Conversion from resistance to susceptibility: a dirty trick by PM 10

The results of whole-body exposure to airborne particulate matter <10 µm on the cornea of BALB/c mice, how their natural resistance to Pseudomonas aeruginosa (PA) keratitis is impacted, and whether plastoquinonyl-decyl-triphenylphosphonium (SKQ1) is protective were examined. Control or PM 10 exposure was performed for 2 weeks; corneal sensitivity, tear production, and Langerhans cell (LC) number were determined; and reverse transcription PCR (RT-PCR) was performed. After exposure, infection with PA, and topical treatment with PBS or SKQ1, clinical score, slit lamp, polymorphonuclear cells (PMN) quantitation, bacterial count, RT-PCR, Western blot, and flow cytometry were performed. PM 10 -exposed corneas showed significantly increased corneal sensitivity, decreased tear production, and a significant increase in conjunctival LC vs. control exposed eyes. PM 10 exposure significantly increased mRNA for proinflammatory cytokines and significantly decreased them for glutathione peroxidase 4 and Nrf2. Disease severity after PM 10 exposure was significantly increased at 3 and 5 dpi and resulted in higher plate counts and PMN numbers in the cornea at 5 dpi. Corneal mRNA levels after infection showed a similar pattern to the uninfected cornea after PM 10 exposure and were selectively confirmed by Western blot. Flow cytometry showed increases in macrophages and T cells in the cornea after infection following PM 10 exposure. SKQ1 significantly improved disease at both 3 and 5 dpi, restored mRNA and protein to control levels, reduced corneal bacteria and PMN number, and prevented increased sensitivity and tear reduction. Whole-body exposure to PM 10 resulted in converting BALB/c mice that exhibit genetic resistance to a susceptible phenotype in response to PA and SKQ1 reversed the majority of these effects and restored resistance.

Ambient air pollution, comprised of particulate matter (PM) (solid and liquid particles suspended in air) and gases, e.g., ozone, NO 2, and SO 2, has reached unparalleled levels worldwide. Airborne particulate matter <10 µm in diameter (PM 10) is a significant environmental pollutant with well-documented effects on human health, with chronic exposure creating a serious challenge to public health. According to the World Health Organization, 92% of the world's population is affected by contaminated air, and in both urban and rural areas, it is projected to cause 4.2 million premature deaths worldwide per year (1). Current research suggests that exposure to PM 10 alters the ocular surface in ways that may increase susceptibility to Pseudomonas aeruginosa (PA) keratitis. Laboratory and animal model data indicate that PM 10 induces oxidative stress and inflammation in corneal epithelial cells (2), reduces cell viability (3), disrupts barrier function (4), and impairs innate defense mechanisms, such as the Nrf2-mediated antioxidant response (2). Cellular protection against this insult depends on Nrf2-driven antioxidant responses and GPX4-mediated suppression of lipid peroxidation; disruption of these pathways compromises epithelial integrity and promotes inflammation, increasing susceptibility to ocular surface damage and infection. In mouse models, PM₁₀ exposure led to earlier corneal thinning/perforation in genetically susceptible C57BL/6 mice. Higher bacterial loads, increased inflammatory cell infiltrate, and elevated proinflammatory cytokines after infection suggest that particulate exposure can exacerbate disease severity and potentially enhance susceptibility to bacterial keratitis (5). These mechanistic findings are supported by epidemiological observations linking higher ambient PM₁₀ levels to increased outpatient visits for keratitis and other ocular surface diseases (6, 7). However, comprehensive human clinical data specifically linking PM₁₀ exposure to an increased risk of Pseudomonas keratitis remain limited, and further in vivo and population studies are needed to fully characterize this relationship.

PA, a gram-negative bacterium, is a common opportunistic pathogen and a contributing agent of microbial keratitis worldwide, resulting in acute ocular pain, corneal opacity, reduced visual acuity, and possibly blindness (8). Traumatic accidents involving the surface of the eye in developing countries (9), use of extended wear soft contact lenses (10), and the presence of preexisting or long-standing ocular conditions, such as dry eye (11), are among the most prevalent risk factors. The immune response to PA keratitis is driven by a complex interplay between Th1 and Th2 immune pathways. BALB/c mice are classically resistant to PA keratitis, typically mounting a Th2-skewed, anti-inflammatory response that limits corneal damage and promotes bacterial clearance (12). If PM₁₀ exposure causes BALB/c mice to develop more severe keratitis, corneal perforation, higher bacterial burden, or a shift toward pro-inflammatory cytokine profiles, this strongly supports the hypothesis that PM 10 actively reprograms host responses, rather than merely exacerbating an already susceptible background. It also provides real-world applicability, as PM 10 exposure may increase infection risk even in individuals without obvious pre-existing susceptibility.

