Lipoic Acid Modulates Nrf2/HO-1 Pathway to Suppress Inflammation in Fungal Keratitis Through Host-Directed Cytoprotective Mechanisms

Summary

Lipoic acid activates the Nrf2/HO-1 cytoprotective pathway in fungal keratitis, reducing levels of inflammatory cytokines and corneal damage. This host-directed therapy demonstrates superior safety profiles compared to conventional antifungals while providing mechanistic insights into the treatment of oxidative stress-driven ocular diseases through endogenous antioxidant pathway enhancement.

Abstract

Lipoic acid (LA), a dithiol antioxidant with an exemplary ocular safety profile, was investigated for its capacity to temper the oxidative-inflammatory milieu that drives fungal keratitis. Human corneal epithelial cells tolerated LA up to 60 µM without viability loss and, upon Aspergillus fumigatus challenge, LA induced a three-fold escalation of nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and a five-fold surge in its downstream effector heme oxygenase-1 (HO-1). Pharmacological silencing of Nrf2 with ML385 or catalytic blockade of HO-1 with tin protoporphyrin IX abrogated these molecular events and reinstated interleukin-1β and tumor necrosis factor-α expression, affirming strict Nrf2/HO-1 dependence. Parallel experiments in a murine keratitis model corroborated the in vitro observations: topical LA diminished corneal opacity and inflammatory scores by 47%, concurrently amplifying corneal Nrf2 and HO-1 expression while halving cytokine transcripts; co-administration of SnPPIX nullified these benefits. Collectively, the data delineate a coherent mechanistic hierarchy in which LA, at clinically attainable concentrations, increases Nrf2 protein levels and enhances nuclear translocation, potentiates HO-1 activity, and thereby quells pathogen-induced cytokinic turbulence. These findings position LA as a readily translatable, host-directed adjunct capable of complementing antifungal chemotherapy and suggest broader therapeutic vistas for Nrf2-centric modulation of sight-threatening corneal inflammation.

Introduction

Fungal keratitis (FK) is now recognized as a pervasive ophthalmic emergency, responsible for more than 1 million culture-proven cases annually and a disproportionate share of corneal blindness in tropical and subtropical regions^1,2. Incidence estimates approaching 6-8 per 100,000 in the United States and even higher in agrarian economies underscore a mounting global threat that preferentially strikes working-age populations after vegetal trauma or contact-lens wear^3,4. Filamentous fungi, most notably species of Fusarium and Aspergillus, dominate the etiological landscape, thriving in warm, humid climates and exploiting ocular micro-injuries to initiate infection⁵.

Despite half a century of clinical use, natamycin 5% remains the only topical agent approved specifically for FK; however, its polyene chemistry confers poor corneal penetration, requires frequent dosing, and is not universally available^6,7. Alarmingly, a successive surveillance study from South Asia has revealed incremental increases in the minimal inhibitory concentrations of both natamycin and triazoles, with Fusarium isolates exhibiting a ≥6% yearly increase in natamycin resistance and a parallel trend for voriconazole⁸. These data align with clinical reports of therapeutic failure and reinforce forecasts that the market for FK therapeutics will expand from USD 0.9 billion in 2023 to USD 1.48 billion by 2033, driven largely by the inadequacies of current pharmacotherapy. Ad-hoc surgical measures, such as conjunctival flaps or penetrating keratoplasty, salvage anatomy in recalcitrant ulcers, but are resource-intensive and vision-limiting⁹. Collectively, these shortcomings underscore the need for host-directed or multitarget strategies that extend beyond fungistatic action alone.

