative stress-induced Nlrp3 inflammasome activation. Interestingly, TXNIP overexpression enhances Nlrp3 activation, while TXNIP depletion inhibits the activation of the Nlrp3 inflammasome [51,56,79]. Furthermore, TXNIP has been found to be the mediator of oxidative stress, thereby damaging cells/tissues via activation of Nlrp3 and NF-ĸB pathways. It has been reported that TXNIP is involved in oxidative damage in HEI-OC1 cochlear hair cells and that blocking TXNIP exerts anti-oxidative stress and anti-inflammatory effects by inhibiting both Nlrp3 and NF-ĸB pathways [55].

In this work, using mouse and human lens/lens epithelial cells (LECs) of different ages, including mLECs and lenses from C57BL/6 mice, we showed that reduced cellular antioxidant capacity in aging LECs promotes oxidative and TXNIP-dependent activation of Nlrp3 inflammasome, leading to inflammatory cell death, pyroptosis. Mechanistically, we identified that aberrant TXNIP expression represses antioxidant gene activity, leading to excessive ROS accumulation–induced Nlrp3 inflammatory responses and cell damage. We observed that TXNIP-overexpressing mLECs exhibited heightened Nlrp3 inflammasome signaling, which was exacerbated by Nlrp3 inflammasome inducers, such as LPS, H_2_O_2_, or UVB, making the cells more vulnerable to inflammatory response-induced cell damage. In contrast, TXNIP-depleted mLECs or TAT-HA-Prdx6-treated LECs/lenses exhibited increased resistance and survival, as well as prevention of lens opacity in response to stressors, such as LPS, H_2_O_2_, or UVB. Collectively, our findings suggest that Prdx6 delivery and/or ShTXNIP may serve as an effective therapeutic strategy to prevent or treat inflammasome activation–mediated inflammatory signaling involved in the etiopathogenesis of age-related diseases.

2. Materials and Methods

2.1. Cell Culture

2.1.1. Mouse Lens/Lens Epithelial Cells (LECs)

All animal procedures complied with the Association for Research in Vision and Ophthalmology (ARVO) “Statement for the Use of Animals in Ophthalmic and Visual Research” and were approved by the Institutional Animal Care and Use Committee (IACUC), University of Nebraska Medical Center (UNMC), Omaha, NE. All animals were maintained under specific pathogen-free conditions in an animal facility. Different ages (4 months (M), 15 M, and 21 M) of C57BL/6 mice were obtained from Charles River Laboratories, MA, USA, and were maintained at a stable temperature (22 ± 2 °C) and humidity (55 ± 5%). C57BL/6 mice were sacrificed by cervical dislocation, and lenses were isolated. Mouse lenses were immediately stored at −80 °C or processed for experimentation [7,8,37].

2.1.2. Human Aging Lens/LECs

Human lens/LECs were isolated from the normal eye of deceased donors of different ages (25 years (y) and 74 y, n = 4 each group) obtained from the Lions Eye Bank, Nebraska Medical Center, Omaha, NE, USA, within 1 to 12 h postmortem. These human aging lens/LECs were used for total protein and RNA isolation, and the experiments were performed. According to regulation HHS45CFR 46.102(f), studies involving material from deceased individuals are not considered human subject research as defined under 45CFR46.102(f) 10(2) and do not require IRB oversight.

2.1.3. Mouse Lens Epithelial Cells (mLECs)

All animals were maintained under specific pathogen-free conditions in an animal facility as indicated in Section 2.2.1. mLECs isolated from C57BL/6 mice were generated and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Atlanta Biologicals, Inc., Flowery Branch, GA, USA), following our published protocol [21]. mLECs were isolated, and Western analysis was carried out to confirm the presence of αA-crystalline [21,81], a specific marker of LECs, which minimizes the variation due to genetic background.

2.2. Quantitation of Intracellular Reactive Oxygen Species (ROS)

2.2.1. ROS Level by H2-DCF-DA in Lens/LECs

Intracellular ROS levels were assessed using the fluorescent probe H_2_-DCF-DA, a nonpolar compound that is deacetylated by intracellular esterase and oxidized by ROS to form fluorescent DCF. (1) Lenses isolated from C57BL/6 mice of 4 M (n = 4) and 21 M (n = 4); (2) aged human lenses of 25 y (n = 4) and 74 y (n = 4); and (3) lenses were isolated form 15 M-old C57BL/6 mice and transduced with TAT-HA-Prdx6 following H_2_O_2_ exposure for 45 h and 90 h in 199 media ex vivo (n = 6 per group). These lenses were immediately stored at −80 °C or processed to measure ROS levels in lens tissues according to our published protocol [7,8,37]. Briefly, mouse/human lenses were thawed on ice and homogenized (100 mg/mL) in freshly prepared homogenization buffer containing 50 mM phosphate buffer, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1µM pepstatin, and 80 mg/L trypsin inhibitor, pH 7.4. H_2_-DCF-DA dye was added to freshly prepared lens homogenate at a final concentration of 30µM and incubated at 37 °C for 30 min. Intracellular fluorescence was recorded at 485 nm excitation (Ex) and emission (Em) at 530 nm using the Spectra Max Gemini EM (Molecular Devices, San Jose, CA, USA).

2.2.2. Quantitation of Intracellular ROS Level by CellROX® Deep Red Reagent

ROS levels were assessed using CellROX^®^ Deep Red Oxidative Stress Reagent (Catalog No. C10422, ThermoFisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions and our published protocol [37,82]. In brief, mLECs transfected with pGFP-vector or pGFP-TXNIP or infected with LV Sh-Control and LV Sh-TXNIP were seeded in a 96-well plate. Twenty-four hours later, cells were exposed to different concentrations of H_2_O_2_, LPS, and UVB. Four hours later, 5 µM of CellROX deep red reagent was added and incubated for 30 min at 37 °C. Cells were fixed with 3.7% formaldehyde for 15 min. Fluorescence intensity was recorded at Ex640 nm/Em665 nm with Spectra Max Gemini EM (Molecular Devices, San Jose, CA, USA).