PM₁₀ exposure can significantly influence Th1/Th2 immunity (13), generally skewing responses toward a Th1-dominant, pro-inflammatory phenotype, with important implications for host defense and tissue damage (14). Experimental studies show that PM₁₀ induces oxidative stress and activation of innate immune pathways, leading to increased production of Th1-associated cytokines while concurrently suppressing Th2-associated mediators. This shift has been observed in both pulmonary and extrapulmonary tissues, including the ocular surface, where PM₁₀ exposure can amplify inflammatory responses to secondary insults such as bacterial infection (3, 15). Th1 immunity, driven primarily by interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), performs a key function in defending against pathogens, including PA (16). T cells and natural killer (NK) cells make IFN-γ to activate macrophages. Once activated, macrophages produce TNF-α, enhance phagocytosis and antigen presentation, and release nitric oxide (NO) and reactive oxygen species (ROS) to kill intracellular pathogens. polymorphonuclear cells (PMN), although more associated with innate immunity, are also recruited in Th1-dominated inflammation and contribute to bacterial clearance and continued influx of macrophages (17).

This study presents data to aid in the crucial understanding of how PM 10 exposure influences the Th1/Th2 balance, as it may alter host susceptibility to PA keratitis. We show that PM 10 exposure can modulate immune responses from a resistant to a susceptible phenotype, affecting corneal sensitivity, tear production, cellular infiltrate, subsequent cytokine and chemokine responses, and the resulting acute disease response, and that these can be reversed by the mitochondrial antioxidant, plastoquinonyl-decyl-triphenylphosphonium (SKQ1).

Mice

Eight-week-old female BALB/cJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and were housed in accordance with the National Institutes of Health guidelines. The mice were humanely treated in compliance with both the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and the Institutional Animal Care and Use Committee of Wayne State University (IACUC 24-03-6665). We elected to use only female mice because of their smaller size, allowing them more space in the compartments of the exposure chamber for freedom of movement. Female mice also exhibit less aggressive tendencies when housed together compared to males, so damage to the infected cornea as a result of fighting would not confound data interpretation. In addition, previous studies (unpublished data) found no difference in the response of male mice to PA challenge.

Whole-body exposure to PM 10

The experiments were performed with PM 10 purchased from the National Institute of Standards and Technology (NIST) [Standard Reference Material (SRM) 2,787, complete analysis and composition included as Supplementary Data Sheet 1]. Exposure was conducted using a whole-body exposure system (CH Technologies, Westwood, NJ, USA) as previously described (5). Using two chambers, divided into 32 compartments, the mice were exposed to PM 10 in one and humidified, double-filtered air (control) in the other. Exposure to PM 10 or control air was carried out for 2 weeks, 3 h/day, 5 days/week. Over the daily 3 h period, the mice were exposed to PM 10 at a concentration of approximately 1,000 μg/m 3, a level similar to that reported in China, India, and parts of the Middle East (18–20).

Corneal sensation

A Cochet–Bonnet esthesiometer (Luneau SAS #10129; Prunay-le-Gillon, France), with a 0.12 mm diameter nylon filament, was used to assess corneal sensitivity before beginning exposure (0 weeks) and again after 2 weeks of control or PM 10 exposure, as described previously (2). A nylon filament was used by a masked observer to touch the corneal surface. A blink response indicated detection of the mechanical stimulus (i.e., a positive sensation response). In total, 10 repeat trials were performed for each filament length. This process was repeated until positive sensation responses were achieved for five or more of the 10 repeat trials, and this value was recorded as the corneal sensitivity threshold. These measurements were also taken at baseline (0 weeks) and after 2 weeks of control or PM 10 exposure from mice treated daily with PBS or SKQ1 (n = 5 mice/group) (see description below under SKQ1 treatment).

Tear secretion

Tear secretion was assessed before beginning exposure (0 weeks) and again after 2 weeks of exposure using a Phenol red thread test (Tianjin Jingming New Technological Development Co Ltd., Tianjin, China) as described previously (2). The edge of the thread was gently placed by a masked observer, touching the lower eyelid for 15 s. The wetted length (mm), indicated by a color change from yellow to red, was immediately measured. Tear secretion was also measured at baseline (0 weeks) and after 2 weeks of control or PM 10 exposure in mice treated with PBS or SKQ1 (n = 5 mice/group), as described below.