Oxidative stress and the resulting inflammatory cascade are now understood to be pivotal determinants of fungal pathogenesis and corneal scarring. Upon fungal invasion, corneal epithelial cells and resident immune populations, including conjunctival mast cells, limbal dendritic cells, and stromal keratocytes, initiate innate immune responses through pattern recognition receptors, such as Dectin-1 and Toll-like receptors¹⁰. Neutrophil-derived reactive oxygen species (ROS) accumulate in infected stroma as part of the antimicrobial oxidative burst, while infiltrating macrophages and T lymphocytes perpetuate chronic inflammation through cytokine amplification¹¹. Paradoxically, filamentous fungi upregulate their own antioxidant circuits (e.g., Yap1-regulated superoxide dismutases) to survive this hostile milieu. Excess ROS in turn activates NF-κB-dependent transcription of IL-1β, TNF-α, and other pro-inflammatory mediators that drive tissue destruction and corneal opacification¹². At the center of endogenous cytoprotection lies nuclear factor erythroid 2-related factor 2 (Nrf2), whose translocation to the nucleus orchestrates expression of heme oxygenase-1 (HO-1) and an array of detoxifying enzymes across multiple corneal cell types, including epithelial cells, keratocytes, and endothelial cells^13,14. Aging, diabetes, and chronic inflammation downregulate this axis, rendering ocular tissues vulnerable to oxidative insults, whereas genetic or pharmacological enhancement of Nrf2 expedites epithelial wound closure and suppresses cytokine surge in diverse corneal injuries¹⁵.

Proof-of-concept studies have begun to exploit this pathway in FK. Natural small molecules such as perillaldehyde¹⁶, gallic acid¹⁷, hydroxytyrosol¹⁸ , and the organoselenium compound ebselen¹⁹ achieve dual antifungal and anti-inflammatory effects by potentiating Nrf2-HO-1 signaling while directly impairing fungal viability. Nanomedicine platforms that co-deliver antifungals and antioxidants further illustrate the therapeutic promise of combining pathogen clearance with host defense reinforcement^20,21. Yet many of these candidates face translational hurdles related to formulation complexity or unproven ocular safety.

Lipoic acid (LA) is a ubiquitous dithiol antioxidant with a well-established systemic safety record and exceptional pharmacodynamic versatility. Based on studies in hepatic and neural tissues^10,12 LA is proposed to directly modify cysteine residues (particularly Cys151, Cys273, and Cys288) on Kelch-like ECH-associated protein-1 (Keap1), disrupting its interaction with Nrf2 and thereby preventing Nrf2 ubiquitination and proteasomal degradation, which results in increased Nrf2 protein levels, enhanced nuclear translocation, and augmented HO-1 transcription. However, direct demonstration of Keap1-LA interaction in corneal epithelial cells warrants future investigation. LA has demonstrated cytoprotective benefits in hepatic, neural, and ocular models of oxidative injury^13,15. Its amphipathic nature facilitates tissue penetration without the membrane toxicity observed with polyenes, and clinical formulations already exist for topical and systemic indications. Nevertheless, LA's capacity to modulate the corneal inflammatory micro-environment and thereby ameliorate FK has not been rigorously interrogated.

The present study, therefore, investigated whether LA could mitigate A. fumigatus-induced keratopathy by activating the Nrf2/HO-1 axis while suppressing pro-inflammatory cytokines. Using human corneal epithelial cells and a validated murine FK model, we delineated the dose-response characteristics, dissected mechanistic dependencies with selective inhibitors, and benchmarked therapeutic outcomes against uninfected controls and infected vehicle-treated groups. To our knowledge, this represents the first systematic investigation to establish lipoic acid's therapeutic efficacy in fungal keratitis through comprehensive dose-response characterization, mechanistic dissection using selective pharmacological inhibitors of both Nrf2 and HO-1, and validation in both cellular and animal models. This work uniquely integrates molecular, cellular, and translational readouts to provide definitive evidence of the Nrf2/HO-1 pathway dependency underlying LA's anti-inflammatory effects. By integrating molecular, cellular, and in vivo readouts, we sought to provide a comprehensive appraisal of LA as a readily translatable, host-directed therapy that addresses the twin imperatives of pathogen eradication and inflammatory restraint in FK.

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee (Protocol number: IACUC-2024-FK-007) and conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Male C57BL/6 mice, aged 8-10 weeks and weighing 20-25 g, were housed in individually ventilated cages under controlled environmental conditions of 22 ± 2 °C and 50%-60% relative humidity with a 12:12 h light-dark cycle. These specific pathogen-free animals were obtained from Charles River Laboratories (see Table of Materials). All mice underwent a 1 week acclimatization period to adapt to the new environment before experimental procedures, with unrestricted access to standard rodent chow and water.