2.3. Mouse and Human Caspase-1 ELISA Assay

Caspase-1 activity was measured in mouse lenses (4 M and 21 M, n = 4 lenses per group and 15 M, n = 6 lenses per group) and human lenses (25 y and 74 y, n = 4 lenses per group) as well as TXNIP-overexpressing mLECs exposed to H_2_O_2_ and/or UVB using caspase-1 ELISA kits (Mouse: Catalog # E4180-100; Human: Catalog # E4588-100; BioVision, Milpitas, CA, USA) according to the manufacturer’s instructions and our published protocol [9]. For standard curve generation, lyophilized recombinant caspase-1 was dissolved in standard/sample dilution buffer to prepare stock concentrations of 2000 pg/mL (mouse) or 5000 pg/mL (human). A series of 2-fold dilutions was prepared to generate a standard curve within the recommended detection range. Cellular extracts were prepared from lenses and mLECs using an ice-cold radioimmunoprecipitation assay (RIPA) buffer and stored at −80 °C. All reagents, standards, and samples were brought to room temperature 30 min before use. After washing the plates with the wash solution, 100 µL of standards or samples were added to the well and incubated at 37 °C for 90 min. Standards or samples were removed, and incubation at 37 °C for 60 min was performed after the addition of 100 µL of biotin-conjugated detection antibody. After three washes with 1× wash solution, 100 µL of streptavidin-HRP (SABC) working solution was added and incubated at 37 °C for 30 min. TMB substrate (90 µL) was added to each well after five washes with 1× wash solution, incubated for 15–30 min at 37 °C, and protected from light. In total, 50 µL of stop solution was added to each well once the color development (blue) was complete. Optical density (O.D.) was measured at 450 nm using Spectra Max Gemini EM (Molecular Devices, San Jose, CA, USA).

2.4. Mouse and Human IL-1β ELISA Assay

Quantification of interleukin-1 (IL-1β) levels in lenses (mouse; 4 M and 21 M, n = 4 lenses per group; human; 25 y and 74 y, n = 4 lenses per group and mouse; 15 M, n = 6 lenses per group) as well as mLECs was performed using the mouse IL-1β ELISA kit (Catalog # ab197742, Abcam, Waltham, MA, USA) and human IL-1β ELISA kit (Catalog # ab214025, Abcam, USA) according to the manufacturer’s instructions and our published protocol [9]. For standard curve generation: lyophilized IL-1β standard was reconstituted in 500 µL sample diluent NS and/or 1× cell extraction buffer PTR to obtain the final concentration of 200 pg/mL (mouse) or 4000 pg/mL (human). A series of eight dilutions was prepared from the stock to generate the standard curve. For sample preparation, (1) A cellular extract was prepared from lenses using RIPA buffer and stored at −80 °C. (2) Cell culture supernatants from mLECs overexpressing TXNIP exposed to H_2_O_2_ and/or UVB were collected at 48 h post-treatment, and debris was removed by centrifugation at 5000× g RPM for 20 min. Aliquoted clear supernatants were stored at −80 °C. For assay, all solutions were equilibrated to room temperature before use. A volume of 50 µL of standard or sample was added to each well, followed by 50 µL of the provided antibody cocktail. Plates were incubated for 1 h at room temperature on a plate shaker. A volume of 100 µL of TMB development solution was added to each well after three washes with 1× wash buffer PT and then incubated in the dark for 10–15 min on a plate shaker set to 400 rpm. A volume of 100 µL of stop solution was added to each well to stop the reaction and mixed for one minute on the plate shaker. The absorbance was quantified at 450 nm using Spectra Max Gemini EM (Molecular Devices, San Jose, CA, USA).

2.5. Mouse and Human IL-18 ELISA Assay

IL-18 protein levels were quantified in mouse lenses (4 M and 21 M, n = 4 lenses per group and 15 M, n = 6 lenses per group) and human lenses (25 y and 74 y, n = 4 lenses per group), as well as mLECs using the mouse IL-18 ELISA Kit (Catalog # ab216165, Abcam, USA) and human IL-18 ELISA Kit (Catalog # ab215539, Abcam, USA) according to the manufacturer’s instructions and our published protocol [9]. To generate a standard curve: lyophilized IL-18 was resuspended in 500 µL sample diluent NS and/or 1× cell extraction buffer PTR to generate a standard stock solution of 5465 pg/mL (mouse) and 8000 pg/mL (human). Serial dilutions were prepared from this standard stock solution to establish a standard curve for each species. Sample preparation from lens tissue: RIPA buffer was used to extract the protein lysate from mouse and human lenses, and was immediately stored at −80 °C. Sample preparation from cell culture supernatant: mLECs were cultured in DMEM containing 0.2% BSA up to 48 h. Supernatants were collected from mLECs overexpressing TXNIP exposed to H_2_O_2_ and/or UVB, centrifuged for 20 min, and clear supernatants were transferred to clean tubes and immediately stored at −80 °C. For the assay, all solutions were brought to room temperature before the assay. 50 µL of standard or sample was added to the assay plate wells, and then 50 µL of antibody cocktail was added. Plate wells were covered and incubated for 1 h at room temperature on a plate shaker at 400 rpm. After three washes with 1× wash buffer, PT and excess liquid were removed. TMB development solution (100 µL) was added, and plate wells were incubated in the dark for 10–15 min on a plate shaker set to 400 rpm. The reaction was stopped by the addition of 100 µL of stop solution, mixed for 1 min, and absorbance was read at 450 nm using Spectra Max Gemini EM (Molecular Devices, San Jose, CA, USA).