Preparation, staining, and quantitation of Langerhans cells

To compare the number of Langerhans cells (LCs) in the conjunctiva, epithelia were collected from mice exposed to control air or PM 10 (n = 5 mice/group) and stained with adenosine diphosphatase as described previously (21). Briefly, the corneas were incubated in 0.02 M EDTA-PBS buffer (pH 7.2) at 37 °C for 1 h to allow separation of corneal and conjunctival epithelia from the underlying stroma. Epithelial sheets were fixed in cacodylate-buffered formaldehyde for 20 min at 4 °C and washed four times. LCs were visualized by incubation in ADPase substrate containing ADPase buffer, 2% lead nitrate, and ADPase (5 mg/mL; Sigma) for 15 min at 37 °C, followed by four washes in Tris-maleate buffer (pH 7.2), and development for 5 min in a 1:10 ammonium sulfide solution. After three washes, epithelial sheets were mounted in glycerol on glass slides and coverslipped. Representative areas were photographed at 20X on a Leica DM4000B microscope Leica Microsystems (CMS GmbH, Wetzlar), and the total number of LCs in 10 images for each exposure group was counted for statistical comparison.

Bacterial culture and corneal infection

PA strain ATCC 19,660 (Manassas, VA, USA) was prepared as described previously (22). In brief, the culture was grown in 25 mL peptone tryptic soy broth medium in a rotary shaking water bath at 37 °C and 150 rpm overnight until an optical density between 1.3 and 1.8 (read at 540 nm) was obtained. The culture was centrifuged at 5,500 × g for 10 min, and pellets were washed and diluted in sterile saline. The mice were anesthetized with ether and placed under a stereoscopic microscope at 40× magnification. Using a 25 5/8-gauge needle, three parallel wounds were made to the corneal surface, and 5 μL containing 1 × 10 6 colony-forming units (CFUs)/μL of the bacterial suspension was applied topically.

Ocular response to bacterial infection

To assess and compare the ocular response to infection, clinical scores published previously by this lab were used. Numerical scores were designated as follows: 0 = clear or slight opacity, partially or fully covering the pupil; +1 = slight opacity, fully covering the anterior segment; +2 = dense opacity, partially or fully covering the pupil; +3 = dense opacity, covering the entire anterior segment; and +4 = corneal perforation or phthisis (23). Each mouse was scored on days 1, 3, and 5 dpi by a masked observer for statistical comparison and photographed at 5 dpi with a slit lamp to record disease.

SKQ1 treatment

SKQ1 (BOC Sciences, Shirley, NY, USA) is a mitochondria-targeted antioxidant designed to accumulate selectively in the mitochondrial inner membrane, where it neutralizes excess ROS at the source (24). Across ocular and inflammatory models, SKQ1 has been shown to preserve mitochondrial membrane potential, reduce mitochondrial ROS production, prevent lipid peroxidation, and maintain ATP generation (25). In the eye, SKQ1 exhibits strong cytoprotective and anti-inflammatory effects, improving corneal epithelial integrity, reducing oxidative damage, and limiting neutrophil-driven tissue injury (26). SKQ1 was used to treat the control and PM 10 -exposed mice using the published dose of 7.5 μM (27). SKQ1 was dissolved in 50% ethanol to obtain a 162 mM stock solution and was diluted daily to a 7.5 μM working solution for topical application (28). The stock solution was stored at −20 °C for up to 6 months with no loss of stability (29). The eyes were treated topically with 5 μL SKQ1 or PBS (control) three times on the day before the first chamber exposure and once each day before control or PM 10 exposure. Treatment with PBS or SKQ1 was continued after infection with PA, beginning at 6 hpi, followed by a single daily topical treatment on days 1–4 dpi.

Bacterial plate count

Viable bacteria were quantified in infected corneas from PBS- and SKQ1-treated, control vs. PM 10 -exposed mice (n = 5 mice/group) as described before (5). Infected corneas were removed at 3 and 5 dpi and homogenized in 1 mL of sterile saline containing 0.25% bovine serum albumin (BSA), serially diluted 1:10, and selected dilutions plated in triplicate on Pseudomonas isolation agar plates (Becton Dickinson, Franklin Lakes, NJ, USA). After overnight incubation at 37 °C, bacterial colonies were counted and reported as log 10 CFU/cornea + SEM.

Myeloperoxidase assay

A myeloperoxidase (MPO) assay was used to quantify neutrophils in the infected corneas of mice exposed to control air or PM 10 and treated with SKQ1 or PBS (n = 5 mice/group) (5). Briefly, individual corneas were removed at 3 and 5 dpi and homogenized in 1 mL of 50 mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethyl–ammonium (Sigma-Aldrich, St. Louis, MO, USA). Samples were freeze–thawed four times, centrifuged, and 100 μL of the supernatant was added to 2.9 mL of 50 mM phosphate buffer containing o-dianisidine dihydrochloride (16.7 mg/mL; Sigma-Aldrich) and hydrogen peroxide (0.0005%). The changes in absorbance were monitored at 460 nm for 4 min at 30 s intervals. The optical densities obtained for each cornea were plotted and the slope of the line was determined and used to calculate units of MPO/cornea. One unit of MPO activity equals approximately 2 × 10 5 neutrophils (30).