CAUTION: Aspergillus fumigatus is a pathogenic organism requiring biosafety level 2 containment. All fungal cultures must be handled in a certified biological safety cabinet using appropriate personal protective equipment, including gloves, laboratory coats, and eye protection. Dimethyl sulfoxide (DMSO) is flammable and can penetrate skin; handle in well-ventilated areas and avoid direct contact. Ketamine and xylazine are controlled substances requiring secure storage and proper documentation per institutional regulations.

Experimental preparation
All required reagents, drugs, and materials were prepared according to the experimental workflow (see Table of Materials and Supplementary Figure 1 for schematic). Racemic (±)-α-Lipoic acid (LA) stock solutions were prepared using racemic α-lipoic acid (1,2-dithiolane-3-pentanoic acid) dissolved in DMSO to a concentration of 60 mM and stored in single-use aliquots at -80 °C to prevent oxidative degradation. The Nrf2 inhibitor ML385 and HO-1 inhibitor SnPPIX were similarly prepared and aliquoted for single-use to avoid freeze-thaw cycles. All cell culture media and supplements were prepared under sterile conditions and pre-warmed to 37 °C before use.

All materials contaminated with fungal cultures, including agar plates, culture tubes, and disposable plastics, were autoclaved at 121 °C for 30 min before disposal in biohazard waste containers. Chemical waste containing organic solvents was collected separately and disposed of through institutional environmental health and safety protocols. Animal tissues and carcasses were incinerated through approved biomedical waste services.

Fungal culture and infection preparation
Aspergillus fumigatus was cultured on Sabouraud dextrose agar at 37 °C for 5-7 days until mature conidial formation was observed. Conidia were harvested by flooding mature cultures with sterile phosphate-buffered saline (PBS) containing 0.05% Tween-80, and conidial suspensions were filtered through sterile gauze to remove hyphal fragments. Conidial concentration was determined using a hemocytometer and adjusted to established concentrations based on published fungal keratitis models. For in vitro experiments, 1 x 10⁶ conidia/mL (multiplicity of infection of 10:1) was selected based on previous studies demonstrating that this concentration induces robust inflammatory responses in corneal epithelial cells while maintaining adequate cell viability for downstream analyses^16,17. For in vivo studies, 1 x 10⁷ conidia/mL was chosen as this inoculum produces consistent, moderate-severity keratitis with reproducible disease scores by day 5 post-infection, providing sufficient dynamic range to assess therapeutic efficacy without excessive mortality^16,19.

Animal treatment and corneal infection
Under general anesthesia (intraperitoneal injection of ketamine 100 mg/kg and xylazine 10 mg/kg), fungal keratitis was induced by creating three 1 mm linear scratches on the central cornea using a sterile 25G needle, followed by topical application of 5 µL of A. fumigatus conidial suspension. Mice were randomly assigned to treatment groups (n=8 per group): control (uninfected), infected control, infected + LA treatment (60 µmol/L topically 4x times daily), and infected + LA + SnPPIX treatment (LA plus 5 mg/kg SnPPIX intraperitoneally daily). Clinical assessment and tissue collection were performed on day 5 post-infection.

Animal handling precautions: All procedures involving anesthetized animals should be performed under aseptic conditions. Monitor animals continuously during anesthesia for respiratory depression. Dispose of contaminated bedding, surgical waste, and infected tissues by autoclaving followed by incineration according to institutional biohazard waste management protocols.

Cell culture and treatment
Human corneal epithelial cells (HCECs) were obtained from ATCC and maintained in DMEM/F-12 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine at equimolar concentrations at 37 °C in a humidified 5% CO₂ atmosphere. Cells were subcultured every 3-4 days upon reaching 80%-90% confluence and used between passages 3-8 to ensure consistency. For experimental treatments, cells were seeded at 5 x 10⁴ cells per well in appropriate plates and allowed to adhere for 24 h before treatment. For all in vitro mechanistic experiments, cells were first exposed to Aspergillus fumigatus conidia at a multiplicity of infection (MOI) of 10:1 for 4 h to establish infection, followed by the addition of LA (60 µM), ML385 (5 µM), or SnPPIX (1 µM) alone or in combination for an additional 24 h before sample collection. This sequential design allows assessment of LA's therapeutic effects on established fungal challenge rather than prophylactic treatment.