2.6. Protein Isolation and Western Blotting

Cellular protein lysates from mouse and human lenses, as well as mLECs, were isolated using an ice-cold RIPA buffer. Western blot analysis was performed as previously described [7,8,21,37]. The membranes were probed with the following antibodies: Prdx6 and TRX1 (LF-PA0011 and LF-PA0187; 1:2000 dilution, Ab Frontier, Seoul, Republic of Korea), TXNIP (# 14715), caspase-1 (# 24232 and # 3866), ASC (# 13833 and # 67824), IL-1β (# 122452), IL-18 (# 57058 and # 54943), gasdermin D (# 39754S) (1:1000 dilution; all from Cell Signaling Technology, Danvers, MA, USA), Nlrp3 (# PA5-79740; 1:1000 dilution, ThermoFisher Scientific, Waltham, MA, USA), IL-1β, and ASC (sc-52012 and sc-271054; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). β-actin (A2066; 1:2000 dilution; Sigma-Aldrich, St. Louis, MO, USA) and tubulin (ab44928; 1:4000 dilution; Abcam Inc., Waltham, MA, USA) were used as internal controls. After incubation with HRP-conjugated secondary antibodies (sc-2354 and sc-2768, 1:2000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), protein bands were visualized using Western blotting with Luminol reagent (sc-2048; Santa Cruz Biotechnology, Dallas, TX, USA) and imaged with a FUJIFILM-LAS-4000 luminescent image analyzer (FUJIFILM Medical Systems Inc., Hanover Park, IL, USA).

2.7. RNA Isolation and Quantitative mRNA Analysis by RT-qPCR

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from mouse lenses (4 and 21 M-old; n = 4 lenses per group), human lenses (25 and 74 y old; n = 4 lenses per group), 15 M-old mouse lenses transduced with TAT-HA-Prdx6 following H_2_O_2_ exposure (n = 6 lenses per group), and TXNIP overexpressing and/or under-expressing mLECs exposed to H_2_O_2_ and UVB. cDNA was synthesized from 0.5 to 5 µg of total RNA using Superscript II RNAase H-reverse-transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative PCR was performed using SYBR Green Master Mix (Roche Diagnostic Corporation, Indianapolis, IN, USA) on a Roche^®^ LC480 sequence detector system (Roche Diagnostic Corporation, Indianapolis, IN, USA). PCR amplification was performed with an initial hot start at 95 °C for 5–10 min, followed by 45–55 cycles of 10 s at 95 °C, 30 s at 60 °C, and 10 s at 72 °C. Gene expression levels of Prdx6, Nlrp3, ASC, caspase-1, IL-1β, IL-18, GSDMD, TXNIP, TRX, and β-actin were quantified using real-time PCR (RT-qPCR). Primer sequences used for RT-qPCR are shown below in Table 1.

2.8. Plasmid Construct and Lentiviral (LV) Infection

Plasmids pGFP (green fluorescent protein)-vector (Clontech, San Jose, CA, USA) and pGFP-TXNIP (Plasmid # 18758, Addgene, Watertown, MA, USA) were purchased. CopGFP control lentiviral (LV) particle (LV GFP-ShControl, sc-108084) and GFP–tagged ShTXNIP linked to lentivirus (LV GFP-ShTXNIP (mouse), sc-44944-VS) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). mLECs were infected with lentivirus, following the company’s protocol and our previously published protocol [9,37,82]. Briefly, mLECs were seeded in a 6-well plate in complete medium. After 24 h, the medium was replaced with 2 mL of fresh medium supplemented with polybrene (5 µg/mL; sc-134220, Santa Cruz Biotechnology, Inc.). GFP-ShControl and GFP-Sh-TXNIP lentiviral particles were added, mixed gently, and incubated overnight. The next day, cells were washed, and viral medium was replaced with fresh complete media. Lentiviral-infected mLECs were subcultured and treated with puromycin dihydrochloride (sc-108071, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for selection of stable cell lines. Puromycin-resistant clones were expanded and maintained as stable lines for the current study.

2.9. Cell Viability Assay

Cell viability under basal or oxidative stress (H_2_O_2_, LPS, and/or UVB) conditions was determined using the MTS assay. TXNIP over-expressed, or under-expressed mLECs were exposed to different concentrations of H_2_O_2_, LPS, and/or UVB, and viability was evaluated using the colorimetric CellTiter 96 ^®^ Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), following the manufacturer’s instructions and the previously described protocol [7,8,9,10,21,37,83]. This colorimetric assay method employs the MTS tetrazolium compound, which is metabolically reduced by viable cells to a water-soluble formazan product. After 24 h of oxidative stress exposure, MTS reagent was added directly to the culture wells, and cells were incubated for 2 h at 37 °C. Absorbance was measured at 490 nm using a microplate reader (DTX 880, Multimode detector, Spectra MAX Gemini, Molecular Devices, San Jose, CA, USA). Cell viability data were normalized to untreated control cells.

2.10. Expression of Recombinant Protein, TAT-HA-Prdx6

Full-length human Prdx6 cDNA was amplified from a human lens epithelial cell (LEC) cDNA library using Prdx6 gene–specific primers: forward primer, 5′-GTCGCCATGGCCGGAGGTCTGCTTC-3′ (contained NcoI site); and reverse primer, 5′-AATTGGCAGCTGACATCCTCTGGCTC-3′, as described previously [83]. The amplified PCR product was purified using the QIAEX II Gel Extraction Kit (Cat No. 20021, Qiagen Inc., Valencia, CA, USA), ligated into a TA-cloning vector (Invitrogen, Carlsbad, CA, USA), and transformed into competent Escherichia coli (E. coli) cells. Plasmids were isolated from individual colonies, and inserts were confirmed by sequencing. Verified Prdx6 cDNA was subcloned into the pTAT-HA expression vector using NcoI and EcoRI restriction sites (a kind gift of Dr. S. F. Dowdy) [84,85].