Reverse transcription PCR

Total RNA was isolated (RNA STAT-60; Tel-Test, Friendswood, TX, USA) from corneas exposed to control air or PM 10, as reported previously (n = 5 mice/group) (31). One microgram of each RNA sample was reverse transcribed using Moloney-murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA, USA) to produce a cDNA template. cDNA products were diluted 1:20 with diethylpyrocarbonate-treated water, and a 2 μL aliquot of diluted cDNA was used for the reverse transcription PCR (RT-PCR). SYBR green/fluorescein PCR master mix (BioRad Laboratories, Richmond, CA, USA) and primer concentrations of 10 mM were used in a total 10 μL volume. After a preprogrammed hot start cycle (3 min at 95 °C), the parameters used for PCR amplification were 15 s at 95 °C and 60 s at 60 °C, with the cycle repeated 45 times. The fold differences in gene expression were calculated relative to naïve controls and normalized to the housekeeping gene 18S rRNA. Similar mRNA analysis was performed on infected corneas from mice exposed to control air or PM 10 treated with PBS or SKQ1. Primer pair sequences used for RT-PCR are shown in Table 1.

Western blots

To investigate potential changes in protein expression, Western blot analysis was performed as previously described (5). Infected corneas from control air- and PM 10 -exposed mice treated with PBS or SKQ1 (n = 5 mice/group) were taken at 5 dpi and placed into ice-cold 0.1 M PBS (pH 7.4), lysed in RIPA buffer with protease and phosphatase inhibitors (SantaCruz Biotech, Dallas, TX, USA), incubated on ice for 20 min, centrifuged at 12,000 × g at 4 °C for 10 min, and the supernatant was collected. Total protein was determined using a BCA protein kit (ThermoFisher Scientific, Rockford, IL, USA). In brief, samples (35 μg) were run on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) system in Tris-glycine-SDS buffer and electroblotted onto nitrocellulose membranes (BioRad). Membranes were blocked for 1 h in 5% MTBST [Tris Buffer Saline containing 0.05% Tween 20 (TBST) and 5% non-fat milk], then probed with primary antibodies: rabbit antimouse tumor necrosis factor alpha (TNF-α, 1:1,000; Cell Signaling Technology, Danvers, MA, USA) and nuclear factor erythroid 2 related factor 2 (NRF2; 1:1,000; Cell Signaling Technology) in 5% BSA in TBST overnight at 4 °C. After three washes with TBST, membranes were incubated with HRP-conjugated antirabbit secondary antibody (1:2,000; Cell Signaling Technology) and diluted with 5% BSA in TBST at room temperature for 2 h. Positive reactivity was visualized with Supersignal West Femto Chemiluminescent Substrate (ThermoFisher Scientific), visualized using an iBright™ CL1500 Imaging System (ThermoFisher Scientific), normalized to β-actin (1:1,000; Abcam, Waltham, MA, USA), and the intensity was quantified using Image Lab 6.1 software (BioRad).

In addition to conventional Western blot analysis, rapid detection of additional proteins was performed using the Protein Simple Abby System (Biotechne, Minneapolis, MN, USA) to facilitate detection of interleukin (IL)-18 (IL-18), IFN-γ, and GPX4 in exposed infected corneas treated with PBS or SKQ1. After removal, whole corneas were ground in V-bottom glass grinders in 200 μL of RIPA buffer, supplemented with a protease cocktail, until the tissue was completely dissolved. The samples were centrifuged at 12,500 RPM to remove any debris, and the supernatant was removed for further processing. Protein estimation of the supernatant was performed using a Bradford Assay (BioRad, USA) and samples diluted with PBS to a concentration of 0.25 μg/μL as per titration experiments and the manufacturer’s guidelines. The samples were further processed for the Protein Simple Abby Western system using the Protein Simple kits and accompanying guidelines. The antibodies, IL-18, IFN-γ (Proteintech, Rosemont, IL, USA), and GPX4 (Abcam), were used as the primary at a dilution of 1:10, with the manufacturer-provided antirabbit HRP-conjugated secondary used as the detection probe. The samples were then re-probed using Abby's RePlex option with HRP-conjugated β-tubulin or β-actin at a dilution of 1:200 (Proteintech, USA) as the housekeeping protein. Densiometric readings were taken via the software provided with the Protein Simple Abby System, Compass for SW (Biotechne), and normalized against β-tubulin or β-actin.