Cell viability assessment (CCK-8 analysis)
Cell viability was assessed using the Cell Counting Kit-8 (CCK-8). Cells were seeded in 96-well plates at 5 x 10⁴ cells per well and incubated for 24 h before treatment. Following drug exposure, 10 µL of CCK-8 solution was added to each well, and plates were incubated for 2 h at 37 °C. Absorbance was measured at 450 nm using a Multi-Mode Microplate Reader. Each experimental condition was performed in octuplicate, and experiments were repeated three times independently. Vehicle control (DMSO) concentration was maintained below 0.1% to avoid cytotoxic effects.

RNA extraction and quantitative real-time PCR
Total RNA was isolated from cultured cells and corneal tissues using the RNeasy Mini Kit according to the manufacturer's protocol. Tissue samples were homogenized in RLT buffer using a tissue homogenizer for 2 min at 50 Hz. RNA quality and concentration were assessed using a spectrophotometer, with samples having 260/280 ratios of 1.8-2.0 used for downstream analysis. First-strand cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit with 1 µg total RNA per reaction. Quantitative PCR was conducted using SYBR Green-based qPCR master mix on a real-time PCR system with initial denaturation at 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Melting curve analysis was performed to verify amplicon specificity. All primers were validated for efficiency (90%-110%) and specificity prior to use. Relative mRNA expression was calculated using the 2^(-ΔΔCT) method with GAPDH as the reference gene.

Protein extraction and Western blot analysis
For total protein extraction, cells and tissues were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktails. Nuclear and cytoplasmic protein fractions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer's protocol. Protein concentrations were determined using the Bradford assay. Equal amounts of protein (30-50 µg) were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, membranes were incubated with primary antibodies overnight at 4 °C, followed by appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence reagent with exposure times of 30-180 s depending on signal intensity. Bands were quantified using ImageJ software, with β-actin serving as the loading control for total proteins and lamin B1 for nuclear proteins.

Clinical assessment and histological analysis
Corneal disease severity was evaluated using a standardized clinical scoring system based on established protocols for murine fungal keratitis²². The scoring system assesses three parameters on a 0-4 scale: corneal opacity (0 = clear, 4 = completely opaque), surface irregularity (0 = smooth, 4 = severe ulceration with perforation), and inflammatory response (0 = no infiltrate, 4 = dense infiltration with hypopyon), yielding total scores from 0 to 12. All clinical assessments were performed by two independent observers who were masked to treatment group assignments, with inter-observer agreement exceeding 90%. Corneal photographs were captured using a slit-lamp biomicroscope equipped with a digital camera. For histological analysis, eyes were enucleated, fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 5 µm thickness. Sections were stained with hematoxylin and eosin for general morphology assessment.

Enzyme-linked immunosorbent assay (ELISA)
Secreted levels of IL-1β and TNF-α in cell culture supernatants and corneal tissue homogenates were quantified using commercial ELISA kits according to the manufacturer's instructions. Cell culture supernatants were collected after 24 h of treatment, while corneal tissues were homogenized in PBS containing protease inhibitors. Samples were centrifuged at 12,000 x g for 10 min at 4 °C, and supernatants were used for ELISA analysis. All samples were analyzed in duplicate, and cytokine concentrations were calculated from standard curves.

Statistical analysis
All experiments were performed with appropriate biological replicates (n ≥6 for in vitro studies, n =8 for animal studies) and repeated at least 3x independently. Data are presented as mean ± standard error of the mean (SEM). Data analysis was performed using GraphPad Prism version 9.0. Specifically, we used the normality test function (Shapiro-Wilk) to assess data distribution, one-way ANOVA with Tukey's multiple comparisons test for experiments with more than two groups, and unpaired Student's t-test for two-group comparisons. Graphs were generated as grouped bar charts with individual data points overlaid as scatter plots, with error bars representing the standard error of the mean.