2.11. Site-Directed Mutagenesis (SDM)

PCR-based site-directed mutagenesis was performed using the Quick-Change Site-Directed Mutagenesis (SDM) Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions [86]. The primers used to generate specific mutations in TAT-HA-Prdx6 are as follows:

Prdx6 C47S (Cysteine 47 to Serine 47);

Forward: 5′-CTTTACCCCAGTGTCCACCACAGAGCTTGGCAGAGC-3′;

Reverse: 5′-GCTCTGCCAAGCTCTGTGGTGGACACTGGGGTAAAG-3′.

2.12. Expression and Purification of Recombinant Protein, TAT-HA-Prdx6WT and TAT-HA-Prdx6-Mutant

TAT-HA-Prdx6-WT and/or TAT-HA-Prdx6-mut (C47S) plasmid DNA were transformed into Escherichia coli BL21 (DE3) cells and plated on LB agar containing 100 µg/mL ampicillin. Single colonies were cultured overnight in 10 mL of LB medium containing ampicillin at 37 °C with continuous shaking at 200 rpm. The overnight culture was used to inoculate 250 mL of pre-warmed LB–ampicillin medium, which was grown at 37 °C with shaking until OD_600_ reached ~0.6. A 0.5 mL sample was collected before induction. Protein expression was induced with 1 mM IPTG, and the culture was incubated for an additional 5 h at 37 °C with vigorous shaking. A second 0.5 mL of the sample was collected post-induction. These samples were pelleted, resuspended in SDS-PAGE loading buffer, boiled at 100 °C for 5 min, and stored at −20 °C for later analysis. After 5 h of incubation, the remaining culture was centrifuged at 4000× g for 20 min to pellet the cells and stored at −20 °C overnight. Recombinant proteins were purified after thawing the pellet for 15 min on ice using the QIAexpress^®^ Ni-NTA Fast Start kit column (Qiagen Inc., Valencia, CA, USA) as described [21,83,84,85,86]. This purified protein was quantified, frozen at −80 °C, and lyophilized for long-term storage.

2.13. Lens Organ Culture, TAT-HA-Prdx6 Transduction, and H2O2 Treatment

Lenses were isolated from 15 M-old mice and cultured in a 48-well plate. Lenses were either transduced with TAT-HA-Prdx6 WT or its inactive-mutant (TAT-HA-Prdx6-mut, mutated at C47) for 3 h, and subjected to H_2_O_2_-induced oxidative stress as indicated in Figures. The lenses were observed routinely. After 45 h and 90 h, lenses were photographed using a Nikon SMZ 745T Trinocular Stereo Microscope (Nikon Instruments Inc., Melville, NY, USA) fitted with a camera and a computer loaded with an analysis software program [83,84]. After 45 h, lenses were used to measure ROS levels and RNA isolation. Protein was extracted at 45 h and 90 h of H_2_O_2_ exposure, and caspase-1, IL-1β, and IL-18 activities were measured.

2.14. Statistical Analysis

All quantitative data were analyzed using Student’s t-test and/or one-way ANOVA where appropriate. Results are expressed as mean ± standard deviation (S.D.) from the indicated number of experiments. Statistical significance between the control and treatment groups was defined as p < 0.05 and p < 0.001.

3. Results

3.1. Increased Lens Opacity and Accumulated ROS Levels with Aberrant Nlrp3 Inflammasome Activation in Aging Lenses

Prdx6 plays an important role in protecting cells against various endogenous and exogenous oxidative stressors by normalizing ROS generation and maintaining redox homeostasis. However, loss of Prdx6 during aging or oxidative stress conditions leads to aberrant accumulation of ROS and spontaneous cell death [9,10,87,88]. To determine the role of Prdx6 in the regulation of Nlrp3 inflammasome activation and its associated inflammatory signaling pathways, we used young and aged lenses as a model system. Lenses are isolated from young (4 M) and aged (21 M) C57BL/6 mice and visualized under a microscope to examine the opacity of lenses. We found significantly increased lens opacity in aged mouse lenses, as evidenced by lens images and densitometric analysis of these lenses (Figure 1A–C). Increased ROS levels were determined in aged lenses using the H_2_DCF-DA fluorescence dye method (Figure 1D). Further, we found enhanced caspase-1 activity in aging lenses (Figure 1E), which was a key component of the inflammasome complex responsible for the release of pro-inflammatory cytokines, inducing GSDMD cleavage. Excessive ROS accumulations in aging cells are known to promote DAMP/PAMP signaling and activation of Nlrp3 inflammasome, resulting in maturation and release of pro-inflammatory cytokines. Similarly, our results showed elevated levels of mature pro-inflammatory cytokines, IL-1β and IL-18 (Figure 1F,G), in response to oxidative stress/aging. Protein expression analysis by Western blotting demonstrated the age-related upregulation of Nlrp3, ASC oligomerization, cleaved caspase-1, mature active IL-1β and IL-18, pyroptosis executor GSDMD and TXNIP, and reduced expression of TRX1 and Prdx6 (Figure 1H). In consistency with upregulated protein expression, results showed significantly enhanced mRNA expression of Nlrp3 and its associated components (Figure 1I). However, our findings revealed that cellular Prdx6 abundance is essential for subsiding aberrant Nlrp3 inflammasome activation–mediated inflammatory signaling and cellular protection.