Flow cytometry

Individual infected corneas from PM 10 - (± SKQ1 treatment, five mice per treatment group) and control air-exposed mice were harvested at 5 dpi and prepared for flow cytometry as described before (32). Individual corneas were harvested into 250 μL RPMI 1,640 medium without serum. Liberase TL (Roche, Indianapolis, IN, USA; 2.5 mg/mL) was added to each tube, followed by incubation at 37 °C for 45 min using a Disruptor Genie (Scientific Industries, Bohemia, NY). Samples were triturated by passing through a 70 μm cell strainer using a 3 mL syringe plunger, and the single-cell suspension was washed with 5 mL RPMI 1,640 containing 10% FBS and pelleted at 315 × g for 8 min in a refrigerated centrifuge. Cells were stained for cell surface CD45, CD3, CD56, and F4/80. To identify specific cell types, the following antibodies were used: PE conjugated rat antimouse CD45 (553,081/561,087, 1 μg per 1 million cells) (BD Biosciences, San Jose, CA, USA), PE-Cy5 conjugated rat antimouse CD3 (17A2, 1 μg per 1 million cells) (Biolegend, San Diego, CA, USA), RealBlue 780 conjugated rat antimouse CD56 (809,220, 1 μg per 1 million cells) (BD Biosciences), and CoraLite Plus 488 conjugated rabbit antimouse F4/80 (CL488-28463, 1 μg per 1 million cells) (Proteintech). Samples were acquired on a Cytek Northern Lights (Fremont, CA, USA) machine and analyzed using FlowJo software (Ashland, OH, USA).

Statistical analysis

GraphPad Prism software (Boston, MA, USA) was used to perform statistical analyses for all data. An in vivo comparison of clinical scores between two groups at each time was conducted using the Mann–Whitney U -test. An unpaired Student's t -test was used to compare the two exposure groups for sensitivity, tear production, LC number, and RT-PCR data. A one-way ANOVA followed by Bonferroni's multiple comparison test was used for plate counts, MPO, RT-PCR, and Western blot data when SKQ1 treatments were employed. Data were considered significant at a p -value of <0.05. All experiments were repeated at least once to ensure reproducibility, and the data are shown as mean + SEM.

Corneal sensitivity and tear production

Changes in corneal sensitivity and tear production are biologically important determinants of susceptibility to ocular infection because they directly regulate the integrity of the ocular surface defense system. The effects of whole-body exposure to PM 10 vs. control on corneal sensitivity and tear secretion at 0 (baseline) and 2 weeks of exposure are shown in Figures 1A,B. Corneal sensitivity before PM 10 or control air exposure was not significantly different; however, a significant increase in sensitivity was found between control air and PM 10 after 2 weeks of exposure (p < 0.01). The PM 10 -exposed mice at baseline, compared to after 2 weeks of exposure, also showed a significant increase in corneal sensitivity (p < 0.01) (Figure 1A). Tear volume significantly decreased in the PM 10 -exposed mice between baseline and after 2 weeks of exposure (p < 0.01). There was no difference in tear volume between the exposure groups at either baseline or after 2 weeks of exposure (Figure 1B).

Langerhans cell number

LCs enhance antigen presentation and T-cell priming, shifting the local immune milieu toward Th1 dominance, resulting in susceptibility. To examine the response of BALB/c mice to control air vs. PM 10 exposure, the number of LCs in the conjunctiva after 2 weeks of exposure was quantitated (Figure 2A). The number of LCs significantly increased in the conjunctiva of mice exposed to PM 10 compared to control air (p < 0.05). Representative images taken from control air- and PM 10 -exposed conjunctiva are shown in Figures 2B,C, respectively.

RT-PCR for cytokines/chemokines and oxidative stress molecules

Oxidative stress reprograms cytokine and chemokine signaling, amplifying pro-inflammatory pathways while suppressing regulatory and antioxidant defenses. RT-PCR was used to characterize gene expression after PM 10 exposure. RT-PCR tested whether PM 10 exposure enhanced pro- or anti-inflammatory and oxidative stress molecules in BALB/c mouse corneas (Figures 3A–L). Exposure to PM 10 significantly elevated relative mRNA levels of TNF-α, IL-1β, C-X-C motif chemokine ligand 2 (CXCL2), IL-6, IL-18, IFN-γ, cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS) (Figures 3A–D,G–J) when compared to control air (p < 0.01 for A, B, p < 0.001 for the remaining). mRNA levels of IL-10 and IL-12 (Figures 3E,F) were unchanged by PM 10 exposure. GPX4 and Nrf2 (Figures 3K,L) were significantly reduced (p < 0.001) in PM 10 -exposed corneas compared with control exposed corneas.

Effect of SKQ1 on corneal sensitivity and tear production

The mitochondrial antioxidant, SKQ1, was used to treat eyes before and during the exposure period to test its effects on sensitivity and tear production (Figures 4A,B). Sensitivity (Figure 4A), significantly increased after 2 weeks of PM 10 exposure (p < 0.001), was returned to control levels by SKQ1 treatment. SKQ1 treatment of PM 10 -exposed corneas also prevented the significant (p < 0.01) reduction in tear production, returning tear levels to those detected at baseline measurement (Figure 4B).