Results

The experimental workflow demonstrated that lipoic acid treatment effectively modulates the Nrf2/HO-1 signaling pathway to suppress inflammation in fungal keratitis models.

Cytotoxicity assessment of Lipoic acid and pathway-specific inhibitors
Initial dose-response studies were conducted to establish optimal concentrations for each compound used in this investigation. LA demonstrated excellent biocompatibility with HCECs, maintaining cell viability at concentrations up to ...

Discussion

The present findings confirm lipoic acid (LA) as a potent inducer of the Nrf2/HO-1 axis that mitigates fungal keratitis (FK) by curbing pro-inflammatory cytokine expression and preserving corneal architecture. Consistent with recent work showing robust Nrf2 activation by dithiolane compounds in oxidative disorders¹⁵, LA produced a five-fold rise in HO-1 transcripts and halved IL-1β/TNF-α secretion, outperforming several phytochemical activators reported for ocular inflammation

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-HO-1	Cell Signaling Technology, Danvers, MA, USA	#8803
Anti-β-actin	Sigma-Aldrich, St. Louis, MO, USA	A5441
Anti-lamin B1	Abcam, Cambridge, MA, USA	ab16048
Anti-Nrf2	Abcam, Cambridge, MA, USA	ab62352
Aspergillus fumigatus	ATCC 204305	N/A
Bradford Assay Kit	Bio-Rad Laboratories	5000006
C57BL/6 mice	Charles River Laboratories	N/A
Cell Counting Kit-8 (CCK-8)	Dojindo Laboratories, Kumamoto, Japan	343-07623
DMEM/F-12 medium	Gibco, Thermo Fisher Scientific	11320033
DMSO	Sigma-Aldrich	D2650
ECL reagent	Thermo Fisher Scientific	32106
Fetal bovine serum	Gibco, Thermo Fisher Scientific	16000044
GAPDH Forward primer	Custom synthesis	Custom
GlutaMAX	Gibco, Thermo Fisher Scientific	35050061
High-Capacity cDNA Reverse Transcription Kit	Applied Biosystems, Thermo Fisher Scientific	4368814
HO-1 Forward primer	Custom synthesis	Custom
HO-1 Reverse primer	Custom synthesis	Custom
HRP-conjugated anti-mouse IgG	Abcam, Cambridge, MA, USA	ab205718
HRP-conjugated anti-rabbit IgG	Abcam, Cambridge, MA, USA	ab205719
Human corneal epithelial cells (HCECs)	ATCC, Manassas, VA, USA	CRL-11135
IL-1β ELISA Kit	R&D Systems, Minneapolis, MN, USA	DY201
Lipoic acid (LA)	Sigma-Aldrich, St. Louis, MO, USA	T1395
ML385 (Nrf2 inhibitor)	MedChemExpress, Monmouth Junction, NJ, USA	HY-100523
NanoDrop 2000 spectrophotometer	Thermo Fisher Scientific	ND-2000
NE-PER Nuclear and Cytoplasmic Extraction Reagents	Thermo Fisher Scientific	78833
Nrf2 Forward primer	Custom synthesis	Custom
Nrf2 Reverse primer	Custom synthesis	Custom
PBS with 0.05% Tween-80	Custom preparation	Custom
Penicillin-streptomycin	Gibco, Thermo Fisher Scientific	15140122
PowerUp SYBR Green Master Mix	Applied Biosystems, Thermo Fisher Scientific	A25741
QuantStudio 5 Real-Time PCR System	Thermo Fisher Scientific	A28140
RIPA buffer	Custom preparation	Custom
RNeasy Mini Kit	Qiagen, Hilden, Germany	74104
Sabouraud dextrose agar	Custom preparation	Custom
Slit-lamp biomicroscope	Custom equipment	N/A
SnPPIX (HO-1 inhibitor)	Frontier Scientific, Logan, UT, USA	F6140
Synergy HTX Multi-Mode Microplate Reader	BioTek Instruments, Winooski, VT, USA	SYNHXTTR
TBS-T (0.1% Tween-20)	Custom preparation	Custom
TissueLyser LT	Qiagen	69980
TNF-α ELISA Kit	R&D Systems, Minneapolis, MN, USA	DY210