3.2. Age-Related Reduction in Prdx6 Was Directly Correlated with Increased ROS and Nlrp3 Inflammasome Activation in Human Aging Lenses

Recent studies demonstrate that a disequilibrium antioxidant defense system and excessive accumulations of ROS-driven dysregulated Nlrp3 inflammasome activation play a key role in the pathogenesis of age-related diseases. Previously, we reported that age-associated loss of antioxidant enzyme, Prdx6, leads to elevated ROS production and impaired redox homeostasis [7,8,9]. The findings from Figure 1 support that an aging lens resulted in heightened ROS accumulation and subsequent activation of Nlrp3 inflammasome–mediated inflammatory response. To examine whether increased ROS generation due to reduced Prdx6 expression in aging promotes similar inflammasome activation in human aging lenses, we quantified intracellular ROS levels using H_2_-DCF-DA dye. As expected, we observed an age-associated increase in ROS accumulation in the aged human lenses (Figure 2A). DCF fluorescence results reflect overall oxidative burden in the cells, as it includes H_2_O_2_, peroxynitrite, superoxide (O_2_^−^), and nitric oxide (NO). ELISA assay revealed the age-related increased activity of caspase-1 and the mature form of pro-inflammatory cytokines, IL-1β, and IL-18 (Figure 2B–D). Immunoblotting and mRNA analysis demonstrated age-dependent elevation in Nlrp3 expression, activation of caspase-1, increased ASC monomer, dimer, and oligomers, enhanced mature IL-1β and IL-18, cleaved GSDMD and TXNIP, with reduction in TRX1 and Prdx6 expression (Figure 2E,F). Taken together, our findings demonstrate that age-related loss of antioxidants Prdx6 and TRX contributes to the increased oxidative load, which may serve as a critical trigger for aberrant inflammasome activation and likely contributes to the onset of age-related lens pathobiology.

3.3. TXNIP-Sensitized mLECs Showed Reduced Cell Viability with Elevated ROS Generation Under Oxidative Stress Conditions

It has been reported that TXNIP interacts with Nlrp3 and activates the Nlrp3 inflammasome pathway [48,56,78,89]. Also, recent studies showed that oxidative stress provokes and stimulates the interaction of TXNIP with the inflammasome protein Nlrp3 [42,48,56,78,80,90]. Thus, we wanted to know if aging LECs with increased ROS show a higher abundance of TXNIP. As shown in Figure 1 and Figure 2, TXNIP expression was increased in aging lenses/LECs. To examine if increased TXNIP levels can counteract survival pathways, TXNIP-overexpressing cells were treated with different concentrations of H_2_O_2_ and/or LPS and/or UVB, as shown in Figure 3. Toward this, mLECs were transfected with either pGFP-TXNIP and/or control pGFP-empty vector plasmids at equal concentrations (Figure 3). Transfection efficiency was confirmed by Western blot and RT-qPCR analyses (Figure 3A,B). Assessments of cell viability and total ROS using MTS assay and CellROX Deep Red staining demonstrated that TXNIP overexpression led to decreased cell survival (Figure 3C,E) and elevated ROS accumulation (Figure 3D,F) under oxidative stress conditions. Results revealed that overexpression of TXNIP or aberrant cellular abundance of TXNIP promotes oxidative damage and impairs cell survival under oxidative stress conditions, indicating that TXNIP’s aberrant levels initiate cell death signaling as reported earlier in other model systems.

3.4. TXNIP Overexpression Enhanced Caspase-1 Activation and Promoted the Release of Pro-Inflammatory Cytokines Under Oxidative Stress

Oxidative stress induces elevated TXNIP, which interacts with Nlrp3 inflammasome protein and activates the Nlrp3 inflammasome complex, which subsequently releases the pro-inflammatory cytokines, IL-1β and IL-18 [56,90]. As shown in Figure 3, TXNIP-overexpressing mLECs were more vulnerable to oxidative stress and showed reduced viability and increased ROS generation. To examine whether increased TXNIP levels contribute to the activation of Nlrp3 inflammasome and its components, TXNIP-overexpressing mLECs were exposed to oxidative stress, as shown in Figure 4. TXNIP overexpressed mLECs showed significantly increased caspase-1 activity in basal conditions, which was further amplified following H_2_O_2_ (Figure 4A) or UVB exposure (Figure 4B). In addition, the ELISA assay demonstrates elevated levels of IL-1β (Figure 4C,D) and IL-18 (Figure 4E,F) in the supernatant of TXNIP overexpressed mLECs, which were remarkably enhanced under oxidative stress conditions. Results revealed the involvement of TXNIP in Nlrp3 inflammasome activation and pro-inflammatory signaling in response to TXNIP overexpression.

3.5. TXNIP Drove the Elevation of Nlrp3 Inflammasome Gene Expression Under Oxidative Stress Conditions

As shown in Figure 3 and Figure 4, TXNIP-overexpressing mLECs indicated the contribution of TXNIP to Nlrp3 inflammasome activation and generation of ROS levels under oxidative conditions. Next, we were interested to know whether TXNIP overexpression enhanced the Nlrp3 inflammasome and its components at the transcription levels. mLECs overexpressing TXNIP were exposed to H_2_O_2_ (Figure 5A) and UVB (Figure 5B), and the mRNA levels of Nlrp3, caspase-1, ASC, proinflammatory cytokines IL-1β and IL-18, as well as pyroptosis executor GSDMD were quantified. Results demonstrated a significant increase in Nlrp3, caspase-1, ASC, IL-1β, IL-18, and GSDMD mRNA following H_2_O_2_ and UVB exposure, as determined by RT-qPCR. Taken together, findings confirmed that TXNIP amplifies inflammasome-associated gene expression, suggesting the role of TXNIP as a positive regulator of inflammasome activation and inflammatory death signaling under oxidative conditions.