Clinical score, bacterial load, and MPO

Treatment of infected corneas with SKQ1 after PM 10 and control air exposure showed significant protection from more severe corneal disease at 3 dpi and prevented perforation at 5 dpi (p < 0.01 for both time points, Figure 5A). Photographs taken with a slit lamp at 5 dpi show dense opacity covering the pupil with hypopyon in PBS (Figure 5B) and SKQ1 (Figure 5C) treated control air-exposed corneas. After PM 10 exposure, 50% of the PBS-treated corneas were perforated (Figure 5D), but SKQ1 treatment diminished the severity of disease and reduced the number of perforations (Figure 5E). The elevated number of viable bacteria in the cornea seen at 5 dpi following PM 10 exposure compared to control air was also decreased to near the control levels after treatment with SKQ1 (Figure 5F, p < 0.05). Topical application of SKQ1 lowered the number of PMN present in the cornea of mice exposed to either control air or PM 10 at 3 (p < 0.05 and p < 0.001) and 5 dpi (p < 0.01 and p < 0.001) to below the numbers enumerated in the control air- or PM 10 - exposed PBS-treated groups at both time points (Figure 5G).

RT-PCR and Western blot analysis

RT-PCR analysis of PBS- vs. SKQ1-treated PM 10 - and control air-exposed infected corneas at 5 dpi (Figures 6A–L). Similar to the untreated, infected corneas, exposure to PM 10 significantly elevated relative mRNA levels of TNF-α (p < 0.001), IL-1β (p < 0.001), CXCL2 (p < 0.001), IL-6 (p < 0.001), IL-18 (p < 0.001), IFN-γ (p < 0.001), COX2 (p < 0.001), and iNOS (p < 0.001) (Figures 6A–D,G–J) when compared to control air and mRNA levels of IL-10 (p < 0.001), GPX4 (p < 0.001), and Nrf2 (p < 0.001) (Figures 6E–L) were significantly reduced by PM 10 compared to control air exposed corneas. SKQ1 reduced levels of mRNA in the control and PM 10 -exposed groups (Figures 6A–D,G–J), returning them to levels equal to or higher than those seen in the PBS-treated groups. SKQ1 increased the levels of mRNA in control and PM 10 -exposed groups (Figures 6E–L), returning them to levels equal to or lower than those seen in the PBS-treated groups. No change was detected in the level of IL-12 between the exposure groups; however, treatment with SKQ1 reduced mRNA levels in both control and PM 10 -exposed samples (Figure 6F).

After exposure to control air or PM 10, treatment with PBS or SKQ1, and infection, Western blots confirmed RT-PCR data by examining protein levels of selected cytokines and oxidative stress molecules (Figures 7A–E). PM 10 exposure followed by infection significantly increased the protein levels of TNF-α (p < 0.001, A), IL-18 (p < 0.01, B), and IFN-γ (p < 0.05, C) and decreased GPX4 (D) and NRF2 (E) (p < 0.001 and p < 0.01, respectively). Similar to the RT-PCR analysis results, treatment with SKQ1 reversed the effects of PM 10 exposure on protein levels.

Flow cytometry

To ascertain the origin of the increased levels of inflammatory cytokines and chemokines found in the PM 10 -exposed corneas after infection, flow cytometry was performed at 5 dpi to identify and quantitate infiltrating inflammatory cells (Figures 8A–L). Hematopoietic cells were identified using a CD45 antibody and further characterized as macrophages, T cells, or NK cells using antibodies for cell-specific markers. Staining for CD45 + cells with F4/80, a macrophage-specific marker, revealed a significant rise in the percentage of macrophages in the PM 10 - vs. control-exposed corneas (p < 0.05, Figures 8A–D). The number of CD45 + cells that were identified as T cells using a CD3 antibody was also significantly higher in PM 10 -exposed corneas compared to those exposed to control air (p < 0.001, Figures 8E–H). The percentage of NK cells (CD45 +, CD56 +) was elevated in the corneas of the PM 10 - vs. control-exposed mice, but the increase was not statistically significant (Figures 8I–L). SKQ1 treatment reduces the frequency of all three cell types after PM 10 exposure.

Epidemiological studies have shown a direct association between long-term exposure to PM 10 (PM with an aerodynamic diameter ≤10 µm) and increased mortality (33–35). PM 10 exposure is associated with an increased incidence of cardiovascular (35) and pulmonary diseases (36), diabetes (37), and cancer (38). Moreover, PM 10 negatively impacts the ocular surface due to direct exposure. PM 10 exposure has been associated with worsened tear film stability in patients diagnosed with dry eye disease (39), higher risk of Sjogren's syndrome (40), and increased emergency room visits for keratitis (6). Thus, our purpose was to investigate the effects of PM 10 on a mouse strain that is Th2-driven (BALB/c), to determine whether a Th2 vs. Th1 (12) phenotype modifies disease outcome, particularly after infection.