3.6. TXNIP Knockdown Conferred Resistance to H2O2, LPS, or UVB-Induced Cellular Damage, and Infectants Showed Reduced Levels of Oxidative Load

As shown in Figure 3, Figure 4 and Figure 5, TXNIP increased expression plays a key role in Nlrp3 inflammasome activation and shows higher sensitivity to oxidative stress. To test whether depletion of TXNIP mitigates oxidative stress-induced cytotoxicity and ROS accumulations, we employed a knockdown approach using lentiviral-mediated delivery of Sh-TXNIP in mLECs (Figure 6) as described in the Section 2. mLECs were stably infected with either LV ShTXNIP and/or LV ShControl. No morphological difference was observed between the groups (Figure 6A). Western blotting and RT-qPCR analyses confirmed the effective silencing of TXNIP in LV ShTXNIP–infected cells (Figure 6B,C). Next, stably infected mLECs cell lines of LV ShControl and LV ShTXNIP were subjected to different concentrations of H_2_O_2_, LPS, and UVB, and cell viability and ROS levels were measured as indicated in Figure 6. TXNIP-depleted mLECs showed resistance against oxidative stress and protected mLECs against H_2_O_2_-, LPS- (Figure 6D), and UVB- (Figure 6F) induced cell death by suppressing ROS production (Figure 6E,G) in comparison to control/untreated cells. Data demonstrates that TXNIP regulates oxidative stress-induced cell death.

3.7. TXNIP-Depletion Inhibited Inflammasome Gene Expression Under Oxidative Stress Conditions

TXNIP overexpression leads to the activation of Nlrp3 inflammasome and release of pro-inflammatory cytokines. Next, we were interested to know whether knocking down TXNIP expression prevents Nlrp3 inflammasome activation. TXNIP-depleted mLECs were subjected to H_2_O_2_ (Figure 7A) and UVB (Figure 7B) exposure for 24 h, and mRNA expression levels were determined using RT-qPCR analysis. In basal conditions, H_2_O_2_ and UVB-exposed LV ShControl-infected mLECs showed a significant increase in Nlrp3, ASC, caspase-1, IL-1β, IL-18, and GSDMD, while this gene expression was significantly low in LV ShTXNIP-infected mLECs in response to H_2_O_2_ and UVB exposure. Taken together, the data indicate that TXNIP is required for activation of the Nlrp3 inflammasome signaling cascade under oxidative stress conditions. However, TXNIP deficiency suppresses H_2_O_2_ and UVB-induced Nlrp3 inflammasome activation, suggesting the regulatory role of TXNIP in Nlrp3 inflammasome activation.

3.8. TAT-HA-Prdx6 Protein Internalized in the Lenses and Prevented H2O2-Induced Lens Opacity

Previously, we have shown that Prdx6 linked to the transduction domain TAT efficiently interlines in lenses and protects against oxidative stress-induced lens opacity [83]. Moreover, the eye lens and lens epithelial cells are well-established models for studying age-related diseases. In this study (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7), we confirmed that aging lenses/LECs facing oxidative stress displayed increased expression of TXNIP and Nlrp3 inflammasomes with their inflammatory proteins. In this context, TXNIP over- or under-expression assays in LECs revealed the key role of TXNIP in activation of Nlrp3 inflammasome and subsequently activation of caspase-1 and the release of mature pro-inflammatory cytokines, as reported for other model systems [41,56,78,90]. Previously, we have shown that Prdx6 is a critically important antioxidant that protects cells by maintaining redox homeostasis, and loss of Prdx6 leads to the activation of cell death signaling pathways. To test whether TAT-HA-Prdx6 can cross the lens capsule and internalize into lenses, we performed ex vivo lens organ cultures using lenses from 15 M-old C57BL/6 mice (~56 years of human age) [91]. Isolated lenses were pre-treated with TAT-HA-Prdx6^WT^ and/or TAT-HA-Prdx6–^inactive mutant^ for 3 h, then challenged with 100 µM H_2_O_2_ as shown in Figure 8. H_2_O_2_ exposure induced lens opacity (Figure 8B(b1,b2,d1,d2)), while TAT-HA-Prdx6 WT–pretreated lenses showed a preventive effect against H_2_O_2_ (Figure 8B(c1,c2,e1,e2)), as evidenced by photomicrographic analysis. Western blot analysis (Figure 8Bi) showed the internalization of the TAT-HA-Prdx6^WT^ and/or TAT-HA-Prdx6–^inactive mutant^ protein into the lens. Densitometry of lens opacity showed a 32% (Figure 8C, b) increase in lens opacity at 45 h, and this further increased to 51% (Figure 8C, d) at 90 h in H_2_O_2_-exposed lenses in the absence of TAT-HA-Prdx6^WT^. Furthermore, TAT-HA-Prdx6^WT^ significantly attenuated ROS accumulation in H_2_O_2_-exposed lenses (Figure 8D), suggesting that Prdx6 supplementation restores redox homeostasis and preserves lens transparency.

3.9. TAT-HA-Prdx6 Suppressed the H2O2-Induced Caspase-1 Activation, Release of Inflammatory Cytokines, and Inflammasome Gene Expression

As shown in Figure 8, TAT-HA-Prdx6 successfully internalizes in the lenses and prevents H_2_O_2_-induced lens opacity by removing the accumulation of ROS. Next, we were interested in determining if TAT-HA-Prdx6 delivery prevents oxidative-induced Nlrp3 inflammasome activation in aging lenses ex vivo. We found that delivery of TAT-HA-Prdx6 into the mouse lenses markedly inhibited H_2_O_2_-induced caspase-1 activity compared to lenses treated with the inactive mutant (Figure 9A). In a parallel experiment, we measured the levels of pro-inflammatory cytokines (IL-1β and IL-18) and found significantly reduced levels of bioactive IL-1β and IL-18 in TAT-HA-Prdx6^WT^-treated lenses (Figure 9B,C). Also, RT-qPCR analysis demonstrated that TAT-HA-Prdx6 delivery suppressed the H_2_O_2_-induced, enhanced expression of Nlrp3, caspase-1, ASC, IL-1β, IL-18, and GSDMD, as well as TXNIP expression, while at the same time restoring the antioxidant gene TRX (Figure 9D). These data indicate that Prdx6 delivery counteracts oxidative stress–mediated inflammasome activation and inflammation, thereby preserving lens integrity. Taken together, this finding suggests that TXNIP knockdown or Prdx6 can be considered as a therapeutic molecule to prevent age and oxidative stress-associated Nlrp3 inflammasome activation and inflammatory cell death.

4. Discussion

Aging and oxidative stress are the biggest factors in the development of many age-related diseases, including ocular diseases [7,8,9,10,37]. Previously, we have demonstrated that aging LECs/LECs facing oxidative stress showed activated Nlrp3-inflammasome signaling with reduced antioxidant defense response [9]. In this study, opaque lenses/lens opacity were observed in aging C57BL/6 mice with reduced expression of antioxidant proteins Prdx6 and TRX1, which are directly linked to elevated levels of TXNIP as well as Nlrp3 inflammasomes and their inflammatory components. Our findings revealed that aging human or mouse lenses/LECs had reduced antioxidant expression, which is directly associated with increased ROS levels and activation of the Nlrp3 inflammasome and its inflammatory molecules (Figure 1 and Figure 2). Similarly, it has been reported that with advancing age, glomerular podocytes undergo aberrant changes caused by inflammation. Furthermore, podocytes isolated from a different cohort showed increased transcripts of Nlrp3, caspase-1, and ASC in aging mice (24 months old, which is equivalent to ~70 human years) [91]. The Nlrp3 inflammasome, such as Nlrp3, caspase-1, IL-1β, and IL-18, was also increased in podocytes of middle-aged humans [76]. Aberrant Nlrp3 activation also has an impact on liver aging [76]. Also, inflammation is a hallmark of aging and negatively affects female infertility. Age-dependent increased expression of Nlrp3 in the ovary was observed in WT mice during reproductive aging [92]. Aberrant expression of Nlrp3, caspase-1, and IL-1β was observed in granulosa cells from patients with ovarian insufficiency, suggesting that the Nlrp3 inflammasome is implicated in the age-dependent loss of female fertility [92]. Consistent with our current results, it has been shown that TRX expression was decreased with increased expression of TXNIP, Nlrp3, cleaved caspase-1, and mature IL-1β in an age-dependent manner in the brains of both male and female C57BL/6 mice [65]. Similarly, increased TXNIP expression was observed in β cells of pancreatic tissue as well as serum samples of aging human subjects, which is associated with the age-dependent onset of cellular senescence [42]. TXNIP is highly expressed in skeletal satellite cells of aging mice [50]. Additionally, TXNIP expression was significantly increased in different cells, such as T cells, monocytes, hepatocytes, and hematopoietic progenitor cells isolated from older human (>55 years) subjects. In the same T cells isolated from older human (>55 years) subjects, TRX enzymatic activity was found to have declined [1]. Multiple stimuli, such as H_2_O_2_, uric acid crystals (MSU), ATP, nigericin, β-amyloid, etc., can induce overproduction of intracellular ROS levels, and this aberrant expression of ROS activates the level of TXNIP in cells by dissociating from TRX. Similarly to our results, increased Nlrp3, cleaved caspase-1, mature IL-1β, and TXNIP levels, and reduced TRX expression were observed in islet cells [64,77,93].

In this study, we examined how TXNIP expression influences Nlrp3 activation and antioxidant defense in aging and oxidative stress conditions. Additionally, we investigated whether TXNIP deficiency or an extrinsic supply of Prdx6 can block or delay oxidation-initiated inflammatory responses and protect the lenses and lens epithelial cells. To accomplish this, we performed the following experiments: (i) we overexpressed mLECs with TXNIP, (ii) depleted TXNIP in mLECs, and (iii) transduced recombinant TAT-HA-Prdx6 to lenses ex vivo. These manipulations allowed us to evaluate Nlrp3 inflammasome activity and associated changes in the absence or presence of oxidative stress. mLECs overexpressed with TXNIP showed more sensitivity against the H_2_O_2_, LPS, or UVB-induced cell damage with increased ROS level and Nlrp3 inflammasome molecules in comparison to empty vector (Figure 3, Figure 4 and Figure 5). TXNIP overexpression enhances the Nlrp3 and its molecules (IL-1β, IL-18, GSDMD, caspase-1, and ASC). These results are consistent with previous reports showing that TXNIP regulates Nlrp3 inflammasome-induced pyroptosis in HT-22 cells. While TXNIP knockdown reduces the expression of Nlrp3 and its molecules at protein and mRNA levels in HT-22 cells [51]. As shown in Figure 6 and Figure 7, knocking down TXNIP showed resistance against oxidative stress and protected LECs by inhibiting the activated Nlrp3 inflammasome pathway. Furthermore, TXNIP plays a crucial role in the induction of inflammatory response by activating Nlrp3, and its expression is regulated by oxidative stress-induced NF-κB activation in macrophages [94]. It is worth mentioning that deep venous thrombosis (DVT) is a common peripheral vascular disease wherein TRX expression level was suppressed with an increase in TXNIP and Nlrp3 in DVT rats [56]. In this context, Si-TXNIP or MCC950 could reduce thrombosis weight, ameliorate pathological changes, and decrease inflammatory reaction. Si-TXNIP or MCC950 inhibited the expression levels of TXNIP, Nlrp3, and IL-1β while elevating the antioxidant TRX levels, thereby suppressing the DVT [56]. TXNIP is highly expressed in the model of hyperuricemia. High doses of uric acid showed reduced cell viability with increased levels of ROS production and elevated TXNIP in HK-2 cells. Metabolomics and transcriptomics analyses showed that TXNIP overexpression disrupts purine metabolism and affects UA synthesis [95]. In vitro, TXNIP knockdown normalized GLUT9 levels and reduced UA uptake in HK-2 cells [95]. Collectively, results of these studies identify TXNIP as a potential therapeutic target for hyperuricemia and related metabolic disorders [95]. Moreover, the redox regulator TXNIP plays an important role in controlling lifespan and resistance to oxidative stress. Increased TXNIP activity shifts cells towards a pro-oxidative state, which promotes DNA damage in Drosophila [1]. As oxidative stress and DNA damage are key drivers of Nlrp3 inflammasome–mediated inflammatory response and its driven age-related cellular decline, elevated TXNIP accelerates this deterioration due to overactivation of Nlrp3 inflammasome via TXNIP [89]. Conversely, reducing TXNIP expression enhances cellular resilience, prevents inflammatory responses to oxidative stress, and promotes an extended lifespan and cellular survival [1,89]. Nonetheless, the Nlrp3 inflammasome regulates innate immunity, and its dysregulation contributes to inflammatory diseases. During aging and under oxidative stress, Nlrp3 inflammasome components and TXNIP levels increase. As observed in our study, excessive TXNIP abundance in cells triggered by oxidative stress activates Nlrp3 signaling, making TXNIP-overexpressing cells more sensitive to oxidative stress. In contrast, TXNIP deficiency reduces Nlrp3 activation and IL-1β secretion, provides resistance to oxidative stress, and protects cells by normalizing ROS production [1,44,64].