Previously, others reported that PM 10 exposure in drops (5 mg/mL, 4X/day) reduced tear volume, leading to tear film abnormality, with a reduction in conjunctival goblet cells in male BALB/c mice (41). When we examined the effects of PM 10 exposure (500–1,000 μg/m 3 in a whole-body exposure chamber), a significant decrease in tear volume was also observed after 2 weeks of exposure in female mice. Our results are also similar to a recent study in female rats exposed to PM in an exposure chamber (500 μg/m 3 for 2 weeks) that showed a significant reduction in tear volume after 14 days (42). Thus, neither the method of delivering the particulate, sex, nor species differences were detectable.

We also observed increased corneal sensitivity after 2 weeks of PM 10 exposure. Increased corneal sensitivity after PM 10 exposure can occur due to several factors, including PM 10 -induced damage to corneal nerves (43), oxidative stress (2), and impaired corneal function (44). This damage can result in a reduced ability of the cornea to detect touch or other stimuli (42). We did not explore whether PM 10 had a direct impact on corneal nerves, which would be of interest because of the neuroimmune interactions that could have been compromised and affected by cytokine production (45). A study in this regard is underway.

Past studies by this lab have shown that inducing LCs into the central cornea before infection exacerbated disease in BALB/c, a Th2-responsive mouse strain normally resistant to PA keratitis, converting a resistant phenotype to a susceptible phenotype (21). Whole-body exposure of this mouse strain to PM 10 led to a significant increase in LCs in the conjunctiva when compared to controls. The peripheral cornea, being proximate to the conjunctiva, possesses immunological components essential to producing an immune response (46), so this effect in the conjunctiva could play a role in the acceleration of disease progression. Increased corneal LC density correlates with epithelial damage, tear dysfunction, and disease severity, supporting LC density as a clinical biomarker (47).

The levels of TNF-α, IL-1β, CXCL2, IL-6, IL-18, IFN-γ, COX2, and iNOS were elevated, while oxidative stress markers GPX4 and Nrf2 were downregulated, a pattern similar to that observed in susceptible C57BL/6 mice after similar PM 10 exposure (5). PM 10 exposure can trigger inflammation in the body, leading to increased production of certain cytokines. PM 10 can cause a considerable elevation in the production of cytokines such as IL-6, IL-1β, and IL-8, according to a study published by the National Institutes of Health (NIH). Experiments using RNA-seq analysis of PM 10 -exposed human peripheral blood mononuclear cells showed that PM 10 was able to alter the expression of 1,196 genes in immune cells, involving the activation of a proinflammatory status and a rise in cytokines and chemokines. In addition, upregulating chemokines includes neutrophil and monocyte recruitment, proposing potential mechanisms for increased inflammation following PM exposure (48).

To determine the effect of SKQ1 on the restoration of sensitivity levels in the absence of infection, we tested its effects at baseline and 2 weeks after exposure. Others have reported that PM 10 induces inflammatory responses, mitochondrial impairment, and can modify tear composition (44). These events impact ocular diseases such as dry eye, conjunctivitis, and keratitis. Preexisting disorders and illnesses, such as dry eye and infectious keratitis, can be exacerbated by PM 10 exposure (49). Our whole-body exposure model does not appear to induce the clinical dry eye disease seen in other PM 10 treatment models (41, 50), but results in the presentation of milder symptoms, including reduced tear production and corneal hypersensitivity (49). SKQ1 is available as an eye-drop formulation (51) and has been shown to not only lessen inflammation but also prevent tissue degeneration and tear quality insufficiency by affecting oxidative stress within the eye. In VISTA-1, a Phase 2b/3 clinical study in the United States (NCT03764735), SKQ1 demonstrated verification of efficacy in diminishing both the signs and symptoms in subjects with dry eye (52). Treatment with SKQ1 in BALB/c mice was also successful in preventing tear reduction and an increase in corneal sensitivity.

We next tested the infected corneal response to SKQ1 treatment following PM 10 exposure. Treatment with SKQ1 reduced disease severity in PM 10 -exposed mice. SKQ1 also significantly decreased plate counts at 5 dpi. SKQ1 has been shown by others to possess antibacterial properties against Gram-positive and some Gram-negative bacteria by targeting bacterial bioenergetics (53). This laboratory tested its efficacy against PA and confirmed that it was also susceptible to SKQ1 (5). This is likely one mechanism, in addition to its host immunomodulatory effects, responsible for our observation of reduced bacteria in the cornea after SKQ1 vs. PBS treatment.

Although more associated with innate immunity, neutrophils are recruited in both Th1 and Th2-dominated inflammation and contribute to bacterial clearance. Neutrophils play a complex role in PA keratitis. While they are initially recruited to combat the bacteria, they also contribute to the progression of disease pathogenesis through the destruction of innocent bystander tissue, resulting in perforation. MPO was increased by PM 10 exposure and reduced by SKQ1. Treatment of infected corneas from C57BL/6 mice exposed to PM 10 with SKQ1 was also effective in decreasing MPO (5). In support of our data, Demianenko et al. showed that treatment with SKQ1 by subcutaneous injection, Bioplen film application containing SKQ1, or SKQ1 supplied daily in drinking water, suppressed PMN infiltration in rats and mice after dermal wounds or aseptic inflammation (54).

After infection, a panel of cytokines/chemokines and oxidative stress molecules was examined using RT-PCR and Western blot after PM 10 or control air exposure and PBS or SKQ1 treatment. The inflammatory environment induced by PM 10 exposure was characterized by an increased expression of cytokines commonly associated with Th1 vs. Th2 responses. IL-10 plays a critical role in modulating inflammatory responses during PA keratitis, as IL-10 deficiency alters neutrophil recruitment and reduces bacterial burden, demonstrating its function in balancing pro- and anti-inflammatory mediators (55, 56). PM 10 exposure is associated with decreased IL-10 expression alongside elevated pro-inflammatory markers, consistent with an inflammatory bias that may contribute to increased susceptibility (3). In contrast, IL-12 promotes Th1-associated cytokines such as IFN- γ and TNF-α, and exogenous IL-12 drives susceptible phenotypes even in normally resistant hosts, linking heightened pro-inflammatory signaling to increased disease severity (57). While these cytokine patterns are consistent with a shift toward a pro-inflammatory response, they should be viewed with caution, as cytokine expression provides only indirect evidence of T-cell polarization. This inflammatory skewing, in conjunction with increased Langerhans cell density and oxidative stress, likely contributes to the loss of immune regulation and increased susceptibility to infection following PM 10 exposure.

Flow cytometry was next employed to determine whether PM 10 exposure caused a modification in type (macrophage, T cell, or NK cells) or number of inflammatory cells in the infected cornea and whether this was the mechanism underlying the changes in disease response. In this regard, this lab demonstrated that macrophages control resistance to PA keratitis through the regulation of PMN infiltration to the cornea, bacteria killing, and the ability to successfully balance the levels of pro- and anti-inflammatory cytokines. Selective depletion of macrophages by injection of liposomes containing dichloromethlyene diphosphonate (clodronate) exacerbated PA keratitis, converting the resistant BALB/c to a susceptible phenotype, supporting the hypothesis that dysregulation of macrophage number after infection is detrimental to disease resolution (58). Macrophages are activated by IFN-γ (elevated by PM 10) to enhance phagocytosis and antigen presentation. IL-18 is produced by macrophages and dendritic cells and possesses similar functions to those of IL-12, including the initiation of IFN-γ production by both T cells and NK cells (59). NK cells and T cells produce IFN-γ and further initiate production of macrophage-derived mediators such as TNF-α, IL-1, IL-6, IL-12, IL-18, and NO, which are also increased after PM 10 exposure. A previous study from this lab used CD8-knockout mice (C57BL/6 background) and antibody depletion of CD4T cells in C57BL/6 mice to show the role of specific T-cell subsets in the susceptibility response to PA keratitis (22).

We demonstrated that PM 10 exposure alters cytokine and chemokine expression, likely reflecting increased inflammatory cell infiltration into the infected cornea. Nrf2 is a key regulator of immune cell function, influencing macrophages, neutrophils, lymphocytes, and neutrophil recruitment during inflammation. Reduced Nrf2 expression contributes to cellular immune activation and modulates immune polarization, particularly within T-cell subsets. Activation of Nrf2 suppresses the differentiation of pro-inflammatory Th1, Th17, NKT1, and NKT17 cells while promoting Th2, Treg, and NKT2 populations (60). In our model, decreased Nrf2 expression shifted the normally Th2-biased response of BALB/c mice toward a Th1-skewed inflammatory phenotype. Nrf2 activation also promotes anti-inflammatory M2 macrophage differentiation (61) and limits the transcriptional upregulation of pro-inflammatory cytokine genes, positioning Nrf2 as an upstream regulator of cytokine production and inflammation (62). Disruption of Nrf2-mediated stress responses increases susceptibility to infection and inflammatory disease; although the mechanisms remain incompletely defined, Nrf2-regulated antioxidant genes likely coordinate innate immune responses and control pro-inflammatory mediator expression (63).