We have shown previously that different kinds of oxidative stress inducers, such as H_2_O_2_, UVB, LPS, paraquat, glutamate, tunicamycin, and cobalt chloride, at various time points showed reduced cell viability and increased ROS levels, lipid peroxidation, and DNA damage in lens epithelial cells as well as neuronal cells [21,82,96]. H_2_O_2_ exposure to HEI-OC1 auditory cells showed reduced cell viability in concentrations as well as a time-dependent manner. It is found that 1 mM of H_2_O_2_ concentration stimulated TXNIP expression in a time-dependent manner and increased total intracellular and mitochondrial ROS levels [44]. Similarly to studies by others, in our study, we have found that H_2_O_2_ exposure activated Nlrp3 with increased pro-caspase-1, and matured proinflammatory cytokines (IL-1β and IL-18), which were inhibited by TXNIP knockdown [55]. Furthermore, in support of our study, another study showed elevated levels of total intracellular ROS and increased expression of Nlrp3, IL-1β, IL-18, caspase-1, and cytoplasmic TXNIP in ARPE-19 cells. At the same time, H_2_O_2_ exposure reduced Nrf2, TRX1, and nuclear TXNIP in ARPE-19 cells, suggesting TXNIP-mediated Nlrp3 inflammasome regulation [39]. Xi et al. showed that TXNIP regulates Nlrp3 inflammasome-induced pyroptosis in HT-22 cells related to aging via the PI3K/Akt and cAMP/PKA pathways [51]. A known Nlrp3 inflammasome inducer, LPS, facilitated ROS production and Nlrp3 inflammasome activation with high expression levels of Nlrp3, ASC, caspase-1, and TXNIP with elevated levels of IL-6, TNFα, and IL-1β in BV-2 cells, which was inhibited by N-acetyl-L-cysteine (NAC) treatment [40]. It has been shown that ROS generation and TXNIP, Nlrp3, ASC, caspase-1, IL-1β, and HMGB1 expression/activity increased in LPS-treated cultured mouse periodontal ligament fibroblasts in a dose-dependent manner [97]. Skin and eyes are constantly exposed to sunlight/ultraviolet radiation, which results in an inflammatory reaction due to excessive ROS production. Studies have shown that ultraviolet B (UVB) induces inflammasome activation in keratinocytes to instigate the cutaneous inflammatory responses [98]. UVB exposure to human keratinocytes promotes Nlrp3 inflammasome activation, leading to IL-1β production [98]. In our study, we found that pretreated TAT-HA-Prdx6 lenses showed significantly reduced lens opacity induced by H_2_O_2_ by inhibiting the Nlrp3–inflammasome signaling (Figure 8 and Figure 9). Our previous and current studies demonstrated that recombinant Prdx6 fused to the TAT protein transduction domain can be efficiently internalized in LECs and intact lenses in vitro and in vivo, and protects the lenses/LECs as well as preventing lens opacity against various oxidative stresses [83,84,85]. We have also shown that a transduction domain–linked Prdx6 protein was internalized into HT-22 and HCN-2 cells, efficacious in alleviating various kinds of oxidative stress (H_2_O_2_, paraquat, glutamate)–induced cellular insults by inhibiting NF-ĸB activation [96]. These studies emphasize that Prdx6 linked to TAT should be an ideal and vital molecule to treat or prevent aging/oxidative stress–induced inflammatory signaling–driven pathobiology.

5. Conclusions

In conclusion, our findings reveal that loss of Prdx6 is a cause for oxidative–driven aberrant expression of TXNIP and activation of Nlrp3 inflammasome–mediated inflammatory cell death (pyroptosis) in aging lenses/LECs. We show that weakened antioxidant defense during aging and under oxidative stress conditions is directly associated with elevated ROS and TXNIP/Nlrp3 expression and activation. Our over- and under-expression experiments reveal that TXNIP plays a central role in the activation of Nlrp3, leading to increased bioactive caspase-1 and secretion of IL-1β and IL-18, which together promote lens and LEC pathobiology, which ultimately contribute to disease progression and disease state. Our study also provides proof-of-concept that TXNIP depletion and TAT-HA-Prdx6 supplementation have therapeutic potential for protecting the lenses/lens epithelial cells and preventing lens opacity. We further propose that local, focal, or systemic application of the transduction protein-linked Prdx6 may effectively reinforce natural Prdx6-mediated cellular defenses and thereby reduce ROS-driven oxidative damage and Nlrp3-mediated aberrant signaling. The findings also provide a background for future testing of Prdx6 as a therapeutic molecule in animal model systems and support its potential use in treating or preventing blinding disorders associated with inflammation or inflammatory cell death (Figure 10).